Astrobiology and Cuatro Ciénegas Basin As An Analog of Early Earth

Cuatro Ciénegas Basin: An Endangered Hyperdiverse Oasis
Valeria Souza
Antígona Segura
Jamie S. Foster Editors
Astrobiology and
Cuatro Ciénegas
Basin as an
Analog of Early
Earth
Cuatro Ciénegas Basin: An Endangered
Hyperdiverse Oasis
Series Editors
Valeria Souza, Universidad Nacional Autónoma de México, Ecology Institute,
Mexico City, Distrito Federal, Mexico
Luis E. Eguiarte, Universidad Nacional Autónoma de México, Ecology Institute,
Mexico City, Distrito Federal, Mexico
This book series describes the diversity, ecology, evolution, anthropology,
archeology and geology of an unusually diverse site in the desert that is paradoxically
one of the most phosphorus-poor sites that we know of. The aim of each book is to
promote critical thinking and not only explore the natural history, ecology, evolution
and conservation of the oasis, but also consider various scenarios to unravel the
mystery of why this site is the only one of its kind on the planet, how it evolved, and
how it has survived for so long.
More information about this series at http://www.springer.com/series/15841

Valeria Souza • Antígona Segura • Jamie S. Foster
Editors
Astrobiology and Cuatro

Ciénegas Basin as an Analog
of Early Earth
Editors
Valeria Souza Antígona Segura
Instituto de Ecología Instituto de Ciencias Nucleares
Universidad Nacional Autónoma de Universidad Nacional Autónoma de
México, UNAM México, UNAM
Mexico City, Mexico Mexico City, Mexico
Jamie S. Foster
Space Life Sciences Lab
University of Florida
Merritt Island, FL, USA
ISSN 2523-7284 ISSN 2523-7292 (electronic)

Cuatro Ciénegas Basin: An Endangered Hyperdiverse Oasis
ISBN 978-3-030-46086-0 ISBN 978-3-030-46087-7 (eBook)
https://doi.org/10.1007/978-3-030-46087-7
© Springer Nature Switzerland AG 2020

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Preface
This is the fifth book of the Cuatro Ciénegas Basin (CBB): An Endangered
Hyperdiverse Oasis series and targets the dynamic field of astrobiology. Cuatro
Ciénegas Basin (CCB) is one of the most important astrobiological sites that exists
on Earth, and in this book we examine how model systems, such as CCB, can serve
as critical platforms to understand the origin, evolution, and distribution of life
throughout the Universe. In this book, we begin with Chaps. 1 and 2 by explaining
the field of astrobiology as well as its development in Mexico. However, as we only
know of one experiment of life, planet Earth, locations such as CCB become critical
to understand what makes a world habitable. Chapter 3 examines the origins of life
and what were the probable conditions where the Last Unique Common Ancestor,
or LUCA, arose. Following, Chap. 4 explains why stromatolites serve as important
reservoirs for the study of life’s biosignatures, and Chap. 5 shows the geographical
distribution of stromatolites in modern times. However, to understand the formation
of stromatolites and their microbial diversity, Chap. 6 examines the rare biosphere
and discusses the hypothesis that in the beginning of diversification, all life was
rare. In Chap. 7, the authors examine bacterial communities living in hot and high-
pressure environments in hydrothermal systems at the ocean floor, which may har-
bor relict physiological capabilities that show microbial life in its extremes and also
may reflect conditions similar to Jupiter and Saturn moons. Meanwhile, Chaps. 8
and 9 show stromatolites in different sites of Latin America, including the Andes
and additional sites in México, with the aim of comparing these important astrobi-
ology-relevant ecosystems. Chapter 10 presents the Mexican contribution to under-
stand the origins of life and their current lines of research that look at the first stages
of the evolution of life. Finally, the closing chapter is about the Archaea at a particu-
lar CCB site and discusses why CCB is such a special site for Astrobiology.
Although CCB is not as extreme of an environment, compared to Mars or the
moons of Jupiter and Saturn, it is a special place for astrobiology due to its long and
ancient history. CCB is a lost world that has survived and retained the ancient lin-
eages inside the deep aquifer in the Sierra under conditions akin to an ancient,
magmatic influenced, ocean where phosphorous was extremely limited, sulfur was
rich, and oxygen was poor. In the case of CCB, under the mountain magma heats the
v
vi Preface
deep aquifer and water, along with deep microbes and minerals, surges to the sur-
face, forming ponds, lakes, rivers and pozas. This unique oasis creates an ecosystem
where stromatolites and microbial mats flourish under the sun. The CCB, therefore,
represents a co-evolving community where the feedbacks between microbial life
and the environment drive the habitability of this unique ecosystem. The underlying
mechanisms that makes CCB an analogue of early Earth and Mars is precisely what
makes life so diverse, the extreme low abundance of phosphorus, a rare element in
the first three billion years of life. Despite the skew in stoichiometry balance, or
maybe because of it, CCB is a hotspot of biodiversity. This amazingly diverse eco-
system has been studied by a large group of scientists and has been the theme of a
series of seven books published by Springer.
We believe that CCB is an ideal analogue of Mars, a place where the ancient
shallow ocean probably was poor in phosphorus and rich in the same prebiotic cos-
mic soup that made the origin of life possible on Earth. It is also possible that if
Mars life evolved, it may also exhibit chemolithotroph metabolisms, paralleling that
of modern sulfate-reducing bacteria and methanogenic archaea. Although the recent
missions to Mars have not been tasked to specifically look for life, these missions
have been quite successful in evaluating the potential habitability of Mars.
Understanding what makes a world habitable is the extraordinary gift that the CCB
deep aquifer can show us and help inform what characteristics or biosignatures to
look for on other worlds. Nevertheless, CCB is succumbing to overexploitation of
its most precious resource, water. Therefore, the ancient life in this system is slip-
ping away in front of us. The overall message of this book is that we can stop deg-
radation of our planet and protect critical habitats that enable us to learn about the
boundaries of life on this planet and beyond. Perhaps, we can save humanity from
destruction if we dare to imagine another society, a society that abides to life and
tries to understand its rules and follow its lessons. In the case of CCB, we as scien-
tists have been working towards improving awareness of this precious ecosystem
through art, education, and science. It is our overarching goal to improve the pub-
lic’s understanding, appreciation, and love for the unique life, beauty of the oasis at
Cuatro Ciénegas Basin, as well as increase awareness that such unique ecosystems
needs to be nurtured and protected.
Mexico City, Mexico Valeria Souza

Mexico City, Mexico Antígona Segura
Merrit Island, FL, USA Jamie S. Foster
Contents
1 What Is Astrobiology? �� 1

Antígona Segura, Sandra Ignacia Ramírez Jiménez,
and Irma Lozada-Chávez
2 Astrobiology and Planetary Sciences in Mexico�� 31
Karina Cervantes de la Cruz, Guadalupe Cordero-Tercero,
Yilen Gómez Maqueo Chew, Irma Lozada-Chávez, Lilia Montoya,
Sandra Ignacia Ramírez Jiménez, and Antígona Segura
3 Was LUCA a Hyperthermophilic Prokaryote?
The Impact-Bottleneck Hypothesis Revisited �� 75
Gilberto P. Morales and Luis Delaye
4 Stromatolites, Biosignatures, and Astrobiological Implications�� 89
Jamie S. Foster, Joany Babilonia, Erica Parke-Suosaari,
and R. Pamela Reid
5 The Global Distribution of Modern Microbialites:
Not So Uncommon After All�� 107
Richard Allen White III
6 The Importance of the Rare Biosphere for Astrobiological
Studies and the Diversification and Resilience of Life on Earth �� 135
Jazmín Sánchez-Pérez, Juan Diego Villar, Nohely Alvarez-López,
Bernardo Águila, Jhoselinne Buenrostro, Luis J. Chino-Palomo,
Marisol Navarro-Miranda, Julián Felipe Cifuentes,
Ana G. Cruz-Cruz, Benjamín Vega-Baray, Mariette Viladomat,
Maria Kalambokidis, Luis E. Eguiarte, and Valeria Souza
vii
viii Contents
7 Bacterial Communities from Deep Hydrothermal Systems:

The Southern Gulf of California as an Example
of Primeval Environments�� 149
Laura Espinosa-Asuar, Luis A. Soto, Diana L. Salcedo,
Abril Hernández-Monroy, Luis E. Eguiarte, Valeria Souza,
and Patricia Velez
8 Andean Microbial Ecosystems: Traces in Hypersaline
Lakes About Life Origin�� 167
Luis A. Saona, Mariana Soria, Patricio G. Villafañe,
Agustina I. Lencina, Tatiana Stepanenko, and María E. Farías
9 Stromatolites in Crater-Lake Alchichica and Bacalar Lagoon�� 183
Luisa I. Falcón, Patricia M. Valdespino-Castillo,
Rocio J. Alcántara-Hernández, Elizabeth S. Gómez-Acata,
Alfredo Yanez-Montalvo, and Bernardo Águila
10 The Origin and Early Evolution of Life on Earth:
A Laboratory in the School of Science�� 203
José Alberto Campillo-Balderas and Arturo Becerra
11 Cuatro Ciénegas as an Archaean Astrobiology Park�� 219
Nahui Olin Medina-Chávez, Susana De la Torre-Zavala,
Alejandra E. Arreola-Triana, and Valeria Souza
Index�� 229
Chapter 1
What Is Astrobiology?
Antígona Segura, Sandra Ignacia Ramírez Jiménez,
and Irma Lozada-Chávez
Abstract Astrobiology is an inherently multidisciplinary field that is focused on

the origins, evolution, and distribution of life throughout the Universe. The question
of whether life extends beyond Earth was a question that used to be answered mostly
based on human imagination reflecting our passions and fears. Philosophers, scien-
tists, and even politicians, such as Winston Churchill, have argued about the exis-
tence (or nonexistence) of alien life in the Universe. For scientists, this ambitious
endeavor begins with Earth, as it represents the only known example of life in the
Universe. Understanding Earth is, therefore, the first step to understanding the
requirements for life to emerge and make a habitable world. In this book, with the
collaboration of scientists from many disciplines, we gather the knowledge about
the requirements, diversification, and characteristics of terrestrial life, as well as the
characteristics of potentially habitable worlds in our Solar System and beyond. In
this chapter, we describe the objectives and strategies of this dynamic field that has
emerged with a multidisciplinary approach, leading us to one of the most exciting
goals: the search for extraterrestrial life.
1.1 Aliens Everywhere
In Mexico, 41% of the population thinks that unidentified flying objects (UFOs) are
evidence of extraterrestrial civilizations (INEGI 2015), while in Germany, the
United States, and Britain, more than a half of the population believes e xtraterrestrial
A. Segura (*)
Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México, UNAM,
Mexico City, Mexico
e-mail: [email protected]
S. I. Ramírez Jiménez
Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos,
Cuernavaca, Morelos, Mexico
I. Lozada-Chávez
Interdisciplinary Center for Bioinformatics, University of Leipzig, Leipzig, Germany
© Springer Nature Switzerland AG 2020 1

V. Souza et al. (eds.), Astrobiology and Cuatro Ciénegas Basin as an Analog of
Early Earth, Cuatro Ciénegas Basin: An Endangered Hyperdiverse Oasis,
https://doi.org/10.1007/978-3-030-46087-7_1
2 A. Segura et al.
life exists (YouGov 2015). Every year, movies, books, and TV shows display extra-
terrestrial beings, some of them are heroes, others are villains, and others just live
their lives until we find them. Aliens have all kinds of shapes, colors, and sizes. In
all cases, what we learn from these mediatic products is not about life elsewhere but
about our humanity in the face of “otherness.” Our mythology about aliens often
includes wrong ideas about scientists and their search for extraterrestrial life. The
image of the all-knowing scientists that fight against the establishment to convince
the rest of the scientific community that extraterrestrials exist is not accurate. The
scientific community has created a science to understand life on Earth and to better
define how to find it elsewhere. This science is called astrobiology. NASA’s defini-
tion of astrobiology is the study of the origins, evolution, distribution, and future of
life in the Universe (Hubbart 2015; NASA 2018). Astrobiology articulates the
knowledge about origins, the characteristics and evolution of life, Earth’s history
(its geology and chemistry), planetary bodies in our Solar System, and planets
around other stars to understand life as a universal phenomenon. For the first time
in human history, we have the expertise and technological tools to test the hypoth-
esis: the origin of life on Earth is the result of a series of natural events; therefore, it
is possible that there is life outside Earth. This chapter describes the main questions,
target places, and strategies used by astrobiology in its quest.
1.2 What Astrobiologists Are Looking for?
The main problem in searching for extraterrestrial life is not the “extraterrestrial”
part but “life” itself, as scientists have no consensus for a definition of life, despite
of having hundreds of proposals (e.g., Trifonov 2011). What we have in practice is
a list of characteristics, for example, reproduction, information, and metabolism,
but we do not know if they should be considered valid everywhere in the Universe,
as it is the case of the laws of physics or chemistry. A definition for life should set a
clear boundary between the objects that belong to a “natural category” and those
that do not, and at the same time, it should not leave anything that belongs to that
“natural category” outside those boundaries (Tsokolov 2009; Mariscal and Doolittle
2018). The problem of defining life is twofold, since it has epistemological and
scientific considerations, which sometimes have led to suggest that such endeavor is
almost impossible, useless, and even counterproductive for the daily work in life
sciences (Cleland 2012; Mix 2015). Epistemologically, our attempts to define life
fail when (Tsokolov 2009) (a) we use undefined (or highly contextualized) terms,
e.g., the term “information” has different meanings in biology and cognitive and
computational sciences, (b) confuse a description or a list of properties with a defi-
nition, or (c) define life arbitrarily in terms of minimal living systems (such as pro-
tocells and viruses). Scientifically, we have only one sample of life in the Universe
so far, the so called N=1 problem, so that our current attempts to define life are
based, and thus biased, on the features, processes, and patterns of “life as we know
it” on Earth. Also, life on a planet or a moon can only exist as a “historical-collective
1 What Is Astrobiology? 3
phenomenon”; we should thus conceptualize life on Earth as a single individual, a

“life-individual” belonging to a universal collection (Hermida 2016; Mariscal and
Doolittle 2018).
To tackle the previous problems, a universal definition of life should embody a
Theory of Life, such as the definition of water – a substance that we recognize as a
transparent and odorless liquid – uses the valence bond and molecular orbital theo-
ries to understand its basic properties. Both theoretical frameworks allow us to
unambiguously define water as a molecule made of one oxygen atom and two
hydrogen atoms (H2O), so that we can identify it throughout the Universe regardless
its physical state as liquid, solid (ice), or gas. During the last 20 years, philosophers
and scientists from diverse fields (particularly astrobiologists) have been working
on the construction of a Theory of Life, a major unifying explanatory framework of
life, which is commonly referred in the scientific literature as “universal biology”
(Hermida 2016; Mariscal and Fleming 2018). The program of universal biology is
not just about finding out which features, processes, and patterns of life on Earth are
universal and which are peculiar to it (Hermida 2016). Among other things, univer-
sal biology should also help us to clearly distinguish the different concepts within
the life definition, so we can guide our understanding of and search for non-terran
life. For instance, most of us are familiar with the concept of organism or cell, or
what we understand for “being alive”; however, “being alive”, “evidence of life”
(e.g., a biosignature), and “life-individual” (e.g., life on Earth) are only incomplete
representations of the concept of life as a phenomenon elsewhere (Benner 2010),
regardless if such phenomenon is uniquely found on Earth or is widespread in the
Universe.
The discovery of at least one other form of alien life would, without doubt, pro-
vide significant advances on the program of universal biology and the creation of a
consensus for a definition of life; but until that happens (if so), cleverly our best
option has been the use of “operational” definitions of life. They are based on the
known characteristics of living systems and serve to build strategies for the search
of life on places different from Earth (Tsokolov 2009; Benner 2010).
Our working definition is based on the most general characteristics of life on
Earth. It seems that life on Earth covers a huge variety of forms, colors, sizes, or
environments that could represent a challenge to classify but looking closer to some
general patterns emerge. All terrestrial living beings undergo Darwinian evolution
since their informational molecules (DNA and RNA) are diverse due to mutation,
which means that everything that has ever lived on Earth has somehow common
ancestors that used these molecules (see Chap. 3, 6, and 10 in this volume). Looking
even closer, the chemical elements used to build useful molecules for life are just a
few; they are famously known as CHONPS for carbon, hydrogen, oxygen, nitrogen,
phosphorus, and sulfur. Carbon is the atom in charge of putting together the rest of
the elements because it has the property of bonding with up to four elements includ-
ing itself, by a variety of chemical bonds. In this way, carbon serves as the central
chemical atom for the matter that makes living beings. Molecules also need to move
to find other molecules and react to finally build living beings. The medium they use
is liquid water. This is true for all life forms on Earth. Thus, we can say that life on
4 A. Segura et al.
Earth has a chemistry based on carbon and requires liquid water. When astrobiolo-
gists say “we are looking for life as we know it,” we are not thinking on eyes, anten-
nas, or gray humanoids, but in carbon-based molecules and liquid water.
But people may think maybe there are other forms of life that we have not imag-
ined yet. First, carbon is the fourth most abundant element in the Universe, and
water is composed of the most abundant element, hydrogen, and the third most
abundant element, oxygen. So, life is not made of anything special. For the second
reason to choose carbon and water, we need to turn to the stars.
The birth of a star – and their planets – starts in places called molecular clouds;
they are made of the leftovers of stars in their final stages of evolution (Fig. 1.1a).
Molecular clouds have two components: dust and gas. Gas is mostly molecular
hydrogen (H2), and the rest is dominated by organic compounds, that is, molecules
made of carbon. Dust is made of silicates; these are molecules of silicon, oxygen,
and other elements like iron and magnesium. About half of our planet is made of
silicates; rocks are silicates. Dust particles have other minor components like inor-
ganic carbon, which is graphite and diamonds smaller than bacteria, called
Fig. 1.1 (a) Orion Nebula; credit: NASA, ESA, M. Robberto (Space Telescope Science Institute/
ESA) and the Hubble Space Telescope Orion Treasury Project Team. (b) Edge-on protoplanetary
disk in the Orion Nebula; credit: Mark McCaughrean (Max-Planck-Institute for Astronomy),
C. Robert O’Dell (Rice University), and NASA/ESA
n anodiamonds. Water is part of the gas and, depending on the temperature, can
cover dust particles as ice.
Molecular clouds are no homogeneous; some parts become denser and contract
to form a disk structure (Fig. 1.1b). In the middle of the disk, the new star will be
formed while the dust in the disk will coagulate to form larger structures; some of
them will originate planets. Then, carbon and water in molecular clouds will become
part of those planets as the natural process of planetary formation. That is why sci-
entists assume that the chemistry of carbon and the availability of liquid water are
our best choices in the current working definition to search for life in other planets.
Despite being a single example of life (based on carbon and liquid water), the study
of life on Earth is relevant for astrobiology because it (1) gives us the tools to under-
stand the requirements for the origins of life and (2) allows us to understand the
most extreme conditions where life can survive. In general, the geological history of
our planet is an example of the evolution of a habitable planet, a blue planet where
life transformed everything through its metabolism. Astrobiologists consider that
extreme conditions in planet Earth are akin to the conditions present in other bodies
of the Solar System without the need of using a spaceship. However, it remains to
be seen to what extent biological adaptations to those extreme extraterrestrial envi-
ronments might be similar or different to those observed on Earth, given that most
complex adaptations in our planet are the results of a long and contingent evolution-
ary history.
1.3 The Limits of Life
Earth has hosted living organisms during the past 4.0–3.5 billion years (Nutman
et al. 2016; Tashiro et al. 2017). This time has been enough for biological evolution
to explore traits that had allowed organisms to colonize every habitat compatible
with the stability of biomolecules and where liquid water is available. Since the
discovery of microorganisms living in the geothermal hot springs of Yellowstone at
temperatures near the boiling point of water and very low acidity values (Brock
2012), we now know that very diverse communities of organisms can thrive in
niches with physical or geochemical conditions that exceed those where the more
familiar forms of life proliferate. These organisms are called extremophiles as they
can grow at harsh conditions of temperature, pH, pressure, dryness, salinity, and
even radiation. Extremophiles are defined by the environmental condition in which
they grow optimally as is shown in Table 1.1. Extremophiles are primarily prokary-
otic, it means single-celled organisms belonging to the Bacteria and Archaea
domains, but a few examples are also found in the Eukarya domain. Nevertheless,
the most extreme organisms belong to the Archaea domain. Extreme environments
are associated with deserts, hydrothermal vents, springs, acid mine drainages and
rivers, permafrost sediments, alkaline lakes, and nuclear reactors, among others
(Mastascusa et al. 2014). Some extremophiles are adapted simultaneously to
6 A. Segura et al.
Table 1.1 Classification of extremophiles as defined by their environment

Environmental Defining growth Representative extreme
parameter Type of extremophile condition environment
Temperature Psychrophile < −15 °C Ice, snow
Thermophile 60 to 80 °C Hot springs
Hyperthermophile 110 to 121 °C Oceanic hydrothermal
vents, hot springs
pH Acidophile pH < 3.0 Mining drainage, volcanic
springs
Alkalophile pH > 9.0 Alkaline lakes
Pressure Piezophile > 10 MPa Deep ocean, e.g., Mariana
Trench
Salinity Halophile > 10% (w/v) NaCl Salt lakes, deserts, salt
mines, salted food,
evaporites
Ionizing radiation Radioresistant 1500 to 6000 Gy Nuclear reactor water core
Desiccation Xerophile aw < 0.6 Deserts, rock surfaces,
hypersaline environments
Heavy metals Metallotolerant High concentrations Contaminated waters, soils
of Cu, Cd, As, Zn
Nutrients Nutritionally limited Oligotroph Pelagic and deep ocean
environments
Rock-dwelling Endolith Resident inside rocks Upper subsurface to deep
subterranean locations
ultiple stresses, and they are recognized as polyextremophiles; common examples

m
are the thermoacidophiles or the haloalkaliphiles.
Extremophiles provide good models for the study of biodiversity on Earth; they
are relevant for the recognition of the boundaries of life, the formulation of theories
about the origin of life, to sustain the search for life in extraterrestrial scenarios, and
to get insights into the potential of other worlds to support life, all of which are
important issues in astrobiology.
Current environmental and theoretical studies suggest that the upper limit of life
might lay near 150 °C, as this is the temperature value where most of the macromol-
ecules used by living organisms, such as the thermophiles and hyperthermophiles,
remain functional. Similarly, thermodynamic considerations suggest that life might
be impossible below −40 °C (Price and Sowers 2004). Extremophiles that are active
at cold temperatures, in saline environments, or at anoxic conditions are of particu-
lar interest for astrobiologists. They can provide clues to assess the habitability
potential of the liquid water salty oceans suspected to exist below the icy crust of
some of the satellites in the outer Solar System, or on the surface and subsurface of
Mars, where sporadic episodes of liquid water were detected below the Martian
polar caps (Orosei et al. 2018) or as oxychlorinated brines have been reported (Ojha
et al. 2015).
Despite ongoing scientific investigations in our planet for most of the recorded
human history, we still find life in unexpected places; and given the number of Earth
ecosystems that still need to be explored in detail, we expect the current boundary
of life to be pushed even further. While considering the possibility for life to origi-
nate and exist on other planetary bodies, it is important to understand the variability
of Earth’s local conditions when compared to the planetary mean. The ability of
earthly microorganisms to colonize extreme environments expands the range of
extraterrestrial bodies that may be candidates for extant life, or that may have har-
bored life in the past, and therefore may retain fossil records. Knowledge of survival
mechanisms in extremophiles will also assist the understanding of how extraterres-
trial life may survive space travel (e.g., in meteorites). Biological studies will also
provide important information about potential biomarkers (Cavicchioli 2002).
Biomarkers may be biological remnants such as the presence and type of lipid rem-
nants (Summons and Walter 1990), the chirality of amino acids and sugars, and
carbon isotope ratios1 (Sumner 2001). Most parameters considered are unlikely to
be extreme over an entire planet, and local or transient conditions might still support
life. Whether or not other planetary bodies such as Mars, Enceladus, or Europa
could or did support life, the search for Earth’s life true limits will inform our explo-
ration of space and could provide insight into processes that have led to the origin
of life on our planet (Merino et al. 2019).
1.4 Extreme Sites on Earth
The search for the suitable conditions for life in the planetary bodies of the Solar
System can be achieved either by studying specific environments on Earth that
exhibit similar conditions as those reported for the planets and satellites in the Solar
System (i.e., planetary field analogues) or by recreating planetary conditions in
well-controlled laboratory setups (i.e., laboratory analogues). Frequently these
approaches are complemented with observations of specific planetary targets.
There are some terrestrial sites with extreme ranges of temperature, salinity, min-
eral content, pH, pressure, and water availability that have been classified as ana-
logues to any of the planetary bodies of astrobiological importance, such as the
rocky planets Venus or Mars, the Jovian moons Europa or Ganymede, and the
Saturn satellites Titan and Enceladus (Marlow et al. 2011; Preston and Dartnell
2014). Terrestrial analogues help to optimize scientific and technological needs
such as the tests of machinery and scientific instruments, as well as exploration
strategies or preparation of astronauts for robotic or manned space missions. A good
analogue site does not replicate all the conditions of another planetary object, but
instead, it mimics specific parameters such as mineralogy, elemental abundances,
organic content, redox potential, water activity, temperature, radiation, mechanical,
and bulk physical characteristics (Marlow et al. 2008; Marlow et al. 2011). Besides,
1
Isotopes are atoms of the same element that have different atomic masses due to the number of
neutrons in their nuclei. For example, carbon has two stable isotopes 12C with six neutrons and 13C
with seven neutrons.
8 A. Segura et al.
analogue sites can be used while the expensive and time-consuming space explora-
tion missions are prepared.
Among the most representative analogue sites relevant to astrobiology are Rio
Tinto in Spain and the cold and hyperarid Atacama Desert in Chile as analogue sites
for the present planet Mars, as well as the black smokers in the deep sea or the
Antarctic lake system in Antarctica as analogues sites for the icy satellites, or the
Alaskan oil fields for Titan’s environment. In Mexico, several analogue sites of
astrobiological interest for different topics have been identified. A detailed descrip-
tion of them can be found in Chap. 2 of this volume.
On the one hand, several microbiological communities have been identified at the
Rio Tinto, in southern Spain, despite its acidic waters having a very high iron con-
centration. This makes Rio Tinto a good electrochemical and compositional ana-
logue of early Mars, in particular, to study the possible role of microorganisms on
the formation of iron oxide and sulfate deposits on the Red Planet (Amils et al. 2007).
On the other hand, the regions of Yungay in Chile and Arequipa in Peru that
forms the Atacama Desert are the driest hot places on Earth, serving as a composi-
tional Martian analogue because of its sulfate and perchlorate mineralogy and low
organic content (Catling et al. 2010).
Also, the Antarctic subsurface lake system is an environment closely linked to
the subsurface liquid water ocean of the satellite Europa. The largest and most com-
prehensively studied is Lake Vostok located beneath Russia’s Vostok Station under
3488 m of ice, with dimensions of 250 km long by 50 km wide, covering an area of
12,500 km2, and a volume of 5400 km3. Lake Vostok waters have been isolated for
the last 15 to 25 million years, so it has been hypothesized that in this sealed envi-
ronment, unusual forms of life could be found. The conditions of the Lake could
resemble those of the ice-covered oceans of the Jovian satellites Europa and
Ganymede and the Saturnian satellite Enceladus (Priscu et al. 1999).
In the vicinity of deep-sea hydrothermal vents, no sunlight reaches (see Chap. 7),
so sulfur compounds are utilized as a source of energy, as well as the conversion of
CO2 and H2 into CH4 (McCollom 1999). Similar environments might exist on the
satellites Europa and Enceladus. Also, sulfate salts have been detected in Europa’s
icy surface, possibly providing a sample of the ocean underneath (Brown and Hand
2013), and in the plumes of Enceladus (Bouquet et al. 2015).
All these facts indicate that the efforts to continue advancing not only in the rec-
ognition of our planet but also with the exploration of the objects in the Solar System
and from other planetary systems as possible niches for life must go on.
1.5 Where Are Astrobiologists Looking for Life?
Planets are the places where all the raw materials for building life may be available
along with several sources of energy ideal to promote the chemical reactions that
life require to emerge and evolve. Carbon is abundant in the Universe, thus we
expect it will be part of planets in general, and then the main requirement is liquid
water. Potentially habitable planets are those that have liquid water. This strategy is
based on the motto “follow the water” (Hays et al. 2015), and its purpose is to iden-
tify the physical conditions or evidence that indicates the past or present presence of
water in a planetary object. For example, the geological record on Mars includes
minerals and structures that required liquid water, although this compound is not
liquid anymore on the surface of the Red Planet (e.g., Carr and Bell 2014; Golombek
and McSween 2014).
The only habitable planet we know now is Earth, and we use it to understand how
a potentially habitable planet may work and change in time. So, astrobiology is not
only “astro” and “biology” but geology too. Unlike the case of life, where there is
only one example, the case of potentially habitable planets has many examples. In
our Solar System, there are eight planets and tens of natural satellites that we can
use to make comparative studies with respect to Earth. This comparative method is
used in order to understand if there is something special about Earth and if there are
other planetary bodies in the Solar System that are or were potentially habitable.
Besides, we also have the exoplanets (i.e., planets around other stars) that may also
able to sustain life.
1.6 T
he Closest Neighbors: Planetary Bodies in the Solar
System
Exploration of the Solar System started using remote observations, that is, using
instruments to measure the movement of planets and telescopes that observe some
details of the surface or the atmospheres of planets. By the 1970s, the exploration
included spacecrafts that flew by many planets, like the Pioneer 10 and 11 missions,
while others were dedicated just to one planet, like the Mariner (USA) and Venera
(Soviet Union) missions. Each of these missions has several probes, even when
many of those probes could not fulfill their purpose, the successful ones opened the
window for a closer look of our Solar System. Venera 7 was the first to send infor-
mation from the surface of another planet, starting the possibility of studying plan-
ets in situ. Venus and Mars were the first planets to be explored, and their
characteristics offered clues for understanding planetary habitability.
1.6.1 Venus, Earth’s Hot Twin
Venus seemed very similar to Earth because of its size and mass (Table 1.2), but its
atmosphere is 92 times denser and composed mostly by carbon dioxide (CO2).
Clouds that cover Venus are made of sulfuric acid instead of water, as the last one is
scarce on Venus and is present only in the gaseous phase at high altitudes in the
atmosphere. Temperatures over 400 °C at the surface eliminate any possibility of
10 A. Segura et al.
Table 1.2 Physical, orbital, and atmospheric characteristics with exploration information of some
of the astrobiological important objects in the Solar System
Earth Venus Mars Europa Titan
General Terrestrial planet Icy satellite Satellite of
description of Jupiter Saturn
Discovery Visible to the naked eye Galileo Christiaan
Galilei, Huygens (1655)
Simon
Marius
(1610)
Mean 1.0 0.73 1.52 5.2 9.5
distance
from Sun
(AU)
Mass (kg) 5.97 × 1024 4.87 × 1024 6.42 × 1023 4.79 × 1022 1.34 × 1023
Equatorial 6378 6052 3397 1561 2575
radius (km)
Mean 5.51 5.24 3.93 3.01 1.88
density (g/
cm3)
Surface 9.81 8.87 3.72 1.31 1.35
gravity (m/
s2)
Orbital 365.26 224.70 686.97 3.55 15.95
period (days)
Rotational 0.997 −243.025 1.026 3.55 15.95
period (days) retrograde
Axial tilt (°) 23.44 2.64 25.19 0.1 0.0
Visual 0.367 0.689 0.170 0.67 0.22
geometric
albedo
Average 288 737 210 102 93.7
surface
temperature
(K)
Atmospheric N2 (78.08), CO2 (96.5), CO2 (95.97), O2 N2 (98.4),
major O2 (20.95), N2 (3.5), Ar (1.93), CH4 (1.4),
constituents H2Ov (1.0), SO2 (0.015), N2 (1.89), H2 (0.2),
(%)
Ar (0.93), Ar (0.007), O2 (0.15), Hydrocarbons,
CO2 (0.04) H2Ov (0.002) CO (0.06) Nitriles
Surface 1.01 92 6.4 × 10−3 1 × 10−11 1.47
pressure
(bar)
Natural Moon None Phobos and
satellites Deimos
(continued)
Table 1.2 (continued)
Earth Venus Mars Europa Titan
Liquid water Oceans, Oxychlorinated Subsurface, No, but liquid
seas, lakes, brines in the global ocean methane seas
rivers, surface, and lakes on the
groundwater subsurface surface.
Probable
subsurface
ammonia-water
global ocean
Spacecraft Venera More than 20 Pioneer 10 Voyager
visitors program successful and 11 program
(1961)a, missions. A (1973, (1977)b,
Mariner 2 complete updated 1974)b. Cassini-
(1962)b, list can be Voyager Huygens
Pioneer consulted in the program (2004)b, c
Venus NASA Mars (1979) ,
b
(1978)b, Vega exploration Galileo

1 and 2 websitee mission
(1985)a, (1996)b
Magellan
(1990)b,
Venus
Express
(2006)c,
Akatsuki
(2010)d
a
Soviet Union Space Program. bNASA: National Aeronautics and Space Agency (USA). cESA:
European Space Agency. dJAXA: Japan Aerospace Exploration Agency. e https://mars.nasa.gov/
mars-exploration/missions/historical-log/
liquid water (Taylor and Hunten 2014; Taylor et al. 2018). Therefore, Venus is not
an actual habitable planet. However, composition of the surface and atmosphere of
Venus provides evidence that once there was liquid water on its surface.
The evolution of Venus from a planet with liquid water to a dry and hot desert is
linked to Sun history. Our star, as all the stars that convert hydrogen into helium in
their cores (aka main sequence stars), is slowly increasing its luminosity. When the
planets formed, the Sun emitted 70% of the energy that emits today. As the Sun
luminosity increased, Venus surface temperature raised too. As liquid water in the
Venusian surface evaporated (water is a greenhouse gas), the temperature kept on
increasing, this is called “runaway greenhouse,” and once it starts, temperature and
water vapor rise until there is no liquid water left on the surface. From this hypoth-
esis, we would expect a steamy atmosphere, but that is not what we see in Venus.
Gaseous water molecules are broken by ultraviolet light, and hydrogen and oxygen
atoms escape to space. Thus, even if Venus has not been habitable for a long time, it
is a good example of what happens with a potentially habitable planet that is too
close to its star.
12 A. Segura et al.
1.6.2 Mars, the Martians, and the Emergence of Astrobiology
Our red neighbor has been the most important planetary body for astrobiology. The
history of Mars exploration goes hand to hand with the first hypothesis and experi-
ments related to the search of life on other planets. On December 9, 1906, The
New York Times published an article entitled “There is life on the planet Mars”
(Whiting 1906). The article presented the research of American astronomer Percival
Lowell who was convinced that Mars was inhabited by a civilization that built
canals to irrigate the Martian equator. Canals were a scientific fact, according to
Lowell. His conclusions were derived from telescope observations that were cap-
tured in drawings (Lowell 1895). Thus, human hands, eyes, and brain were respon-
sible for obtaining, registering, and analyzing the evidence, and humans make
mistakes. There was controversy about the existence of the canals, and as the
observing instruments were improved, the evidence of canals vanished (Sagan and
Fox 1975), but not the idea that Mars had life (Salisbury 1962).
Once photographs were used for astronomy, it was evident that Martian surface
seasonally changed becoming darker in spring. This led to the idea that there was
vegetation on Mars. A Russian astronomer, Gavriil Adrianovich Tikhov (1875–1960),
funded the Sector for Astrobotany of the Kazakhstan Academy of Sciences. His idea
was that vegetation could be detected in the reflected light of a planet, so he mea-
sured the light reflected by several plants and compare them with Mars observations
(Sullivan and Carney 2007). He published two books, Astrobotany in 1949 and
Astrobiology in 1953 – this was the first major work to hold the name “astrobiol-
ogy” (Cockell 2001). In 1962, one of the most important scientific journals, Science,
published the paper “Martian Biology” where Salisbury proposed a biogeochemical
cycle for Mars (Salisbury 1962). By then it was known that the surface temperature
of Mars was below zero degrees Celsius most of the time and its atmosphere was
tenuous and composed by CO2 (see Table 1.2). Salisbury’s idea was tested by
another research group who concluded: “Thus, whatever environments may be
encountered during future explorations of the solar system there is every prospect
that those which are not too extreme relative to Earth, for example, those expected
on Mars may well support forms of life that we could recognize readily” (Siegel
et al. 1963).
The first experiment designed and performed to search life on other planet trav-
eled in two of the four spacecrafts from the Viking mission; both landers arrived on
Mars surface on 1976, Viking 1 at Chryse Planitia and Viking 2 at Utopia Planitia
(NASA Viking missions). They carried several experiments to study the surface and
atmosphere of Mars, three biology experiments to detect different possible meta-
bolic pathways in presumed Martian organisms and an instrument for the search of
organic material on the surface. The three experiments happened to be positive,
almost. Part of the response in every experiment was consistent with life, but at the
same time, some other part of the experiment was negative. Even more, the search
for organic material in the Martian soil was negative. The conclusion was that
n on-biological chemical reactions were the best explanation for the overall results;
mainly a highly oxidized chemical compound was responsible for what was
observed in the experiments (Jakosky et al. 2007). In 2008 the Phoenix lander
(NASA Phoenix Mission) identified perchlorates in the arctic plains of Mars (Hand
2008). Perchlorates (ClO42−) are chemical species made of one chlorine and four
oxygen atoms; these are highly oxidized compounds. Quinn and collaborators
(Quinn et al. 2013) replicated the biology experiments under conditions like those
reported on Mars, including perchlorates, and found that reactions with these com-
pounds could explain the results of all Viking biology experiments (see Chap. 2 for
more details on the detection of organics in Mars).
1.6.2.1 Martians on the Rocks
For the scientific community, the consensus is that Viking experiments did not
found evidence of life on Mars. But this did not close the case for Martians; in fact,
for some authors they may be already on Earth, but we just did not see them before
because they traveled in rocks instead of spaceships. In 1996, NASA announced an
upcoming paper with strong evidence of life on Mars. David McKay and collabora-
tors (1996) analyzed the Martian meteorite ALH84001 and found structures, miner-
als, and chemical compounds consistent with biological activity. So, let’s start with
the rock. We have more than a couple of hundreds of rocks on Earth that came from
Mars; they are called Martian meteorites (NASA 2012; Baalke). They were expelled
from Mars surface by impacts with asteroids and traveled through space until they
fall on Earth. We know they came from Mars because the gases trapped in their
minerals have the same isotopic signature as those measured in Mars atmosphere by
the Viking landers. What is more, we know how much time these Martian meteor-
ites were in space because their surfaces changed due to the constant interaction
with high-energy particles called cosmic rays. The oldest of the Martian meteorites
is ALH84001, a volcanic rock formed 4 billion years ago in Mars (Lapen et al.
2010), which was ejected from Mars 14 million years ago (Eugster et al. 1997) and
was finally falling at Antarctica 13,000 years ago. The rock has carbonates, com-
pounds formed under the action of liquid water, 3.8 billion years old.
The research group led by McKay presented the following evidence: (1) pres-
ence of ovoid and tubular structures with sizes of about 100 nm, (2) carbonate glob-
ules especially associated to ovoid and tubular structures, (3) ringed carbon
molecules called polycyclic aromatic hydrocarbons (PAH), and (4) a magnetic min-
eral called magnetite, with a shape similar to the fossil remains of magnetotactic
bacteria on Earth. The conclusion was: “Although there are alternative explanations
for each of these phenomena taken individually, when they are considered collec-
tively, particularly given their spatial association, we conclude that they are evi-
dence for primitive life on early Mars” (McKay et al. 1996). After the paper
publication, evidence accumulated in favor of a non-biological origin of the struc-
tures and carbon compounds. The agreement among the scientific community has
14 A. Segura et al.
been since then that there are no Martian fossils in AL84001. Despite not finding
life on Mars (or in the rock that came from Mars), the Viking experiment and the
ALH84001 structures were important because they opened discussions regarding
what we should expect as a proof of extraterrestrial life, something for which we
have not an agreement yet and that thus remains as an open research in astrobiology.
1.6.2.2 Discovering Mars
Twenty years after Viking, the next successful missions were NASA’s Mars Global
Surveyor (NASA Mars Global Surveyor Mission) and Pathfinder (NASA Pathfinder
Mission). Mars Global Surveyor obtained high-resolution images and altimeter
measurements to map the entire Martian surface. Pathfinder mission carried a
robotic rover named Sojourner used to study the surface of Mars in an unprece-
dented way. The rover was able to move around and was directed from Earth to
choose the rocks and soil to be photographed and analyzed with its instruments.
Viking orbiters and Mars Global Surveyor showed gullies and debris flows pro-
duced by a liquid on the surface, Sojourner registered rounded pebbles and cobbles
at the landing site, and other observations suggested conglomerates formed by run-
ning water.
All the geologic evidence pointed out a very different Mars compared to the
present, cold dusty desert. Liquid water on Mars implies a denser carbon dioxide
atmosphere that provided higher surface pressure and an enhanced greenhouse
effect. From the observation with telescopes, scientists knew there was frozen water
in the Martian poles, and missions have found indirect and direct evidence of frozen
water beneath the surface of the Red Planet. Putting together the pieces collected by
a century of observations, spacecraft and rover missions, we are certain that liquid
water was present on Mars surface since 3800 billion years ago. The Red Planet was
potentially habitable just by the time life on Earth was arising; even more, both
planets shared similar conditions: active volcanoes, liquid water on the surface, an
atmosphere of carbon dioxide and nitrogen, and availability of elements such as
sulfur and phosphorus, fundamental for making life on Earth. Then, the question is
whether life arose on Mars as it did on Earth. If there was life on Mars, what hap-
pened? Has it been hiding somewhere in the subsurface? If it was extinguished
when or after Mars lost its atmosphere, did it leave some evidence of its presence?
We do not have answers to these questions, yet. The European Space Agency (ESA)
and NASA have their eyes and instruments on Mars, like Odyssey (NASA) and
Mars Express (ESA), which have been orbiting the Red Planet for more than
10 years. Other countries are joining the Mars exploration, such as India, which
successfully launched Mangalyaan, an orbiter inserted in Mars orbit in September
2014 and was still operational by September 2019, although it was originally
planned to last only 6 months. Finding life on Mars is still an open goal for new
missions, as well as the possibility of a human mission (ESA; NASA’s Mars
Exploration Program).
1.6.3 Life in the Cold: The Icy Satellites
Icy moons circle around the giant planets, Jupiter and Saturn. In general, they are
made of combinations of iron, rock, and water. The densest moons, like Io, Europa,
and Ganymede, have iron cores and rocky mantles. Over the rocky mantles, Europa
and Ganymede have water ices. Other satellites, like Callisto, Titan, and Enceladus,
are made of rock and ice only. Despite, being too far from the Sun, to be warm
worlds covered by oceans, their interiors may provide the ingredients for the origin
and evolution of life.
1.6.3.1 Europa, Ganymede, Callisto, and Enceladus
The Jupiter system has been studied by several missions, but just Galileo was dedi-
cated to exploring the Jovian system for 14 years (NASA Galileo Mission). Europa
and her sibling satellites Io, Ganymede, and Calisto were discovered by Galileo
Galilei and allegedly by Simon Marius at the start of the seventeenth century. Europa
may be the best place in the Solar System to look for currently existing life beyond
Earth. The Voyager twin spacecrafts were the first to take detailed images of Europa
in 1979; they revealed a bright surface crossed with numerous bands, ridges, cracks,
and a surprising lack of large impact craters, suggesting that something had erased
them. Voyager’s data also indicated that Europa’s icy surface had slowly migrated
eastward with respect to the satellite’s tidal axes because a ductile or liquid layer
exists between the icy surface and the deeper interior. To complement this explana-
tion, sophisticated theoretical models of tidal heating reinforced the idea of a global
subsurface ocean within Europa (Prockter and Pappalardo 2007). The Galileo mis-
sion detected a magnetic field induced by the satellite’s motion through Jupiter’s
field, creating eddy currents in a briny water layer beneath the surface, enriched by
sulfates and carbonates salts (Chyba and Phillips 2002), and probably with chlori-
nated compounds (Hanley et al. 2014). Charged particles irradiation of the surface
materials can create oxidants that can be transported to the subsurface ocean and
could serve as a fuel for simple forms of life (Phillips and Pappalardo 2014). On the
other hand, Ganymede, the largest, more massive, and the only satellite in the Solar
System that possesses a magnetic field, also hosts an internal liquid water ocean
extended by perhaps 800 km. General characteristics of these satellites and other
bodies relevant to astrobiology are summarized in Table 1.2. Callisto, the third-
largest satellite in the Solar System, shows the oldest and most heavily cratered
surface, meaning no signs of geological activity, but leading to partial differentia-
tion and possibly to the formation of a subsurface ocean 100–150 km deep. Because
of its low radiation levels, Callisto has been considered a most suitable place for a
human base for future exploration of the Jovian system (Troutman et al. 2003). A
variety of organic functional groups, such as hydrocarbons, nitriles, or aldehydes,
have been detected in these worlds. Carbon dioxide concentrations as high as
0.2 wt% (weight percent) have been also reported, and the accumulation of other
16 A. Segura et al.
organics provided by cometary impacts has been proposed as another source of

biogenic elements for these worlds (Chyba and Phillips 2002).
A return mission to Europa is the only way to gather the critical data required to
answer the highest priority astrobiological questions about this ocean world. The
Europa Clipper mission will fly by the satellite repeatedly observing it with a pay-
load specifically designed to address potential habitability, this understood as the
ability for a planetary environment to support life forms analogous to known ter-
restrial ones. The mission consists of an ice-penetrating radar to search for water on
the subsurface, an infrared spectrometer to identify molecular compounds, a stereo
camera for mapping and topography, a neutral mass spectrometer to identify atmo-
spheric constituents, a magnetometer along with Langmuir probes to measure the
induced magnetic field constraining the salinity and thickness of the ocean, and the
spacecraft’s radio system to undertake gravity measurements. All these instruments
would enable the mission to seek evidence of subsurface water chemistry compati-
ble with habitability, and active geological processes driven by tidal flexing and
heating (Phillips and Pappalardo 2014).
Around Saturn, a little moon surprised scientists. Enceladus has a radius of
252 km, and it is expelling water and dust from its interior through plumes that feed
Saturn’s ring E, the largest and most tenuous of the rings. What is amazing is that
such a small body has energy sources large enough to produce the observed jets.
The output material indicates an environment that may be suitable for the origins of
life (McKay et al. 2008) if life can emerge in sites similar to hydrothermal vents
(Deamer and Damer 2017). Future missions may sample the plume in search for
signatures of life (Tsou et al. 2012).
1.6.3.2 Titan
Titan was discovered in 1655 by the Dutch astronomer Christiaan Huygens. In 1908
the Spanish astronomer Josep Comas i Solà observed a limb darkening, now inter-
preted as the first evidence of the atmosphere. By 1925 Sir James Dean demon-
strated that some of the constituents of the protosolar nebula would not have escaped
from Titan gravitational field despite its small size and weak gravity (Table 1.2,
Coustenis 2014). In 1944, the Dutch-American astronomer Gerard P. Kuiper
detected, by infrared spectroscopy, the strong absorption bands of methane in the
atmosphere (Kuiper 1944). Pioneer 11 was the first probe that took images of Titan
by 1979 and determined that it was a very cold place to support life. In 1980, the
Voyager 1 mission made a flyby close to Titan, 4400 km from the surface, and
recovered information that allowed the calculation of the density, mass, and compo-
sition of the satellite, as well as the temperature of the atmosphere. Unfortunately,
atmospheric haze prevented the direct imaging of the surface. Titan has also been
observed by ground-based and Earth-bound observatories like the Canada France
Hawaii Telescope (CFHT), the Keck, the Very Large Telescope (VLT), the Hubble
Space Telescope (HST), and the Infrared Space Observatory (ISO) to extract com-
plementary information from its non-homogeneous surface and the complex organic
chemistry developing on its substantial atmosphere. Cassini-Huygens, one of the

most ambitious and recent space missions, reached Titan in 2004 to gather both
remote sensing and in situ data that has deepened immensely our knowledge of the
satellite. The Cassini orbiter took the highest-resolution images of Titan’s surface
during a close flyby at 1200 km, discovering patches of light and dark terrain. The
closest flyby at 880 km, in June 2010, allowed the identification of lakes and seas on
the surface of the north polar region (Porco et al. 2005). On January 14, 2005, Titan
became the most distant body from Earth to have a space probe, Huygens, landed on
its surface. During the descent, Huygens photographed pale hills with dark rivers
running down to a dark plain. After landing, a dark plain covered with small rocks
and water-ice pebbles was photographed. The surface is dark because it consists of
a mixture of water and hydrocarbon ices (Porco et al. 2005). A rediscovering of
Titan had begun.
Under a hazy orange atmosphere, there is a hidden world. Dunes extend for kilo-
meters, round white rocks lie over frozen ground, lakes evaporate, clouds are
formed, rain lasts for days, and faults and mountains break the surface. All this may
sound very similar to Earth, but we need to look deeper. On Titan, the atmosphere
is made of molecular nitrogen and methane (Table 1.2, Coustenis 2014), while on
Earth the atmosphere is molecular nitrogen and molecular oxygen, with just 1.7 ppm
of methane. At a temperature of 180 °C below zero, the surface is made of frozen
water; the same is for rocks; in our planet, rocks and surface are made of silicates, a
combination of oxygen, silicon, and other metals like iron and magnesium. In Titan,
silicates are mixed with water in the core of the satellite. Lakes and clouds are not
made of water but methane, ethane, and other hydrocarbons. Methane (CH4) and
nitrogen molecules (N2) are broken 1000 km over the surface by ultraviolet photons
and high-energy particles; then carbon, nitrogen, and hydrogen recombine forming
large hydrocarbon molecules that further aggregate to form a haze that covers the
entire planet and scatters orange light, all of which produce the global view of Titan.
Haze falls on the surface forming dunes. Some of the mountains have craters, and
they may be volcanoes where instead of lava, water-ammonia (H2O-NH3) mixtures
flow. Thus, this is a planetary body with active geology and a constant exchange
between the atmosphere and surface. Not only carbon is present, but complex
hydrocarbon and nitrile molecules are, and though there is no liquid water, there is
methane. Is life possible on Titan?
The major problem for life on Titan is the temperature. Living organisms need a
boundary between them and the environment; this ensures their control over the
processes needed for obtaining energy, for growing and reproduction. On Earth, the
boundaries are cellular membranes made of lipids; these molecules have the prop-
erty of having hydrophobic and hydrophilic ends, that is, one part that likes water
but the other does not. Hydrophobic ends in lipids are the reason for the formation
of a small capsule when a drop of oil falls on water. Membranes must be a flexible
boundary; on the one hand, they should separate the cell from the environment, but
they should also allow nutrients to enter and products of metabolism to exit. What
happens at low temperature is that lipids lose their flexibility, they freeze, so that
they are not useful to form membranes. Stevenson and collaborators (2015) proposed
18 A. Segura et al.
a membrane composition that may work for this cold world. Instead of long hydro-
carbon molecules like lipids, small nitrogen-bearing hydrocarbon molecules may be
more appropriate. They ran numerical models that calculated the chemical and
physical properties of molecular structures using the most common hydrocarbons
with nitrogen found in Titan. The result was the azotosome, a capsule built of acry-
lonitrile (CH2CHCN), a molecule made of just seven atoms, three carbon, four
hydrogens, and one nitrogen linked using single, double, and triple bonds. For now,
this is hypothetical as we are not planning to go back to Titan soon (for more on
Titan exploration, see Chap. 2 of this volume).
The temperature in Titan is low at the surface, but underneath, as the pressure
increases, the temperature does too. Then, hypotheses for life on Titan include the
interior of the satellite planet. In the outermost layers of the core, life could use sili-
cate compounds (Bains 2004); the temperature is below 120 °C (the maximum tem-
perature for the growth of organisms called hyperthermophiles), although high
pressures may be a problem, being 10 thousand times the pressure at Earth’s sur-
face. Over the silicate core, there is the mantle, a layer made of water ice where fluid
inclusions rich in nutrients may exist. Models of Titan’s interior predict an aqueous
ammonia ocean between the mantle and the crust. In there, organisms may use sul-
fur compounds for their metabolism. All these niches together have a volume of
4 × 1010 km3, that is, the double of the terrestrial biosphere (Norman and Fortes
2011). Although carbon-based informational molecules and liquid ammonia would
allow – in principle – for evolution to occur by means of natural selection, if there
is life on Titan, we expect something other than “life as we know it.”
1.7 Habitable Planets Around Other Stars
The Sun is not the only star with planets; we have detected thousands of planets
around other stars; we called them extrasolar planets or “exoplanets.” Some may be
similar to the planets we see in our Solar System, rocky planets like Earth, Mars,
and Venus, or icy worlds like Neptune and Uranus, or hydrogen and helium giants
as Saturn and Jupiter, but most of the exoplanets we have found have characteristics
not seen in our Solar System, which provides a new opportunity for the research of
planetary interiors. Yet the question that matters for astrobiology is: are exoplanets
habitable?
Unlike planets in our Solar System, spacecrafts cannot explore exoplanets. The
closest one circles Proxima Centauri, a star that is 4.2 light years away. Then our
only possible strategy to look for life in exoplanets is using telescope observations,
which are limited because we cannot observe any details, but only general proper-
ties of those planets like their bulk atmospheric composition. Therefore, the strategy
is to look for life that has changed their planet on a global scale, so that the planetary
surface must be habitable. This means that liquid water must be on the surface
which is limited by pressure and temperature conditions, both provided by the

atmosphere. While the weight of the atmosphere provides the pressure, greenhouse
gases help to keep the energy from the star to warm the planet. Earth, for example,
would be frozen without its atmosphere; the greenhouse gases made this planet
habitable although too much of them will make it inhospitable for life. Thinking on
this we build the concept of the “habitable zone,” defined as the region around a star
where a rocky planet with atmosphere can maintain liquid water on the surface. A
rocky planet is made of an iron core and silicate mantle and crust, just like Earth,
Venus, and Mars. This condition is needed because we are assuming there is a sur-
face; planets like Jupiter or Neptune have no surface. Besides, it has been shown
that silicate surfaces might have contributed to the organization of organic mole-
cules for the origin of life (e.g., Negron-Mendoza and Ramos-Bernal 2005).
Atmospheres on rocky planets are produced by volcanic outgassing: nitrogen (N2),
carbon dioxide (CO2), hydrogen (H2), and water. Small planets, like the rocky plan-
ets in the Solar System, do not have enough gravity to keep hydrogen molecules, so
that their atmospheres will be composed of CO2 and N2. Thus, we are looking for
habitable planets that had evolved life under particular conditions, these are not
habitable planets for humans.
Numerical models calculate the habitable zone for a star with a given luminosity
(amount of stellar energy emitted per unit of time), assuming planets with atmo-
spheres made of water, CO2, and N2 (Domagal-Goldman and Segura 2013). Once
the habitable zone is obtained, we can tell which exoplanets lie in the habitable zone
of their star. Of course, this is just the start; we need to know if the planets are rocky
and have atmospheres dense enough to keep the surface above the water freezing
point. From the observations available, we can derive the planetary radius and/or its
mass, but we don’t have instruments to detect their atmospheres. Mass and plane-
tary radius constrain the likely composition of a planet, but there is a range of com-
positions that can result in the observed mass and radius, particularly for planets
more massive than Earth and less massive than Neptune, called sub-Neptunes.
Those planets may be small versions of Neptune, mini-Neptunes, or big versions of
Earth, that is, very large rocks called super-Earths, or ocean planets. Maybe we
think of our planet as an ocean planet because it apparently has lots of water, but less
than 1% of its mass is water. Ours is a dry rock; for ocean planets 10, 20, or even
50% of their total mass is water. We do not know if such planets may be habitable.
Super-Earths, on the other hand, may be habitable because their atmospheres are
probably made of hydrogen (H2), which is used for organisms on Earth and has been
hypothesized to sustain life on such exoplanets (Seager et al. 2012; Seager et al.
2013a; Seager et al. 2013b). A short list of exoplanets has been selected as poten-
tially habitable planets by the Planetary Habitability Laboratory (2019), we do not
know yet if those planets have atmospheres, or water, or are of rocky composition,
their selection is based on the habitable zone calculated by Kopparapu et al. (2013),
and assuming that planets smaller than 1.5 Earth radius or 5 Earth masses may
be rocky.
20 A. Segura et al.
1.7.1 A
Habitable Planet Around our Closest Stellar
Neighbor?
Proxima Centauri is the Sun’s closest stellar neighbor; its only detected planet is
named Proxima Centauri b; for short, the star is called Proxima and the planet
Proxima b. This star is one tenth of the solar mass, and its luminosity is only one
thousandth of the solar luminosity; it is classified as an M dwarf or red dwarf.
Proxima b is 30% more massive than Earth (Anglada-Escudé et al. 2016), but its
radius cannot be measured (Jenkins et al. 2019), so that its composition cannot be
constrained (Bixel and Apai 2017), although this has not diminished the interest on
Proxima b. The reason is that this exoplanet lies in the middle of Proxima’s habit-
able zone. Proxima b is 0.05 AU (astronomical unit = Sun-Earth distance) from its
star; its “year” is of 11 days since this is the time needed to complete one orbit
around Proxima. For comparison, Mercury, the Sun’s closest planet, is at 0.4 AU
from the Sun, and its year lasts 88 days.
What is interesting about this planetary system are the properties of Proxima.
As many red dwarfs, this star is “active,” which means that the stellar atmosphere
produces high-energy particles and radiation capable of breaking molecules
(X-rays and extreme ultraviolet = XUV) much larger than those emitted by a star
like the Sun. For a planet in the habitable zone of a red dwarf, XUV radiation can
heat the planet’s upper atmosphere as a result; the atmosphere will flow away from
the planet so that there will be no liquid water on the surface without an atmo-
sphere (Luger and Barnes 2015). And it gets worse. Proxima’s habitable zone has
moved since the planet was formed. All stars start as protostars, when their cores
are not hot enough to initiate the conversion of hydrogen into helium; this stage is
known as the pre-main sequence. This period lasts a few million years in stars like
the Sun, but for less massive stars, it can be extended over one billion years. During
this stage the luminosity of the star changes, being larger at the start of the pre-
main sequence; therefore, the limits of the habitable zone move. For Proxima the
calculated inner limit of the habitable zone started beyond 0.2 AU (Barnes et al.
2018); by the time we suppose Proxima b was formed, the planet was in the zone
where it received too much energy for having liquid water at the surface. If Proxima
b had water, it was likely lost by the process defined earlier as the runaway green-
house (see Sect. 1.6.1).. Under this scenario, habitability does not look promising,
but there is some hope according to a recent study by Barnes and coauthors (2018).
If Proxima b started with a hydrogen atmosphere equivalent to one thousandth
Earth masses, then some of that atmosphere may have survived, preserving the
planet’s habitability potential (Barnes et al. 2018). Meadows et al. (2018a) have
calculated what we expect to see with future telescopes for the scenarios predicted
by Barnes et al. (2018). So, we can test our hypothesis soon and tell if they were
correct or wrong.
1.8 Signatures of Life
Once we have identified habitable planets, we need strategies to recognize the sig-
natures of life. In our Solar System, we can go to the sites and directly analyze the
surface and atmosphere and even drill or crash into a planetary body to study what
lies beneath the surface to search for present or extant life, or products of life such
as chemical compounds or structures (see Chap. 4 for the use of stromatolites as
biosignatures). Methane (CH4) offers an illustrative case of a signature that can be
measured on-site (in situ) or with the use of telescopes and shows all the complexity
behind the production and identification of a compound as a signature of life.
On Earth, CH4 is biologically produced by bacteria–called methanogens–living
in anoxic extreme environments, such as hydrothermal vents and animal digestive
tracts (including humans). Methanogens are important in the formation of microbial
mats and stromatolites, since they are also present in anoxic marine and freshwater
sediments (Montoya et al. 2011). At least one billion tons of CH4 is being formed
and consumed by microorganisms in a single year (Zheng et al. 2018). However,
CH4 is also abiotically produced by diverse geochemical processes on our planet,
such as volcanic degassing and serpentinization at oceanic hydrothermal vents
(Martin et al. 2008). Methanogens are evolutionarily restricted to the Archaea
domain so far, although a novel light-dependent pathway for methane production
using bacterial iron-only nitrogenase has been recently reported on the proteobacte-
ria Rhodopseudomonas palustris (Zheng et al. 2018). Similar to biogenic O2, meth-
ane is released to the environment as a waste product from the reduction of
low-molecular-weight organic compounds such as CO2 with H2, or acetate, or other
methylated compounds. This process – known as methanogenesis – is thought to be
one of the earliest metabolisms on Earth, very close to the origin of life indeed
(Martin et al. 2008; Weiss et al. 2016).
Carbon isotopic composition is essential to understand the origin of methane,
and it could also be implemented in planetary landers designed to look for signs of
life. Biogenic methane usually, but not always, contains a higher percentage of the
lighter carbon isotope (12C), while abiotic methane contains relatively more of the
heavier isotope (13C). Because methanogens require oxygen-free (anoxic) condi-
tions and a low redox potential for growth and activity, methanogenesis will occur
on modern Earth when other electron acceptors such as oxygen, nitrate, and sulfate
are depleted biotically or abiotically from the environment. Under pervasive extreme
conditions (such as high salinity, temperature, or pH), methanogens can further face
bioenergetic constraints to use less or non-competitive substrates to produce meth-
ane (with different carbon isotopic composition). For instance, in hypersaline envi-
ronments, such as the ones located at Guerrero Negro’s salt flats in Baja California
or at the sulfate-rich sediments of Tirez Lagoon in Spain, the diversity of substrates
and phylogenetic types (phylotypes) producing methane is considerably reduced
and bioenergetically constrained within methanogenic archaea (Lozada-Chávez
et al. 2009; Montoya et al. 2011, Chap. 8 in this volume). Also, carbon isotopic
boundaries of biogenic methane are found to change with salinity differences in the
22 A. Segura et al.
microbial mats, as a probable consequence of methanogens tolerating different

salinities by using different substrates (Potter et al. 2009). Thus, identifying life
diversity and characterizing their geochemical profiles on extreme environments
will help astrobiologists to understand the environmental contexts for false positive
and negative mechanisms of biogenic O2/CH4 production on possible habit-
able worlds.
With such goal in mind, microbiologist Lilia Montoya and evolutionary biologist
Irma Lozada-Chávez investigated the bioenergetic constraints of methanogenesis
under hypersaline conditions at the athalassohaline2 and sulfate-rich sediment of
Tirez lake in Spain, an analogous metabolic system for Mars and Europa (Montoya
et al. 2011). For such a task, sediment samples were taken at different depths from
winter and summer seasons, their physicochemical parameters were measured, and
DNA was extracted from sediments (a very tough task to perform!). They sequenced
two functional gene markers that encode highly conserved enzymes used to produce
methane (mcrA, in Archaea) and sulfate (aprA, in Archea and Bacteria) to assess the
phylogenetic diversity, abundance, and metabolic capacity at Tirez lake. In contrast
to ribosomal genes, these functional genes work as metabolic fingerprints (from a
huge pool of DNA sequences), by providing an unambiguous link between the
genetic identity of an uncultured microorganism and its metabolic activity (Lozada-
Chávez et al. 2009; Alvarado et al. 2014; Watanabe et al. 2016). They found few but
constant phylogenetic types performing methanogenesis and sulfate reduction/oxi-
dation at Tirez lake. Strikingly, high salt and sulfate concentrations impose a com-
petition between methanogens and sulfate-reducers that use H2 and acetate as
common substrates. By using non-competitive substrates (e.g., methylamine and
methanol), other methanogenic phylotypes are not only able to coexist with sulfate-
reducers in such extreme conditions, but they also are potentially producing from
twice to six times more energy than their counterparts. According to other studies
(e.g., Potter et al. 2009), such extra energy is likely used by these methanogens to
maintain osmotic balance, which allows them in turn to tolerate higher salinities of
sodium chloride (NaCl). These findings have motivating astrobiological implica-
tions (now more than ever) since rich salts of sulfate and NaCl have been recently
confirmed on the surface of Europa (Trumbo et al. 2019).
1.8.1 Fingerprints of Life in Exoplanets
We can tell if a planet is habitable using its spectrum. The amount of energy emitted
or reflected (Fig. 1.2) by a planet in different wavelengths (colors for visible light)
tell us how much light reflects the surface and the composition of the atmosphere, if
there is one. Another possibility is to use the transmitted spectra, which is the light
2
An athalassohaline lake is a saline lake not of marine origin, but from evaporation of freshwater
in a system dominated by calcium, magnesium, and sulfate (as opposed to sodium and chloride in
the ocean).
Fig. 1.2 Reflected and emitted planetary spectrum. The spectrum forms when the light emitted or
reflected by the planetary surface is absorbed by the atoms or molecules in the atmosphere
of the star that crosses the planetary atmosphere when the planet is in front of the
star from our point of view. If there is an atmosphere, specific wavelengths will be
absorbed by the atmosphere depending on the atmospheric composition. Each atom
and molecule emits or absorbs energy in specific wavelengths that can be used to
identify it in a spectrum. Then, we can tell a planet is potentially habitable if we
identify carbon dioxide and water vapor in its spectrum. But a habitable planet is not
necessarily inhabited, for identifying life on a planet we need a biosignature or a
phone call, more specifically a radio call.
A biosignature is a pattern in a planetary spectrum that results from a biological
agent (Schwieterman et al. 2018). In particular, we are interested on gaseous biosig-
natures which result from metabolic activity. Our approach is to focus on the
“inputs” and “outputs” regardless the specific biochemical machinery that uses
those inputs and produces the outputs. This is based on the fact that life on Earth
harnesses energy from chemical reactions where we know the reactants and the
products (e.g., Seager et al. 2012). We can find our best example of a biosignature
in our planet. When Earth was formed, volcanic outgassing generated an atmo-
sphere of CO2, N2, and H2O. More than 3.8 billion years ago, life emerged on Earth,
and around 2.2 billion years ago after a global glaciation, life made the most dra-
matic global change in the history of this planet. Unicellular microorganisms called
cyanobacteria recollect light with pigments and use it to convert CO2 and H2O into
glucose; the leftover of this process known as photosynthesis is oxygen (O2). These
photosynthetic organisms contaminated Earth for the first time producing a global
climatic change. No doubt that cyanobacteria initiated big changes on our planet;
from an oxygen-free atmosphere, we ended today with an atmosphere containing
24 A. Segura et al.
21% of oxygen. These organisms are still producing oxygen as well as plants do. If
they were extraterrestrials looking at our planet’s spectrum, they could clearly see
the water and oxygen absorption bands (Fig. 1.3).
Therefore, for most astrobiologists (e.g., Meadows et al. 2018b), oxygen is con-
sidered the strongest biosignature so far, due to the following:
(a) Reliability: O2 has been produced by life on Earth since at least 3.7 Ga, accord-
ing to the stromatolites oldest fossils (Nutman et al. 2016), and has no signifi-
cant geological sources.
(b) Survivability: O2 has risen over time to become the second most abundant gas
in our atmosphere (~ 21% by volume), with major biological consequences at
planetary scale, including the evolution of complex multicellular life (Lozada-
Chávez et al. 2011) and a biomass dominance of land plants on Earth (~80% of
550 gigatons of Earth’s biomass of carbon), which strikingly contrast the bio-
mass of bacteria (~15%) and archaea (~1.3%) being produced mainly at deep-
anoxic subsurface environments (Bar-On et al. 2018).
(c) Detectability: O2 is accessible to distant observation by several methods, includ-
ing transit spectroscopy. However, oxygen is not the only biosignature worth
astrobiological investigation. With the identification of methane (CH4) in the
atmospheres of Mars and Titan, as well as in the Enceladus plumes, CH4 has
gained astrobiological interest as another potential biosignature gas (Young
et al. 1997; Taubner et al. 2018).
Fig. 1.3 Earth spectrum. Absorption of oxygen (O2) is evident at 0.76 microns (μm), while water
band absorption dominates most of the visible (0.35 μm to 0.7 μm) and near-infrared (0.7 μm to
5 μm). Data from Robinson et al. (2011), image from the VPL Spectral Explorer (http://depts.
washington.edu/naivpl/content/vpl-spectral-explorer)
The question is if a potential biosignature can have false positives, that means it
could be produced by non-biological processes that we have not identified. Oxygen,
for example, is present in Mars and Venus atmospheres as a result of the breakup of
their most abundant atmospheric gas, CO2, by ultraviolet (UV) light. The difference
between that oxygen and ours is that we can predict the abundance of that O2 from
a given amount of CO2 and UV light flux. Then, for our planet the only explanation
for the 21% O2 filling its atmosphere is life; this implies that unicellular life can
globally change a planet such as to be detectable with a telescope. We have created
a strategy to detect unicellular life with a telescope.
The strategy sounds straightforward, but it is not. We have identified just a few
compounds that may work as biosignatures (e.g., Domagal-Goldman et al. 2011;
Seager et al. 2012; Schwieterman et al. 2018), and there is another caveat, planetary
spectrum won’t be anything like the one in Fig. 1.3. Instruments to characterize the
atmosphere of potentially habitable planets are not yet available. Once they become
available, the measured spectra won’t be as detailed as the ones we calculated with
models. On top of the fuzzy observations, here we have the same problem as in the
Martian meteorite or the Viking experiments, how will we distinguish a biologically
produced signature from one that was created from other processes? This is a key
problem for astrobiologists. Current research in biosignatures to answer this ques-
tion is focused on (1) understanding false positives for life, that is, planetary pro-
cesses that may produce detectable amounts of compounds that can be confused
with those produced by living things, and (2) generating observational strategies to
distinguish between both scenarios (Meadows 2017; Catling et al. 2018; Kiang
et al. 2018; Fujii et al. 2018; Walker et al. 2018).
1.9 What About Intelligent Life?
We may expect something more spectacular than gases produced by bacteria or hol-
low prints left by extinct life. Although that would be a breakthrough, we may have
the fantasy of communicating with alien civilizations. Astrobiologists have that
dream too. In the late 1950s, Frank Drake had recently earned a Ph.D. and was
working at the National Radio Astronomy Observatory (NRAO) located at West
Virginia, United States. Radio telescopes are antennas that detect and emit light
with wavelengths from centimeters to meters. Radio waves travel fast, and they are
not absorbed by the dust between the stars. Also, Earth’s atmosphere is transparent
to radio waves making easy to detect them from the ground. Then, Drake had the
idea of searching for signals of extraterrestrial (ET) civilizations with the telescopes
at Green Bank that were under construction. He was granted permission by the
interim director, as long as there was no press involved. Just before starting the
project OZMA, the first campaign to search for ET signals, two high energy physi-
cists at Cornell University published a paper proposing the use of radiofrequencies
for interstellar communications with other civilizations (Cocconi and Morrison
1959). They started the manuscript by stating that there were no theories to calculate
26 A. Segura et al.
if life may emerge on other planets, but they made a good point at the end by saying:
“The probability of success is difficult to estimate; but if we never search, the chance
of success is zero.”
Certainly, until now, such theory does not exist, but we can organize our igno-
rance. In 1961, Frank Drake proposed an equation that has been used to calculate
the number of technological civilizations in our galaxy. The “Drake Equation,” as it
is known, does not actually provide an exact number but helps us to identify all the
factors that may be involved in the development of other civilizations (SETI Institute
2019). The Equation has been modified to include different factors, for example,
Sara Seager, professor at the Massachusetts Institute of Technology, changed the
Equation to calculate how many planets around low-mass stars may have detectable
biosignatures (Seager 2018).
At first, the efforts for searching extraterrestrial civilizations were called SETI:
Search for Extraterrestrial Intelligence. This name may be misleading. We do not
have a definition for intelligence, neither a strategy to detect it remotely. What we
can do is to search for extraterrestrial technologies that can either be artifacts, par-
ticles, or electromagnetic waves (Tarter 2001, 2007). Since project OZMA, several
searches have been conducted by professional and amateur astronomers; published
reports may be accessed at https://technosearch.seti.org/. Until now, we have not
found evidence of alien technologies, but the search has just started.
1.10 Our Habitable Planet
For the first time in the history of humanity, we can perform experiments that may
lead us to find life in other planets or moons. Larger telescopes on the ground and in
space will focus on potentially habitable planets, and spacecrafts will continue to
explore the bodies of our Solar System. For now, there is only one habitable planet
we know of: Earth. As the Sun increases its luminosity as part of its natural evolu-
tion, Earth will lose its oceans and become not habitable for any kind of life; that
future is millions of years ahead. For now, and the close future, no matter what we
do, Earth will still be habitable in the broad sense. Global warming will extinguish
many species, but others will survive and thrive. We have learned that there is life
that will be happy in hot waters or rivers contaminated by mining products. A revi-
sion of Earth’s history shows us that life survived when our planet was totally frozen
and after the impact of large rocks from the space. Human activity may be only one
more of those catastrophes where thousands of species will disappear and new will
emerge. Life, as the phenomenon that has dominated Earth for at least 3.5 billion
years, won’t vanish because of us.
But we should be worried anyway, although the fate of life is not on our hands,
the destiny of humanity is. Global warming and pollution do influence human life,
and we do not have any other place to go. This is our only home, and we can either
use our knowledge to keep it habitable for a few million years more or accelerate
our extinction. The Universe does not care.
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Chapter 2
Astrobiology and Planetary Sciences
in Mexico
Karina Cervantes de la Cruz, Guadalupe Cordero-Tercero,
Yilen Gómez Maqueo Chew, Irma Lozada-Chávez, Lilia Montoya,
Sandra Ignacia Ramírez Jiménez, and Antígona Segura
Abstract A small community of scientists in Mexico has been contributing to the

study of planetary bodies in our Solar System and around other stars, including their
potential for habitability. Here, we present particular aspects of this research told as
a journey: from the first attempts to reproduce cells and the laboratories where the
first Mexican astrobiologists were educated to the sites in Mexico where scientists
are studying the extremes of life and likely environments of other planets. We jump
to space rocks that narrate the history of the Solar System. Then, we move to Mars
and the debate of organics and the Viking experiment to continue with the hidden
water oceans of the icy satellites and Titan, an exotic orange satellite with methane
lakes, hydrocarbon dunes, and water ice rocks. Our journey continues toward other
All authors contributed equally to the text, listed in alphabetical order.
K. Cervantes de la Cruz
Departamento de Física, Facultad de Ciencias, Universidad Nacional Autónoma de México,
Mexico City, Mexico
G. Cordero-Tercero
Instituto de Geofísica, Universidad Nacional Autónoma de México, Mexico City, Mexico
Y. Gómez Maqueo Chew
Instituto de Astronomía, Universidad Nacional Autónoma de México, Mexico City, Mexico
I. Lozada-Chávez
Interdisciplinary Center for Bioinformatics, University of Leipzig, Leipzig, Germany
L. Montoya
Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, Mexico
S. I. Ramírez Jiménez (*)
Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos,
Cuernavaca, Morelos, Mexico
A. Segura
Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México, UNAM,
Mexico City, Mexico

32 K. Cervantes de la Cruz et al.
stars where we search for planets beyond our Solar System, known as exoplanets,
that have shown a surprising diversity more familiar to science fiction with hot
Jupiters, lava worlds, mini-Neptunes, super-Earths, and potentially habitable worlds.
2.1 Astrobiology and Planetary Sciences in Mexico
The exploration of other worlds is a young endeavor, telescopes and meteorites

were our only chance to study other planets until the first missions were launched
and successfully orbited and landed other bodies of our Solar System after the
1960s. Astrobiology is even younger, consolidating its purpose and methods during
the last decade of the past century (see Chap. 1 in this volume). But for both, plan-
etary sciences and astrobiology, their roots can be traced from Earth sciences,
astronomy, and the study of the origin of life. After all, the planet and life we have
on hand to study are our own. Mexico has a long tradition in those sciences. Here
we will review part of that history and the most recent projects lead by Mexican
scientists that are contributing to the understanding of the Solar System, planets
around other stars, and life as a universal phenomenon.
2.1.1 Plasmogenesis: A Hypothesis for the Origins of Life
Alfonso Luis Herrera was a dangerous man. He was convinced that life was the
result of a mixture of chemical compounds that could be reproduced in a laboratory
(Herrera 1919). He was the author of the first biology textbook in Mexico and one
of the main promoters of Darwinism in this country at the start of the twentieth
century (Ledesma-Mateos and Barahona 2003). In 1902, Herrera established the
first general biology course in Mexico at the Escuela Normal, a school dedicated to
teachers’ education (Ledesma-Mateos and Barahona 2003). These activities were
noticed by powerful politicians and religious people leading to the cancellation of
the course in 1906 because it was considered “dangerous to the young people and to
beliefs” (Ledesma-Mateos and Barahona 2003).
Plasmogenesis was born from Herrera’s idea that the functions and structures of
living beings could be explained from physicochemical laws (Negrón-Mendoza
1995). He proposed this new science in 1904 and dedicated 30 years to experiments
focused on “producing artificial cells intended to mimic as perfectly possible the
structures of nature.” (Herrera 1919). In 1942, he reported the formation of “sul-
phobes,” cell-like microstructures formed by evaporation of mixtures of inorganic
compounds (Negrón-Mendoza 1995). His approach to the origins of life prevails
until today and precedes Oparin’s book, The Origin of Life (Oparin 1924) and
Miller’s experiments (Miller 1953). For astrobiology, this view of life means that
2 Astrobiology and Planetary Sciences in Mexico 33
life is a physicochemical phenomenon that can happen on other planets. Today

those ideas may seem logical, but more than 100 years ago he was certainly ahead
of his time.
2.1.2 Origins in the Lab
Alicia Negrón liked science, she does not remember why; her family was into poli-
tics, so she did not have role models, except Marie Curie. Chemistry was her pas-
sion despite the bad teachers during most of her basic education. A high school
teacher changed the pattern and convinced Alicia that organic chemistry was what
she wanted to study. She graduated as chemical pharmacobiologist at the Universidad
Nacional Autónoma de México (UNAM) in 1968; by then, she was interested in the
chemical evolution of life and decided to study a PhD in the laboratory of Cyril
Ponnamperuma, an expert of the chemical evolution of life, that was involved with
NASA in the Viking and Voyager programs and was one of the founders of the
International Society for the Study of the Origin of Life (ISSOL) that became The
International Astrobiology Society in 2005. In his laboratory, they analyzed organ-
ics from meteorites and lunar rocks brought by the Apollo missions (Navarro-
González 1998). In 1976, Alicia got a job at the UNAM, where she started the
Laboratory of Chemical Evolution.
Alicia taught at the school of chemistry (Facultad de Química) and the school of
sciences (Facultad de Ciencias); she was a very good teacher and students soon
arrived at the new laboratory. Over time, the laboratory grew with more instruments,
space, two colleagues, and students. Nowadays, there are between 15 and 20 stu-
dents at any time, working at her laboratory from undergraduate to graduate pro-
grams of chemistry, biology, physics, and Earth sciences. In her laboratory, Dr.
Negrón explores the role of high-energy radiation like gamma ray and particles like
cosmic rays in the chemical evolution of life. She has proposed that clays are key on
the formation and organization of the molecules that led to first cells. One of the
many students who arrived at the Laboratory of Chemical Evolution was Rafael
Navarro-González.
Rafael was a bachelor student when Cyril Ponnamperuma visited the
UNAM. They had a casual talk after Ponnamperuma’s conference, and he invited
the student to come to Maryland to study in the chemistry graduate program after he
finished his undergraduate studies under the supervision of Alicia Negrón. Then he
fulfilled his goal of earning a PhD with Ponnamperuma as his advisor in 1989. He
returned to UNAM in 1994 to the Laboratory of Plasma Chemistry and Planetary
Studies, which had just started operations. In 1995, Mexico had the first student to
earn a degree with a thesis focused in astrobiology, Alfredo Romero Delgado from
the biology bachelor program at UNAM. After Alfredo, many other students found
in Navarro’s laboratory the place to start their career in astrobiology.
2.1.3 The Mexican Society of Astrobiology
For most authors on this chapter (as being part of the Society’s Executive Councils),
keeping alive a Society dedicated to boost astrobiology in Mexico has been a huge
lifetime challenge. The Sociedad Mexicana de Astrobiología (SOMA, www.soma.
org.mx) is a nonprofit, self-sustained organization comprised of a multidisciplinary
group of researchers, teachers, students, and anyone else within the general public
interested in supporting the development of astrobiology in Mexico. Its headquar-
ters were initially located at Instituto de Ciencias Nucleares at the UNAM campus
in Mexico City. SOMA’s roots date back to November 2000, when the Sociedad
Mexicana de Ciencias de la Vida en el Espacio (Mexican Society of the Sciences of
Life in Space) was created by Mexican researchers and people dedicated to the
popularization of science. By February 2002, the Society adopted its current name
and identity. Without a doubt, SOMA can be considered the major self-organized
assembly of astrobiologists in Mexico, not only it harbors between 50 and 100
members each year from most country states, but it has also established partnership
agreements with the NASA Astrobiology Institute and the European Astrobiology
Network Association since 2012.
During its almost 20 years of existence, SOMA has managed to hold 11 National
Meetings, five sessions of the Mexican School of Astrobiology, nine research semi-
nars to celebrate the “Day of Astrobiology,” and multiple science outreach events
every year.1 With the exception of the National Meeting, SOMA’s activities are free
of charge. The National Meeting and the Mexican School of Astrobiology (EMA)
represent two of SOMA’s major academic activities, taking place every 2 years to
promote national and international research collaborations, as well as transdisci-
plinary training and discussions, on a wide range of subjects related to Astrobiology.
We are particularly proud of three projects. First, a new strategy for the last EMA –
held last September 2019 in Puebla at the National Institute of Astrophysics, Optics
and Electronics (INAOE) – was enforced toward teaching the scientific background
and research being developed for two complementary topics in astrobiology (e.g.,
exoplanets and extremophiles), through an intense schedule of courses, research
talks, outreach activities, training on field sites, computational analysis, laboratory
experiments, and telescope observation. Second, a collective effort of 10 years will
conclude on 2020 with the publication of the first academic book on astrobiology in
Spanish for Latin America, entitled “Astrobiología: una visión transdisciplinaria de
la vida en el Universo,” in a co-edition between UNAM and Fondo de Cultura
Económica. Lastly, we are moving forward to actively engage Mexican astrobiolo-
gists on research collaborations by documenting and providing information about
field sites of astrobiological interest.
1
SOMA activities are supported by SOMA members, public Mexican Universities, and other
national and international institutions. Most funds are provided by the Mexican National Council
of Science and Technology (CONACYT).
2.1.4 F
ield Sites of Astrobiological Research Interest
in Mexico
On her own words, Janet Louise Siefert – an Associate Research Professor at Rice
University and the NASA Astrobiology Institute – became an astrobiologist after
10 years of being a full-time real housewife (Siefert 2011). Along with Valeria
Souza and Luis Eguiarte at UNAM, she has dedicated 20 years of research to under-
stand the biological diversity and ecological structure of the Cuatro Ciénegas Basin
(CCB), Coahuila, one of the most enigmatic Mexican field sites (Souza et al. 2012).
Mexico is within the world’s top five mega diverse countries, so the CCB is just one
of the almost 30 field sites of astrobiological interest that SOMA has been able to
empirically track throughout the country. These field sites are extreme environments
located throughout a large diversity of natural settings, such as lakes, ponds, lagoons,
basins, oceanic hydrothermal vents, geothermal fields, caves, volcanoes, and phre-
atic sinkholes (cenotes). Some of them are shown in Fig. 2.1. Unfortunately, less
than a third of such field sites has been studied for a full microbial characterization.
Moreover, some of the developed studies do not include the expertise of Mexican
researchers, although others have been cleverly performed by Mexican scientists
with international collaboration of well-known astrobiologists, like Janet Siefert,
Christopher P. McKay (NASA Ames Research Center), and Purificación López-
García (French National Centre for Scientific Research).
Astrobiologists develop diverse interests by exploring the geochemical profiles
and characterizing the phylogenetic types (phylotypes) of these field sites, mainly
represented by members of the “rare biosphere” (see Table 2.1). But in general, we
all are searching for any signal or process in the environment pointing out to a self-
sustained disequilibrium in the geochemical cycles. After careful examination, such
disequilibrium might indicate that life is or was present on such environment. For
instance, some extreme environments are considered analogous metabolic ecosys-
tems for Mars, Europa (one of the Jupiter’s moons), Enceladus (one of Saturn’s
moons), or Earth’s earliest life. Other environments instead might host extremo-
philes or biosignatures that can help us to unambiguously detect past or present life
on another world (see Chap. 1). Roughly speaking, a biosignature is any detectable
substance (e.g., element, isotope, molecule) or phenomenon that provides scientific
evidence of extant or ancient life. On the other hand, extremophiles are organisms
able to metabolically operate under physical (e.g., pressure, temperature, radiation)
or geochemical (e.g., pH, salinity, desiccation) extremes, and whose adaptive
responses and survival thresholds have evolved to grow optimally under one or
more of these extreme conditions. Hydrothermal vents, microbial mats, and micro-
bialites embrace most astrobiological interests: analogous terrestrial metabolisms,
biosignatures, and extremophiles. In contrast to hydrothermal vents (Martin et al.
2008), microbial mats and microbialites occur in several environments throughout
Mexico (Beltrán et al. 2012). Some examples are shown on Fig. 2.1 and are also
described in detail throughout the book. Cuatro Ciénegas is in the Chihuahua Desert.
The basin is surrounded to the northwest by the mountain range Sierra La Madera,
Fig. 2.1 Examples of field sites of astrobiological research interest in Mexico. CENTRAL PANEL
shows the geographical location of the field sites on the satellite map of Mexico (retrieved from
Google Maps 2019, with information of NASA, NOAA, and INEGI). UPPER PANEL shows the
landscape views of some of the field sites explored by astrobiologists and students from the
to the west by Sierra de la Fragua, to the south by Sierra de San Marcos, and to the
north and northeast by Sierra de Alamillos and de la Fragua. These mountains pro-
tect the ponds in Cuatro Ciénegas. The mountain ranges (sierras in Spanish) retain
the oasis humidity and prevent from light pollution generated by the municipality of
Monclova (Falchi et al. 2016). Cuatro Ciénegas night sky is illuminated only by the
Milky Way, the Moon, and sometimes by a shooting star, a bolide, or a superbolide.
Next, given its astrobiological importance, we will review the difference between
these phenomena, their relationship with the objects that create them, and their ori-
gin. Afterwards, we will discuss some of the history of the meteorites that fell in
Coahuila and the importance of protecting them as a national heritage.
2.2 The Rocks That Fell from the Sky
In the context of the class Geology and Planetary Atmospheres taught at the School
of Science (UNAM), the first subject that we teach is the difference between the
terms: asteroid, meteoroid, micrometeoroids, meteor, and meteorite. According to
Rubin and Grossman (2010) given the size of the material, micrometeoroids size is
between 10 μm and 2 mm, meteoroids is between 2 mm and 1 m, while asteroids
and comets are bodies of more than 1 m; all these objects are currently in orbit. The
term meteor refers to the phenomena that occur in the atmosphere, when a solid
body enters the planetary atmosphere at speeds larger than 7 km/s and the atmo-
sphere exerts resistance-causing fusion of the material and even partial ionization.
When a micrometeoroid enters the atmosphere, it creates a narrow light track called
shooting star. When a meteoroid enters the atmosphere, it generates a ball of fire or
bolide brighter than the planet Venus. But when an asteroid enters the atmosphere,
a superbolide is generated, accompanied by fragmentation of the material and
shockwaves, and if the asteroid has a diameter larger than 50 m, it creates an impact
crater on the surface. Micrometeorites (10 μm – 2 mm) and meteorites (>2 mm) are
solid objects originated in a celestial body but that are found on the surface of planet
Fig. 2.1 (continued) Mexican School of Astrobiology (EMA). Locations are: (A) Figueroa
Lagoon, Baja California (Photo credit: Paco Beretta, III EMA 2015); (B) Guerrero Negro salt flats,
Baja California; (C) Pozas Azules, Cuatro Ciénegas Basin, Coahuila (Photo credit: National Park
Service, USA, nps.gov); (D) Cenote “El Zacatón,” Tamaulipas (Photo credit: Art Palmer,
from astrobio.net); (E) Crater-lake Rincón de Parangueo, Guanajuato (see photo on Fig. 2.4); (F)
Los Azufres, Michoacán (Photo credit: Raúl López Mendoza from cambiodemichoacan.com.mx);
(G) Paricutín and Sapichu volcanoes, Michoacán; (H) Alchichica crater-lake, Puebla (Photo credit:
students from IV EMA 2019); (I) “Luna Azufre” Cave, Tabasco (Photo credit: Michael Tobler); (J)
Bacalar Lagoon, Quintana Roo (see photo on (i)); (K) Pescadero Basin, Gulf of California (Photo
credit: Ronald Spelz from MBARI, USA, mbari.org). DOWN PANEL shows cross-sections of
extant microbialites and biomats. (i) oxygen being released by stromatolites under water (seen as
bubbles over the green biomat) at Bacalar Lagoon (Photo credit: Kamila Chomicz from nytimes.
com). (ii) spongy microbialites from Alchichica crater lake (Photo credit: Beltrán et al. 2012,
Aquat. Microb. Ecol.). (iii) hypersaline microbial mat at Figueroa Lagoon, different layers of col-
ors are made up by different phylotypes. (Photo credit: Ruth Quispe, III EMA 2015)
Table 2.1 Description of some of the most studied field sites with astrobiological research interest
in Mexico
Site coordinates and Microorganisms Astrobiological
Site description detected interests
(A) 30°38′41.3″ N; 116°01′08.3″ Thick, laminated, Mars: A model to
Figueroa W endolithic, and redefine the isotopic
Lagoon, A closed hypersaline lagoon, hypersaline biofilms boundaries of biogenic
Ensenada, almost completely filled with comprised methane (a
Baja silica particles and gypsum. predominantly of biosignature), helpful
California The main source of water is the oxygenic and to test for false
(a.k.a. ocean, seeping through the anoxygenic negatives returned
Mormona dunes at high tide and forming phototrophs, sulfate- from measurements of
Lagoon) (1) permanent puddles or and methane-reducing methane on Mars and
ephemeral pools (with meters bacteria. Cyanobacteria other planetary bodies
or kilometers in diameter) such as Microcoleus are
(B) 28°06′16″ N; 114°09′10″ O living in water 2–3
Salt flats at A 250 m2 area of shallow times as salty as
Guerrero (~1 m) evaporating basins, with seawater. See references
Negro, Baja high salinity (50–130 ppt). for a full list of found
California Gypsum deposits are formed in phylotypes
(1) saltern seawater concentration
ponds and in columnar
microbial mats located on
natural anchialine pools
(K) 23°38.5′ N; 108°23.6′ W Surface-attached Europa: A model
Guaymas A system of hydrothermally microbial mats, diverse system to study
Basin, active environments (e.g., prokaryotic and anaerobic microbial
Gulf of Pescadero and Alarcón Rise eukaryotic microbes, communities (methane
California vents) that includes vent and symbiont-harboring oxidizers, and sulfate
(2) plumes, seeps, and anoxic invertebrates reducers) that catalyze
sediments, each of them See references for a full a sulfate- and
exhibiting a wide range of pH list of found phylotypes methane-driven
and temperatures anaerobic carbon cycle
and that is compatible
with stable carbon and
sulfur isotope evidence
for early Earth
ecosystems
(C) 26°50′53.19″ N; 102°08′29.98″ One of the most Mars: A proxy for the
Cuatro W microbial diverse sites late Precambrian, when
Ciénegas A large number of highly on Earth for viruses, eukaryotic organisms
Basin diverse springs (>300, e.g., bacteria, and archaea, diversify and became
(CCB), Pozas Azules), spring-fed including several more complex. It was
Chihuahuan streams, and terminal ancient species and suggested as the most
Desert, evaporitic ponds form an endemic species (e.g., analogous target to
Coahuila (3) inverse archipelago, in which 220 new Bacillus Gale crater from the
aquatic systems are separated linages). See references MSL/Mars 2018
by sparse desert vegetation, for a full list of found mission
microbial crusts, and salty phylotypes
soils. It is a phosphorus-limited
environment
(continued)
(D) 22°59′35.10″ N, 98°09′55.96″ Low diversity in water Europa: A model for
Cenote W column (1–273 m): chemoautotrophic,
“El The world’s deepest limestone, mostly photosynthetic anaerobic sulfide
Zacatón,” vertical phreatic sinkhole anoxygenic sulfide oxidation (independent
Tamaulipas (cenote). It has 105 m in oxidizers Gamma- and of photosynthetically
(4) diameter, 318 m deep, and a Epsilon-proteobacteria, derived carbon) in
17 m deep limestone rim/cliff Chlorobi; deep, dark,
band above the water; Crenarchaeota. High extraterrestrial aqueous
elemental sulfur clouds develop diversity in wall systems with a
in sunlight. Its hydrothermal biomats (1–273 m): hydrothermal feed
activity has considerably chemoautotrophic source. It is
calmed down, but there are anoxygenic sulfide investigated by the
signs of an unusually oxidizers Chlorobi, Deep Phreatic Thermal
well-mixed environment: a Chloroflexi, and Explorer (DEPTHX),
pervasive scent of sulfur hovers Deltaproteobacteria; an autonomous robotic
around the cenote, and its Crenarchaeota, underwater vehicle
water has a constant Methanomicrobia, designed to explore
temperature of 30 °C anaerobic methane signs of life in the deep
oxidizers (ANME-1), ocean under the icy
and the candidate crust of Europa
phylum Azufrosa
(E) 20°25′53.4″ N; 101°15′00.7″ Hygroscopic Mars and Enceladus:
Crater-lake W cyanobacteria in the Analogous microbial
Rincón de A Quaternary maar non-lithified crustal ecosystems for extant
Parangueo, (<0.137 Ma) located at the layers of individual and ancient life.
Guanajuato central part of the TMBV. A thrombolites. Characterization of
(5) perennial lake was present until Microbialites and extremophiles and
the 1980s. Nowadays, most of endolithic microbial biosignatures
the lacustrine area has been mats in hypersaline
transformed in a subaerial ponds, including
alkaline soil surface, but since phylotypes from
2009, there is always at least a bacteria (Cyanobacteria,
remnant hypersaline and Proteobacteria),
alkaline (pH 9–10) pond inside Euryarchaeota, and
the crater Eukarya (Chlorophyta,
Bacillariophyta,
Rotifera)
(continued)
(F) 19°46′51.7″ N; 100°39′23.6″ Mesophilic, Analogous microbial
Los W hyperthermophilic and ecosystems for extant
Azufres, A geothermal field located acidophilic and ancient life on
Michoacán within the silicic volcanic microorganisms. other worlds.
(6) complex in the TMVB Anoxygenic Characterization of
(3000 m a.s.l.). It comprises phototrophs in biomats extremophiles and
many natural hydrothermal (Rhodobacter), biosignatures
springs, fumaroles, and boiling colorless sulfur
mud pools (pH 5.5–7.4, oxidizers in mud and
25–300 °C). It has the highest water
natural arsenic concentrations (Acidithiobacillus,
in America (24 mg/L) Lysobacter); sulfate and
sulfur reducers
(Thermodesulfobium,
Desulfurella);
Acidithiobacillus,
Thiomonas
(G) 19°29′36.8″ N; 102°15′04.4″ Thin biomats over Analogous microbial
Paricutín W mineral crusts: ecosystems for extant
and Sapichu The Paricutín is the most photosynthetic bacteria and ancient life on
volcanoes, recently active monogenetic (Cyanobacteria, other worlds.
Michoacán cinder cone in the MGVF (at Chloroflexia class), Characterization of
(7) 2808 m a.s.l.). It has vapor Actinobacteria, extremophiles and
steam vents that are slightly Proteobacteria, biosignatures
acid to neutral (pH 5.3–7.9), Ktedonobacteria,
with different temperatures Acidobacteria;
(soil: 27–87 °C; air: 13.6–56 anaerobic bacteria:
°C), and high concentrations of Clostridia, Bacilli.
metals such as iron and arsenic Suggested metabolisms
include sulfur
respiration, nitrogen
fixation,
methanogenesis, carbon
fixation, photosynthesis
(H) 19°24′51.5″ N; 97°24′10.9″ W Living columnar and Mars: Microbial
Alchichica A soda, athalassohaline spongy microbialites ecosystems analogs for
Lake, crater-lake located at down to at least 14 m extant and ancient life
Puebla (8) 2300 m a.s.l., and a depth of deep. Dominant on Mars. Identification
64 m. It is a hyposaline phylotypes are oxygenic and characterization of
(8.5–10 g/liter), alkaline (pH (cyanobacteria, green biosignatures that can
8.9–9.3), and volcanic algae, and diatoms) and be used to detect extant
monomictic lake, i.e., stratified anoxygenic phototrophs life in Martian
during most of the year, the (Alphaproteobacteria, carbonates
oxygenated surface water Chloroflexi);
mixing with deep anoxic water heterotrophic bacteria
only occurs during the winter (sulfate reducers) and
season. Its water is eukaryotes (fungi and a
oversaturated with magnesium huge diversity of
and calcium carbonates protists); Crenarchaeota
(Mg:Ca = 40):
Na+ > Mg2+ > K+ > Ca2+
(continued)
(I) 17°27′09.8″ N; 92°46′41.6″ W Chemoautotrophic Analogous microbial
“Del These are mostly permanent bacteria in snottites. A ecosystems for extant
Azufre” and dark caves. Inside the “Del particular dominant and ancient life on
“Luna Azufre” cave, several springs phylotype is sulfur- other worlds.
Azufre” discharge water that is rich in oxidizing Characterization of
Caves, hydrogen sulfide (H2S). Its Acidithiobacillus spp. extremophiles and
Tabasco (9) water sulfide is oxidized to (pH 0–1.5) biosignatures
colloidal sulfur, which gives
the water a milky appearance.
“Luna Azufre” is a non-sulfidic
cave
(J) 18°46′03.9″ N; 88°18′24.7″ W Microbialites include Analogous microbial
Bacalar An oligosaline karstic coastal, domes, ledges, and ecosystems for extant
Lagoon, freshwater system (15 m deep, oncolites and exhibit a and ancient life on
Quintana pH 7.8, 25–28 °C) with the high content of organic other worlds.
Roo largest (10 km) and oldest carbon, as well as C:N, Characterization of
(a.k.a. (from early Holocene) C:P, N:P ratios. It has a extremophiles and
“Lagoon of occurrence of extant high genetic diversity biosignatures
the seven microbialites. Ca2+ cation (equally to that found in
colors”) concentrations are close to CCB). Dominant
(10) marine values (Mg:Ca = 0.24), phylotypes are
and HCO3 values exceed Cyanobacteria,
average marine concentrations: Proteobacteria, and
Ca2+ > Mg2+ > Na+ Crenarchaeota. See
references for a full list
of found phylotypes
(1) Ley et al. (2006); Vogel et al. (2009); Potter et al. (2009); Tazaz et al. (2013); Harris et al.
(2012); Valdivieso-Ojeda et al. (2014). Main Mexican PIs: Jacob A. Valdivieso-Ojeda, Miguel
A. Huerta Diaz, Francisco Delgadillo-Hinojosa
(2) Teske et al. (2003); Goffredi et al. (2017). Main Mexican PIs: Elva Escobar Briones, Ronald
M. Spelz Madero
(3) Souza et al. (2012); Centeno et al. (2012); Tapia-Torres et al. (2016); De Anda et al. (2018);
Valdespino-Castillo et al. (2018). Main Mexican PIs: Valeria Souza Saldivar, Luis Eguiarte Fruns,
Gabriela Olmedo Álvarez, Luisa I. Falcón
(4) Krajick (2007); Sahl et al. (2010)
(5) Montoya et al. (2017); Chacón et al. (2018); Sánchez-Sánchez et al. (2019). Main Mexican PIs:
Elizabeth B. Chacón, Lilia Montoya Lorenzana, Luis M. Cerca Martínez
(6) Brito et al. (2014); Chen et al. (2018). Main Mexican PIs: Elcia M. Souza Brito, Luis E. Servín
Garcidueñas
(7) Medrano-Santillana et al. (2017); Brito et al. (2019). Main Mexican PIs: Elcia M. Souza Brito
(8) Navarro et al. (2010); Couradeau et al. (2011); Beltrán et al. (2012); Valdespino-Castillo et al.
(2018). Main Mexican PIs: Elva Escobar Briones, Javier Alcocer Durand, Luisa I. Falcón, Rosaluz
Tavera Sierra
(9) Tobler et al. (2008); Jones et al. (2016)
(10) Gischler et al. (2008); Centeno et al. (2012); Valdespino-Castillo et al. (2018). Main Mexican
PIs: Luisa I. Falcón
The capital letters within parentheses (depicting the name of each field site) correspond to the ones
showing their geographical location on the map and landscape views on Fig. 2.1, numbers in
parentheses are for references, and PIs listed in the Table footnote. Abbreviations, PIs principal
investigators, a.s.l. above the sea level, ppt parts per thousand, TMBV Trans-Mexican Volcanic
Belt, MGVF Michoacán-Guanajuato Volcanic Field
Earth. In the following section, we present the general classification of meteorites

and the importance of reporting if one is found or if a fall is witnessed.
2.2.1 The History of the Solar System Written in Rocks
Some meteorites are shiny and heavy, others dark and light, some others are difficult
to distinguish from a rock. Each of those characteristics is the result of processes
that happen before, during, and after the formation of the planets of the Solar
System. Classifications are the first step to understand those processes. For meteor-
ites, one type of classification is based on the processes they have experienced,
leading to two main groups, undifferentiated and differentiated.
Undifferentiated meteorites are chondrites, which at some point were part of par-
ent bodies that were not affected by processes of fusion and differentiation, evident
by the uniform and fine dispersion of the metal (iron and nickel alloys) and iron
sulfide (troilite) grains (Fig. 2.2). The most characteristic components of these
meteorites are chondrules. If a full analysis of each of the inner planets (Mercury,
Venus, Earth, and Mars) were done, their composition would be essentially chon-
dritic; thus, chondrites contain all the elements present in the inner planets. The
carbonaceous meteorites are those that contain the highest quantity of hydrated
minerals and the abiotic organic compounds. The most ancient components of the
Solar System are calcium and aluminum inclusions (CAIs), which are dated to
4567 million years (Myr) and are contained in this type of meteorites. Given the
accretion process, the chondrules and the calcium and aluminum inclusions
Fig. 2.2 Classification of meteorites according to the differentiation of their parent body. Examples
of meteorites are (a) Allende chondrite, (b) Acapulco primitive achondrite, (c) Brenham stony-
iron, (d) Gibeon iron. (Photos of meteorites: Martha Garay and Ana Emilia Pérez)
agglomerate with small olivine and pyroxene crystals to form bodies that grew to
form rocks, planetesimals, and planetary embryos until they formed the planets as
we know them. Planetesimals or part of planetary embryos that fragmented are
represented by the asteroids and fragments of comets that we know today. The
source of chondrites are the asteroids, although some authors propose that comets
are the source of some carbon chondrites and micrometeorites.
Differentiated meteorites include the achondrites, the mixed meteorites, and the
metallic meteorites, all of them come from bodies that were large enough for their
self-gravity to induce a chemical differentiation (Fig. 2.2). The complete fusion of
the interior caused the separation of the silicate and metal liquids, each component
with a distinct density, in such a way that the light silicate liquid surrounds a metal-
lic core of iron and nickel that concentrates in the center of the body because it is the
heaviest, and then, they cooled passing from liquids to solids. Silicates are com-
pounds of silicon, oxygen, and cations such as iron, magnesium, calcium, sodium,
and potassium. The crust and mantle of Earth are made of silicates.
The cooling of these bodies was followed by collisions in space. Thus, the frag-
ments of the core are now what we know as metallic meteorites; the mixed meteor-
ites were part of the transition zone between the metallic core and the silicate
mantle; finally, the achondrites are the fragments of what was the mantle and crust
of the parent body. The differentiated meteorites support the theory that the interior
of the rocky planets are also separated in a metallic core in the center of the planet,
surrounded by a mantle and covered by a silicate crust. The source of these meteor-
ites is some asteroids and certain planetary surfaces such as Vesta, the Moon,
and Mars.
Have meteors been sighted close to Cuatro Ciénegas? Recently, there have been
no reports of fallen material, but the Meteoritical Bulletin Database reports five
meteorites found in the entire state of Coahuila. These are an ordinary chondrite
named La Esmeralda and five metallic meteorites named El Burro, Coahuila,
Coahuila II, Puente de Zacate, and Acuña.
2.2.2 F
rom the Sky to Mexico: Mexican Meteorites
and Their Study
In Mexico, meteorites are being studied in three different epochs marked by the
publication of different catalogs. Antonio del Castillo, an engineering geologist,
established in Mexico the study of meteoritics since 1864 with the description of the
iron meteorite Yanhuitlán up to the publication of the catalog (1889) and a meteorite
distribution map (1893). When the first catalog had been finished, the engineer-
ing geologist José Haro published, in 1931 a second catalog.
Meteorites in Mexico were studied outside of the territory during the period
encompassing 1930 to 1978. In 1969, in Pueblito de Allende, Chihuahua (320 km to
the west of Cuatro Ciénegas), an event that revolutionized the meteoritic science at
a global level occurred. Between 0105 and 0110 Central Standard Time, people
located in the South of the United States of America (in Nuevo Mexico and Texas)
observed the entry of a bright bolide, while the inhabitants of Parral, Chihuahua,
heard a very strong noise followed by a strong air blast (King et al. 1969). The
explosion created thousands of fragments that were dispersed over an ellipse of
more than 50 km in elongation. The original rock is a carbonaceous chondrite CV3
(Fig. 2.2a) and by that time the knowledge about the fragments was limited. The
Smithsonian Institution collected more than two tons of material. The smithsonian
was able to distribute four kilograms of pulverized material to the laboratories that
would later receive the lunar samples (Mason 1975). As chondrites are the precursor
material of rocky planets, the evolution of the planetary bodies is studied comparing
the differences in the chemistry of the rocks from a planetary body with respect to
the initial chondritic material. The scientist found calcium aluminum inclusions, a
high-temperature mixture of minerals that are more than 4567 million years old; it
means the beginning of the Solar System (Connelly et al. 2012). The study of
Allende drove the start of cosmochemistry, at the intersection of geology, planetary
science, astronomy, and astrophysics. A big piece of the Allende rock is in display
in the Museo de Geología in downtown Mexico City (Mason 1975).
In August 11, 1976, at 11:00 a.m. local time, near to the Acapulco beach,
Leodegario Cardenas heard an airplane-like sound, and when he turned-up his face
to the sky, he saw a bolide falling to him. Fortunately, the rock fell near to his
farmed land and he picked-up a 10-cubic centimeters blacked sample. A few years
later, he visited Arcadio Poveda, a prominent Mexican astronomer, and the geolo-
gist Gerardo Sánchez Rubio. Sánchez Rubio reported the event to the Scientific
Event Alert Network (SEAN 1978) and to the Meteoritical Bulletin (Sánchez Rubio
1978). This rock has a chondritic composition, but chondrules are not present
(Fig. 2.2b). The rock is a primitive achondrite, and it was a missing link between
chondrite parental bodies and the evolved planet, that way a new group of meteor-
ites was born: the acapulcoite primitive achondrite group (Palme et al. 1981).
In 2013, the teacher Elvia Muñiz took a rock to the Museum of Geology because
she thought it could be a meteorite. After a special procedure, the Widmanstatten
structures appeared, these structures are made by two minerals of iron and nickel
(taenite and kamacite) that seem interlaced like the threads in an artisan fabric (this
pattern is shown in Fig. 2.2d). This 2.3 kg rock was an iron meteorite. It was regis-
tered as the Mexican meteorite number 100 (Cervantes et al. 2017). The study of
this piece of cosmic material allowed, for the first time, in Mexico, to determine
abundances of some elements such as Ge, Ga, Mo, Ir, Pd, Sn, Co, CR, Pt, In, and
Re. This meteorite is a medium octahedrite called Tequisquiapan, and its name is
due to the nearest principal town, located in Queretaro State, in Central Mexico.
2.2.3 Understanding Alien Rocks: Research in México
On October 4, 1990, a group of researchers of the UNAM participated in the

Colloquium of the Meteorites (Ruvinovich-Kogan 1992), during which planetary
science subjects, such as impacts, meteoritics, and planetary geology, were
discussed. Since then, the study of meteoritics in Mexico has grown, for example,
between 2003 and 2019, and 21 students have graduated with thesis about meteorit-
ics. In 2001, Sánchez Rubio and collaborators published a catalog of the meteorites
of Mexico in which they report Allende CV3 and Acapulco (acapulcoite). In this
catalog, the present location of most of the Mexican meteorites is reported, in par-
ticular at the collections of Instituto de Geología and Instituto de Astronomía, both
at UNAM.
In 2003, a meteoritics group was created and led by researcher Fernando Ortega
Gutiérrez to study mainly the ages of some Mexican meteorites (Hernández-Bernal
and Solé 2010), the possible sources of chondritic meteorites (García-Martínez and
Ortega-Gutiérrez 2008) and the formation and thermic evolution of minerals in
ordinary chondrites (Cervantes-de la Cruz et al. 2010; Alba-Aldave et al. 2009). The
group also studies the formation of structures within meteorites, such as chondrules
(Cervantes-de la Cruz et al. 2015) and chondrites (Reyes-Salas et al. 2010; Corona-
Chávez et al. 2018). Magnetism of planetary bodies that is found as a remanent in
meteorites has also been studied in Mexico (Flores-Gutiérrez et al. 2010a, b;
Urrutia-Fucugauchi et al. 2014).
2.2.3.1 A Multidisciplinary Group to Study Meteorites
Geologists study rocks and astronomers study stars, their expertise seem very far
away from each other, but for meteoritics, we need both. Geologists can tell what a
rock is made of, the processes it has been through, and its origin. Astronomers are
in charge of understanding how planets form, in particular those in our Solar System.
Meteorites formed during the first stages of our planetary system and are our best
samples available on Earth to understand what happened. In 2013, Karina Cervantes
de la Cruz, a geologist and Antígona Segura, an astronomer and both authors of this
chapter, formed an interdisciplinary group to study the evolution of minerals in
meteorites, since their formation until the years that they spend on Earth. The group
included two experimental physicists dedicated to quantum optics and undergradu-
ate and graduate students of Earth sciences, engineering geology, and astrophysics.
The group has dedicated to:
1. Study the evolution of minerals in chondritic meteorites from their formation
and their transformation due to heating processes in the parental bodies. These
heating processes are due to the radioactive decay of elements such as 26Al. Heat
transforms minerals from one crystalline form to another, and the process occurs
from the edges of the mineral toward the center (Cervantes-de la Cruz et al.
2010, Alba-Aldave et al. 2009).
2. Understand the origin of chondrules. Chondrules are spheres of micrometer to
millimeter sizes. They are composed of olivine and pyroxene, which are silicate
minerals rich in iron, magnesium, and calcium (Montoya-Pérez 2016, 2019).
Their spherical form and igneous texture suggest that the minerals were melted
at some point and that they cooled down very quickly. Scientists have not agreed
on how chondrules were formed. Our approach is to create chondrules in the

laboratory by melting silicates with an experimental device designed and created
for such purpose called Citlalmitl (Cervantes-de la Cruz et al. 2015).
3. Study the terrestrial weathering and the degree of deterioration due to atmo-
spheric and climatic processes in iron meteorites (González-Medina 2019,
López-García 2019).
2.3 Planetary Collisions
Six planetary processes have been recognized on planetary bodies with solid sur-
faces: tectonics, volcanism, impact cratering, weathering, mass wasting, and inter-
action with surface fluids (this last one includes eolian, fluvial, and glacial processes)
(Melosh 2011). Not all of these processes have occurred on planetary surfaces, and
some of them occurred in the past but not currently. For example, on Mars, water
flowed on its surface, but this process ended in the Noachian, a geological Martian
period that lasted between 4100 and 3700 billion years ago.
Among the previous processes, the only one that has happened along all the his-
tory of our planetary system on all (or almost all) planetary surfaces is impact cra-
tering. This is evident from the heavy cratered surfaces of Mars, Mercury, the Moon,
Calisto, and even asteroids. Counts of impact craters on the Moon’s surface have
shown that from 4500 to 3800 billions of years ago, there was a heavy impact bom-
bardment of objects, and that from ~3800 billions of years ago to the present time,
the rate of impacts has been more or less the same, even though some authors think
that the cratering rate in the inner Solar System decreased by a factor of two to three
during the past 3 billions years, but it increased during the last 500 millions years,
probably due to the catastrophic disruption of a large main-belt asteroid (Michel and
Morbidelli 2013).
Studying impact craters has been very useful to understand rheology of planetary
crusts, assess relative and absolute ages (and as a consequence to know about the
geological histories of planets and moons), and recognize the role of impacts on the
life on Earth (Osinski and Pierazzo 2013). When an asteroid or a cometary nucleus
impacts on a planetary body, different situations can happen. Impacts on airless
bodies produce impact craters from microns to hundreds or thousands of kilome-
ters, depending on the impactor kinetic energy. On the other hand, if the target has
an atmosphere, then the impactor interacts with it and a meteor or a fireball is pro-
duced. Due to the drag pressure that the atmosphere exerts on the impactor, it can be
fragmented into pieces, each one of them produce meteors and can fragment again
(Hills and Goda 1993; Ceplecha et al. 1993). If the impactor (or its fragments) has
enough kinetic energy, its impact on planetary surfaces produces impact craters
(Passey and Melosh 1980), otherwise if the original body or its fragments survive
their passage through the atmosphere and we can collect them, they are called
meteorites.
Fig. 2.3 Possible situations when asteroids or cometary nucleus impact planetary surfaces
On the morning of June 30, 1908, an object of several tens of meters long burst
over the Siberian taiga at a height between 6 and 10.5 km, with an energy between
10 and 15 megatones. The object did not touch the surface, but the shock wave
destroyed 2150 ± 50 km2 of taiga (Farinella et al. 2001). As far as we know, this is
the only event of this type registered until now, but there could be more in the future.
Fig. 2.3 shows a diagram that summarizes the phenomena that can happen due to
impacts of asteroids or cometary nucleus with planetary surfaces.
2.3.1 Impact Studies from Mexico
At UNAM, we have studied several of the previous phenomena, in particular, impact

craters, meteorites, and atmospheric bursts. In the next paragraphs, we briefly
describe some of this work.
Examining the frequency distribution of diameters of the fragments produced by
an impact by Dohnanyi’s distribution (Dohnanyi 1969), the relation between the
maximum diameter of the fragments ejected from the primary craters and their
ejecta velocities done by Vickery (1987), and the total mass of ejecta with escape
velocity due to an impact (O’Keefe and Ahrens 1977; Melosh 1989), Cordero
Tercero and Poveda calculated the number of escaped fragments larger than 2 cm
and 10 cm as a result of the excavation of Chicxulub impact crater. They compared
the ratio of the number of Chicxulub ejecta with a specific size to the number of
NEOS of the same diameter and concluded that it is possible that some fragments
of Chicxulub ejecta entered to Earth atmosphere after circulating the inner Solar
System for some time (Poveda and Cordero 2008). The falling frequency of this
material was larger in the past than nowadays. It is possible to find Chicxulub
meteorites on the Moon and even on Earth, but in the latter case, it would be more
difficult to recognize them. This one was one of the first works done on this topic,
and their authors realized how interesting it is, as well as the possibility to under-
stand interactions between cosmic bodies (Poveda and Cordero 2008).
In 1931, the priest Fedele da Alviano reported that in the morning of August 13,
1930, people that lived near Curuça River, near the border between Brazil and Peru,
witnessed the fall of three objects together with the falling of dust, earth tremors,
and terrible sounds (L’Osservatore Romano 1931). This news was known by Leonid
Kulik who wrote a short note about it and called it “The Brazilian Tunguska” (Kulik
1931). Fifty-eight years later, Vasilyev and Andreev (1989) made some comments
to Kulik’s note and wrote a kind of invitation to South American scientists to study
this event. Bailey et al. (1995) analyzed the three previous papers, suggested that
this event could be produced by a fragment of Perseids, and reiterated the invitation
to South American colleges. Finally, Reza and colleges studied Curuça event using
remote sensing images, seismic records, and fieldwork (Reza et al. 2004). After
analyzing these works, Cordero and Poveda started a Sherlock Holmes style
research in which they study each line of evidence done by the original Da Alviano’s
report and the Bailey’s translation (Cordero and Poveda 2011). In this work, they
used information about nuclear weapons, scaling laws, empirical data, and numeri-
cal simulations to study several topics related to Curuça event: a possible impact
crater related to this event, height of explosion, energy of explosion, seismic records,
sounds, and its possible relation with the Perseids meteor shower. Their conclusions
were that neither the circular feature found by Reza and coauthors nor the seismic
record obtained by them from the San Calixto seismic observatory in La Paz,
Bolivia, are related to Curuça event. Moreover, they have evidences (not published
yet) that the mentioned circular feature is not an impact crater. Other conclusion
was that the object that produced the Curuça event could be a 9 m long object that
exploded at a height of 6 km releasing an energy less than 9 kilotons of TNT and
entered the Earth’s atmosphere at 11.2 km s−1. So, this event was three orders of
magnitude less energetic than Tunguskas’s.
On February 10, 2010, around 15:50 local time, inhabitants near the border
between the States of Hidalgo and Puebla, located in central Mexico, witnessed a
phenomenon similar to the one in Curuça: they heard a strong blast overhead, felt
seismic tremors, and roof and windows vibration, but only a few persons saw a
bolide. This was the first time Karina Cervantes de la Cruz, Eduardo Gómez, and
Guadalupe Cordero went to field to investigate the fall of a cosmic object. After they
interviewed more than 100 persons in 13 towns, they found only 12 persons that saw
the bolide. With the information obtained from them, Karina Cervantes used a Wulff
(equal-angle) stereo net to delimit the path of the meteor, finding that the object fall
followed an azimuth between 55° and 90° with an angle to the horizontal between
16° and 47°. Comparing Curuça inhabitant narrations to Puebla-Hidalgo’s, it is pos-
sible that the energy of the explosion was several kilotons. In addition, they realized
that people fear these events and that civil protection and military agencies spend
resources looking for something that can hurt people (Cordero et al. 2011).
One year later, on February 22, another object fell oriented more to the north-
west, around the border between Zacatecas and Aguascalientes states. Again, peo-
ple were afraid about this phenomenon, and it was more difficult to find witnesses
of this event; that was the reason Cordero, Cervantes-de la Cruz, and Gómez started
to plan the creation of the Mexican Meteor Network (or “Citlalin Tlamina” that
means “meteor” in Nahuatl). With the support of CONACYT and the initial advice
of Josep Trigo and José María Madiedo to choose appropriate cameras and lenses,
the Citlalin Tlamina team started to work on the Network. The objectives of Citlalin
Tlamina are to (a) study the fragmentation of meteoroids and small bodies in the
Earth’s atmosphere, (b) determine values of important parameters used to model the
dynamics of objects through the atmosphere, (c) understand the interaction between
the atmospheric shock wave and the surface, and what information this process
provides about the height and energy of the explosion, (d) determine trajectories and
orbital parameters of cosmic objects, (e) recover meteorites to study them, and (f)
give information to population and Civil Protection Department to get its help and
to avoid fear among inhabitants. Moreover, they want to install observational sta-
tions that can be functional, easy to manage, and to maintain and that operate with-
out the help of an operator near them. One of the first thing was to design a platform
to support video cameras (Whatec 902). The first design was very large and difficult
to manage (Cordero-Tercero et al. 2016). With the collaboration of Fernando
Velázquez Villegas, Alejandro Farah Simón, and Rodrigo García Fajardo, several
designs were made. After several attempts, they got a compact, easy-to-manage
platform made from 27 aluminum pieces that put together like a LEGO set. This
platform is stable under vibrations (García Fajardo 2017).
During the same time of design and construction of the platform, the group also
worked on (a) understanding the use of UFOCapture, the software used to record
meteors; (b) understanding the way to find the path of a meteor from data of two or
more stations; (c) programming data mentioned in (b) to determine pre-entry orbit,
path through the atmosphere, and possible area to recover meteorite(s); (d) checking
how to obtain meteor spectra; (e) testing how to recognize seismic record from air
shock waves; (f) thinking how to protect the video cameras at the same time that
keeping them at optimal conditions of temperature avoiding the point of dew. In
addition, Citlalin Tlamina can be used to collect other kind of data such as clouds,
bird migrations, and lightning. Much work needs to be done, but their creators
expect to solve soon the technical problems they have. This project will permit us to
study the sky with feet on surface at a low cost.
2.4 Mars: The History of the Search for Organics
Mars is a red, cold desert, but it was not always like that. In the past, 3900 billion
years ago, it had liquid water on its surface, therefore a thicker atmosphere and a
surface temperature above freezing (see Chap. 1). Comparing Earth and Mars at that
time, they look very similar with active volcanoes, liquid water on the surface, and
an atmosphere composed by carbon dioxide and nitrogen. On Earth, life emerged

during that time; did the same happen on Mars? Part of Mars’ exploration has
focused on understanding its past and present geologic conditions and their habit-
ability potential. Several scientific controversies have resulted from such research;
in this section, we will focus on one that changed NASA’s strategy to study organic
matter on Mars and was starred by the Mexican scientist, Rafael
Navarro-González.
In 1976, two probes landed on the Martian surface, Viking 1 and 2. They carried
the first experiment to search for organic matter on Mars. Organics are carbon com-
pounds that can be related to life, or not. Their presence in a planetary surface is
interesting to astrobiology regardless of their source. When they do not come from
life, they can be precursors of processes that result in the origin of life. For the
Viking landers, analysis to detect organic matter was linked to the search for life
(see Chap. 1). Although the experiments showed contradictory results that seem to
indicate the presence of life, organics were not detected, and the conclusion was that
there was no life at the Viking’s landing sites. Mars was abandoned for 20 years and
a new search for organics for more than 35 years; thus, there was enough time to
think about the experiments and find out that maybe we missed something.
2.4.1 Mars on Earth: Clues on the Search for Organic Matter
The story starts in the Atacama Desert, the most arid place on Earth with tempera-
tures between −2 °C and 32 °C, located at the south of Peru and north of Chile.
Different from other deserts, its central region has no life because it never rains, and
the air is totally dry. In that sense, it is similar to Mars, else the soil is oxidized,
although not with iron oxides but sulfur and nitrogen oxides. This is what astrobi-
ologists call an analog site, a place on Earth that shares some physical or chemical
characteristics of other planetary body (see more in Chap. 1). Even when there is no
life, organics arrive to the Atacama Desert carried by the wind. In 2001, Rafael
Navarro-González and Christopher McKay (NASA Ames) took their instruments to
Atacama and analyzed the soil to detect organics. Navarro-González used the same
technique of the Viking landers. Organics were no detected although they knew they
were there. Other researchers joined the quest and the same technique was repeated
in other analog sites: the Antarctic Desert; the Lybian Desert; the Mojave Desert;
Rio Tinto, Spain, and Panoche Valley, California. For most of them, the Viking tech-
nique did not work for detecting organics. For the Viking analysis, samples were
heated at 500 °C, Navarro-González and collaborators used the same temperature
and ran another set of experiments at 750 °C. Higher temperature allowed to detect
organics in several sites, indicating that an oxidizing reaction during the analysis
was responsible for not detecting organics on Mars (Navarro-González et al. 2006).
After the publication of these results, the head of the team that build the instru-
ment that analyzed the Martian soil was not happy. The Viking’s analysis instrument
was a huge achievement when it was built because the technology existed, but it had
to be small enough to fit in the Viking landers. All the miniaturization work and the
testing of the instrument were led by Klaus Biemann. His answer to the results of
Navarro-Gonzalez and collaborators was published in 2007 (Biemann 2007) in the
same scientific journal. He claimed that “the experiments reported by Navarro-
Gonzalez et al. (2010) are poorly conceived, executed, and interpreted….” The team
did not respond, but that was not the end of the story. In 2009, it was announced that
Phoenix, a lander that arrived at the Martian Arctic, found perchlorates on the soil.
Navarro-González’ team reproduced their 2006 experiments but now including
these chlorinated compounds in the samples (Navarro-González et al. 2010). They
found that after heating at 500 °C, organic compounds reacted with perchlorates
producing chloromethane, a molecule that was detected in the Viking analysis. The
only problem is that chloromethane was used to clean the Viking instruments, so
there were two possibilities: Viking detected martian organics or detected its own
cleaning product. Biemann and Bada (2011) argue that cleaning products and not
organic compounds were detected by Viking instruments, Navarro-González and
McKay (2011) replied to every one of the critics and stand by their results.
2.4.2 Curiosity Is What Is Needed
In 2006, NASA was planning Curiosity, a rover the size of a small car, with several
instruments to study Mars, including search for organics. Navarro-González and
collaborators results changed the planned instruments for this purpose, and Rafael
was included in the curiosity team. The rover landed on Mars in 2012 at the Gale
Crater formed when a meteor hit Mars, about 3.5–3.8 billion years ago, and there
was evidence of liquid water in the past (Mars Exploration Program and Jet
Propulsion Laboratory). In 2014, the first results that hinted to the presence of
organic compounds in Mars, as predicted by Navarro-González and collaborators in
2010, were published. The detected organics contained chlorine acquired by reac-
tions during the analysis process (Ming et al. 2014; Freissinet et al. 2015). Curiosity
continued his work on Mars, and in 2018, a team lead by Jennifer L. Eigenbrode
(Eigenbrode et al. 2018) announced the detection of sulfur-bearing organic matter
preserved in a 3-billion-year-old mudstone. Presence of organics in Mars is now
confirmed beyond doubt, where it comes from is something else. Several sources
are possible, from meteoritical materials to geologic processes. In any case, we can
say that all elements for the origins of life were in Mars, about four billion years ago.
2.5 Icy Worlds: Europa and Enceladus
An exciting debate connected to Astrobiology was published in Nature journal:

“Mars habitability” as the focus of interest (Lowell 1908). This was due to Mars
atmosphere ability to retain water. The atmospheric centered idea of habitability
was preponderant; indeed, it brought up the “habitable zone” concept, which is

defined as the circumstellar region where a terrestrial planet (iron core and silicate
mantle and crust) with an atmosphere can retain liquid water on its surface
(Segura 2018).
During the late 1970s, with the first observations of hydrothermal systems at the
Galapagos Spreading Center, the “habitable zone” concept was expanded (Dick
2013). The concomitant Voyager spacecraft discovery of a very young state of the
icy satellite’s surfaces in 1979, with very few impact craters on Europa surface,
promoted the development of theoretical studies (e.g., Squyres et al. 1983), which
supported the existence of a liquid water ocean, under an ice crust, that is still liquid
nowadays (Scipioni et al. 2017).
2.5.1 Europa
The Jupiter Galilean system (Jupiter and the satellites Io, Europa, Ganymede, and
Callisto) has been subject to intense study since its discovery by Galileo Galilei in
1601. Galilean satellites are far from being inactive because of their viscoelastic
properties combined with the fact that they undergo periodic tidal deformations.
However, it has been calculated that radiogenic heating, a product of radioisotope
decaying, was enough to differentiate Europa leading to a water layer (about
100 km). Tidal heating, resulting after satellite deformation, is responsible for keep-
ing most of its thick water crust melted, which maintains its global ocean (Nimmo
and Pappalardo 2016). The dissipation of deformation energy is a valuable source
of heat in satellites from outer Solar System and help to explain, for example, Io’s
volcanic activity and the plausible hydrothermal systems on Europa and Enceladus.
Therefore, the warming of icy satellites as a sequel of tidal heating is valuable
enough to redefine the habitable zone concept.
The geological features obtained by Galileo orbiter flybys, in 1997, informed
immediately about the occurrence of tectonic, cryovolcanic, and diapiric processes
(Cordero and Mendoza 2004) in Europa. The discovery was enough to support a
two-year extension of the Galileo Europa Mission (GEM) to 1999 in the search of
a consistence with the presence of liquid water beneath the water ice crust. At first,
the satellite’s magnetic field induced by Jupiter’s was the conclusive evidence for an
Europan ocean because it is consistent with a global electrically conducting shell
and suitable to be salt water (Nimmo and Pappalardo 2016). Hubble Space Telescope
(HST) observations of Europa surface, performed in 2012, showed the first evidence
of water plumes of a tidal origin, because of their magnetic and plasma wave signa-
tures (Table 2.2). Afterward, the HST water plumes interpretation was confirmed
with data available from the Galileo Magnetometer and the Stratospheric
Observatory for Infrared Astronomy (SOFIA) Spectrograph (e.g., Roth et al. 2014).
Ocean physical–chemical features such as pH, salt composition and concentration,
and redox state are also a matter of debate. At first, Galileo Near-Infrared Mapping
Spectrometer (NIMS) pictured a view of a hypersaline ocean because of the finding
Table 2.2 Europa and Enceladus habitability-linked features that promote them as the best
candidates, among icy satellites, for habitability. Despite that some properties constraint habitability
requirements (liquid water, chemical energy source, and organic carbon molecules), Europa and
Enceladus are still in the astrobiology roadmap
Europa Enceladus References
Radius and 1565.0 ± 8.0 252.1 ± 0.2 Vance et al.
layers (km) Water cortex (106), mantle Water cortex (62) and core (2018)
(980), and core (475) (190)
Foundation for a The inference of eruptive The discovery of geysers and Roth et al.
liquid water water emissions with the thermal emission at (2014)
ocean Hubble Space Telescope Enceladus’ south polar region Nimmo and
Imaging Spectrograph (STIS), (along the tiger stripes) linked Pappalardo
though they are infrequent to tidal forces. (2016)
when compared with The detection of silica-rich Postberg
Enceladus jets. particles originating from the et al. (2011)
Galileo spacecraft detection of plume suggests a
a Jupiter-induced magnetic hydrothermal rock-water
field and clearly distinguished interaction beneath the ice
from an internal field layer
Ocean salts MgSO4 > > Na2SO4, Na2CO3 NaCl > NaHCO3 + Na2CO3 Hibbitts
or KCl > NaCl Chlorides > carbonates et al. (2019)
Sulfates > > carbonates > > > sulfates Glein et al.
chlorides (2015)
Ocean salinity 282 3–8 Hand and
(g kg−1) Chyba
(2007)
Zolotov
(2007)
Ocean pH It is not expected an extreme Between 8 and 12 Johnson
pH et al. (2019)
Mg amount dependent Glein et al.
(2015)
Zolotov
(2007)
Are organics No, however, organics have Yes, hydrocarbons (C2 to C5 Hand and
confirmed? been used as a possible and C7 to C15) and ammonia Carlson
explanation to Galileo have been detected in the (2015)
Solid-State Imager (SSI) data plume Postberg
et al. (2018)
Laboratory Precipitation of inorganic Production of gas species (H2, Sekine et al.
chemical iron-sulfide membranes under NH3, CO2, CH4, N2) by (2015)
simulations hydrothermal solutions and serpentinizing-like reactions Barge and
generation of a membrane White
potential (2017)
Example of Simple (unicellular), Simple (unicellular), Zolotov and
suitable life hydrogen-sulfate reduction hydrogen-methanogenesis Shock
style (2003)
Taubner
et al. (2018)
(continued)
Europa Enceladus References
Laboratory Halotolerant bacteria growth Methanogenic archaea growth Avendaño
biological under varying sulfate and salt under hydrothermal-like et al. (2015)
simulations concentrations conditions (varying fluid Taubner
composition) et al. (2018)
Environmental Vostok Lake, East Antarctic Wadi al Natrun, Egypt Lorenz et al.
analogs ice sheet Kulunda Lake, Russia (2011)
Tirez Lake, Spain Mono Lake, USA Glein et al.
Borup Fiord Pass, Canada Rincon de Parangueo, Mexico (2015)
Lost City hydrothermal field, Lost City hydrothermal field,
Mid-Atlantic Ridge Mid-Atlantic Ridge
Samail ophiolite, Sultanate of Samail ophiolite, Sultanate of
Oman Oman
Habitability To be established by NASA’s Sufficiently established by Hendrix
status Europa Clipper mission Cassini-Huygens mission et al. (2018)
Next confirmed NASA’s Europa Clipper None
mission ESA’s JUICE (Jupiter Icy
moons Explorer)
of hydrated minerals, while magnesium sulfates and sodium carbonates provided

the closest match to NIMS reflectance spectra. Recently, it has been stated that chlo-
ride salts were omitted because of Galileo NIMS sensitivity bias, to clear statements
new spectra were obtained with HST and sodium chloride (NaCl) was confirmed;
consequently, the actual statement is that salt water system (SO4-Cl content) relies
on the hydrothermal circulation extent (Trumbo et al. 2019).
In 2002, Lilia Montoya performed the first experimental test for the suitability
for life in Europa at the Instituto de Ciencias Nucleares (ICN), Universidad Nacional
Autónoma de México (UNAM). The research group was headed by the chemist and
astrobiologist, Rafael Navarro-González; he and Montoya worked in collaboration
with the astrophysicists and astrobiologists Wanda L. Davis and Christopher
P. McKay (NASA Space Science and Astrobiology Division). The experimental
approach evoked hypothetical hydrothermal systems with a pressurized fluid-
circulating reactor. Covered temperatures included those common of low tempera-
ture (100 °C) and high temperature hydrothermal systems (375 °C), other
experimental variables included mineral and fluid composition. Retrieved mole-
cules from the gas phase were identified as hydrocarbons, a diverse set of C2 to C7
isomers (Montoya et al. 2002). This approach underlined the likely of hydrothermal
systems to be a source of chemicals and energy to organisms (by hydrocarbon cleav-
age, hydrogen oxidation, and methane synthesis) in Europa seafloor.
Later, as part of Montoya’s postgraduate investigation, she studied the life asso-
ciated with the Europa analog system Tirez Lake, an athalassohaline lake located in
Castilla La Mancha, Spain (Fig. 2.4). The experimental research was concreted
thanks to the mentoring of the geomicrobiologists and astrobiologists, Ricardo
Amils, and Irma Marin and the association of laboratories from the following
research institutions: Centro de Biología Molecular Severo Ochoa (CBM, CSIC),
Fig. 2.4 Photo collage of the icy satellites and their analog sites and organisms. (1a) Europa’s
Rhadamanthys Linea captured by Galileo spacecraft. The region is covered by characteristic fea-
tures, such as ridges and lenticulae. (2a) Saturn’s moon Enceladus Great Southern Land acquired
by NASA’s Cassini spacecraft, the “tiger stripe” fractures have been green colored. (1c, 1d) Europa
analog environment Tirez Lake in Castilla La Mancha, Spain. (2b, 2c) Enceladus analog environ-
ment Rincón de Parangueo in Guanajuato, Mexico. (1b) Epifluorescence micrograph of a Tirez
sediment sample treated with a modified version of the fluorescent in situ hybridization protocol
(CARD-FISH). Hybridization treatment highlights the Bacteria cells in green. (1e) Amplified view
of Tirez sulfate-reducing colonies. When the colonies exhibit a dark precipitate (iron sulfide, FeS2),
the sulfate-reducing activity is confirmed. (1f) Epifluorescence micrograph of a Tirez sediment
sample treated with FISH protocol to highlight the sulfate-reducing bacteria in orange. (2d) Bright
field micrographs of the Cyanobacteria Aphanotece sp. and (2e) an anaerobic isolate. Both organ-
isms were found in Rincon de Parangueo water samples. (1a) Image credit: NASA/JPL-Caltech/
University of Arizona. (2a) Image credit: NASA/JPL/Space Science Institute. (1b-1f) Image credit:
L. Montoya. (2b-2e) Image credit: M.E. Hernández-Zavala
the Unidad de Microbiología Aplicada at the Facultad de Ciencias (FC, Universidad

Autónoma de Madrid), and the Centro de Astrobiología (CAB, INTA-CSIC). Tirez
microbial inventory supported the idea of viable life based on methanogenesis and
sulfate reduction in Europa ocean because this lake has already been validated as a
reliable analog for Europa due to a low Cl/SO4 rate and the presence of magnesium
and sodium as main cations (Lorenz et al. 2011). A subsequent research took into
account the ephemeral condition of Tirez Lake to analyze the resilience (ability to
recovery after a disturbance) of methanogenic and sulfate-reducing populations,
and was developed in a collaboration with the evolutionary biologist, Irma Lozada-
Chávez, from the Interdisciplinary Center for Bioinformatics at the Leipzig
University, Germany. They showed that Tirez Lake harbors resilient halophilic
(extremophiles characterized by their preference for hypersaline conditions) sulfate
reducers but not all methanogens share this condition. Also, bioenergetics play an
explanation to the hydrogen-methanogenic un-resilience since a low energy yield.
Therefore, salinity is a significant parameter to be considered in evaluating the hab-
itability in Europa (Montoya et al. 2011).
After the “burst” of information provided by Galileo Mission, it became perti-
nent to define biomarkers ad hoc to Europa; so compatible solutes were an obvious
option. The obviousness of compatible solutes, i.e., low molecular weight and
organic molecules responsible for cell internal stability, came from the fact that they
are widespread in those extremophiles linked to icy satellites studies (halophiles,
psychrophiles, and hyperthermophiles), including methanogens and sulfate reduc-
ers, and these are present in all domains of life. As a consequence, in collaboration
with the geophysicist, Javier Ruiz, from the Facultad de Ciencias Geológicas at the
Universidad Complutense at Madrid, they proposed the search for compatible sol-
utes in the specific Europan features “chaos” and “lenticulae” regions because their
origin has been explained as a result of a diapiric process, i.e., plumes of warm ice
ascending through the ice crust and disrupting the surface (Ruiz et al. 2007). In
Mexico, in collaboration with the planetary chemist, Sandra I. Ramírez-Jiménez,
from the Centro de Investigaciones Químicas, Universidad Autónoma del Estado de
Morelos (CIQ, UAEM), they started the strategy of analyzing the compatible sol-
utes from cultured bacteria. They observed that prokaryotes synthesize compatible
solutes and grow (tolerate) under the common salt sodium chloride (NaCl) and sur-
prisingly when changing the NaCl to sulfate salts (Na2SO2 or MgSO4), displaying a
particular correlation between the chemical identity of salts and that of the accumu-
lated compatible solutes (Avendaño et al. 2015).
2.5.2 Enceladus
Cassini mission performed a close flyby above the south pole of the Saturn’s small
moon Enceladus in 2015. Thereafter, by displaying jets of water vapor emanating
probably from the southern large fractures named as “tiger stripes,” this icy and
active moon gained a place in the astrobiology roadmap. Cassini’s Magnetometer
corroborated the ocean profile. Consequently, the subject of interest was transferred
from a tidally induced plume to fields, such as parallel evidences of a liquid water
ocean, ocean composition, and suggestions of hydrothermal activity. Two Cassini
detections were substantial to infer the hydrothermal activity in the interphase
cortex-mantle: silica nanoparticles and molecular hydrogen at disequilibrium abun-
dance (Vance 2018). Enceladus’ ocean has already been described as global, instead
of a huge aquifer, and its salt content is certain when compared with Europa’s.
Enceladus’ observations have inferred an alkaline ocean and an anion content far
from sulfate (SO4) but close to chloride-carbonate (Cl-CO3) (Glein et al. 2015).
Given the close understanding of Enceladus “oceanographic characteristics,” the
closely resembling environments were immediately the next step: search the over-
lapping hypotheses of habitability, biosignature, and biomarker detection. Probably,
the best terrestrial analog environment alike to Enceladus is the Lost City at the
Mid-Atlantic Ridge. Some hydrothermal systems have been proposed, other than
Lost City, as long as they fit with the low temperature (i.e., a temperature ≤ 100 °C)
and neutral to alkaline pH (Ramírez Cabañas 2017). Low-temperature mid-ridge
hydrothermal systems are also known as white smokers; they are very distinctive for
being serpentinization sites. During serpentinization reactions, ultramafic minerals
react with water and release molecular hydrogen, leading to hydrothermal fluids
with reducing conditions. Molecular hydrogen (H2) is a powerful reducing agent
and source of energy for chemolithotrophs when an oxidant such as carbon dioxide,
oxygen, sulfate, or iron (III) is present. Therefore, it has been growing the consensus
that Enceladus might be habitable for chemotrophic organisms, i.e., capable of
extracting energy from chemical reactions instead of light as plants or cyanobacte-
ria. Besides making hydrogen, serpentinizing reactions act as scaffold to complex
organic synthesis, examples are Fischer-Tropsch type reactions (FTT). The FTT
reactions have been used to explain the finding of organic macromolecules emitted
from Enceladus plume ejects (Postberg et al. 2018).
Continental soda lakes, such as Wadi al Natrun (Egypt) and Mono Lake (USA),
have been proposed as analog and accessible sites to inquire Enceladus’ habitabil-
ity. The Mexican maar crater lake Rincon de Parangueo in Guanajuato was pro-
posed as an analog system to Enceladus after a collaboration with the team of
researchers from the Instituto Potosino de Investigación Científica y Tecnológica
(Ipicyt) in Mexico: the geophysicist Alfredo Ramos and the geoscientist M. Elizabeth
Hernández-Zavala from the División de Geociencias Aplicadas and the biotech-
nologist Ángel G. Alpuche-Solís from the División de Biología Molecular (Montoya
et al. 2017). This analogy contemplated especially the research developed by the
geoscientist, Hernández-Zavala (2016), regarding the hydrogeochemical and micro-
bial characterization of this maar crater lake. M. Elizabeth Hernandez-Zavala
reported the coexistence of low redox values in the water in concomitance with
diverse archaea and bacteria (Fig. 2.4). These data offered astrobiological implica-
tions because from the bioenergetic point of view, the anaerobic organisms are the
most constrained extremophiles; therefore, their finding in Rincon de Parangueo
widens the habitability window in the Saturn satellite. Theoretical and experimental
strategies to ascertain Enceladus habitability have been already explored (Table 2.2).
However, to accomplish the purpose of being objective in assessing such habitabil-

ity and in accordance with the concept of quantitative habitability plentifully devel-
oped by the biogeochemist and astrobiologist Tori M. Hoehler (NASA Ames
Research Center), Lilia Montoya proposed this quantitative approach to Enceladus
habitability. This inquire has been developing at the Facultad de Ciencias Biológicas
(FCB) at the Universidad Autónoma del Estado de Morelos (UAEM) with the
involvement of the biologist Carlos A. Soriano and the marine biologist Elva
Escobar-Briones from the Instituto de Ciencias del Mar y Limnología (ICMyL,
UNAM). Fortunately, data let them to approach a habitability index, i.e., a quantita-
tive value that allows the comparison between analog sites, e.g., Europa vs Enceladus
(Soriano-López et al. 2017). In advance, their estimates, constrained by salinity, pH,
and hydrothermal vent temperature and pH, have revealed that Enceladus has a
higher habitability index compared to Europa. Among the icy satellites, Enceladus
promises a better understanding of the distribution of life in Universe, indeed a
search-for-life mission to it will be a priority in the Solar System exploration.
Undoubtedly, efforts devoted to gain a better understanding of both satellites will be
well rewarded.
2.6 Titan
Titan is the biggest of the 62 confirmed satellites of Saturn, second in size among
the satellites in the Solar System, and the only one that holds a dense, cold, and
dynamic atmosphere in which a continuous abiotic synthesis of organic compounds
occurs (Fig. 2.5a). The major atmospheric constituent is molecular nitrogen (N2),
the same compound that makes the majority of the terrestrial atmosphere, followed
by methane (CH4), the smallest of the organic molecules. Titan is the only other
world aside from Earth with a permanent presence of liquids on the surface, and an
active hydrological cycle running, but with methane and ethane taking the place of
water (Coustenis 2014) because of its unique conditions of average surface tem-
perature of about 94 K (−179 °C) and a surface pressure of 146.7 kPa (1.45 atm)
(see Chap. 1 for more on Titan potential habitability).
Titan is a satellite of astrobiological importance as many of the processes that
occur on a planetary scale can be considered close analogs to ancient terrestrial
processes that can be monitored remotely and hopefully in situ.
2.6.1 The Voyager and Ground-Based Observations Era
Exogenous and endogenous energy sources promote the chemical transformation of

the dense and mild-reducing atmosphere of Titan originating gaseous hydrocarbons
and nitriles, which after different polymerization processes yield solid aerosol par-
ticles that grow by a variety of mechanisms and fall to the surface.
Fig. 2.5 (a) Titan in false colors. Image credit: NASA/JPL. (b and c) Views from the infrastruc-
ture at Laboratorio de Simulación de Ambientes Planetarios (LSAP) at CIQ, UAEM. (d) Analytical
platform at LSAP. (Images (b–d) credit: Sandra I. Ramírez Jiménez)
The study of the organic chemistry happening on Titan requires direct observa-
tions, generation of models, in situ sampling, as well as laboratory simulations and
analysis of the synthesized products, all of these from atmospheric and surface pro-
cesses. As part of a strategy to run experiments in conditions as close as possible to
Titan’s conditions, a laboratory devoted to the simulation of Titan’s atmosphere was
created at Centro de Investigaciones Químicas (CIQ) at the Universidad Autónoma
del Estado de Morelos (UAEM) (Fig. 2.5b–d). Prior to the arrival of Cassini-
Huygens, several experiments were performed in collaboration with laboratories at
the Universidad National Autónoma de México (UNAM) and Université Paris-Est
Créteil (UPEC).
One of the first systematic studies of Titan’s atmosphere was performed at
Laboratoire Interuniversitaire des Systèmes Atmosphériques (LISA) at UPEC,
where an experimental setup was developed to study the chemistry of Titan’s upper
atmosphere at low pressure under a liquid nitrogen temperature regime. Calculation
of energy yields using a simple mixing atmospheric model was performed (Coll
et al. 1999). A total of 44 hydrocarbons and 26 nitrogen-containing gas-phase com-
pounds were synthesized. The detection of butynedinitrile (C4N2) was reported for
the first time. A yellow solid, analog to Titan’s hazes, was also recovered. Its
spectroscopic characterization indicated absorption bands in the infrared corre-

sponding to C ≡ N bonds; pyrolysis analysis reported some benzene derivatives,
and a few hydrocarbons and nitriles, while electron microscopy observations
showed that the haze had a quasi-homogeneous spherical distribution (Coll
et al. 1999).
In the meantime, the role played by corona discharges developing among pre-
dicted methane clouds in Titan’s troposphere was studied at Instituto de Ciencias
Nucleares (ICN) at UNAM. Detailed argumentation was presented to consider the
likelihood of electrical activity in Titan. The experiments were done at room tem-
perature and high pressure. The results demonstrated that even weak electric fields
could form a variety of saturated hydrocarbons and nitriles, some of them under-
estimated by photolysis or radiolysis experiments, together with molecules of
astrobiological importance such as hydrogen cyanide (HCN) (Navarro-González
and Ramírez 1997). The first evidence of methane clouds in Titan was presented
in 1998 (Griffith et al. 1998) and was confirmed by different techniques in the
following years (Griffith et al. 2000; Roe et al. 2002; Brown et al. 2002; Gibbard
et al. 2004).
To have a better representation of Titan’s scenario, a quantitative comparison
of the products arising when a simulated atmosphere was irradiated by different
energy sources was performed. A laser-induced plasma (LIP) represented the
entrance of high-velocity meteors into Titan’s atmosphere. Gamma radiation
simulated the effect of Saturnian electrons going through Titan’s stratosphere
and of galactic cosmic rays ionizing neutral molecules in their way to the sur-
face. Arc and corona discharges were used to simulate the development of elec-
trical activity within the tropospheric methane clouds (Ramírez et al. 2001). The
evaluated energy sources demonstrated to be good abiotic nitrogen fixers as
nitriles were synthesized in all cases. Corona discharges preferentially produced
saturated and methyl-branched hydrocarbons, while conjugated hydrocarbons
and aromatic compounds were found in arc discharge experiments. The LIP orig-
inated mainly alkynes, unsaturated nitriles, and benzene derivatives. Gamma
rays produced saturated and unsaturated hydrocarbons, as well as aromatic
compounds.
An important outcome from these experiments was a light-yellow solid deposit
observed in the reactor walls for all of the energy sources, except for the gamma
radiation. This solid can be considered an analog of the aerosols that form the haze
layers detected in Titan’s stratosphere, also known as tholins in the jargon of experi-
mentalists. The characterization of the aerosol analogs by infrared spectroscopy
confirm the C ≡ N absorption observed in the analogs synthesized at LISA, but
show additional signatures attributed to aliphatic -CH3, -CH2-, C=C, C ≡ C bonds;
C-N amines and imines bonds; and aromatic C-H, =C-H bonds. All these types of
bonds were confirmed by nuclear magnetic resonance spectroscopy (Ramírez
et al. 2004).
2.6.2 The Cassini-Huygens Era
The Cassini-Huygens space mission involved a collaboration between the American,

European, and Italian Space Agencies to send the Cassini probe (CP) to study
Saturn, its rings and natural satellites, as well as the Huygens lander (HL), the first
spacecraft ever to successfully land on a moon in the outer Solar System, and the
farthest from Earth. Their scientific instruments revolutionized our understanding of
the Saturn system and helped in lifting Titan’s veil providing a wealth of new
information.
The Cassini Plasma Spectrometer (CPS) on board the HL detected heavy nega-
tive ions (10,000 Da), meaning high molecular weights for the organics contributing
to the haze layers found in the stratosphere (Waite et al. 2007; Wahlund et al. 2009;
Coates et al. 2009). The results from the gas chromatograph and mass spectrometer
(GC-MS), and the aerosol collector and pyrolizer (ACP) experiments on HL sug-
gested that Titan’s haze was made of refractory organics, containing C, N, and H
atoms and releasing, when pyrolyzed at 600 °C, HCN and NH3 (Israel et al. 2005).
These results strongly suggest that the complex organic materials constituting the
aerosol particles are similar to some of the Titan’s aerosol analogs produced in ter-
restrial laboratories, thus validating their role as analogs.
The existence of a subsurface liquid water ocean containing a few percent of
ammonia was one of the most striking results from the Cassini-Huygens mission.
Several lines of evidence suggest that Titan’s interior contains ammonia (NH3)
mixed with water, in the form of a liquid layer below a rigid water ice crust that
leads to the possibility that outgassing or erupting fluids could bring NH3 to the
surface (Tomasko and West 2010; Fortes 2000; Tobie et al. 2005; Lorenz et al. 2008;
Béghin et al. 2009; Sotin et al. 2010). If this is the case, a chemical transformation
of the aerosols on the postulated NH3-H2O mixtures introduced onto the surface as
a cryovolcanic eruption, or as a result of crustal melting following an impact crater-
ing event, is expected. To understand the role that aqueous ammonia plays in the
chemical transformation of atmospheric aerosols laboratory analogs, a N2:CH4
(98:2) mixture was irradiated in a low-temperature continuous flow regime by a DC
cold plasma discharge at LISA. The aerosol analogs were exposed to basic hydroly-
sis conditions (0.8–6.5 M NH4OH) at 277, 253, and 96 K for 10 weeks. The hydro-
lysis products were identified by the CG-MS platform at CIQ after their
transformation into volatile MTBSTFA derivatives. Seven amino acids and urea
were detected demonstrating an interesting fate for the aerosols gravitationally
deposited on Titan’s surface, where in spite of the low temperatures, chemistry can
lead to the abiotic synthesis of organic and biologically attractive compounds. What
is more, based on an aerosol mass flux of the order of 2–3 × 10−14 g cm−2 s−1
(Tomasko and West 2010), one milligram of aerosols can be deposited over an area
of 1 cm2 on Titan’s surface over 1000 years. If only 0.1% of these aerosols is trans-
formed into amino acids, the outcome would be on the order of one microgram per
cm2, a quantity detectable in situ with current analytical technology (Ramírez
et al. 2010).
2.6.3 The Future for Titan Exploration
It is true that when trying to find the answer to a specific question, more questions
are generated, and Titan’s environment is not the exception. To fully understand the
global chemistry of Titan, laboratory protocols that support informed models to
help to develop more effective in situ analytical instrumentation are required. New
proposals in each front are now being prepared to document the likelihood of signa-
tures of water-based or hydrocarbon-based life, as well as the habitability of Titan,
one of the astrobiological jewels of the Solar System.
2.7 Planets Around Other Stars
Exoplanets are planets that orbit stars that are not our Sun. The word “exoplanet” is
derived from the words extrasolar that comes from the Latin words extra (beyond)
and solar (the Sun), and the Greek word planet (wanderer), which although has
existed for many centuries, its meaning is still evolving (e.g., IAU resolution regard-
ing Pluto on 24 August 2006). The names of exoplanets are given by the name of the
star followed by a lower-case letter (starting with “b” for the first planet to be dis-
covered around a given star, “c” for the second, etc.). For example, the closest star
to our Sun is Proxima and the exoplanet around it is Proxima b (Anglada-Escudé
et al. 2016).
The first known exoplanet system (PSR + 1257 12) was discovered in 1992 with
three Earth-mass planets orbiting around a rapidly rotating neutron star or pulsar
(remnant of a supernova explosion; Wolszczan and Frail 1992; Wolszczan 1994). It
was discovered by measuring pulses (hence pulsar) that are beamed periodically
every few milliseconds toward Earth. The deviation from a very precise period of
these pulses is what allows us to know that these planets exist and from which we
can measure their masses. Until then everything we knew of planets was based on
our Solar System, and its discovery was tantalizing. However, at this point, we did
not believe this discovery was relevant to the Solar System, since the planets
revolved around a neutron star and not a “normal” star like our Sun. Furthermore,
the pulsar planets would need to survive the supernova explosion or would need to
form a second generation of planets, created from the material that is left over after
the supernova.
It was a few years later that Mayor and Queloz (1995) discovered the first planet
around a star like the Sun (51 Pegasi), by measuring the star’s velocity changes in
time due to the planet tugging on the star as the planet orbits around it. Modeling the
change in the star’s velocity allows the measurement of planet mass. This discovery
blew our minds, because 51 Pegasi b is a gas giant exoplanet that goes once around
its star in a little over 4 days. Mayor and Queloz earned half of the 2019 Nobel Prize
in Physics for finding 51 Pegasi b, the first planet around a star like our Sun. We do
not have examples of this kind of planets in our Solar System. This new and
unexpected type of planet is named hot Jupiter as planets in this class have a mass
like Jupiter, but orbit once around their star every few days receiving from it up to
10,000 times the irradiation that the Earth receives from the Sun (e.g., Demory and
Seager 2011).
In the last decade, the number of known planets outside the Solar System has
sky-rocketed. We went from celebrating the discovery of the first few hundreds of
known exoplanets in 2010 (like the 98th exoplanet with a measured size, WASP-38
b; Barros et al. 2011) to having confirmed more than 4000 exoplanets (accessed on
November 4, 2019; Schneider et al. 2011). The sizes of the planets are measured
from the amount of starlight that the planet blocks as it passes in front of the star
eclipsing it; such a planet is called a transiting planet, and when the planet crosses
the star, it is called a transit. This boom has been the result of a worldwide effort
encompassing the blossoming of facilities and research groups (e.g., Bakos et al.
2002; Pepe et al. 2004; Pollacco et al. 2006), the development of new strategies and
analysis techniques (e.g., Siverd et al. 2012; Gillon et al. 2016, 2017; Talens et al.
2017), and the start of operations of new observing instruments and facilities (e.g.,
the first satellite dedicated to exoplanets NASA’s Kepler was launched in 2009,
Borucki et al. 2010; Quirrenbach et al. 2014; Ricker et al. 2014). In fact, in 2011, a
group of Mexican astronomers (Curiel et al. 2011) applied a genetic algorithm to
velocity data of the star Upsilon Andromedae and were able to newly identify a
fourth planet in the planetary system. Upsilon Andromeda d is very similar to our
own Jupiter, with a mass of at least 1 Jupiter mass and taking ~10.5 years to orbit
once around its star (Jupiter takes 11.86 years to go around the Sun once). Mexican
astronomers have continuously participated in large consortiums in the finding of
new exoplanetary systems and studying them (e.g., Gómez Maqueo Chew et al.
2013a, b; Barragán et al. 2018).
Thus, understanding the physical processes that are involved in the formation
and evolution of our own Solar System was not enough to explain the diversity of
exoplanets and environments in which they have been found. A clear example,
besides hot Jupiters, is the population of planets with masses around 10 Earth
masses that have between 1 and 4 times the diameter of the Earth. Depending on
whether they are expected to have rocky compositions, they are called Super-Earths,
or if they are expected to have significant atmospheres of hydrogen and helium, they
are called Mini-Neptunes (Howard et al. 2010; Fulton and Petigura 2018). This
population is not known to exist in our Solar System, but among the known exo-
planets, Super-Earths and Mini-Neptunes are the most numerous (Petigura et al.
2013). One of the most important exoplanet discoveries was the discovery of
Kepler-16, the first planetary system around two stars (Doyle et al. 2011); finally
science caught up to science fiction’s Tatooine from Star Wars.
So far, the field of exoplanets has been without a doubt observationally driven,
meaning that breakthroughs in scientific knowledge have been pioneered by dedi-
cated astronomical observations. To this end, the first telescope in Mexico fully
dedicated to exoplanets was installed at the Observatorio Astronómico Nacional in
San Pedro Mártir, Baja California, in late 2018 (see Fig. 2.6). This Swiss-Mexican
project, named SAINT-EX (Search And characterIsatioN of Transiting
Fig. 2.6 On the left, the SAINT-EX dome is shown at sunset just before starting astronomical
observations, and the telescope can be seen through the shutter. On the right, the SAINT-EX tele-
scope (in yellow) and the equatorial mount (in light blue) are shown from inside the dome. Both
photos were taken over the summer of 2019 at the Observatorio Astronómico Nacional de San
Pedro Mártir, in Baja California, México by E. Cadena
EXoplanets), consists of the installation and operation of a fully robotic and

remotely operated telescope with a 1-meter primary mirror on an equatorial mount
equipped with a state-of-the-art CCD camera (González et al. 2018). The SAINT-EX
has been in the testing phase throughout 2019, and it has begun astronomical obser-
vations to search for new transiting exoplanets around the smallest, least massive
stars in our Solar Neighborhood. The SAINT-EX team is led by B.O. Demory, as
Principal Investigator, and by Y. Gómez Maqueo Chew, as Project Coordinator, and
is composed of D. Queloz (recipient of the 2019 Physics Nobel Prize), W. Benz,
F. Bouchy, K. Heng, M. Gillon, and L. Sabin, as well as students and postdocs. The
team will search for new exoplanets by measuring the light from the target stars as
a function of time to identify periodic dimmings due to the transiting exoplanets
crossing in front of their stars. Any new exoplanet discoveries from SAINT-EX will
provide optimal targets to observe with the next generation of space telescopes (e.g.,
NASA’s James Webb Space Telescope) and explore the atmospheres of rocky plan-
ets outside of our own Solar System.
2.8 Habitable Worlds and Signatures of Life
In 2003, Antígona Segura was working as a postdoctoral researcher in Penn State

University in the group lead by James Kasting, an expert on planetary habitability.
Her first work was to make the experiment of placing Earth around other stars to
understand how the signals of life on Earth would change. They were not really
moving Earth but using models to simulate a planet just like ours irradiated by two
stars, one hotter than the Sun, an F star, and other one cooler, a K star. Of course,
they used the Sun (a G star) to know if their simulations were reproducing our
planet’s atmosphere. Different stars mean different amounts of ultraviolet (UV)
light for those “earths,” and thus changes on the destruction and creation of chemi-
cal species in the atmosphere. The chemical species they were interested in were the
ones produced by life because they may be useful in the future to detect life on other
planets, and those chemicals are named biosignatures (see Chap. 1).
Earth’s atmosphere contains enough oxygen produced by photosynthetic bacte-
ria to be detectable by telescopes, but oxygen is not always a biosignature (see
Chap. 1), for example, this compound is present in small concentrations in the atmo-
spheres of Mars and Venus produced by the breakup of CO2 molecules by UV with-
out the intervention of life. Back to the experiment Segura was conducting as her
first job in Penn State University, they knew hotter stars produce more UV than the
cooler stars, then all the compounds produced by splitting molecules with this radia-
tion would change their concentrations in the atmospheres of the earths they were
simulating. Results published in 2003 (Segura et al. 2003) showed which com-
pounds of the simulated earths would be detectable in planets around those stars if
chemicals were produced by life at the same rates than on Earth. To extend this
work, Segura chose the coolest stars, M dwarfs. These stars were not studied before
mainly for two reasons, one is that the model crashed when Segura used the flux of
these stars and the other is that for most M dwarfs, UV light was not detect-
able. There were measurements only for those identified as “active”. This means
that one part of their stellar atmosphere, the chromosphere, produces more X-rays
and UV than other stars. This kind of radiation may make a planet around an M
dwarf an unsuitable place for life, so non-active stars would be better, but there were
no measurements available of UV emission for those stars classified as non-active.
What they did was to take a hypothetical UV emission calculated by a numerical
model to represent a non-active M dwarf and the emission of a very active one,
called AD Leonis. And then the surprises started.
In 1965, Lovelock suggested that the simultaneous presence of O2 and methane
(CH4) in a planetary atmosphere may be a good indicator for life. The reason is that
a series of chemical reactions starting with O2 would destroy CH4. In our planet, life
produces one billion kilograms of CH4 every year, but its abundance in the atmo-
sphere is only 1.7 parts per million. This amount of methane would be hardly detect-
able by a telescope on a planet around another star. But when Segura used that
production of methane in the simulated earths around M dwarfs, this compound
built up in the atmosphere, thus the model crashed. Antígona learned how to solve
the problem, and with her collaborators showed that the particular UV emission of
these M dwarfs changed the production of a compound that was responsible for
methane destruction. As a result CH4 along with other atmospheric gases produced
by life were more abundant and therefore more likely to be detected. It this way
methane can be used as a potential biosignature (Segura et al. 2005).
We are interested on M dwarfs because they are about 75% of the stars in our
galaxy, and most of the potentially habitable planets are around M dwarfs. A habit-
able planet is one that is capable of hosting life, although science has not yet a defi-
nition for life. Not having a definition may seem like a problem for identifying
habitable planets, but scientists are experts in solving problems. What we do is to
take the most general characteristics of life in the only inhabited planet we know:
Earth. Although living beings on Earth may look very different from each other –
think for example in a fungus, a bacterium, a shark, and a tree – looking closer, life
is made with a few elements: hydrogen, carbon, oxygen, nitrogen, sodium, magne-
sium, potassium, calcium, phosphorus, and sulfur (see Chap. 1). The first four in the
list are highly common in the universe, and carbon is the element that binds the rest
of the elements to form the molecules that build living beings. For carbon molecules
to find each other and react to create new structures, they require a substance where
they can move. On Earth, this substance is liquid water. Then, the generalization of
Earth’s life is carbon and liquid water. The distance from the star where a rocky
planet with water and atmosphere can have liquid water on its surface is called the
Habitable Zone (HZ) (Chap. 1).
After the 2005 work by Segura and collaborators demonstrated the importance of
UV to predict the biosignatures for planets around M dwarfs, there was a renewed
interest on these stars (Scalo et al. 2007; Tarter et al. 2007; Billings 2011; Shields
et al. 2016) strengthened by the detection of several planets in the habitable zone of
M dwarfs including our closest neighbor, Proxima Centauri (Anglada-Escudé et al.
2016). Because the HZ of M dwarfs is 10–100 times closer than the Sun’s habitable
zone, it was important to measure how much high-energy radiation may reach the
surface of potentially habitable planets around them. So, we needed to measure the
UV of non-active stars (Hawley 2005). Observations with the Hubble Space
Telescope finally showed us that UV emission of “non-active” M dwarfs was higher
than we expected (Walkowicz et al. 2008), and later the MUSCLES treasury survey
started gathering a catalog for the emission (from infrared to X-rays) of M dwarfs
with exoplanets detected (France 2016). During the last decade, the astrobiology
community has been discussing if planets would be habitable and life can emerge
and evolve in those harsh conditions.
M dwarfs have flares, sudden discharges of energy that increase the UV emission
of active M dwarfs 10–100 times during a few hours every day. This UV could
potentially destroy the ozone (O3) in a planetary atmosphere like the present Earth,
representing a problem for planetary habitability. Segura worked on this problem
when she moved to Pasadena, CA, hired by JPL to collaborate in the Virtual
Planetary Laboratory lead by Dr. Victoria Meadows. This time, she modified the
code to include the flux of a flare from AD Leonis finding that actually, their simu-
lated earths did not receive much more UV energy than Earth in the past when life
emerged, and there was no ozone in the atmosphere (Segura et al. 2010). By the end
of 2006, she was hired at the UNAM to continue her work on M dwarfs, now as a
full-time professor.
Since Antígona moved to Mexico, along with students and collaborators, they
have been exploring some of the problems for habitability and life detection on
planets around M dwarfs. For example, with Jesús Zendejas and Alejandro Raga,
they explore the atmospheric loss of planets produced by the interaction with the M
dwarfs stellar wind. Without the protection of a magnetic field, planets around
active M dwarfs will be left with no atmosphere in one billion years (Zendejas et al.
2010). With Lisa Kaltenegger, they made predictions about the atmosphere needed
for a planet called GJ 581 d to be potentially habitable (Kaltenegger et al. 2011).
This exoplanet was the first found in the habitable zone; although it was later dem-
onstrated that the signal was produced by stellar activity and not by an exoplanet
(Robertson et al. 2014), the community learned about how the different assumptions
and models used changed the predictions for the abundance of CO2 that this exo-
planet may need to be habitable (Domagal-Goldman and Segura 2013).
High-energy radiation is not necessarily a hazard for life. Our planet was not
protected from UV irradiation during its first 2 billion years and anyway life
emerged and thrived. Then, Segura leads a group of students who is trying to cover
all the possibilities. Arturo Miranda and David Ramos explored the chemistry of
atmospheres with more CO2 and less O2 under the UV light of M dwarfs to under-
stand how biosignatures may change for these cases. Marion Zulema Armas is
working on the possibility that the UV light was the driver for building the starting
materials for life in planets in the HZ of M dwarfs. Laura Ribeiro Do Amaral is
using a model called VPLanet to find out if an atmosphere will survive to the X-rays
from the frequent and energetic flares of an M dwarf. For now, we cannot detect
atmospheres of exoplanets around M dwarfs; new instruments are being planned for
doing the task. This research will help to choose the best targets to observe with
those instruments and to interpret observations. We expect to discover habitable
planets and maybe find the signatures of life in one of them to tell us we are not
alone in the universe. A happy thought even if our neighbors are just gas-producing
bacteria.
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Chapter 3
Was LUCA a Hyperthermophilic
Prokaryote? The Impact-Bottleneck
Hypothesis Revisited
Gilberto P. Morales and Luis Delaye
Abstract In the Origin of Species, Darwin wrote “The affinities of all the beings of
the same class have sometimes been represented by a great tree. I believe this simile
largely speaks the truth.” Modern comparative genomics has revealed that the intu-
ition of Darwin was correct. A set of highly conserved genes and cellular functions
indicate that all life on Earth is related by common ancestry. These genes were
inherited from the last universal common ancestor or LUCA. The functions coded
by these genes suggest that LUCA was a rather complex cell already endowed with
a genetic code and a protein translation apparatus. One of the questions regarding
the nature of LUCA is whether it was a hyperthermophile. Here, we review recent
evidence derived from the molecular fossil record on the temperature preferences of
LUCA. We suggest that current evidence on the nature of LUCA and its immediate
predecessors are compatible with the impact-bottleneck hypothesis – the proposal
that during the early evolution of life, a meteoritic impact eliminated all life on
Earth except for prokaryotes capable of living at high temperatures. If our interpre-
tation of the data is correct, it would indicate that early life was resilient to the rough
environmental conditions of the Archean, a relevant result from the point of view of
astrobiology because it would exemplify the persistence of life in harsh
environments.
3.1 Early Life as Revealed by Molecular Sequences
The appearance of early life on the primitive Earth was preceded by a diversity of
astronomical and geochemical events that together set the conditions for the origin
of life. From the prebiotic point of view, the timing and the specific conditions that
led to the origin of organic molecules and the assembly and evolution of these into
G. P. Morales · L. Delaye (*)

Departamento de Ingeniería Genética, CINVESTAV, Irapuato, Guanajuato, Mexico

76 G. P. Morales and L. Delaye
the first cellular systems are still active areas of research (Patel et al. 2015). However,
from a biological perspective, there is strong evidence indicating that extant cells
were preceded by simpler systems where RNA performed both catalytic and heredi-
tary functions (Becerra et al. 2007). These primitive systems based on RNA first
evolved protein synthesis leading to RNA/protein cells and then DNA synthesis
(Freeland et al. 1999).
As extant cells use DNA as their hereditary material, parsimony indicates that
the last universal common ancestor (or simply LUCA) had a DNA-based genome.
However, our ability to infer the gene content and therefore the nature of LUCA is
largely determined by the structure and the evolutionary process that generated the
universal Tree of Life (ToL). By using the information contained in the small sub-
unit (SSU) of rRNA, Woese and Fox proposed that all life on Earth can be divided
in three main lines of descent; these are Archaeobacteria (today Archaea), Bacteria
and the nucleo-cytoplasm component of Eukarya (Woese and Fox 1977a).
Incidentally, Woese and Fox also provided evidence of the bacterial origin of organ-
elles, thus supporting the hypothesis proposed by Lynn Margulis for the origin of
mitochondria and plastids (Sagan 1967). In the same year, Woese and Fox also
proposed that all three cellular domains diverged from an ancestral entity they
named progenote. They defined the progenote as a primitive entity “to recognize the
possibility that it had not yet completed evolving the link between genotype and
phenotype” (Woese and Fox 1977b). Several years later, Woese and coworkers for-
mally proposed that all life on Earth should be classified on the basis of the SSU
rRNA phylogeny into Archaea, Bacteria, and Eukarya and that the root of such ToL
was located in the bacterial branch (Woese et al. 1990). Although biologists have
largely adopted this phylogenetic scheme to represent the evolutionary relationships
between all living beings, there have been attempts to improve this view of the ToL
(Fig. 3.1).
Early criticisms to the three-domain view of the ToL suggested that it was inac-
curate to represent the evolution of genomes by using a single gene encoding the
small subunit rRNA (SSU rRNA) molecule because genes among prokaryotes were
often transferred between lineages. Accordingly, the evolution of life on Earth was
better represented by a net rather than by a bifurcating tree (Doolittle 1999). For
instance, Fournier et al. (2009) used the metaphor of the “coral of life” to better
depict the ToL due to the highly connected network of the coral dead layers that
were analogous to horizontal gene transfer (HGT) events. In a similar fashion,
Koonin et al. (2009) used the metaphor of a forest of life to summarize the phyloge-
netic history of multiple genes along evolution.
Other modifications to the scheme proposed by Woese and Fox concerned the
position of eukaryotes in the ToL. Early on, James Lake proposed that eukaryotes
branched from a group of Archaea named by him as Eocytes (Lake et al. 1984). The
Eocytes were later identified as Crenarchaeota. Interestingly, a modified version of
this hypothesis has received support from recent phylogenetic analyses. These anal-
yses suggest that Eukarya evolved from within the Archaea. Specifically, that
Eukarya are closely related to a lineage of Archaea known as TACK: Thaumarchaeota,
Aigarchaeota, Crenarchaeota and Korarchaeota (Spang et al. 2015).
3 Was LUCA a Hyperthermophilic Prokaryote? The Impact-Bottleneck Hypothesis… 77
Fig. 3.1 Different proposals of the Tree of Life (ToL). Circles represent common ancestors of
Archaea (A), Bacteria (B), and Eukarya (E) domains. LUCA last universal common ancestor,
LBCA last bacterial common ancestor, LAECA last archaeal-eukaryal common ancestor, LACA last
archaeal common ancestor, LECA last eukaryotic common ancestor. The arrows represent the hori-
zontal gene transfer process
An alternative version of the origin of eukaryotes (and the treelike structure) was
proposed by Rivera and Lake (2004) where the ToL is substituted by a ring of life.
In this scheme, Eukarya originated by a fusion between a Gram-negative prokaryote
and an Eocyte, thus giving rise to a ringlike structure. Nevertheless, the ring of life
structure has been criticized for being biologically unsound (Forterre 2015). More
recently, there have been attempts to add a fourth domain of life to the ToL con-
formed only by viruses (Boyer et al. 2010). However, the viral fourth domain of life
hypothesis has not resisted more careful phylogenetic analyses (Moreira and López-
García 2015).
As mentioned above, it is crucial to know the structure of the ToL to be able to
infer the biology of LUCA. For instance, if the ToL is governed by horizontal gene
transfer (HGT), then it is not possible to infer the set of genes present in LUCA
simply by looking at universally conserved genes (Doolittle 1999). This issue is
because a widely distributed gene could have its origin in one cellular lineage and
then be horizontally transferred to the other two lineages early in the evolution of
life. One possibility to identify traits inherited from LUCA in the presence of HGT
would be to look at those universally (or nearly universal) conserved genes having
three domain phylogenies (i.e., phylogenies where Archaea, Bacteria, and Eukarya
show as monophyletic). The first attempt in this direction resulted in 50 gene fami-
lies, most of them related to the ribosome (Harris et al. 2003). The existence of
ribosomes indicates that LUCA was capable of protein synthesis and therefore was
more complex than a progenote.
3.2 The Nature of LUCA and Its Conditions of Existence
In relation to its optimal growth temperature, living organisms could be classified as

psychrophiles (≤15 °C), mesophiles (~15–45 °C), thermophiles (~ 45–80 °C), and
hyperthermophiles (≥80 °C) (Gaucher et al. 2010). The ToL, as revealed by rRNA,
enabled the ancestral states of present-day characters to be inferred. By mapping the
location of heat-loving prokaryotes along the ToL, Stetter proposed in 1996 that
LUCA was a hyperthermophile (Stetter 1996). Accordingly, the deepest and short-
est branches of the tree were occupied by prokaryotes living at >70 °C: Aquifex and
Thermotoga (among the Bacteria) and Pyrodictium, Pyrobaculum, Desulfurococcus,
Sulfolobus, Methanopyrus, Thermococcus, Methanothermus, and Archaeoglobus
(within the Archaea).
However, at the end of the last century, an intense debate began regarding the
temperature at which LUCA lived. At that time, the prevailing view was that LUCA
was a hyperthermophile (Stetter 1996). Nevertheless, statistical analysis of rRNA
suggested that LUCA was a mesophile or a thermophile at best (Galtier et al. 1999).
This analysis was based in the correlation between G + C content of rRNA stems
and known optimal growth temperature of prokaryotes (Galtier and Lobry 1997).
The above analysis was contested by Di Giulio (2000) by using a series of rules
based on parsimony to infer the G + C content of rRNA in LUCA. According to Di
Giulio, the universal ancestor was a hyperthermophile. A few years later, the posi-
tion of hyperthermophiles at the base of the ToL was questioned by Brochier and
Philippe (2002). By removing fast-evolving unreliable sites from the multiple align-
ment of rRNA sequences, Brochier and Philippe (2002) obtained a ToL in which
Aquifex and Thermotoga were no longer the deepest branching taxa. Instead, the
Planctomycetales occupied the basal position among Bacteria. This result suggested
that hyperthermophilic bacteria adapted to high temperatures by receiving genes
from heat-loving Archaea through HGT. However, the basal position of
Planctomycetales was questioned also by Di Giulio (Di Giulio 2003; Barion et al.
2007), and, more recently, novel hyperthermophilic bacteria have been discovered
branching at the base of the bacterial tree, earlier than the appearance of Aquifex and
Thermotoga (Takami et al. 2012; Colman et al. 2016).
Later on, Boussau et al. (2008) implemented a similar approach to Galtier et al.
(1999) but this time using 456 organisms sampled from Bacteria, Archaea, and
Eukarya. In addition to the analysis of G + C content in rRNA stems, Boussau et al.
(2008) also used the correlation between optimal growth temperature and the fre-
quency of the amino acids: IVYWREL (Zeldovich et al. 2007) in 56 nearly univer-
sal proteins to infer the optimal growth temperature of LUCA. Both rRNA and
amino acid ancestral reconstruction resulted in a non-hyperthermophilic
LUCA. Their analysis also revealed that the last bacterial common ancestor (LBCA)
and the last archaeal common ancestor (LACA) were both hyperthermophilic. This
result was confirmed by Groussin et al. (2013), who analyzed the phylogenetic sig-
nal present in molecular sequences implementing improved ancestral reconstruc-
tion models for rRNA and amino acid sequences.
3.3 R
everse Gyrase: A Clue to Trace the Evolution
of Heat-Loving Microbes
The discovery of the enzyme reverse gyrase in Sulfolobus acidocaldarius, which is

active only at temperatures >55 °C, immediately raised the suspicion that this mol-
ecule is necessary to prevent denaturation by heat of genomic DNA with poor G + C
content (Kikuchi and Asai 1984). It also suggested that other hyperthermophiles
would require the enzyme for living at high temperatures. Several years after the
discovery of reverse gyrase, Forterre (2002) took a genomic comparative approach
to look for hyperthermophile-specific proteins. Surprisingly, he found that the only
hyperthermophile-specific protein was reverse gyrase.
In principle, it would be straightforward to see if the phylogenetic distribution
and evolution of reverse gyrase is consistent with its presence in the genome of
LUCA. Recently, Weiss et al. (2016) attempted a reconstruction of the gene comple-
ment of LUCA by looking for those gene families that fulfill two criteria: (1) the
protein should be present in at least two higher taxa of Bacteria and Archaea, and
(2) its tree should recover bacterial and archaeal monophyly. The results revealed
355 gene families that putatively were present in LUCA, among which there was
the reverse gyrase. Therefore, Weiss et al. (2016) suggested that LUCA was indeed
a hyperthermophile. However, the selection criteria used by Weiss et al. (2016) were
criticized by Gogarten and Deamer (2016) as perhaps being biased toward genes
“with a limited distribution and utility in today’s organisms” like those used by cells
to live at higher temperatures. Gogarten and Deamer argued that genes used by most
cells are more likely to be horizontally transferred between Archaea and Bacteria
(Fig. 3.2a) and thus not selected by the methodology of Weiss et al. (2016), whereas
genes used only by thermophiles are likely to be selected by the specified criteria if
“these were transferred between the domains and, subsequently, between orders of
Fig. 3.2 False positives and negatives when inferring the genome of LUCA. (a) Highly conserved
genes showing evidence of HGT are not identified to be inherited from LUCA and thus represent
false-negative cases; (b) genes originated in one lineage (e.g., in Archaea: yellow star) and subse-
quently transferred to Bacteria are identified as inherited from LUCA and therefore represent false
positives. Purple branches denote hyperthermophilic prokaryotes
thermophiles within the same domain” (Fig. 3.2b). The result, according to Gogarten
and Deamer, was preliminary and highly biased towards a set of genes present in a
minority of genomes in the tree of life.
This result was in close agreement with a recent result found by Catchpole and
Forterre (2019) with respect to reverse gyrase evolution. In their analysis, which
comprises the largest reverse gyrase dataset to date (376 sequences), archaeal and
bacterial lineages do not form monophyletic groups, thereby contradicting the result
presented by Weiss et al. (2016). Moreover, the short branch lengths separating
archaeal from bacterial lineages in the reverse gyrase tree are not consistent with the
long branches separating Archaea from Bacteria in phylogenies made from univer-
sal markers like 16S rRNA gene, RNA polymerases, or EF-G transcription factors,
thus suggesting that reverse gyrase has not been inherited from the universal ances-
tor (Fig. 3.3). This analysis reinforces the idea that reverse gyrase evolved after the
time of LUCA, perhaps in Archaea, and was later transferred to hyperthermophilic
Bacteria.
3.4 A
ncestral Protein Reconstruction: A Paleobiochemistry
Experimental Approach
Ancestral sequence reconstruction enables the testing of hypotheses concerning the

past histories of protein families. From multiple alignments of homologous
sequences and tree topology, ancestral sequences can be inferred at each node in the
tree by using a model of sequence evolution. Once inferred, ancestral sequences can
be synthesized in vitro to study their properties. This approach has been used to
reconstruct ancient proteins of earliest organisms and trace back the changes in
temperature of the ancient biosphere (Gaucher et al. 2008; Akanuma 2017).
For example, the unfolding temperature of nucleoside diphosphate kinase (NDK)
correlates with the optimal environmental temperature of its host organism
(Akanuma et al. 2013). In an attempt to infer the temperature preferences of LUCA,
Akanuma et al. (2013) reconstructed the ancestral protein sequence of bacterial and
archaeal NDK with a homogeneous model of sequence evolution. They found that
the ancestral bacterial and archaeal NDK sequences had a thermal stability equiva-
lent to or greater than those of extant homologous from thermophilic organisms.
Later on, Akanuma et al. (2015) repeated their analysis now with a nonhomoge-
neous model of sequence evolution and excluding hyperthermophilic sequences
from their dataset. They found that the ancestral proteins from Bacteria and Archaea
are still thermally stable.
In their analysis, Akanuma et al. (2013) were not able to directly reconstruct the
NDK sequence of LUCA because of the lack of a root in the NDK tree. To infer the
NDK sequence of LUCA, Akanuma and coworkers used the following heuristic
approach (Akanuma et al. 2013). The ancestral archaeal and bacterial NDK differed
by only 24 residues. They reasoned that the NDK of LUCA would contain some
combination of these 24 residues. To test the hypothesis of a heat-loving LUCA,
they first took the ancestral bacterial NDK (because it had the lowest unfolding
temperature) and identified those substitutions among the 24 variants, which
Fig. 3.3 Phylogenetic history of reverse gyrase. This enzyme is likely to have originated in the
Archaea and vertically inherited in this domain. However, in Bacteria the phylogeny is more con-
sistent with multiple HGT events. The short branch separating Archaea from Bacteria and the lack
of monophyly of the domains do not suggest the presence of reverse gyrase in LUCA. Node num-
bers refer to 100 bootstrap replicas
decreased its thermal stability when introduced individually. By this they found
seven destabilizing substitutions. Second, they introduced all seven substitutions
simultaneously into the ancestral bacterial NDK and measured its thermal stability.
They found that the unfolding temperature of this NDK was 94 °C, which
corresponds to an environmental temperature of 75 °C. They suggested that this is
the lowest estimate for the environmental temperature of LUCA.
Is the inference made from the NDK analysis reliable to infer the environmental
temperature of LUCA? In the first place, we should ask whether NDK was indeed
present in LUCA. Given the function of NDK, it is very likely to be a very ancient
enzyme already present in LUCA. However, the phylogenetic analysis of NDK pre-
sented by Akanuma et al. (2013) shows the Archaea domain as polyphyletic.
Although the polyphyly of Archaea in NDK phylogeny is likely the result of HGT
after the split of Archaea from Bacteria, other positions of the root should be
explored more thoughtfully. In any case, ancestral protein reconstructions of more
enzymes are needed to evaluate if there is a general trend of increased thermal sta-
bility of the LUCA proteome.
3.5 When Did LUCA Live?
The solar system began to form ~4.56 billion years ago (Gya) and the Earth-Moon
system originated when the proto-Earth collided against Theia ~4.53 Gya (Crawford
2013). During the following ~600 million years, the Earth experienced a series of
events including the Late Heavy Bombardment. The formation of the Moon marks
a maximum constraint for the age of life on Earth. The oldest, however disputed,
evidences of ancient life include microfossils from the Nuvvuagittuq Belt in Quebec,
Canada (3770–4280 Gya) (Dodd et al. 2017), cell-like inclusions (3800 Gya) (Pflug
and Jaeschke-Boyer 1979), stromatolites (3700 Gya) (Nutman et al. 2016), and iso-
tope data from the supracrustal belt in West Greenland (>3700 Gya) (Mojzsis et al.
1996; Rosing 1999) and a controversial evidence from preserved carbon in 4100
Gya zircons (Bell et al. 2015). However, the oldest indisputable evidence of life
(according to Betts et al. (2018)) is the microfossils from the Strelley Pool Formation
from Australia, having ~3.4 Gya (Sugitani et al. 2015).
Based on a recently calibrated two-domain phylogenetic tree (i.e., a tree where
Eukarya branches from within the Archaea and the root are located between Archaea
and Bacteria), Betts et al. (2018) suggest that LUCA lived >3.9 Gya; this is before
the Late Heavy Bombardment ended. How does this time estimate accommodate
with the inferences on the nature of LUCA and its predecessors? The impact-
bottleneck hypothesis suggests that a large meteorite impact capable of heating the
oceans by 100 °C eliminated all life on Earth except hyperthermophilic microorgan-
isms capable of living at such high temperatures (Gogarten-Boekels et al. 1995). In
this scenario, LUCA was not necessarily hyperthermophile, but the ancestor of the
lineages that survived the hecatomb. Accordingly, the surviving lineages were
hyperthermophilic Archaea and Bacteria (Nisbet and Sleep 2001). In fact, computer
simulations of the Late Heavy Bombardment suggest the Earth was not completely
sterilized during this time by meteoritic impacts (Abramov and Mojzsis 2009;
Grimm and Marchi 2018). Additionally as discussed by Gogarten-Boekels (1995),
the long branches connecting Archaea and Bacteria (i.e., the absence of deep
branching lineages) can be explained by the catastrophic extinction made by a large
meteorite impact.
Interestingly, the impact-bottleneck hypothesis fits the results provided by
Boussau et al. (2008) and Groussin et al. (2013) regarding the prediction that the
ancestor of Bacteria and Archaea was hyperthermophilic microbes while LUCA
was a thermophile at best. It also fits the prediction made by the ancestral recon-
struction of the NDK Akanuma et al. (2013). However, in this last case the ancestral
reconstruction of the NDK suggests LUCA was a hyperthermophile.
Therefore, the timing provided by the molecular clock analysis is consistent with
LUCA living before the Late Heavy Bombardment ended (Betts et al. 2018)
(Fig. 3.4). With this result, it remains to be seen whether the position of hyperther-
mophiles in the bacterial tree is basal or not (Brochier and Philippe 2002; Barion
et al. 2007) although the discovery of recent hyperthermophiles in the base of the
bacterial tree suggests that this is the case (Takami et al. 2012; Colman et al. 2016).
3.6 Conclusions and Perspectives
Here, we show that the impact-bottleneck hypothesis accommodates various infer-

ences regarding the growth temperature of LUCA and its immediate predecessors.
This kind of argumentation used to reveal evolutionary history, where several facts
are coordinated by a central explanation, is called consilience (or concordance of
several) and was used by Darwin to suggest that all domestic pigeons originated
from the wild Columba livia (Gould 2002). It can be said that the same kind of
argumentation was used to infer the existence of the RNA world (Lazcano 2016).
However, a close fit of the hypothesis to the data does not prove that the hypothesis
is right. As suggested by the philosopher of biology Elliott Sober, if you hear a loud
noise in the room above, you could suggest it was made by gremlins playing bowl-
ing in the attic; however, it is almost certainly not the case (Page and Holmes 1998).
Therefore, in the case of the impact-bottleneck hypothesis discussed here, we can
only expect that further data is still consistent with it. For instance, if hyperthermo-
philes don’t lie at the base of the bacterial or archaeal branch (Brochier and Philippe
2002; Raymann et al. 2015), then the meteorite-bottleneck hypothesis loses explan-
atory power and is more likely to be wrong. Only further data will tell.
Fig. 3.4 Timeline of the early evolution of life on Earth. Bars represent a time estimated of every
process. Data taken from the following sources: solar system formation ∼4.6 Gya (Banerjee et al.
2016); Earth formation ∼4.56 Gya (Maruyama and Ebisuzaki 2017); Moon formation ∼4.53 Gya
(Crawford 2013); first oceans ∼4.3 Gya (Lyons et al. 2015); a Hadean geomagnetic field ∼4.2 Gya
(Tarduno et al. 2015); duration of the Late Heavy Bombardment (from ∼4.1 to ~3.8 Gya) (Lowe
and Byerly 2018); plate tectonics initiation ∼ 4.4 Gya (Wilde et al. 2001); first continents formed
at ∼4 Gya (Hastie et al. 2016); oldest evidence of hydrothermal vent precipitates ∼3,7–4,2 Gya
(Dodd et al. 2017); timeframe for the origin and early diversification of life of ~300 million years
(Lazcano and Miller 1996); emergence of LUCA ∼3.9 Gya, LBCA ∼3.4 Gya, LACA ∼3.4 Gya,
and LECA ∼1.8 Gya (Betts et al. 2018); sulfate reduction (∼3.0 Gya) and methanogenesis (∼2.6
Gya) initial activity (Lyons et al. 2015); great oxygen event from ∼3.8 to ∼2.3 Gya (Lyons et al.
2014); Cyanobacteria-like structures at ~3.4 Gya (Schopf 1993). Archaeal (A), Bacterial (B), and
Eukarya (E) domains were evolved through time. The average temperature curve of the Earth (at
the bottom) is only for illustrative purposes and shows the increment of the temperature during the
impact-bottleneck process
Acknowledgments The authors would like to thank María González for critical reading of the
manuscript.
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Chapter 4
Stromatolites, Biosignatures,
and Astrobiological Implications
Jamie S. Foster, Joany Babilonia, Erica Parke-Suosaari, and R. Pamela Reid
Abstract For millennia, humanity has looked to stars and wondered, “Are we alone
in the universe?” Although this question was initially the purview of philosophers,
now, with leaps in scientific and technological advances, we have changed the
nature of this question from existential to empirical. Today, the question “Are we
alone?” serves as a crux to the field of astrobiology. To search for life elsewhere in
the universe, we must first understand how life originates and evolves on Earth but
also how biology leaves behind residual signatures of its existence. To address these
questions, many astrobiology researchers have targeted stromatolite-forming com-
munities as model ecosystems to explore how microbe–mineral interactions, under
a range of environmental conditions, can lead to the formation of biosignatures.
Stromatolites are depositional structures formed by the activities and interactions of
microbes and have a fossil record dating back billions of years. Due to their long
evolutionary history and abundance on the modern Earth, research on the biologi-
cal, chemical and geological processes of stromatolite formation have provided
important insights into the field of astrobiology, including the diversity and preser-
vation of biosignatures. In this chapter, we examine the range of biosignatures found
in stromatolites and how these markers improve our understanding of the past, pres-
ent, and future of life in the context of astrobiology. We also discuss whether stro-
J. S. Foster (*) · J. Babilonia

Department of Microbiology and Cell Science, Space Life Sciences Lab, University of
Florida, Merritt Island, FL, USA
E. Parke-Suosaari
Rosenstiel School of Marine and Atmospheric Science, University of Miami,
Miami, FL, USA
Department of Mineral Sciences, National Museum of Natural History, Smithsonian
Institution, Washington, DC, USA
Bush Heritage Australia, Melbourne, VIC, Australia
R. P. Reid
Rosenstiel School of Marine and Atmospheric Science, University of Miami,
Miami, FL, USA

90 J. S. Foster et al.
matolite research can play a role in the future exploration of habitable worlds in our
own solar system and beyond.
4.1 S
tromatolites Are Ideal Models
for Astrobiology Research
Stromatolites are controversial. Scientists argue about their age, their origin, and
whether modern systems can serve as adequate analogs to understanding the earliest
habitats on Earth. Despite the academic discord, both fossil and living stromatolites
represent valuable resources to understand not only Earth’s past but also current
feedbacks that occur between life and its environment. Stromatolites are a type of
microbialite and are one of the most prevalent and most recognized constituents of
the Precambrian carbonate platforms (Grotzinger and Knoll 1999; Reid et al. 2011;
Suosaari et al. 2016b, 2019). Stromatolites are laminated, lithified organosedimen-
tary structures that have formed as a result of the sediment trapping, binding, and
precipitating activities of microbes in response to their local environment (Awramik
et al. 1976). As products of interactions between microbes, minerals and the envi-
ronment, stromatolites are a quintessential embodiment of linkages between the
biosphere and the geosphere. The interplay between biological and geological activ-
ities resulting in stromatolite formation has had a profound influence on the evolu-
tion and function of life on this planet.
For billions of years, stromatolite-forming microbial communities have played a
major role in regulating global cycles of major elements and sedimentation via pro-
duction and decomposition of organic matter, trapping and binding of sediment, and
precipitation of calcium carbonate. As such, stromatolites are ideal model systems
in which to investigate key astrobiological questions regarding the interactions
between microbial life and the environment as well as how these interactions shape
habitability.
4.2 Long Live the Stromatolites and Their Biosignatures
Stromatolites have an extensive geological record extending deep into the Archean.
Recent discoveries have pushed the boundaries of well-organized microbial ecosys-
tems back as far as 3.7 Ga, with reports of stromatolite-like ecosystems within the
Isua supracrustal belt located in southwest Greenland (Nutman et al. 2019).
Although the biogenicity of this site has been challenged (Allwood et al. 2018),
newly published geological and geochemical evidence reinforces the idea that the
Isua structures are likely biogenic in origin (Nutman et al. 2019). The fossil record
indicates that by the late Archean, stromatolites became increasingly common,
forming expansive carbonate reef systems reaching a peak in abundance and
4 Stromatolites, Biosignatures, and Astrobiological Implications 91
diversity during the Proterozoic (Awramik 1991; Reid 2011). For example, well-
characterized stromatolite-rich carbonates have been found at the boundary of the
late Archean and Proterozoic eons (2.5 Ga) in the Campbell and Subgroup of South
Africa (Sumner and Bowring 1996; Kamber and Webb 2001) as well as within the
laminated 3.4 Ga formations of the eastern Pilbara Craton of Western Australia
(Lowe 1980; Walter et al. 1980; Van Kranendonk et al. 2003), thus providing impor-
tant insights into dynamic changes of Earth’s environment at key evolutionary
time points.
Due to the long evolutionary history of stromatolites, these microbial ecosystems
serve as important reservoirs for biosignatures. Biosignatures are patterns or prod-
ucts made by the activity of life and not by abiotic processes (for review see Hays
et al. 2017; Chan et al. 2019). As the field of astrobiology is hindered by the fact that
we only have one representative biosphere to develop tools and hypotheses about
the nature of life in the universe, cognitive biases that impact our ability to recog-
nize and detect extraterrestrial life can be enormous. With those biases in mind, it is
likely that all life generates some type of residual biosignature, and stromatolites
can provide a valuable ecosystem to explore the processes by which biosignatures
form under a dynamically changing environment (Fig. 4.1). Here, we briefly discuss
the patterns (e.g., morphology and fabrics) and products (e.g., minerals and mole-
cules) of stromatolites as they pertain to the study of biosignatures across different
spatial scales, as well as the role that stromatolite research can play in searching for
the constraints of habitability beyond Earth.
4.3 S
tromatolite Patterns as Biosignatures: Morphology
and Fabrics
Morphological analysis of stromatolites can provide important insight into the feed-
backs, or cause-and-effect relationships, between biotic and environmental evolu-
tion. Spatial self-organization has important implications regarding ecosystem
function, specifically regarding resilience and productivity (Rietkerk and van de
Koppel 2008). By organizing themselves into regular spatial patterns, ecological
communities can concentrate local resources to create a more robust ecosystem that
can resist environmental perturbations.
Stromatolites are ideal ecosystems to explore the feedbacks between life and the
environment and how these feedbacks can lead to morphological biosignatures. The
dominance of stromatolites in the geologic record, and their persistence over time,
suggests that the stromatolite-building microbial communities have a selective
advantage that benefits community structure to withstand disturbances. As stromat-
olites grow, they generate geomorphological features capable of influencing bio-
logical processes, which in turn exacerbate physical processes and induce net
negative feedbacks (depending on the density of the organisms), leading to regular
morphological patterns (Rietkerk and van de Koppel 2008; Budd et al. 2016). These
Fig. 4.1 Stromatolites biosignatures occur across multiple scales and serve as valuable resources
for astrobiology research. Stromatolites biosignatures can be loosely categorized as “patterns and
products.” Characteristics, such as overall morphology and fabric structures, can provide insight
into the environmental inputs, whereas micro- and mesoscale products of stromatolite growth,
such as precipitated carbonate and metabolic outputs, can elucidate the underlying biological pro-
cesses of biosignature, and ultimately stromatolite formation. Together, these patterns and prod-
ucts can provide important insight into the formative environmental and biological influences
across multiple spatial and temporal scales
self-organizing patterns may be indicative of life processes, thereby serving as

important biosignatures.
Spatial patterns in the environment may reflect feedback loops that are depen-
dent on scale (i.e., short or long distances) (Rietkerk and van de Koppel 2008) and
homogenous conditions that lack strong environmental gradients (Turing 1952). For
example, Turing’s (Turing) activator–inhibitor principle suggests that resulting pat-
terns are not imposed on the system, but instead emerge as a result of various feed-
backs. Positive feedbacks, where organisms help each other survive through
changing the local environment, dominate the ecosystem at short distances; and
therefore, aggregation of microbes may provide key functions for the community,
such as facilitating nutrient retention and helping to reduce community losses to
predation or wave action (Liu et al. 2012). Negative feedbacks, where organisms
deplete resources and are in competition with each other, dominate at larger dis-
tances (Rietkerk and van de Koppel 2008). Alternatively, when the environment is
heterogeneous or heavily grazed, structures may lose self-organizing patterns or
pattern formation can be obscured (Rietkerk et al. 2004; Rietkerk and van de Koppel
2008). Short-distance positive feedbacks may increase the sharpness of organiza-
tional patterns, but without the coupling of long-distance negative feedback, forma-
tion of regular patterns may not be possible (van de Koppel and Crain 2006).
An ideal ecosystem to explore the role of biogenic spatial patterns as biosigna-
tures has been the stromatolites of Shark Bay in Western Australia (Suosaari et al.
2016a, 2019). The first documented examples of modern microbial build-ups with
sizes and shapes comparable to Precambrian forms were discovered in 1954 in a
hypersaline embayment of Shark Bay, Western Australia, known as Hamelin Pool
(Playford et al. 2013). With a shoreline of 135 km made up almost entirely of micro-
bial mats and stromatolites, Hamelin Pool is an iconic location hosting the world’s
most extensive and diverse modern marine stromatolite system.
A recent field-intensive mapping effort was conducted to examine stromatolite
distribution within the site (Suosaari et al. 2016a). This mapping program differed
from previous studies (Jahnert and Collins 2012) in that stromatolites were classi-
fied on the basis of morphology rather than microbial mat type or surface texture
(Fig. 4.2). Additionally, high-resolution single-beam sonar data were combined
with Landsat 8 satellite imagery to produce a detailed bathymetry map, which
revealed a unique geographic distribution of morphologically distinct stromatolite
structures around the margins of the Pool (Suosaari et al. 2019). This geographic
zonation allowed the differentiation of eight “Stromatolite Provinces,” each with
distinct patterns of stromatolite morphology and unique shelf physiography
(Suosaari et al. 2016b, 2019).
The microbial surface mats that generate the underlying stromatolite structures
in Hamelin Pool produce exopolymeric substances (EPS), which enhance sediment
cohesiveness and stability (Blanchard et al. 2000), and in turn help structures accu-
mulate and grow above wave-base. When elevated above sediment abrasion, and
with enough accommodation space, the surface area increases and adjacent struc-
tures may merge together. Stromatolites can also influence currents, with decreased
water depth increasing water flow by channeling the flow between structures.
Fig. 4.2 Morphological self-organizing patterns may be indicative of life processes, thereby serv-
ing as important biosignatures. The different Provinces of Hamelin Pool exhibit highly variable
morphologies that are dependent on location with the pool. The first column displays depth con-
tours from a selected area within the Provinces of Nilemah, Carbla, Spaven, and Booldah (Bar,
1 km). The second column shows an example of sub-centimeter scale 2D imagery collected via
UAV platform and processed using Fluid Lensing (Bar, 2 m). Column three displays highly dis-
tinctive morphological structures extracted structures from the fluid-lensed imagery (Bar, 1 m).
Column four provides representative field photos of the targeted Provinces
Exposed stromatolite surfaces can also stabilize sediment, promoting microbial

growth, further increasing ecosystem stability. Runnels, or drainage channels, focus
the increased water flow, preventing colonization of additional microbial communi-
ties. Observations of stromatolite morphologies (Fig. 4.2; Suosaari et al. 2016a,
2019) suggest that these lower level feedbacks may lead to distinctive self-organiz-
ing patterns (Camazine et al. 2003; Budd et al. 2016) and are highly influenced by
the energy regimes of the local environment (Suosaari et al. 2016a, 2019).
Whereas the morphology and physiography of stromatolites provide important

insight into the environmental conditions of the ecosystem, the internal fabrics of
stromatolites can help delineate the biological influences on formation and serve as
yet another distinctive biosignature (Logan et al. 1974; Reid et al. 2000, 2003). For
decades, analyses of the microfabrics in stromatolites have been successfully used
to interpret the nature and metabolisms of the microbial mat communities that pro-
duce them (Visscher et al. 1998; Reid et al. 2000, 2003; Pace et al. 2018). Mineral
precipitation in lithifying microbial mat ecosystems is a complex, intricate process
that is dependent on the saturation index as well as the presence of potential nucle-
ation sites (e.g., EPS) within the mat community (Dupraz et al. 2009). Net precipita-
tion occurs only if the processes of enhancing carbonate precipitation (i.e., microbial
guilds that promote precipitation) outweigh the processes that enhance the dissolu-
tion of carbonate minerals (i.e., microbial guilds that promote dissolution).
Additionally, features within the microfabrics, such as laminae, can form from the
successive cycling of microbial surface communities resulting in depositional
events of cemented and uncemented sediments coupled with the formation of
micrite (Reid et al. 2000). The specific characteristics of the microfabrics, whether
they were the product of trapping and binding or micrite precipitation, are highly
informative of the type of microbial community that made the structure. For exam-
ple, in Bahamian stromatolites, fused micritized sand grains are the product of
endolithic coccoid cyanobacteria. Endoliths can cross through organic matrix mate-
rial (e.g., EPS) and bore into the sand grains, which becomes infilled with micrite,
essentially fusing the grains and forming cemented layers (Reid et al. 2011). The
nature and characteristics of the microfabrics can then be used as an important bio-
signature to infer the biological processes associated with its formation over time
thereby improving the interpretation of the fossil record (Frantz et al. 2015; Pace
et al. 2018).
4.4 Stromatolite Products as Biosignatures: Minerals
Of the more than 5300 mineral species found on Earth, the vast majority are the
products of interactions and alterations by life, with approximately only 1500 min-
erals, the result of purely abiotic processes (Chan et al. 2019). However, with only
a single biosphere as a reference point, one cannot assume that the processes by
which these biogenic minerals form on Earth are the same on other planets and
moons. For example on Earth, the reservoirs of hydrocarbons, or fossil fuels, are
typically derived from geologically altered biomass and found in Earth’s interior,
whereas on Titan, the lacustrine hydrocarbon deposits are located on the surface and
are thought to be abiotic in origin, a product of photochemical interactions with
Titan’s nitrogen-rich atmosphere (Lorenz et al. 2008; Cornet et al. 2015). Therefore,
the presence of a given mineral cannot be considered solely as evidence for life;
however, it can inform the environmental context and conditions in which the min-
eral formed and may serve as an indirect biosignature of life.
Many of the deposited minerals found in stromatolites, such as carbonates, halite,

and gypsum, are found on other terrestrial worlds, such as Mars (Aubrey et al. 2006;
Masse et al. 2012; Niles et al. 2013) and do not directly indicate life. However,
chemical alterations of minerals, such as isotope fractionation, can be used in con-
junction with the depositional setting to help characterize whether the mineral is
biotic in origin and may provide insight into the metabolism that facilitated the
process. Isotopic fractionation in stromatolites has long been used to understand the
biological processes associated with its deposition; however, the fractionation pro-
files can vary considerably depending on the isotope examined and the environmen-
tal context (Andres and Reid 2006; Breitbart et al. 2009; Planavsky et al. 2009;
Chagas et al. 2016; Louyakis et al. 2017). For example, stable sulfur isotopes have
been used to understand and reconstruct stromatolite formation in both ancient and
modern systems due to pronounced fractionation patterns caused by sulfate-
reducing microbes (Kilburn and Wacey 2011; Bontognali et al. 2012). Sulfate-
reducing microbes preferentially use 32S relative to 34S, thereby releasing sulfides
that are depleted in the heavier isotope. Microbes can also deplete sulfur of various
oxidation states, which can also impact the isotopic fractionation values (Bontognali
et al. 2012; Weber et al. 2016). Microbial-induced transformations of sulfur are
ancient (Shen et al. 2001) and sulfur-metabolizing microbes have been linked to
stromatolite formations that date back 3.45 Ga (Bontognali et al. 2012). In modern
stromatolites, the formation of the laminae characteristic of stromatolites is also
highly correlated with sulfur cycling. For example, in the Bahamian stromatolites of
Highborne Cay and Little Darby Island, heterotrophic processes, such as sulfate
reduction and sulfide oxidation coupled with photosynthesis, are major drivers of
carbonate precipitation (Visscher et al. 1998; Reid et al. 2000; Andres and Reid
2006; Casaburi et al. 2016).
Carbon isotopic shifts can also provide important insight into how microbial
communities cycle CO2 and influence the δ13C signatures, thereby recording past
metabolic activities and influences on the environment (Andres et al. 2006).
Stromatolites are ideal models to examine how shifts in δ13C become recorded into
the rock record under different environmental conditions, such as lacustrine or
hypersaline. A comparison of the open marine stromatolites and thrombolites of
Highborne Cay, Bahamas, which are only a few meters away from each other,
revealed pronounced differences in δ13C values (Andres et al. 2006; Planavsky et al.
2009; Louyakis et al. 2017). The intertidal thrombolites exhibited much heavier
values compared to the adjacent stromatolites and likely reflect the larger role of
heterotrophic processes in the formation of the subtidal stromatolite laminae.
Carbon and sulfur fractionation patterns, coupled with their geological context may
be valuable indirect biosignatures for the search for life beyond Earth. Sulfur depos-
its and carbonates have been found on the surface of Mars and derived meteorites
(Aubrey et al. 2006). Additionally, close spectral matches of sulfate minerals have
been observed on more distant moons, such as Europa (McCord et al. 1999; Orlando
et al. 2005), indicating the importance and the need to more fully understand
microbe–mineral interactions on Earth to help delineate what constitutes a biosig-
nature under a given environmental context (Chan et al. 2019).
Another mineral found in stromatolites that has the potential to serve as a valu-
able biosignature is magnetite. A hallmark of modern living stromatolites is the
trapping and binding activity of surface microbial mat communities (Reid et al.
2000). The abundance of EPS material on the mat surface can sequester sediment,
which effectively helps stabilize the stromatolite-forming communities in high-
energy wave environments and under steep angles (Braissant et al. 2007; Dupraz
et al. 2009; Bowlin et al. 2012; Flood et al. 2014). In sedimentary environments,
including carbonate-depositing stromatolite systems, fine detrital magnetite can
be found, which is susceptible to external magnetic fields (Lund et al. 2010;
Petryshyn et al. 2016). As stromatolites accrete, the trapping, binding, and pre-
cipitation activities can cause distinctive patterns in the magnetite abundance,
which might not otherwise occur in the absence of a microbial mat community
(Petryshyn et al. 2016). Research on microbialites from Tahiti (Lund et al. 2010)
revealed a distinctive pattern of detrital magnetite grains bound within the frame-
work of the microbialite and not in the surrounding abiotic carbonate grains, sug-
gesting that the microbialite-forming communities, even at steep angles, were
able to effectively generate a distinctive mineral biosignature pattern (Petryshyn
et al. 2016).
4.5 S
tromatolite Products as Biosignatures:
Organic Molecules
In addition to the type of mineral present, minerals within stromatolites can also
serve as hubs or attachment points for molecular biosignatures (Chaçon 2010;
Alleon and Summons 2019). Minerals have long been thought to serve as hot spots
for the origin of life (Hazen and Sverjensky 2010), as the crystalline surfaces of
minerals can serve to concentrate, protect, and catalyze certain biological reactions
(Roling et al. 2015). Stromatolites represent an ideal ecosystem to examine how
microbe–mineral interactions can lead to the preservation of a diverse range of
organic molecular biosignatures. Molecular biosignatures can been considered
those organic compounds that have retained their original chemical structure pro-
viding information about the organisms that made the molecule and environmental
factors that may have led to its synthesis (Ourisson et al. 1982; Summons et al.
2008). There has been extensive research on using modern stromatolite-forming
mat communities to identify various organic molecular biosignatures, in particular
with lipids (Summons et al. 1999, 2013; Jahnke et al. 2004, 2008; O’Reilly et al.
2017); however, other molecules could serve as viable targets including nucleic
acids as well as primary and secondary metabolites.
The search for suitable candidates for molecular biosignatures can be exceed-
ingly difficult as the potential for biologically derived molecules to be preserved
over geological time can vary between environmental conditions and the chemical
structure (Summons et al. 2008). Moreover, the reconstruction of the community
dynamics that led to the synthesis and deposition of the targeted molecule may be
difficult to trace over evolutionary time. The billions of years of evolution and the
reshaping of genomes through horizontal gene transfer, primarily at the hand of
viruses, has recast the “Tree of Life” into a far more tangled “Web of Life”
(Olendzenski and Gogarten 2009; Koonin and Wolf 2012). Therefore, the search for
molecular biosignatures needs to expand to include not only individual molecules
that identify a given organism or targeted metabolism, but also ecosystem-level
suites of metabolic products that can provide community-level information about
the biological processes or conditions that formed the molecular pattern (Noffke
et al. 2013).
Despite these challenges, several advancements in multi-omics techniques
have been used to survey stromatolite-forming mats at the ecosystem level to
identify organic molecules and their distributions within the community as
potential biosignatures. The semi-closed nature of stromatolites coupled with
their high levels of productivity and rapid cycling of major elements renders
stromatolites amenable to meta-omics and systems biology approaches to iden-
tify potential organic molecular biosignatures. Stromatolites have been subject
of numerous multiomic analyses to more fully characterize the genomes, tran-
scriptomes, and metabolomes of stromatolite-forming taxa in a diverse range of
habitats (Desnues et al. 2008; Breitbart et al. 2009; Khodadad and Foster 2012;
Mobberley et al. 2012, 2015; Peimbert et al. 2012; Edgcomb et al. 2013, 2014;
Saghaï et al. 2015; White et al. 2015, 2016, 2018; Cerqueda-Garcia and Falcon
2016; Ruvindy et al. 2016; Warden et al. 2016; Casaburi et al. 2016; Louyakis
et al. 2017, 2018; Babilonia et al. 2018; Gomez-Acata et al. 2019). For example,
a comparative metagenomic analysis of the stromatolites of Hamelin Pool
revealed distinctive genetic enrichments within the stromatolite-forming mats
that strongly correlated with water depth within the pool (Babilonia et al. 2018).
For the intertidal stromatolite communities, there was an enrichment in photo-
synthesis-related genes, whereas the subtidal mats showed enrichments in more
heterotrophic pathways, such as genes associated with sulfate reduction
(Fig. 4.3). These gene patterns, and their products, create distinctive biomark-
ers, or indicators of the biological state or condition in which they form, to
elucidate the potential adaptive strategies and synergistic interactions between
stromatolite-forming communities and their environment. Whether these dis-
tinctive genetic or metabolic biomarkers within different populations of stro-
matolites can be preserved and used as biosignatures over time remains to be
determined. Regardless, the plethora of deciphered genes, metabolites and path-
ways are helping to lay an important foundation to understand the biological
mechanisms associated stromatolite formation and how these cellular processes
are impacted by perturbations to the environment.
Together, these recent advances in high-throughput microbiome and exposome
analysis techniques coupled with the breakthrough field of paleogenomics represent
a dynamic frontier for future molecular biosignature research. Although the applica-
tion of paleogenomics, or the study of ancient nucleic acids, to microbial ecology is
Fig. 4.3 Comparative metagenomics can create distinctive patterns of gene enrichments that pro-
vide insight into the environmental conditions that drive biological patterns. A direct examination
of the metagenomes derived from an intertidal and subtidal stromatolite-forming microbial mat in
Hamelin Pool, Shark Bay, Western Australia. Colors reflect clusters of pathways group by general
function (i.e., KEGG pathways). A positive log2-fold change designates significantly gene abun-
dances in the subtidal stromatolites, whereas a negative log2-fold change designates differential
abundance within intertidal stromatolite-forming mats. These patterns can provide insights into
potential organic molecular biosignature targets at the community or ecosystem level (Modified
from Babilonia et al. 2018)
still in the early stages, with most research focusing on host–microbe interactions
(Shapiro and Hofreiter 2014; Gorgé et al. 2016), efficient technical and computa-
tional screening capabilities to examine long-lived residual molecules under extreme
conditions is beginning to emerge. Stromatolite, both fossilized and living, serve as
valuable test beds across temporal and spatial scales to expand our search parame-
ters for molecular signatures of life.
4.6 U
sing Stromatolites to Understand the Constraints
of Habitability
For the field of astrobiology, stromatolites serve as valuable resources to explore the
intersection between the biosphere and geosphere as it relates to Earth’s habitability.
Even if stromatolites, as we know them, do not exist beyond Earth, the processes by
which they form, including microbe–mineral interactions and the subsequent feed-
backs that alter the environment, are likely to occur wherever life is present. In real-
ity, there are no perfect modern analogs for the ancient Earth or extraterrestrial
worlds, such as Mars or Europa; however, just as on Earth, the majority of life
elsewhere in the universe is expected to be microbial and likely exist as part of a
structured ecosystem. Thus, the results from stromatolite research are highly infor-
mative for future life detection missions. In particular, studies of modern stromato-
lites allow the science community to address fundamental questions in astrobiology
that go beyond just the detection of life. What is the role of biodiversity in biogeo-
chemical processes and what are the effects of geochemical processes on evolution-
ary dynamics? How do microorganisms modify and adapt to their abiotic
environment and how does that, in turn, alter environmental habitability? What con-
strains a seemingly “equilibrium” state and what are the forces that produce ecosys-
tem and evolutionary transitions?
The search for life beyond Earth is, therefore, likely to be a search for microbial-
based ecosystems, such as stromatolites, and their fingerprints. Understanding the
emergent properties of a microbial ecosystem, i.e., those taxa, genes and functions
that are important for a particular niche, can have a big impact on our understanding
of that environment including how physical and chemical environmental parameters
are affected by interacting microbial communities. The co-evolution between micro-
bial communities and their environments result in “ecosystem signatures,” from which
we can begin to develop generalizable predictions about the habitability of understud-
ied or unknown environments. The study of microbial ecosystems, such as stromato-
lites, enables us to explore fundamental questions regarding ecosystem functionally to
tell the story of life on Earth and, perhaps one day, the universe.
Acknowledgments The authors would like to thank Brooke Vitek for providing an aerial image
of the Hamelin Pool. This work was supported in part by a NASA Exobiology and Evolutionary
Biology award (NNX14AK14G) to JSF and RPR as well as a NASA NESSF fellowship
(80NSSC17K0497) to JB.
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Chapter 5
The Global Distribution of Modern
Microbialites: Not So Uncommon After All
Richard Allen White III
Abstract Modern microbialites, a specialized microbial mat that precipitates car-

bonates, have been thought over the last two decades to be extremely rare, with
some in Shark Bay, Western Australia; Highborne Cay, Bahamas; and Cuatro
Ciénegas Basin, Mexico. Like their fossilized ancient cousins, the stromatolites rep-
resent the oldest evidence for life on Earth (~3.4 Gya), and modern microbialites are
prevalent across the globe and have colonized most environments. Many of these
microbialite-forming ecosystems have been used as models for astrobiology and
NASA mission analogs, including Shark Bay, Pavilion, and Kelly Lakes. This per-
spective will discuss the global distribution of modern microbialites, their use as
astrobiology/NASA mission analogs, and the wealth of information already yielded
from ‘omics studies obtained from these ecosystems.
5.1 Physical Classification and Brief History
The term microbialite refers to a microbially derived organosedimentary carbonate

structure and is thought to be Earth’s earliest evidence of life (Grotzinger and Knoll
1999; Schopf 2006; Dupraz et al. 2009). Microbialites are classified into two super-
groups according to their macrofabric carbonate layout, as well as gross morphol-
ogy (Perry et al. 2007; Riding 2011). The first supergroup contains internally
laminated carbonate macrofabric stromatolites (thinly layered piles), oncolites (lay-
ered, but spherical), and leiolites (amorphous mesofabric) (Perry et al. 2007; Riding
2011; Fig. 5.1). The second supergroup contains unlaminated carbonate macrofab-
ric thrombolites (irregular clots) and dendrolites (branched projections that are tree-
like) (Perry et al. 2007; Riding 2011; Fig. 5.1). The classification of microbialites is
complicated by their plasticity in nature, with many gradations existing between
R. A. White III (*)

Plant Pathology, Washington State University, Pullman, WA, United States
RAW Molecular Systems (RMS) LLC, Spokane, Washington, United States
Australian Centre for Astrobiology, University of New South Wales, Sydney, NSW, Australia

108 R. A. White III
Fig. 5.1 Microbialite morphology and carbonate macrofabric layout. (Adapted from Riding 2011)
these basic morphologies. Among living forms, stromatolites and thrombolites are
the most common (Dupraz et al. 2009; Fig. 5.2).
Microbialites have been intensely studied for over one hundred years (Kalkowsky
1908). The first mention of the term stromatolite, or “stromatolith,” was used to
describe early Triassic stromatolites (Kalkowsky 1908; Riding 2011; Fig. 5.1). Pia
(1933) was the first to classify oncolites as “unattached, regularly or irregularly
spheroidal, concentrically to semiconcentrically laminated bodies” (Fig. 5.1).
Oncolites were reclassified as “a category of stromatolite structure,” thus removing
5 The Global Distribution of Modern Microbialites: Not So Uncommon After All 109
Fig. 5.2 Microbialite morphology examples. (a) A fossilized stromatolite (~505 Mya). Burgess
shale in British Columbia (Photo courtesy of Dr. David Turner, Beatty Biodiversity with permis-
sion). Gray scale bar 7 cm. (b) A close-up of the fossilized stromatolite (10× from scale bar). (c)
Clinton Creek thrombolite from Clinton Creek, Yukon Canada (Photo courtesy of Dr. Ian Malcolm
Power (University of British Columbia) with permission). Gray scale bar 3 cm. (d) A close up of
the Clinton Creek thrombolite (10× from scale bar)
them as a separate category (Pia 1933; Logan et al. 1964). Aitken (1967) was the
first to use the term “thrombolite” from the Greek meaning “thrombos or blood
clot,” from their irregular, clot-like arrangement (Fig. 5.1). Burne and Moore (1987)
effectively provided the modern definition we use today, as structures “that are
accreted as a result of a benthic microbial community trapping and binding detrital
sediment and/or forming the locus of mineral precipitation.” The definition was
broadened from one that included biotic factors to that of a “benthic microbial com-
munity,” took into account abiotic factors, such as “detrital sediment,” and added
geologic context (“forming the locus of mineral precipitation”).
5.2 Microbialites as the Oldest Persistent Ecosystems
Microbialites represent the oldest known persistent ecosystems and potentially the
earliest evidence of life (Schopf 2006; Grotzinger and Knoll 1999). The geologic
record indicates that microbialite diversification occurred concurrently with the
emergence of life ~3.50 billion years ago (Gyr), followed by a period of dominance
extending until ~542 million years ago (Mya) following the Cambrian explosion
(Allwood et al. 2006; Dupraz and Visscher 2005; Schopf 2006; Rothschild and
Mancinelli 1990; Fig. 5.3). With the age of the Earth estimated to be ~4.54 billion
years (Gyr), the appearance of microbialites in the fossil record at ~3.85–3.5 Gyr
suggests that microbialites have persisted for ~85% of geological history (Patterson
1956; Dupraz and Visscher 2005; Fig. 5.3). Modern microbialites first appear when
110 R. A. White III
Fig. 5.3 Timeline of microbialite and global atmospheric oxygen concentration over geological
time. (The figure was adapted from Canfield 2005 and Dupraz and Visscher 2005. The oxygen data
were provided by Dr. Sean Crowe (University of British Columbia) with permission, including
data from Lyons et al. 2014 and Crowe et al. 2013. Oxygen data are expressed as %PAL or present
atmospheric levels)
cyanobacteria emerge (~3.0 Gyr), persisting for roughly 66% of Earth’s geologic
history (Dupraz and Visscher 2005; Fig. 5.3). Thus, modern microbialites are the
best available representation of Earth’s earliest life forms, and their biology may
grant insight into the oldest aspects of the biosphere (Grotzinger and Knoll 1999;
Dupraz and Visscher 2005).
5.2.1 Global Distribution of Microbialites
Microbialites have adapted to radiate into many environmental conditions, includ-

ing extremes of temperature, pH, and salinity (Dupraz and Visscher 2005).
Microbialites are globally distributed and can be found in the open ocean (Reid
et al. 2000; Khodadad and Foster 2012; Mobberley et al. 2013), freshwaters (Ferris
et al. 1997; Laval et al. 2000; Gischler et al. 2008; Breitbart et al. 2009), hypersaline
lagoons (Goh et al. 2009; Allen et al. 2009), tropical lagoons (Sprachta et al. 2001),
hot springs (Bosak et al. 2012), and remnant mining sites (Power et al. 2011a, b). To
a lesser extent, they have also colonized terrestrial environments, such as soils
(Maliva et al. 2000) and caves (Lundberg and Mcfalane 2011).
Modern microbialite-forming ecosystems have been found globally, suggesting
that they are not as rare as previously thought. North America and Antarctica harbor
many examples of freshwater microbialites, with Cuatro Ciénegas and Yellowstone
microbialites being the most studied (Breitbart et al. 2009; Bosak et al. 2012). In
North America, microbialites are found in the Yukon (Power et al. 2011a),
Southwestern British Columbia (Laval et al. 2000; Ferris et al. 1997), Yellowstone
National Park in Wyoming (Bosak et al. 2012), Pyramid Lake Nevada (Arp et al.
1999), and in Mexico at Cuatro Ciénegas (Breitbart et al. 2009), Lake Pátzcuaro
(Bridgwater et al. 1999; Bischoff et al. 2004), and Lake Alchichica (Couradeau
et al. 2011). Antarctica is a haven for microbialites, with many found in pristine
condition with little human influence. Antarctica has four ecosystems containing
conical stromatolites or thrombolites: Lake Untersee (Andersen et al. 2011), Lake
Joyce (Hawes et al. 2011), Lake Hoare (Squyres et al. 1991), and Lake Vanda
(Andersen et al. 2011).
The microbialites of Central and South America are found in some of the most
extreme environments, including lakes containing toxic levels of heavy metals.
Microbialites are found in Central and Mesoamerica, including Laguna Bacalar,
Mexico, which borders with Belize (Gischler et al. 2008). In South America, micro-
bialites are found in Brazil in the hypersaline lakes of Lagoa Vermelha and Lagoa
Salgada (Vasconcelos et al. 2006; Spadafora et al. 2010), in northern Chile in La
Brava (Farías et al. 2012), and at the southern tip of Chile (Graham et al. 2014).
Laguna Socompa on the border of Chile and Argentina is an example of an extreme
ecosystem that contains microbialites, as it contains high levels of arsenic
(18.5 mg L−1) and iron (1 mg L−1) (Farías et al. 2012).
Diverse microbialites, including rare hydromagnesite forms and the largest
microbialite ever recorded, are found in Europe. The Ruidera pools in Spain hold a
great diversity of freshwater stromatolites (Santos et al. 2010), and Lake Van,
Turkey, contains the largest microbialites on record (Kempe et al. 1991; López-
García et al. 2005). Salda Gölü Lake, in southern Turkey, also holds the only other
example of hydromagnesite microbialites outside of Lake Alchichica, Mexico
(Braithwaite and Zedefa 1994; Couradeau et al. 2011).
Microbialites in Oceania are rare; however, they include modern examples of
coral replacement. Hypersaline lakes found in Lake Stonada, Indonesia (Arp et al.
2004), Kiritimati Atoll in the central Pacific (Schneider et al. 2013), and the caldera
lakes of Niuafo’ou Island, Tonga (Kazmierczak and Kempe 2006) contain micro-
bialites. Microbialites in the tropical lagoons of French Polynesia and New
Caledonia exist side by side with dead and live corals, illustrating direct competition
(Sprachta et al. 2001).
Western Australia has the oldest fossilized examples of microbialites at roughly
3.45 Gyr old (Van Kranendonk et al. 2008), and Shark Bay harbor is one of the few
examples of marine stromatolites (Goh et al. 2009; Allen et al. 2009). Two lakes in
Australia have documented microbialites, including Lake Thetis (Grey et al. 1990)
and Lake Clifton (Konishi et al. 2001; Warden et al. 2016). Other lakes in Australia
that contain thrombolitic microbialites include Lake Richmond, Lake Walyungup,
and Lake Cooloongup, which are protected and endangered; however, no publica-
tions exist describing them.
While China and Russia, the largest combined landmass on the planet, have
many examples of microbialite fossils, no studies describe modern versions, or if
112 R. A. White III
studies are available, they have not been translated into English (Wu et al. 2014). In
terms of African microbialites, a recent study found some in southern Africa (Smith
et al. 2011), and fossils have been found throughout the continent. However, Africa
remains largely unstudied when it comes to modern microbialites.
5.2.2 Proposed Microbialite Formation Hypotheses
There is much debate on whether the formation of ancient microbialites dating back
~3.4 Gya in Western Australia was purely abiotic (Lowe 1994; Grotzinger and
Rothman 1996) or a combined biotic–abiotic event (Dupraz and Visscher 2005;
Schopf 2006; McLoughlin et al. 2008; Dupraz et al. 2009).
5.2.2.1 Abiotic (Spectator Hypothesis)
The abiotic model stresses that ancient microbialites and possibly even modern ver-
sions are formed completely by abiotic processes (Lowe 1994; Grotzinger and
Rothman 1996; McLoughlin et al. 2008). Lowe suggested that the Western
Australian stromatolites within the Warrawoona group (3.5–3.2 Gya) lack the struc-
tural evidence of biotic processes and therefore are of abiotic origin (1994).
Moreover, a laboratory spray deposition experiment showed that stromatolites could
be generated abiotically using enamel spray paint with metal-oxide pigment parti-
cles (McLoughlin et al. 2008). A purely abiotic model would suggest that the
microbes and/or pathways would be the same between the microbialite and their
surrounding environment, as the chemistry alone is driving the formation of the
microbialite: microbes would merely be “spectators.” Thus, the abiotic model would
suggest that microbes have no role in the formation of microbialites.
Abiotic factors are certainly critical to microbialite formation. These include
hard water (≥121 mg CaCO3 L−1) with free divalent cations (mainly Ca2+ and/or
Mg2+), high alkalinity (≥121 mg CaCO3 L−1), stable alkaline pH water (pH ≥ 8),
clear water with visible light penetration, and dissolved inorganic carbon (DIC)
(Dupraz et al. 2009). No carbonate-derived microbialite-forming ecosystem has
been found without at least hard water, high alkalinity, and a stable alkaline pH
(pH ≥ 8); carbonates cannot form without these factors.
5.2.2.2 Biotic-Driven Hypothesis
The biotic link to the origin of microbialites has been debated for over one hundred
years since it was first suggested by Kalkowsky (1908). Lowe (1994) rejected any
biotic role in the formation of the Australian Warrawoona group stromatolites
(3.5–3.2 Gya). Buick et al. (1995) responded that Lowe violated Descartes’ fourth
rule of science, “omettre rien,” directly translating to “omit nothing,” suggesting
that Lowe (1994) omitted critical examples relating to a combined hypothesis. The
examples not included by Lowe (1994) were the post-Isua δ13C isotopic data, and
various microfossil examples illustrating that biotic–abiotic processes are involved
in the formation of microbialites (Buick et al. 1995). Since that time, sulfur isotopic
measurements in the Australian stromatolites from ~3.45 Gya suggest negative δ34S
isotopic fractionation, which can only be explained by biotic processes (Bontognali
et al. 2012, Fig. 5.3). Ancient microbialites spanning ~3.5–3.0 Gya formed under
anoxygenic processes, as sufficient oxygen was not present to drive oxygenic pho-
tosynthesis, whether cyanobacteria were present or not (Crowe et al. 2013;
Planavsky et al. 2014; Fig. 5.3).
Biotic metabolism in modern microbialites is an active and highly complex inter-
action with abiotic factors. Both oxygenic and anoxygenic metabolisms within the
surface microbial mat on microbialites are highly productive (5000–6200 mg bio-
mass cm−2 day−1) and similar to the primary productivity of tropical forests (6000 mg
biomass cm−2 day−1) (Stal 2000; Centeno et al. 2012). Carbonates and free calcium
are initially trapped by adhesive biofilms and mats that are often cyanobacterial in
origin (Dupraz and Visscher 2005; Dupraz et al. 2009). The cyanobacterial mat is
then degraded by heterotrophic bacteria that liberate HCO3− and Ca2+, thereby
increasing the saturation index and producing nucleation points for carbonate pre-
cipitation (Dupraz and Visscher 2005; Dupraz et al. 2009). Cyanobacterial mats
contain exopolymeric substances (EPS) that serve as a location of mineral nucle-
ation, while providing a heterotrophic microenvironment favorable for organomin-
eralization via dissimilatory sulfate reduction (Dupraz and Visscher 2005; Dupraz
et al. 2009). Light is another critical factor in the biotic–abiotic relationship of
microbialite formation. Many microbialite-forming ecosystems are either in shal-
low locations or have clear light visibility with high light penetration allowing for
photoautotrophic microbial mat growth.
The concept that microbes have no role in microbialite formation does not
account for evidence that specific mineral precipitation is a function of microbial
presence. For the abiotic hypothesis, the question is, why would only certain miner-
als form, and rates of precipitation differ, with the presence of microbes under simi-
lar abiotic conditions? The Atlin wetland in British Columbia, Canada, contains
carbonate-precipitating microbial mats. A microcosm experiment showed that abi-
otic–biotic microcosms promoted carbonate formation at a faster rate and resulted
in a completely different mineral (dypingite) than the abiotic microcosm (nesque-
honite) (Power et al. 2007). This suggests that both biotic and abiotic forces are
required to form particular minerals associated with lithifying microbial mats,
including microbialites (Power et al. 2007). Microbialite formation is likely a
consequence of complex interactions between both biotic and abiotic factors

(Dupraz et al. 2009). However, much is still unknown regarding the effect of micro-
bial community interactions on the formation of microbialites.
114 R. A. White III
5.2.3 Major Members of the Microbialite Microbiome
Microbialite-forming ecosystems, which include microbial mats, contain highly

diverse microbial consortia, including both cellular (bacterial, archaea, and eukary-
otes) and viral (capsid based) organisms.
5.2.3.1 Alphaproteobacteria
Alphaproteobacteria are abundant in marine, freshwater, and artificial marine-

derived microbialite-forming systems (Havemann and Foster 2008; Breitbart et al.
2009; Goh et al. 2009; Khodadad and Foster 2012 ; White III and Suttle 2013) and
cyanobacterial nonlithifying mats (Varin et al. 2012). Pozas Azules II within Cuatro
Ciénegas and the high-altitude, hypersaline arsenic-rich stromatolites of Lake
Scompa within the Argentinean Andes have a high percentage of Alphaproteobacteria
sequences (~12–15%), mainly belonging to the Rhodobacterales (Breitbart et al.
2009; Farías et al. 2012). The rare hydromagnesite microbialites of Lake Alchichica
and the oncolites of Rios Mesquites had cyanobacterial sequences that outnumber
the Alphaproteobacteria sequences (Breitbart et al. 2009; Couradeau et al. 2011).
Alphaproteobacteria contain members (e.g., Rhodobacterales) that are photohet-
erotrophic, which could influence the formation of microbialites by anoxygenic
photosynthesis and microbialite dissolution through fermentation (Dupraz and
Visscher 2005; Fig. 5.4). Anoxygenic photosynthesis would been the only type of
photosynthesis within the Precambrian stromatolites prior to the origin of cyano-
bacteria, and alphaproteobacterial anoxygenic phototrophs could have filled that
metabolic niche (Bosak et al. 2007). Alphaproteobacteria appear to be important for
microbialite nitrogen fixation, even in the presence of cyanobacterial nitrogen fixa-
tion, likely because of diel cycles (Havemann and Foster 2008; Fig. 5.4).
5.2.3.2 Planktonic Cyanobacteria
Cyanobacteria in microbialites exist as planktonic cells and mat builders. The

planktonic unicellular cyanobacteria are found in the surrounding water.
Cyanobacterial calcification is well documented (Dittricha et al. 2003; Obst et al.
2009). Synechococcus sp., a model planktonic cyanobacterium, has been shown in
multiple studies to precipitate carbonates using oxygenic photosynthesis (Thompson
and Ferris 1990; Dittricha et al. 2003; Obst et al. 2009; Fig. 5.4). Synechococcus sp.
is known to cause “whiting events,” in which calcium carbonate rains down through
the water column (Thompson et al. 1997). These “whiting events” are a photosyn-
thetically driven alkalization process that precipitates calcium carbonate principally
around the Synechococcus cells, which serve as nucleation points of precipitation
(Dittricha et al. 2003; Obst et al. 2009). These planktonic cyanobacteria provide the
Fig. 5.4 Generalized equations for microbialite formation and dissolution. Includes examples of
key taxa involved or substrates (ethanol only) during day (light) and night (dark) cycles. (White III
et al. 2016)
“bricks,” in the form of either magnesium- or calcium-based carbonates, which are

the building blocks of microbialites (Jansson and Northern 2010).
5.2.3.3 Filamentous Cyanobacteria, Mat Builders
If the planktonic cyanobacteria provide the “bricks” to make microbialites, then

mat-building filamentous cyanobacteria provide the “mortar” that stabilizes the
structure. Filamentous cyanobacteria are fundamental in modern microbialites, as
they provide nutrients and structure and directly act in the formation processes.
Filamentous cyanobacteria, like their planktonic counterparts, influence carbonate
precipitation through photosynthesis-induced alkalinization (Dupraz and Visscher
2005; Fig. 5.4). Mat-building cyanobacteria produce extracellular polymeric sub-
stances (EPS), which trap Ca2+ ions near dissolved CO32−, and provide nucleation
points for precipitation (Dupraz and Visscher 2005; Dupraz et al. 2009). Commonly
found genera of filamentous mat-building cyanobacteria in microbialite-forming
sites include Nostoc, Pseudanabaena, Schizothrix, Dichothrix, and Lyngbya, (Laval
et al. 2000; Dupraz et al. 2009).
116 R. A. White III
Mat-building filamentous cyanobacteria also provide an essential energy source

for heterotrophic bacteria in an oligotrophic water column (Lim et al. 2009). These
heterotrophic bacteria in turn mediate further lithification and buildup of the micro-
bialite structure (Omelon et al. 2013). Heterotrophs release calcium and/or magne-
sium ions that are bound to cyanobacterial EPS mat by degradation (Dupraz and
Visscher 2005). This process allows for more free interaction between cations and
CO32− anions and leads to carbonate precipitation (Dupraz and Visscher 2005).
Phosphorus, nitrogen, and various vitamins and cofactors are critical nutrients
required for primary production by filamentous cyanobacteria; these nutrients can
be sequestered in the mats and then released by heterotrophic degradation, which
feeds further primary production. These cycles of autotrophic and heterotrophic mat
breakdown are balanced; otherwise, the structures would likely dissolve and the
mats would die. Through primary production, the microbialite cyanobacterial mat is
an oasis in a desert-like oligotrophic water column.
5.2.3.4 Sulfur-Oxidizing Bacteria (SOBs)
Sulfur-oxidizing bacteria (SOBs) are a double-edged sword for microbialite forma-

tion, as they directly cause dissolution of microbialites, but byproducts of this dis-
solution provide intermediate substrates that allow for colonization by
sulfate-reducing bacteria (SRBs; Fig. 5.4). Microbialites are often found in highly
oxygenic water (Dupraz et al. 2009), and SOBs remove high amounts of oxygen,
allowing for further lithification via sulfate reduction by anaerobic sulfate-reducing
bacteria (Dupraz and Visscher 2005). SOBs are chemolithotrophs that promote dis-
solution of microbialites through the oxidation of hydrogen sulfide, which in turn
releases calcium ions from the microbialite calcium carbonate matrix (Fig. 5.4).
The sulfur oxidation pathway encodes the sox gene cluster that is responsible for
aerobic sulfur oxidization (Friedrich et al. 2005). Sox genes are conserved across
Proteobacteria, including Gamma-, Beta-, and Alphaproteobacteria (Friedrich et al.
2005). Beggiatoa sp., a ubiquitous filamentous sulfide-oxidizing gammaproteobac-
terium, would be an excellent candidate for the study of the removal of oxygen from
the highly oxygenated surface mat of microbialites (Dupraz and Visscher 2005).
The sox pathway encodes four protein complexes SoxYZ, SoxAX, SoxB, and
SoxCD, which oxidize reduced sulfur species, including sulfide (HS−), elemental
sulfur (S), and sulfite (HSO3−) (Friedrich et al. 2005). The diversity and abundance
of SOBs, and whether Sox-related pathways are found in active microbialite-
forming ecosystems, are still unclear. It is also unclear whether sulfur oxidation
pathways are a signature of healthy (actively forming) or failing (actively dissolv-
ing) microbialites.
5.2.3.5 Sulfate-Reducing Bacteria (SRBs)
Sulfate-reducing bacteria (SRBs) are a crucial part of the heterotrophic microbial

consortia responsible for lithification in microbialites (Visscher et al. 2000; Dupraz
and Visscher 2005; Gallagher et al. 2012; Fig. 5.4). Sulfate reduction is an ancient
metabolism, predating oxygenic photosynthesis (Canfield et al. 2000). Sulfate
reduction was occurring in microbialites prior to the great oxidation event and the
appearance of cyanobacterial mats (Canfield et al. 2000; Wacey et al. 2011;
Bontognali et al. 2012; Fig. 5.3).
SRBs reduce sulfate under anaerobic conditions, but also exist in active layers of
highly oxygenated mats, which presents a metabolic paradox (Dupraz and Visscher
2005; Dupraz et al. 2009). SRBs in microbialites must be oxygen tolerant, find
anoxic microenvironments, or form tight consortia with oxygen-depleting anoxic
organisms, such as SOBs (Dupraz and Visscher 2005). Recent rate measurements
suggest that SRBs within microbialites must be oxygen tolerant, as measurable
amounts of dissimilatory sulfate reduction occur in the highly oxygenated surface
layers of the cyanobacterial mat (Baumgartner et al. 2006; Gallagher et al. 2012).
SRBs may mediate carbonate precipitation in microbialites by altering the satu-
ration index and increasing alkalinity though dissimilatory sulfate reduction
(Baumgartner et al. 2006; Gallagher et al. 2012; Fig. 5.4). Active microbialite
growth is thus suggested to be related to the heterotrophic breakdown of
cyanobacterial-derived mats, which provide a rich and complex carbon source
(Dupraz and Visscher 2005). SRBs need electron donors for functional dissimila-
tory sulfate reduction: these include organic carbon, as acetate, lactate, ethanol, or
formate, or molecular hydrogen (Baumgartner et al. 2006, 2009; Gallagher et al.
2012). The concept of lithifying mats and nonlithifying mats could be explained by
the rates of dissimilatory sulfate reduction and related to the abundance of metaboli-
cally active SRBs (Dupraz and Visscher 2005; Gallagher et al. 2012). However, this
process and the underlying mechanism are not well understood.
SRBs that use the highly conserved dissimilatory sulfate reduction (or dsr) path-
way are mainly found among Proteobacteria, but are found also in nonproteobacte-
rial lineages. Specifically, the majority of SRB-related taxa are within the class
Deltaproteobacteria and fall within the orders Desulfovibrionales, Desulfobacterales,
and Syntrophobacterales. Nonproteobacterial lineages that have SRBs include the
taxa Nitrospira, Thermodesulfobacteria, Thermodesulfobiaceae, and Firmicutes
(mainly Clostridia). The dsr pathway consists of sulfate adenylyltransferase (sat),
adenylylsulfate reductase with the A and B subunit (apsAB), and dissimilatory sul-
fite reductase with alpha and beta subunits (dsrAB) (Zhou et al. 2011). The pathway
transports sulfate (reactant) with an electron donor (organic carbon, acetate, lactate,
formate, hydrogen, and/or ethanol), and through various enzymatic steps, it yields
sulfide (Zhou et al. 2011). The diversity and occurrence of the dsr pathway in micro-
bialites is not known, but it is critical to our understanding of microbialite formation
(Baumgartner et al. 2006; Gallagher et al. 2012).
118 R. A. White III
5.2.3.6 Firmicutes
Firmicutes are Gram-positive bacteria that are abundant in a multitude of environ-

ments, including microbialites (Breitbart et al. 2009). Bacillus spp. were shown
over 40 years ago to produce carbonates as a “general phenomenon” (Boquet et al.
1973). Firmicutes are known to precipitate calcium carbonate with or without the
presence of cyanobacteria, with at least one operon (lcfA) geared toward carbonate
precipitation (Barabesi et al. 2007).
Firmicutes have many mechanisms for facilitating carbonate precipitation. Seven
different Bacillus species are known to precipitate carbonates, including B. subtilis
(Barabesi et al. 2007), B. sphaericus and B. lentus (Dick et al. 2006), B. megate-
rium, B. cereus, and B. thuringiensis (Dhami et al. 2013), and B. amyloliquefaciens
(Lee 2003). Bacillus spp. are able to induce carbonate formation through the metab-
olism of urea (Hammes et al. 2003), heterotrophic ammonification of amino acids
(Castanier et al. 1999), carbonic anhydrase (Dhami et al. 2014), EPS formation
(Ercole et al. 2007), and the lcfA operon (Barabesi et al. 2007). Even at low abun-
dance, members of the Firmicutes have metabolic versatility geared toward carbon-
ate precipitation, which may have a role in the formation of microbialites (Paerl
et al. 2001; Breitbart et al. 2009; Omelon et al. 2013).
Microbial mats including microbialites have colored pigmented layers (orange to
yellow) (Bottos et al. 2008). Little is known about nonphotosynthetic pigmented
heterotrophic bacteria in microbialites, although many studies have focused on the
oxygenic phototrophs in these layers and suggested that the coloration is related to
carotenoid pathways (Nübel et al. 1999; Lionard et al. 2012). Diversity studies
(based on 16S rDNA) have found firmicutes in these layers (Bottos et al. 2008;
Lionard et al. 2012), and firmicutes synthesize carotenoids that give rise to many
different colors of bacteria, including orange (Köcher et al. 2009; Klassen 2010). A
recent study has screened for cultivatable isolates of nonphotosynthetic, pigmented,
heterotrophic members of the Firmicutes, which found isolates relating to the
Exiguobacterium genus (White et al. 2013b, 2019). The Exiguobacterium genus
appears to found in many microbialite-forming sites including Pavilion Lake (White
et al. 2019) and Cuarto Ciénegas (Rebollar et al. 2012). The functional roles of these
nonphotosynthetic, pigmented, heterotrophic Exiguobacteria are unknown, and
they may function in detoxification of heavy metals in microbialite-forming ecosys-
tems (White et al. 2019).
5.2.3.7 Actinobacteria
Representatives of the phylum Actinobacteria appear to be rare within microbialite-

forming ecosystems, and little is known about their abundance, diversity, and role.
Actinobacteria have been found in smooth and pustular pre-stromatolitic mats in
Shark Bay and the freshwater stromatolites of Ruidera Pools, Spain (Allen et al.
2009; Santos et al. 2010). Their presence in microbialites does not entail a f unctional
role, but the knowledge gap points to a need for both culture-dependent and culture-
independent studies.
Actinobacteria have also been identified in the pigmented layers in microbial
mats (Bottos et al. 2008; Lionard et al. 2012), and it has been suggested that they
may be responsible for the coloration (Klassen 2010). Although carotenoid biosyn-
thesis also occurs in Actinobacteria, little work has been completed on nonphoto-
synthetic pigmented heterotrophic members of the Actinobacteria to determine if
they may contribute to the colored layers found in microbial mats and microbialites.
Recently, a paper describing an Agrococcus (White III et al. 2013a, b, 2018) sug-
gests that it may be involved detoxification of heavy metals.
5.2.3.8 Eukaryotes
Eukaryotes in microbialites are present in low abundance (<1%), by physical counts

(Power et al. 2011a) and by sequence abundance (Couradeau et al. 2011; Khodadad
and Foster 2012). Even at low abundance, metazoans have been associated with the
global decline of microbialites (Awramik 1971; Garrett 1970, Figure 1.3). Metazoan
grazing following the Cambrian explosion has been correlated to a drop in stro-
matolite diversity about ~500–800 Mya (Awramik 1971; Garrett 1970; Fig. 5.3).
However, the metazoan grazing hypothesis is hindered by a lack of fossil evidence;
as well, protist grazers presumably predated the Cambrian microbialites far earlier
(Bernhard et al. 2013). Another major problem is that unicellular or soft body graz-
ers would leave no obvious fossils, possibly contributing to a virtually nonexistent
fossil record (Bernhard et al. 2013).
The question remains, why do modern microbialites exist if grazing is the cause
of their decline? This is puzzling, as metazoan grazing occurs even under the oligo-
trophic conditions in which many microbialites are found (Elser et al. 2005).
Possibly, metazoan grazing is controlled by abiotic factors, such as low nutrients,
high temperature, high salinity, and high pH. Cyanobacteria make cytotoxic com-
pounds that can inhibit and kill metazoans, thus cyanobacterial mats in microbial-
ites could also biologically control metazoan grazing (Neilan et al. 2013; Wu et al.
2014). The type of microbial mat that exists on the surface of a marine microbialite
is suggested both to alter the microbialite fabric (whether stromatolitic or thrombo-
litic) and to control the metazoan community structure (Tarhan et al. 2013). Thus,
the microbial community also likely plays a role in controlling metazoan grazing
(Tarhan et al. 2013).
In contrast, photoautotrophic eukaryotes (e.g., algae and other protists) could aid
in the organosedimentary formation process via photosynthesis-induced carbonate
precipitation (Fig. 5.4). Even at low sequence abundances of eukaryotes (<1%) in
marine microbialite ecosystems (Highborne Cay, Bahamas), it is suggested that
foraminifera influence the microfabric by stabilizing carbonate grains, and their
reticulopods, once encased, act as sheaths that effect the lamination of microbialites
(Bernhard et al. 2013; Edgcomb et al. 2013). Thus, foraminifera may help to explain
the changes in the microbialite microfabric observed in the late Precambrian
120 R. A. White III
(Bernhard et al. 2013). In modern microbialites, foraminifera are suggested to move

carbonate grains during diel vertical migration, disrupting lamination critical for
stromatolite formation, and promote clot formation or thrombolitic structure
(Bernhard et al. 2013). Foraminifera were found in microbialites in the Highborne
Cay, Bahamas, including thrombolites and stromatolites; however, the highest
diversity was observed in thrombolite mats or mats that were biologically turbated
(Bernhard et al. 2013; Edgcomb et al. 2013). Metazoans, Stramenopiles, Alveolata,
Amoebozoa, and Rhizaria were found to be active metabolically in various micro-
bialites across Highborne Cay and Shark Bay (Edgcomb et al. 2013).
5.2.3.9 Viruses
The role of viruses in microbialite communities has remained elusive. Viruses are
the most prevalent “organisms” on Earth, with an estimated 1030 viruses in the ocean
and around 106–107 viruses ml−1 in most freshwater systems (Suttle 2005). Viruses
are agents of cell lysis and thus play a role in carbon cycling on a global scale (Suttle
2005). Additionally, new research has found that many viruses harbor auxiliary
metabolic genes, including genes that encode the core proteins involved in photo-
synthesis (psbA, psbD) and phosphate starvation (phoH) (Clokie et al. 2010;
Thompson et al. 2011). Viral auxiliary metabolic genes have also been shown to
redirect their host’s metabolic potential to fuel viral replication (Clokie et al. 2010;
Thompson et al. 2011). Some marine cyanophages encode a Calvin cycle inhibitor
(cp12) that inhibits the carbon-fixation actions of Rubisco (Ribulose-1,5-
bisphosphate carboxylase/oxygenase), and a viral-encoded transaldolase (talC)
redirects the host metabolism to the pentose phosphate pathway, benefiting viral
replication (Clokie et al. 2010; Thompson et al. 2011). This evidence suggests that
viruses affect the carbon metabolism of an ecosystem and may influence the chem-
istry of microbialite formation or dissolution.
5.2.4 Previously Studied Microbialite-Forming Ecosystems
Shark Bay in Western Australia, Highborne Cay in Bahamas, and the Cuatro
Ciénegas basin of Mexico, including Pozas Azules II and Rios Mesquites, are the
most studied microbialite-forming assemblages. The best studied for microbiome
studies in freshwater include Pavilion and Kelly Lake and Clinton Creek.
5.2.4.1 Shark Bay Microbialites
The oldest fossil microbialites are found in Western Australia, dating back ~3.5 Gya
(Van Kranendonk et al. 2008). Near these fossil microbialites are active, modern
versions within Shark Bay at Hamelin Pool in Western Australia. These are the main
example of hypersaline marine microbialites (Goh et al. 2009; Allen et al. 2009;
Suosaari et al. 2016). Shark Bay microbialites range in morphology from columnar,
club, spheroidal, domal, nodular to irregular-shaped morphotypes (Burns et al. 2004).
Shark Bay microbialite diversity has been assessed by three 16S rDNA amplicon
studies (Burns et al. 2004; Goh et al. 2009; Allen et al. 2009). Currently, only 412
rDNA sequences are available on GenBank from all bacterial surveys of the Shark
Bay microbialites. These sequences reveal the distribution of the various bacterial
taxa, indicating that Alphaproteobacteria (20%), Actinobacteria (16%), and
Cyanobacteria (15%) dominate, with the rest of the sequences belonging to
Planctomycetes (11%), Gammaproteobacteria (10%), and the Firmicutes (9%)
(Burns et al. 2004; Papineau et al. 2005; Allen et al. 2009; Goh et al. 2009). Papineau
et al. (2005) concluded that Shark Bay microbialites are highly diverse, with 90% of
sequences belonging to bacteria, and that Shark Bay microbialites have novel
Proteobacteria representing 28% of the sequences, followed by Planctomycetes
(17%) and Actinobacteria (14%), respectively.
Shark Bay is novel compared to other microbialite-forming ecosystems as it has a
relatively high abundance of archaea (~10% of 16S rDNA sequence), which are usu-
ally <1% of the microbial community in other microbialites (Burns et al. 2004;
Papineau et al. 2005; Allen et al. 2009; Goh et al. 2009). The archaea in Shark Bay
microbialites are principally halophiles (Papineau et al. 2005; Allen et al. 2009; Goh
et al. 2009). Many unclassified haloarchaea have been detected only in Shark Bay
microbialites and not in the surrounding seawater, indicating that Shark Bay micro-
bialites are a potential reservoir for these archaea (Papineau et al. 2005; Allen et al.
2009; Goh et al. 2009). Metagenomic data are beginning to emerge for Shark Bay
microbialites (Babilonia et al. 2018; Ruvindy et al. 2016; Wong et al. 2012) to com-
plement the amplicon libraries (Burns et al. 2004; Papineau et al. 2005; Goh et al.
2009; Wong et al. 2015, 2017), and a RNA-based study on active eukaryotes (Edgcomb
et al. 2013) is available for comparison. Recently, a virome for Shark Bay has also
been published, describing novel ssDNA viral genomes (White III et al. 2018).
5.2.4.2 Highborne Cay Microbialites
Like Shark Bay, Highborne Cay microbialites are found in shallow depths. They
have a high diversity of colored mats covering the microbialites, ranging pink,
green, brown, and black (Reid et al. 2000; Myshrall et al. 2010; Mobberley et al.
2013). Highborne Cay stromatolite-forming mats have been classified into three
types (I–III) based on morphology (Reid et al. 2000). Type I mats are considered
young and are dominated by filamentous cyanobacteria with few heterotrophic bac-
teria. They have low diversity (~128 OTUs) and low sulfate reduction, photosynthe-
sis, and aerobic respiration rates and have been described as nonlithifying mats
(Reid et al. 2000; Myshrall et al. 2010; Khodadad and Foster 2012; Mobberley et al.
2013). Type II and III mats are considered older lithifying mats with the beginning
of carbonate lamination; both have higher rates of sulfate reduction, photosynthesis,
and aerobic respiration rates than type I mats (Reid et al. 2000; Myshrall et al. 2010;
122 R. A. White III
Khodadad and Foster 2012; Mobberley et al. 2013). Type II and III mats have higher
bacterial diversity (~181 OTUs) than type I mats (Reid et al. 2000; Myshrall et al.
2010; Khodadad and Foster 2012; Mobberley et al. 2013).
Previous bacterial diversity studies based on 16S rDNA amplicons of Highborne
Cay microbialites reveal that bacteria assigned to the Proteobacteria dominate all
three mat types (52.0%), followed by Planctomycetes (12.1%), Cyanobacteria
(11.6%), and Bacteroidetes (10.4%) (Myshrall et al. 2010; Mobberley et al. 2013).
Within the Proteobacteria, Alphaproteobacteria are dominant, making up to 42% of
the proteobacterial sequences, followed by 4.9% for both the Delta- and Gamma-
proteobacterial lineages (Myshrall et al. 2010; Mobberley et al. 2013). The sur-
rounding water communities differ from those in the stromatolitic mats I–III, with
more taxa assigned to the Alphaproteobacteria and Gammaproteobacteria in the
water, and no Deltaproteobacteria detected (Myshrall et al. 2010; Mobberley et al.
2013). Like Shark Bay, Proteobacteria are the dominant group in Highborne Cay,
with Alphaproteobacteria being the most abundant OTU in the pink thrombolitic
mats (Myshrall et al. 2010; Mobberley et al. 2013).
Metagenomes for both the microbial fraction and the viral fraction have been
published for Highborne Cay (Desnues et al. 2008). However, because the sur-
rounding environment was not sampled, including the water, it is hard to know
which taxa are specific to the microbialite. Also, multiple displacement amplifica-
tion (MDA) was used to obtain DNA for sequencing, which is known to affect
quantitative results due to amplification bias (Abulencia et al. 2006; Yilmaz et al.
2010; Abbai et al. 2012).
The Highborne Cay viral metagenome was dominated by ssDNA microphages,
likely due to MDA bias toward circular templates, which enriches these viral geno-
types and skews quantitative results (Kim and Bae 2011). Moreover, these phages
are present in the surrounding seawater (Desnues et al. 2008). Nonetheless, some
ssDNA viral genotypes in Highborne Cay were found to be highly specific, suggest-
ing that microbialites act as islands of viral diversity (Desnues et al. 2008).
Highborne viral metagenomes had contigs that were similar to those in the Sargasso
Sea or Cuatro Ciénegas. No Caudovirales phage sequences were detected, which is
unusual, and may be the results of bias from MDA (Desnues et al. 2008).
5.2.4.3 C
uatro Ciénegas Microbialites: Pozas Azules II and Rios
Mesquites
Cuatro Ciénegas, or the “four marshes,” is found in the northern Mexican state of
Coahuila. Cuatro Ciénegas is home to Pozas Azules II or the “blue pools,” which
have thrombolites, and Rios Mesquites (“mesquite tree rivers”), which have onco-
lites (Breitbart et al. 2009). Cuatro Ciénegas has metagenomic data sets for both the
viral and microbial fractions, but not samples from the sediment or water (Desnues
et al. 2008; Breitbart et al. 2009).
Comparing the viral metagenomic data in Highborne Cay and Cuarto Ciénegas
suggests that microbialites act as islands of novel viral genotypes and reflect an
ancient marine origin that has persisted in a freshwater ecosystem (Desnues et al.
2008). However, high abundances of ssDNA viruses within both systems are an
artifact of the bias of MDA toward circular templates, which enriches for ssDNA
sequences and skews quantitative results (Abulencia et al. 2006; Yilmaz et al. 2010;
Abbai et al. 2012). Pozas Azules II is similar to Highborne Cay, but it has more
dsDNA phage sequences, including sequences that are similar to common marine
phages, which could be due to cross-contamination from marine samples.
5.2.4.4 Pavilion Lake
Pavilion Lake (50.8°N, 121.7°W) lies in Mable Canyon between Squamish and
Lillooet, BC, and contains active thrombolite-forming microbialites (Laval et al.
2000). Pavilion Lake thrombolitic microbialites range from ~2000 to 12,000 years
in age, with the corrected age maximum at ~4600 years and the uncorrected age
maximum at ~12,300, based on Uranium-Thorium dating (Laval et al. 2000). These
age estimates put the initial formation of thrombolites in Pavilion Lake after the end
of the last ice age (Laval et al. 2000).
Pavilion Lake is a dimictic, alkaline (pH 8.4), oligotrophic (mean total phospho-
rus 3.3 μg L–1), freshwater lake with hard water (mean calcium carbonate 182 mg L–1)
(Laval et al. 2000; Lim et al. 2009; Brady et al. 2014; Omelon et al. 2013). Pavilion
Lake microbialites consist primarily of calcite thrombolites, which change in mor-
photype as a function of depth, as follows: shallow domes resembling “open lettuce-
beds” at ~10 m, intermediate domes resembling “cabbage heads” at ~20 m,
intermediate-deep domes with hollow conical “asparagus-like” outcroppings at
25 m, and deep domes that possess “artichoke leaflets” at ≥25 m (Laval et al. 2000;
Brady et al. 2014; Omelon et al. 2013; Fig. 5.5).
Morphological characteristics of the cyanobacterial community in Pavilion Lake
microbialites have been described (Laval et al. 2000). The microbialites have thin
(>5 mm) cyanobacterial-derived mats, comprising coccoids (Gloeocapsa spp.),
rods (Synechococcus spp.), and filamentous cyanobacteria (Fischerella spp. and
Pseudoanabaena spp.) (Laval et al. 2000). The water column of Pavilion Lake is
dominated by Synechococcus spp. (8.3 × 104 cells per ml), the picoplankton known
for causing carbonate precipitation “whiting events” (Thompson et al. 1997).
Pavilion Lake microbialites have round filamentous balls of cyanobacteria that
are >2 mm at 15–20 m, which decrease in size with depth (Fig. 5.6, Brady et al.
2010). These filamentous balls of cyanobacteria are called “nodules,” which range in
pigmentation from purple to green to black. The purple cyanobacterial nodules were
morphologically classified as either Oscillatoria sp. or Calothrix sp. (Laval et al.
2000). Recent clone library analysis suggests that the green nodules are Leptolyngbya
sp. and the purple nodules are Tolypothrix sp. (Russell et al. 2014). Tolypothrix spp.
and Calothrix spp. are morphologically similar, and their phylogenetic relationships
are not well understood (Berrendero et al. 2011). Green and purple nodules create a
microenvironment that has a higher pH and higher dissolved oxygen and has active
lithification of calcite within the filaments (Brady et al. 2009, 2010). The nodules are
124 R. A. White III
Fig. 5.5 Pavilion microbialite morphologies. Red scale bar is ~10 cm across for both. (Photo
courtesy of Donnie Reid (Nuytco research) with permission)
Fig. 5.6 Pavilion Lake nodular cyanobacterial morphologies. Scale of the photo on the right at
~10 cm across. (Photo courtesy of Tyler Mckay (UC Davis))
only found at 15–25 m depths, which makes it unlikely that they are major players
in the formation of the overall microbialite structure; otherwise they would be found
at all depths.
The diversity and abundance of microbial taxa and the metabolic potential of
Pavilion Lake microbial communities are unknown. Only a single metagenomic
study is available for freshwater microbialites, which did not account for the sur-
rounding environment (e.g., sediments and water column) (Breitbart et al. 2009). Is
the microbialite microbial community and metabolic potential the same or different
from that in the surrounding environment? This knowledge will either support pre-
vious studies that microbial community and metabolic potential are microbialite
specific or will provide evidence of microbes from the surrounding environment
(Breitbart et al. 2009; Khodadad and Foster 2012; Mobberley et al. 2013). A metage-
nomic study from Pavilion Lake suggests the metabolism, viral defense genes, and
microbial abundances differ between surrounding water column, sediments, and
microbialites (White III et al. 2016).
5.2.4.5 Kelly Lake
Kelly Lake (51.1°N 101.5°W) is located outside Clinton, BC, also within Mable
Canyon, where Pavilion Lake is also located. Whispering Pines tribe among the
Secwepemc (Shuswap) first nation having settled within Clinton, BC, since the last
ice age nearly 10,000 years ago. Very little oral tradition exists from the Whispering
126 R. A. White III
Pines tribe relationship with Kelly Lake to outside their community. Kelly Lake was
named after Edward Kelly, and irishman in 1863 after a large ranch near the lake
(Akrigg and Akrigg 1997)
Kelly Lake is ~40 km from Pavilion Lake, at an elevation of 1.07 km (Ferris
et al. 1997). Kelly Lake is a smaller (1.5 × 0.3 km), is more shallow (maximum
depth 35 m), and has lower water column visibility (<10 m Secchi disk depth)
than Pavilion Lake (Ferris et al. 1997). The microbialites in Kelly Lake are mainly
thrombolites, but stromatolites are also found and have morphologies similar to
Pavilion Lake thrombolites (Ferris et al. 1997; Fig. 5.3). Kelly Lake has lower
hardness of water (mean CaCO3 ~163 mg L–1) than Pavilion Lake, is less oligo-
trophic than Pavilion Lake (mean phosphorus ~5.3 μg L–1), and has half the salt
(Na+/K+), total nitrogen, sulfate, and sulfide than Pavilion Lake (Ferris et al. 1997;
Lim et al. 2009).
Cyanobacterial composition within Kelly Lake was been morphologically char-
acterized Synechococcus sp., in the water column are ~105 ml–1 in Kelly Lake
(Ferris et al. 1997). Morphologically, cyanobacteria in Kelly Lake are classified as
coccoids (Gloeocapsa sp.), rods (Synechococcus sp.), and filaments (Fischerella sp.
and Pseudoanabaena sp.), similar to those in Pavilion Lake (Ferris et al. 1997).
Ferris et al. suggest that nodules and mats were present; however, current samples
(2011) had no visible mat but rather a deep black layer on most of the microbialites
(1997). Mat thickness was not measured in the study by Ferris et al., so it is unknown
whether they had similar mat thickness or mats at all (1997). However, due to Kelly
Lake proximity to Pavilion Lake, it is suggested that cyanobacterial mats of Kelly
Lake microbialites at least at some point were similar to those of Pavilion Lake. The
lake has changed since the 1997, and the high amounts of invertebrates on Kelly
Lake microbialites were alarming in our 2011 sampling trip. These high amounts of
metazoans indicate that their grazing could have reduced cyanobacterial mat in
Kelly Lake. Modern Kelly Lake microbialites (at ~20 m) have deeply encrusted
coccoid cyanobacteria (potentially Gloeobacter sp.) underneath the covering black
layer of iron sulfide. Small green nodules were found on microbialites tree near the
surface, but these were quite rare. Kelly Lake appears to be very different than
Pavilion Lake. At present, no molecular survey exists for Kelly Lake.
5.2.4.6 Clinton Creek
Clinton Creek has a subarctic climate and is located near the Arctic Circle, ~77 km
northwest of Dawson City, Yukon, Canada (64°26′42″N and 140°43′25″W). It is a
flooded open pit from an abandoned asbestos mine, which was active from 1969 to
1978 (Power et al. 2011a). Clinton Creek has served as a model research site for
carbon sequestration studies within industrial mining sites (Power et al. 2011a).
Sediment mineralogy is highly variable, containing quartz, muscovite, kaolinite,
and chrysotile, as well as minor aragonite and trace calcite (Power et al. 2011a).
Aragonite will preferentially lithify abiotically over calcite due to the inhibiting
effect of the high magnesium to calcium ratio (Power et al. 2011a). Clinton Creek
microbialites have a columnar thrombolitic morphology that is primarily composed
of aragonite with spherulitic texture (Power et al. 2011a). The water in the open pit
is subsaline (>3.0 g L−1), alkaline (average pH 8.4), and supersaturated with arago-
nite (Power et al. 2011a). It is oligotrophic (undetectable phosphorus) and has low
iron (~0.04 mg L−1), and light penetrates to the bottom (Power et al. 2011a). The
water is very hard (>200 mg L−1 and >75 mg Ca L−1) and has high alkalinity
(>190 mg HCO3− L−1) (Power et al. 2011a). There is minimal nutrient input into the
open pit due to the lack of surrounding soil and human activity over the last 35 years
(Power et al. 2011a).
Power et al. (2011a) were the first to microscopically and isotopically character-
ize the microbial community of the Clinton Creek microbialite, which was morpho-
logically described to have benthic diatoms (e.g., Brachysira spp.), filamentous
algae (e.g., Oedogonium spp.), dinoflagellates, and cyanobacteria. Power et al.
(2011a) noted the low microbial growth rates and suggested that the low sediment
input had little influence geologically, but they did not examine microbe–sediment
interactions.
The microbial community structure and metabolic potential of the Clinton Creek
microbialites and their surrounding environment are uncharacterized, as are micro-
bialite systems from cold regions in general. As well, the metabolic potential of
freshwater microbialites, especially from cold environments, is mostly uncharacter-
ized; only one freshwater microbialite metagenomic dataset exists, which is from a
tropical environment and which was biased by using MDA for DNA amplification
(Breitbart et al. 2009). Prior metagenomic studies, whether marine or freshwater,
did not examine the composition or metabolic potential of the adjacent sediments or
surrounding waters; such sampling would provide a context for identifying the
microbes and processes that are confined to the microbialites. In Clinton Creek, the
microbialite and sediment communities that were compared using metagenomics
determined that sediment and microbialite metabolic potential and taxonomic abun-
dances differed as well the microbialite metagenomes were more similar to polar
microbial mats than over modern stromatolites from marine systems (White III
et al. 2015). Photosynthetic was enriched in microbialites over sediments as well in
Clinton Creek due to higher cyanobacterial abundances (White III et al. 2015).
5.3 Conclusions
Microbialites represent modern laboratories to test hypotheses related to ancient

ecosystems that have global distributions. We can elucidate key questions using
microbialites, such as “how does life assemble” and “how do communities of
microbes form.” Microbialites represent the past, present, and future of life on this
planet. Finally, with modern techniques, we can learn from their history.
128 R. A. White III
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Chapter 6
The Importance of the Rare Biosphere
for Astrobiological Studies
and the Diversification and Resilience
of Life on Earth
Jazmín Sánchez-Pérez, Juan Diego Villar, Nohely Alvarez-López,

Bernardo Águila, Jhoselinne Buenrostro, Luis J. Chino-Palomo,
Marisol Navarro-Miranda, Julián Felipe Cifuentes, Ana G. Cruz-Cruz,
Benjamín Vega-Baray, Mariette Viladomat, Maria Kalambokidis,
Luis E. Eguiarte, and Valeria Souza
Abstract Let’s travel in our mind eye to either early Earth or Mars: life had already
evolved, but it was most probably patchy and hard to find; it was likely rare. We can
even imagine the same process in exoplanets or moons, life starting as an organic
oddity that eventually starts to evolve by Darwinian evolution, consuming resources
to grow and reproduce. Under that scenario, we can speculate that after the last
universal common ancestor, LUCA, diversified locally, life kept on diversifying in
J. Sánchez-Pérez · B. Águila · M. Navarro-Miranda · M. Viladomat · M. Kalambokidis ·

L. E. Eguiarte · V. Souza (*)
Instituto de Ecología, Universidad Nacional Autónoma de México, UNAM,
Mexico City, Mexico
J. D. Villar · J. F. Cifuentes
Pontificia Universidad Javeriana, Bogotá, DC, Colombia
N. Alvarez-López
Escuela Nacional de Estudios Superiores, Unidad Morelia, Universidad Nacional Autónoma
de México, UNAM, Morelia, Michoacán, Mexico
J. Buenrostro · A. G. Cruz-Cruz
Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México,
UNAM, Tlalnepantla, Estado de México, Mexico
L. J. Chino-Palomo
Facultad de Medicina, Universidad Nacional Autónoma de México, UNAM,
Mexico City, Mexico
B. Vega-Baray
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México,
UNAM, Mexico City, Mexico

136 J. Sánchez-Pérez et al.
a multitude of non-abundant lineages that were locally adapted and perhaps may
explain why we see such early radiation in the tree of life. Therefore, life started as
a rare event and the rare biosphere, that represent most of the taxonomic and func-
tional diversity in actual microbial communities, probably represents the way
communities have organized themselves since the beginning of the conquest of the
Earth. In this chapter, we will explain what it means to have a low abundance in a
community, how we study rarity, and why it is relevant for astrobiology.
6.1 W
hat Does It Means to Be Rare and Its Importance
in Microbial Communities?
For most of the last century, the understanding of microbial diversity was still based
solely on the identification of phenotypic and morphometric characteristics through
the use of microscopy by culture-dependent experimentation (Lynch and Neufeld
2015). In recent years, sequencing of the 16S rRNA gene has been broadly used for
the study of microbial communities. The first extensive work that incorporated these
new methods took place in the North Atlantic Ocean in the early 2000s (Sogin et al.
2006). This study revealed that most microbial communities had a heterogeneous
and asymmetric distribution of species, where relatively few dominant species
coexist with a high number of low-abundant species (Sogin et al. 2006; Nemergut
et al. 2011). It was therefore believed that niches with a wide range of microorgan-
isms at low frequencies were rare, due to the low performance of these species in
different environments. Today, nearly 15 years later, shotgun metagenomics has
confirmed that in most communities, the existence of the rare biosphere is a general
feature of microbial communities. This technique not only seeks to characterize
species, but it also estimates the magnitude of the functional potential of the com-
munity, in particular showing us the potential genomes of non-cultivable and unde-
scribed microorganisms (Scholz et al. 2012).
Due to the initial low coverage of the sample, in the first studies of microbial
communities, the phylotypes with extremely small populations were found only in
specific niches, such as in marine sponge parenchyma (Taylor et al. 2012), or
in amphibian skin (Walke et al. 2014), among others. One group (Gobet et al. 2012)
identified low-abundance bacterial taxa in coastal sands in response to nutrient
stress. Likewise, another group (Bowen et al. 2009) found a large composition of
low-abundance species within marsh sediments, which were highly distinct from
samples of the nearby water column. As a result, it was erroneously assumed that
these species were not well adapted and therefore not important for the function and
structure of these communities. However, through further studies, this vision
changed.
First, let’s specify that there are different definitions and approaches to measur-
ing rarity. Though rarity concepts were initially applied to plants and animals, they
are nonetheless easily extrapolated to microorganisms. The first type of rarity
6 The Importance of the Rare Biosphere for Astrobiological Studies… 137
is defined by an organism with low abundance, which describes species that are
found at a low frequency despite the size of the distribution area. Similarly, species
can also be considered rare if it is only found in very specific niches; many of the
existing examples include extremophilic microorganisms. Finally, there are organ-
isms that are delimited by the geographical area in which they live, being almost
endemic to a site.
The first kind of rarity we will explore is the low abundance. There are different
ways in which a species can become low in abundance, such as through stochastic
mechanisms that cause an abrupt change in the population (e.g., being uncompeti-
tive in a new environment) or the result of a recent migration. In contrast, low abun-
dance can also be a life strategy for many species either because that way they
escape predators or because they use low abundance resources that may be provided
by the rest of the community. For this reason, having low abundance allows for the
survival of organisms in a trade-off mechanism, and it is very possible that rarity
confers an adaptive advantage. This effect has been seen at the genotypic level with
a phenomenon called frequency-dependent negative selection, where a genotype fit-
ness is inversely proportional to its frequency in the population. This mechanism
causes a genotype to initially increase its frequency when they are rare and reduce
it when they become common. This process of succession of genotypes prevents
one genotype from becoming fixed, thus maintaining diversity at different levels, as
seen in mitochondria (Kazancıoğlu and Arnqvist 2014), clonal populations (Weeks
and Hoffmann 2008), and among different cell strains (Minter et al. 2015). Moreover,
predator and parasite dynamics can also modify the frequency of a bacterial popula-
tion (Dybdahl and Lively 1998), where rare organisms are less predated compared
to common ones (Winter et al. 2010).
The large proportion of rare species found in diverse ecosystems (Galand et al.
2009; Sogin et al. 2006; Lynch and Neufeld 2015) has led to further study the role
of these organisms in microbial communities, as well as their ecological relevance.
This rare biosphere, defined as all the organisms with a low frequency, is a relevant
aspect of the microbial communities, as it includes the greatest amount of diversity
within the community (Locey and Lennon 2016; Elshahed et al. 2008) and, in some
cases, the largest proportion of the community (Sogin et al. 2006; Lynch and
Neufeld 2015). Although, it is possible that the rarity of some species is a byproduct
of processes like local extinction or low competitiveness in the community,
many rare taxa could remain rare in the community over long periods (De Anda et al.
2018). This constancy indicates that they may play an important role in the com-
munity. If these organisms are not a stochastic product of migration or extinction
and are preserved in microbial communities, then: What role do they play within the
community?
One of the possible roles of the rare biosphere may be the contribution of
diversity contained as a stability factor. Diversity is associated with the resil-
ience of a system and functional capacity; so species perform functions in a
given environmental condition. This aspect may be counterintuitive, given the
high functional redundancy expected (Rousk et al. 2009). With so many
organisms sharing functions, the rare biosphere does not seem necessary to
maintain them; however, species with functions considered irrelevant could
become important in another environmental condition, either by adding a new
trait to the community or participating in new interspecific interactions (Fetzer
et al. 2015). The rare biosphere could, therefore, function as a seed bank. By
preserving diversity, different metabolic functions can be conserved over time,
becoming beneficial when different environmental conditions arise. This is why
the community, through its seed bank, can react and effectively cope with dif-
ferent environmental fluctuations.
Furthermore, rare organisms seem to have a different role in the maintenance of
communities compared to abundant species, since they react to different stress fac-
tors (Zhang et al. 2018). For example, one of the most important roles reported is
the response to pollution by degradation of organic compounds (Zhang et al. 2018;
Aanderud et al. 2015; Wang et al. 2017). The degradation of organic compounds is
carried out through a network of complex metabolic pathways that are shared and
coupled along with several taxa (Fuentes et al. 2014). Many of these functions are
associated with the rare biosphere since experimental studies have reported that the
capacity to degrade pollutants and toxins is reduced when rare microorganisms are
removed from wastewater (Hernandez-Raquet et al. 2013).
Additionaly, rare biosphere, with its large repertoire of functions, seems to be
part of the complex metabolic pathways associated with biogeochemical cycles. By
providing new substrates, these cycles play a key role in the assembly of communi-
ties. Low-abundant species of green and purple sulfur bacteria appear to be crucial
for the absorption of carbon and nitrogen in aquatic communities (Musat et al.
2008). Likewise, there are several examples associated with different nutrient
cycles, such as the use of alternative carbon sources as a nutritional substrate
(Hernandez-Raquet et al. 2013; Mallon et al. 2015; Matos et al. 2005; Franklin et al.
2001) and in the different phases of the nitrogen cycle, specifically in nitrification
(Griffiths et al. 2004), nitrogen uptake (Musat et al. 2008), and denitrification
(Philippot et al. 2013).
Another role of the rare biosphere in a micorbial community is against invasive
species (Vivant et al. 2013). As a large part of the diversity, low-abundant organ-
isms manage to occupy many niches within the community. Therefore, the limited
availability of niches and resources succeeds in suppressing the invasion of new
organisms into the community (van Elsas et al. 2012). The establishment of an
invasive species is dependent on the amount of resources not consumed by local
species, as well as the rate at which both consume the existing nutrients: in other
words, who is consuming nutrients faster (Tilman 1999). Thus, when a niche is
not occupied by the native species, the invasion by an exogenous species can be
favored (Pedrós-Alió 2012). In this way, the greater the diversity and the greater
the number of organisms that consume different things, the less likely it is that
there will be excess resources to enable the entry and establishment of an invasive
species.
6.2 D
ynamism in the Community: Succession Between
Common and Rare
Currently, metagenomic analyses have shown the presence of thousands of new spe-
cies in all the explored ecosystems, which are in low abundance in the community
(Pedrós-Alió 2012). This ability to describe the different species that inhabit a par-
ticular community made it possible to compare their presence and abundance in
different locations. On some occasions, members of the abundant species in one
community may be part of the rare biosphere in another. In this case, certain species
may be abundant in a particular environment, but when dispersed to other commu-
nities, they become rare (Lynch and Neufeld 2015). The succession between abun-
dance and rarity can then be determined by environmental conditions and biotic
interactions.
The rare biosphere can respond to environmental fluctuations in two ways: (1)
the “conditionally rare”, whose frequency is determined by certain environmental
factors, so when there is a favorable change in environmental conditions, its popula-
tion frequency increases (Coveley et al. 2015; Sogin et al. 2006); and (2) the “func-
tionally rare”, who after a favorable environmental fluctuation, does not increase
their growth rate but rather increase their transcriptional activity (Hausmann et al.
2019). Whatever their response is (increase in number or increase in activity), this
low-abundant genetic diversity becomes crucial to maintaining ecosystem functions
during stressful conditions (Shade et al. 2012). Therefore, understanding the pro-
cess of succession and exchange between rare and common species would provide
a great deal of information about the dynamics of the communities, including how
they are structured, their response to environmental fluctuations, and the resilience
of the community provided by the seed bank of low-abundance organisms that are
preserved.
6.3 Who Came First? The Common or the Rare?
It is possible that the origin of rarity began shortly after the origin of life, the extraor-
dinary event that took place on ancient Earth, from which emerged the last universal
common ancestor (LUCA). Due to the abrupt changes that occurred during the
Archean (Pester et al. 2010), an early diversification by adaptive events likely trans-
pired on early Earth. Considering the impossibility of finding chemical fossils as old
as LUCA, no direct evidence has been found to corroborate this diversification
event, so, logic has to take the place of evidence to fill the void in data.
Following such logic, the idea that rare species have a key function in regulation,
survival, biogeochemical coupling, and community assemblages across all ecosys-
tems may offer indirect evidence for the role that rarity may have had in the early
events of life’s evolution. These key-stone roles of the rare biosphere support the
hypothesis that species rarity has an important role in the temporal stability of the
long-term community assemblage (De Anda et al. 2018; Pepe-Ranney et al. 2012).
To elucidate the origin of the rare biosphere in the structuring of primitive com-
munities, analogous communities must be studied. Thus, the study of microbial
mats and stromatolites is an excellent approach to recapitulate the assembly of these
ancestral niches. These complex communities are perhaps the oldest niches of
which there is fossil evidence, showing evidence of life from more than 3.7 billion
years ago (Brasier et al. 2002; Knoll et al. 2016).
Modern stromatolites and microbial mats contain finely structured communities
of specialized organisms organized by micro-regions and functions. In the outer-
most layers of these structures live photosynthetic organisms, such as microalgae,
cyanobacteria, and diatoms, while in inner layers live anoxygenic photosynthetic
bacteria, sulfate-reducing bacteria, and methanogenic archaea. While in the deepest
layers, there are a large variety of heterotrophic and anaerobic microorganisms
(Wong et al. 2015). Stromatolites and microbial mats coexist with a great amount of
diversity represented by the rare biosphere, and this seems to have been vital in giv-
ing them stability for millions of years, as they coped with great environmental
changes. In this way, they probably worked as diversity hot spots and seed banks
during catastrophic events for life (De Anda et al. 2018; Reeder and Knight 2009).
Further studies are needed to determine the role of rare species in ancestral models,
such as microbialites or microbial mats, to determine if rarity is the product of
biotic/abiotic interactions or if rarity is the main determinant that gives stability to
communities over long periods of time (Ruiz-González et al. 2019).
6.4 R
are Biosphere and Extremophiles: The Importance
of Ancestral Niches for Astrobiological Studies
Today, through technological innovations, life has been found in environments pre-
viously considered uninhabitable (Singh et al. 2019). The microorganisms that
inhabit these extreme environments are adapted to them, surviving conditions such
as extreme temperatures (Galand et al. 2009; Goordial et al. 2016; Russell and
Fukunaga 1990), high salt concentrations (Ventosa et al. 2015), low nutrient avail-
ability (Plümper et al. 2017; Drummond et al. 2015), high atmospheric pressure, or
abnormal pH (Gaboyer et al. 2019; Florentino et al. 2016), among others. For astro-
biology, these adaptations are particularly interesting, as they extend the possibili-
ties of detecting life elsewhere in the cosmos.
The first evidence of life on Earth was found in the traces of isotopically light
carbon in 4.1 billion-year-old rocks (Tashiro et al. 2017; Dodd et al. 2017); soon
thereafter, fossils of microbial communities in stromatolites were discovered. In the
case of astrobiology, the ancestral niches built by microbial communities that form
stromatolites and microbial mats are a study model of interest (Merino et al. 2019;
Clements 1936), especially because these communities are currently found in
extreme conditions where life can develop slowly, without the fast-growing algae
shading the sun from the microbial phototrophs that are part of such communities.
It is, therefore, possible to imagine that ancestral species were likely more similar to
the communities of current extremophilic microorganisms, as similar environmen-
tal conditions existed on ancient Earth (Rampelotto 2013).
Since the only experiment of life that we know is on Earth, we assume that at the
beginning of life, the organisms were associated to environmental conditions as
high concentrations of salts, highly variable temperatures, high incidence of UV
rays, and a lack of easily accessible nutritional/energy sources (Anbar and Knoll
2002). These conditions led organisms to specialize toward different carbon sources
to survive, which subsequently caused a peak in diversification in the highly oligo-
trophic conditions of early life (Souza et al. 2012). For this reason, it is important to
understand the modification and superposition of niches associated with historical,
biotic, and abiotic factors, as well as the interactions that determine the abundance
of a species in a given habitat.
With this in mind, there is a principle called resource allocation that is based on
the costs of staying alive; any organism is limited by its resources, so its survival
depends on how it uses resources for its maintenance cycle, growth, and reproduc-
tion (Sánchez-Silva et al. 2005). This principle is closely related to the adaptations
acquired by extremophiles. Among these examples are thermophiles with special-
ized membrane lipids that make them more compact and resistant to denaturation at
high temperatures (Stetter 1999), as well as psychrophiles who avoid cell freezing
by utilizing a greater amount of unbranched fatty acids, an antifreeze intracellular
fluid called DMC, and adaptations in metabolic regulation (D’Amico et al. 2006).
Acidophiles, on the other hand, employ sulfate as an electron acceptor, as is the case
in acid mine drainage and hydrothermal vents, maintaining homeostasis by ion
pumping to regulate internal cell pressure with respect to their external environment
(Mosier et al. 2013). Similarly, halophiles are osmotically protected with organic
solutes, constantly keeping their cell membrane hydrated (Oren 2002). Extremophile
studies have been carried out in various extreme environments on Earth, such as in
Alaska, Antarctica, hydrothermal vents, hot springs, the ocean floor and its associ-
ated faults, and hypersaline lakes in Australia, among other model ecosystems
(Galand et al. 2009; Goordial et al. 2016; Plümper et al. 2017; Salas et al. 2015).
However, all that is known about the physiology of these extremophilic organisms
is from a few cultivated strains; the role of the diverse rare biosphere in these
extreme ecosystems largely remains unknown.
The interest of astrobiology in the rare biosphere is centered on the fact that
many of the celestial bodies of which their environmental conditions have been
described (e.g., the moon, Mars, Titan, and numerous asteroids) have extreme envi-
ronments potentially habitable by organisms similar to those found among the rare
terrestrial biosphere. Mars is particularly interesting as there is evidence that a few
billion years ago its environmental conditions were very similar to those of early
Earth (Zhang et al. 1993). So far, although the missions to Mars have focused more
on chemical description rather than the search for life, the abiotic components dis-
covered, such as the presence of surface solid water, underground liquid, and
methane (a simple molecule that on Earth is the product of methanogenesis of

Archaea), support the idea of an ancestral or recent existence of extraterrestrial
microbial life (Vago et al. 2017). Another candidate for this type of life is Titan,
Saturn’s largest moon, which has a dense and nitrogen-rich atmosphere. Titan’s
atmosphere is similar to Earth’s in terms of its prevalence of nitrogen, and beneath
the atmosphere, the moon likely has methane, ethane, and other hydrocarbons in the
liquid state – icy and frozen on the surface but potentially warm in the layers closest
to internal magmatic systems. This points to the presence of both atmospheric and
surface components that could provide sufficient raw material for biosynthesis,
enabling the possibility of an independent and persistent origin of life on Titan
(Clarke and Ferris, 1997). On the contrary, even on planets without an atmosphere
or with chemically unfavorable atmospheres for life, it is hypothesized that we
could still discover microorganisms inhabiting their inner layers.
For the aforementioned reasons, among the most important discoveries for astro-
biology in recent times are those that describe microbial life forms hidden in the
depths of the Earth that thrive in conditions independent of those in the surface
biosphere. According to Belilla and coauthors (2019), new organisms are found
every year in environments that were previously thought to be uninhabitable: such
as water systems within gold mines with estimated ages of tens of millions of years
(Lin et al. 2006); hydrothermal vents deep in the sea (Sogin et al. 2006); lagoons
with high temperatures, acidity, or alkalinity (Ward et al. 2006); hypersaline and
oligotrophic lagoons (Souza et al. 2012); and even in places in particular the mete-
oric alkaline water that emanates from a well intersecting quartzite fractures kilo-
meters underground (Moser et al. 2005). In such unique systems, it seems that
finally the limits of life have been reached (Belilla et al. 2019) – environments
where only organisms with unique abilities can inhabit, isolated and independent of
Earth’s biosphere and sunlight suggest the possibility of finding homologous iso-
lated biospheres on other planets, proliferating despite hostile surface conditions.
However, access to samples and avoiding contamination represent some of the big-
ger challenges for the study of these microorganisms (Lin et al. 2006). In addition, being
so adapted or specialized to a specific environment with unique metabolisms and inter-
actions makes traditional isolation and culture methods impossible (Al-Awadhi et al.
2013; Zapka et al. 2017). It was not until the development of sequencing technologies
and the emergence of metagenomic analysis methodologies that it was possible to study
the rare biosphere in greater depth (Lynch and Neufeld 2015).
6.5 C
uatro Ciénegas, a Model for the Study of Rare
Biosphere and Astrobiology
The extraordinary oasis in the Cuatro Ciénegas Basin (CCB) is particularly diverse
(De Anda et al. 2018; Taboada et al. 2018), not only because each site that has been
explored is unique with a large beta diversity (Souza et al. 2006), but also because
most of its microbial diversity is part of the rare biosphere, giving each microbial
mat, stromatolite, sediment sample, water sample, or soil sample a unique signature
(Lee et al. 2017; Souza et al. 2018). The reason behind such a high diversity is its
ancestry and unique geological history, making CCB a true lost world that survived
different mass extinction events throughout Earth’s history. Yet, today in the
Anthropocene, CCB risks destruction, as the wetlands are being drained for local
agricultural use (Souza et al. 2018).
This “lost world” characteristic is not merely a metaphor; it is of great astrobio-
logical relevance. The ancestral lineages survived in these extremely oligotrophic
conditions, which allowed ancient microbes to co-evolve along with their commu-
nity, resulting in an extraordinary codependency. For this reason, they survived:
they could grow and reproduce given the “extended genotype” of the community. It
is, therefore, possible that this feature can explain the resilience of life on Earth,
helping us explore age-old questions: Does life exist on other planets or the moon?
Perhaps, in other planetary systems? If so, how did it survive and diversify? It is
highly probable that the survival of the fittest is a universal rule; however, the fittest,
in this case, is the one that can coexist and collaborate, not the one that outcompetes
the neighbor. May this be a lesson of survival for us all.
6.6 Conclusions
New sequencing technologies, such as the sequencing of highly variable regions of

the 16S rRNA gene and shotgun metagenomics, have been crucial in understanding
the ecology of microbial communities today. The use of these tools has made it
increasingly possible to demonstrate the presence of rare species in communities,
identify them, and understand their role within the community. These molecular
tools are the reasons why the growth and contributions of research in this topic have
led to a rethinking of the first hypotheses about microbial communities and their
rare biosphere.
This rare fraction includes a large number of taxa, often exceeding those that
dominate the community, maintaining a consistent composition despite its low
abundance. There are, however, taxa that are unique to a site, providing a new col-
lection of lineages. This rarity exists mainly due to the particular qualities of the
habitat to which it belongs, as is the case with many extremophiles, or even the
exclusivity of the biogeographic region where they are found, as is the case of sev-
eral microorganisms in Cuatro Ciénegas. Regardless of the type of rarity being stud-
ied, there is still much to learn about the role of these organisms in microbial
communities, as well as the evolutionary and ecological mechanisms that lead a
species to be considered rare.
From a functional point of view, the role and importance of the rare biosphere
in ecological niches warrant investigation. From the advantages for a species to
remain at a low frequency to speculating on the reason why a community main-
tains its rare biosphere, there are many interesting avenues to explore further.
Thanks to the large amount of diversity found in a set of rare species, one of their
most important roles is to function as a seed bank. Together, this pool of species
conserves functions that may be necessary during an environmental change,
ensuring the survival of the community. This, in turn, can increase the frequency
of non-abundant species, making them part of the common biosphere, which cre-
ates a dynamic exchange between the abundant and rare. On the other hand, there
are some occasions where the rare biosphere holds particular functions required
in low abundance, which may play a key role in the regulation, assembly, and
survival of the community. This dynamic exchange between rare and abundant,
along with the presence of indispensable functions for community assembly and
conservation, add a further degree of complexity to the interactions of the com-
munity, giving rise to new questions about the structure of microbial
communities.
The diversity and ability to survive in extreme conditions make the rare bio-
sphere a good study model for astrobiology. Through its role as a seed bank of meta-
bolic functions, the rare biosphere offers functions of vital importance, such as the
degradation of hydrocarbons or the fixation of other carbon sources. In this way,
metabolic diversity enables their survival in extreme conditions, such as at high or
low temperatures, high levels of salinity, or nutrient shortages. Additionally, these
types of conditions are those that most closely resemble a primitive environment on
Earth or the environments of other planets. Because of this, the rare biosphere is an
excellent study model for investigating the origins of life on Earth, as well as the
possibility of life emerging elsewhere.
One hypothesis about the first forms of life after LUCA, is that life could
continue due to the rapid diversification that filled the many available niches. It
is possible that at the beginning of this diversification, there were no abundant
species; rather, there was a large diversity of functions and a low popula-
tion abundance. In this way, it is parsimonious to suggest that at the beginning,
there were generalist organisms that performed different functions, that rapidly
evolve into the current diversity we see today. However, given that there is a
diversity of functions and most species are specialists, the possibility of their
survival alone is lower, even when considering that there were likely low levels
of competition. The rare biosphere can then be closely linked to life in a com-
munity, with high levels of cooperation. In this way, specialists were able to
cover all types of nutrient cycling and be able to survive together as a commu-
nity, despite abrupt environmental changes suffered by Earth throughout its
various geological ages. Thus, evolving communities in stromatolites or micro-
bial mats may have been the key to the survival of life. Similarly, microbes that
did not live in communities could have survived because they took refuge in
places where their ancestral niches were conserved, as can be observed in hydro-
thermal vents, deep rocks, blocks of permafrost, and more. These microorgan-
isms are extremophiles, which due to the specificity of the habitat in which they
are found are considered rare.
Acknowledgments This project was funded by PAPITT-DGPA grant IG200319 to VS and

LEW. We want to thank Laura Espinosa-Asuar and Erika Aguirre-Planter for technical assistance.
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Chapter 7
Bacterial Communities from Deep
Hydrothermal Systems: The Southern Gulf
of California as an Example of Primeval
Environments
Laura Espinosa-Asuar, Luis A. Soto, Diana L. Salcedo,

Abril Hernández-Monroy, Luis E. Eguiarte, Valeria Souza, and Patricia Velez
Abstract Deep hydrothermal systems result from the magmatic and tectonic activ-
ity of the ocean floor. This deep extreme biosphere represents a unique oasis of life
driven by sulfur-based chemosynthesis instead of photosynthesis. The organisms
inhabiting these systems are adapted to cope with harsh environmental conditions
such as the absence of sunlight, high temperatures and hydrostatic pressures, and
elevated concentrations of hydrogen sulfide, as well as high concentrations of heavy
metals. Therefore, this biome is different from any other environment on modern
Earth. As expected from such conditions, chemoautotrophic prokaryotes are the
leading primary producers at the base of the food web considered as an analog to the
oldest signs of life on Earth. Herein, we discuss prokaryotic diversity and commu-
nity structure from the newly discovered hydrothermal systems in the Alarcón Rise
(AR), the Pescadero Basin (PB), and the Pescadero Transform Fault (PTF) at the
mouth of the Gulf of California, Mexico, using 16S rRNA gene amplicon Illumina
sequencing. Despite the spatial proximity of the studied vent systems (<100 km),
L. Espinosa-Asuar (*) · L. E. Eguiarte · V. Souza

Mexico City, Mexico
L. A. Soto
Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México,
D. L. Salcedo
Posgrado en Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México,
A. Hernández-Monroy · P. Velez (*)
Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de
México, UNAM, Mexico City, Mexico

150 L. Espinosa-Asuar et al.
they differ considerably in their physical, chemical, geological settings, and biotic
characteristics. Our results indicated that beta prokaryotic diversity is associated to
the sampling source, suggesting a strong effect of environmental conditions in shap-
ing microbial distribution. The most abundant phyla were Proteobacteria,
Bacteroidetes, Actinobacteria, Chloroflexi, and Epsilonbacteraeota. Also, we found
evidence on the oxidation of methane as a prevalent process in PB and PTF, since
methylotrophic bacteria and Atribacteria were abundant, in contrast to AR basalt-
hosted system. Bacteria associated with the sulfur cycle, in particular sulfur com-
pounds reducing and sulfur compounds oxidizing bacteria predominated in all
samples, confirming the importance of sulfur supporting vent communities. It is
possible that vent systems played a significant role in the origins of life on Earth.
Hence, they represent useful models when searching for life elsewhere in the
universe.
7.1 Introduction
Since the hydrothermal systems were discovered in the Galapagos Ridge, ca.
40 years ago (Corliss et al. 1979), they have proved to be a key feature on the ocean
seafloor where magmatic and tectonic activity occurs. Presently, more than 720 vent
systems are known from mid-ocean ridges and volcanic arcs (Beaulieu and
Szafranski 2018). The seawater percolating through porous sediments or basaltic
rocks entering in contact with the magmatic chamber originates the emission to the
seafloor of hot fluids (>200 °C), containing an enriched mixture of chemical reduced
compounds such as sulfide, hydrogen, methane, manganese, and iron (Brazelton
2017; Dick 2019). When these hot fluids come in contact with the cold and well-
oxygenated deep water they precipitate, forming complex edifices shaped as chim-
neys or small spires on the seafloor surface. These hydrothermal fluids represent the
chemical energy that sustains one of the most enigmatic forms of marine life found
along the plate tectonic boundaries of the ocean floor: the hydrothermal vent com-
munities (Dick 2019). The existence of these life forms that are derived from geo-
thermal energy is considered a good model to search for living conditions in other
planets or moons (Barge and White 2017).
Vent communities are epitomized by a unique assemblage of invertebrate species
that flourish under extreme environmental conditions of temperature, darkness,
pressure, and toxicity (Hessler and Kaharl 1995). These ephemeral biotic assem-
blages depend strictly on the dynamics of the hydrothermal fluid itself, lacking tem-
poral stability: if the fluid emission ceases or is obliterated by tectonic processes,
the supply of chemical energy contained in electron donors such as S−2, Fe2+, H2,
and Mn2+ is no longer available (Baker et al. 1989). These invertebrate deep-sea
communities are possible due to the unseen majority, i.e., the prokaryotic chemo-
lithoautotrophic microorganisms (Pjevac et al. 2018); these microbial communities
constitute the primary producers in the vent community, comprising mesophiles,
7 Bacterial Communities from Deep Hydrothermal Systems: The Southern Gulf… 151
thermophiles, and hyperthermophiles, generally from Phylum Aquificae, and Class

Deltaproteobacteria, Epsilonproteobacteria, and Gammaproteobacteria (Dick 2019).
Hydrothermal vent systems harbor an ecosystem based on microbial communi-
ties with reducing power of hydrogen sulfide or dissolved hydrogen gas as a source
of electrons (Deamer and Damer 2017). Here, processes associated with serpentini-
zation (where mineral components in the olivine undergo an exothermic reaction
that produces hydrogen gas and heat; Kelley et al. 2005) offer further parallels with
the biochemistry of ancient autotrophic cells (Herschy et al. 2014): hydrogen and
sulfur provide a source of chemical energy from solutes and minerals at different
redox states and pH ranges (Wächtershäuser and User 1992; Zierenberg et al. 2000)
that probably reflect the most ancient forms of autotrophy. The associated microbial
consortium in these extreme environments includes methanogenic and sulfo-
oxidizing prokaryotes (Sievert et al. 2007) that transform inorganic energy resources
into organic compounds employing a diversity of complex metabolic pathways to
fix CO2, via at least six known different metabolic pathways (Hügler and Sievert
2011). In nearly all the hydrothermal systems, denitrification processes coupled to
sulfur compounds oxidation and hydrogen oxidation play a key role (Bourbonnais
et al. 2012, 2014; Li et al. 2018).
As described by Martin et al. (2008), the chemistry of life is the chemistry of
reduced organic compounds, and this is particularly true for hydrothermal sys-
tems. These chemically reactive environments provide suitable conditions for
sustained prebiotic syntheses, and in consequence, researchers interested in the
origin of life have paid attention to hydrothermal systems (Corliss et al. 1981;
Baross and Hoffman 1985; Russell et al. 1993; Russell and Hall 1997; Zierenberg
et al. 2000; Kelley et al. 2001). In particular, the sulfide chimneys of the vent
systems are primordial environments evoking early Earth conditions, with reac-
tive gases, dissolved elements, and sharp thermal and chemical gradients (Martin
et al. 2008).
In addition, it has been suggested that a hydrothermal vent origin could allow life
to begin in the Enceladus Ocean (in the sixth largest moon of Saturn with possible
hydrothermal activity), on Europa (moon orbiting Jupiter with possible hydrother-
mal activity), and even indirectly on early Mars (Hsu et al. 2015). This conjecture
has been recently explored (Deamer et al. 2019), leading to a fascinating framework
aiming to understand aqueous environments likely to be present on the prebi-
otic Earth.
In 2000, a milestone was reached with the discovery of the unique hydrothermal
field at 700–800 m below the sea surface, the Lost City hydrothermal field (LCHF),
which is characterized by carbonate chimneys (Von Damm 2001). This system has
been considered as crucial to understanding the origin of life since the ultramafic
underpinnings of LCHF have a similar chemical composition to lavas that erupted
into the primordial oceans on early Earth (de Wit 1997; Kelley et al. 2001, 2005).
Remarkably, in recent years, a new carbonate vent system was discovered at
3800 m depth in the Pescadero Basin (PB), Southern Gulf of California, Mexico, in
the northernmost segments of the East Pacific Rise (EPR) (Paduan et al. 2018). This
site harbors an unexplored microbial diversity (with the EPR and Guaymas Basin as
the nearest known hydrothermal vents prokaryote communities; Teske et al. 2002;
Dhillon et al. 2003; Campbell et al. 2013; He and Zhang 2016; Dombrowski et al.
2018). So, in the present study, we examined the prokaryotic diversity and commu-
nity structure from hydrothermal systems in the Pescadero Basin (PB) and the
neighboring Alarcón Rise (AR), and Pescadero Transform Fault (PTF) systems
using a 16S rRNA gene amplicon Illumina sequencing approach to contribute to our
knowledge on the diversity and functioning of these prebiotic analog sites.
7.2 Materials and Methods
7.2.1 Study Area
The Gulf of California (also known as Sea of Cortes) is a semi-enclosed basin

approximately 1000 km long and 150 km wide on average. It lies between the pen-
insula of Baja California and the northwestern continental region of Mexico, and it
is connected to the Tropical East Pacific at its southern end (Álvarez-Borrego 1983).
There is a series of basins along the gulf, in which the depth increases to the
south. The Pescadero Basin (PB) system is located at the entrance of the Gulf, the
high-temperature vent field located there is at 3700 m depth, and it is a sedimentary
basin with mounds and chimneys composed of white and brown hydrothermal cal-
cite, with heights ranging from 12 to 25 m, and low mounds spread over an area of
approximately 0.2–0.5 km (Paduan et al. 2018). End-member fluids contained high
concentrations of aromatic hydrocarbons, hydrogen, methane, and hydrogen sul-
fide. The temperatures reach up to 290 °C, and their pH has been measured at 6.5
(Goffredi et al. 2017; Paduan et al. 2018). The biotic community is dominated by
the siboglinid tubeworm Oasisia aff. alvinae (Goffredi et al. 2017).
At the southeastern of PB, seepage through sediments and outcrops occurs along
the Pescadero Transform Fault (PTF), extending for 60 km. The low-temperature
vent systems are located at 23.64° and 2400 m in depth (Goffredi et al. 2017;
Salcedo et al. 2019). Only discrete venting was observed in volcanic rubble and
sediments, emanating at temperatures of 5°C (Clague et al. 2018), which supports
scattered dense patches of the coexisting siboglinid tubeworms of the species
Escarpia spicata and Lamellibrachia barhami (Goffredi et al. 2017). This commu-
nity is less complex and diverse than the one in PB, as only 15 taxa have been identi-
fied and the trophic web seems to be quite short (Salcedo et al. 2019).
The Alarcón Rise (AR) is the northernmost bare-rock segment of the EPR, and it
is adjacent to the southeastern tip of Baja California (Clague et al. 2018). This local-
ity hosts young basalt high-temperature vents with black smokers with a maximum
temperature of 360 °C. The fluids have high concentrations of hydrogen sulfide,
little to no hydrogen, methane, or hydrocarbons. The chimneys formed by hydro-
thermal fluids are polymetallic sulfide deposits. The dominant species is the sibogli-
nid tubeworm Riftia pachyptila (Goffredi et al. 2017).
7.2.2 Sampling
The sediment samples were taken during the expedition Vents and Seeps conducted
in 2015 by Monterey Bay Aquarium Research Institute (MBARI), in the Southern
Gulf of California onboard the R/V Western Flyer, equipped with the remotely oper-
ated vehicle (ROV) Doc Ricketts. The sampling was performed using short push
cores (30 cm) operated by the articulating manipulator arm of the ROV at easy
access randomly chosen sites. Samples were taken from three different deep-sea
extreme ecosystems: high-temperature vents in PB (three cores) and AR (one core),
and low-temperature vents at PTF (two cores). Surficial sediments subsamples
(10 cm depth) were obtained from the push cores and deep- frozen at −80 °C.
7.2.3 T
otal DNA Isolation, Construction of 16S rRNA Gene
Libraries, and Illumina Sequencing
The entire community DNA was isolated from six sediment core samples collected
in the three studied vent systems (Fig. 7.1), using the FastDNA®SPIN kit (MP
Biomedicals, Irvine, CA) for soils, according to the manufacturer’s instructions. An
approximately 280-bp of the V3-V4 region of the 16S rRNA gene from 357F primer
W E
PB_8 PB_3
S
PB_1
PTF_4 PTF_2
AR_5
GULF OF
CALIFORNIA 60 km
MEXICO
Fig. 7.1 Location of the three studied deep hydrothermal vent systems in the southern Gulf of
California, Mexico
(Rudi et al. 1997) was commercially amplified and sequenced using the Illumina
MiSeq 300 platform at the Laboratorio de Servicios Genómicos, LANGEBIO.
7.2.4 B
ioinformatic and Phylogenetic Analyses of 16S rRNA
Amplicon Data
Single End (SE) forward sequences were quality filtered and dereplicated using the
QIIME 2 bioinformatics platform version 2019.7 (Bolyen et al. 2018). Initial
sequences were demultiplexed and truncated at the 10th bp from the left, and the
280th bp from the right. Divisive Amplicon Denoising Algorithm 2 (DADA2) was
then used to filter by sequence quality, denoise, create a sample/operational taxo-
nomic units (OTU) table, and remove chimeras. OTUs were then taxonomically
assigned using the Silva-132-99-nb-classifier database. Those OTUs that occurred
<10 times across all six samples were removed. The final OTU table was rarefied to
20,000 sequences to make unbiased diversity comparisons and used for all subse-
quent analyses.
QIIME 2 was then used to obtain alpha and beta biodiversity scores. To test the
correlation between the Jaccard Dissimilarity Index values and geographical dis-
tances, a Mantel test was performed using Mantel Function as implemented in
Vegan R library v1.42 R (Oksanen et al. 2013), and the UpSet plot was prepared by
R library UpSetR (Conway et al. 2017).
7.3 Results and Discussion
7.3.1 Alpha Prokaryotic Diversity Patterns
The analysis generated 564,117 reads after quality trimming and a chimera check,
comprising 7818 OTUs, with a range of 44,368–121,337 reads per site. QIIME2
taxonomic assignment showed 60 different bacterial phyla (Fig. 7.2), a high number
in relation to the adjacent hydrothermal vent system in Guaymas Basin where 29
bacterial phyla were reported (Dombrowski et al. 2018). These diversity results also
surpass those reported for the East Pacific Rise with 37 bacterial phyla (He and
Zhang 2016). Overall, this suggests that vent fields in the southern Gulf of California
are diverse systems in terms of microbial communities, but this statement should be
taken with caution since these studies used different methodological approaches
(e.g., genomes and 454 pyrosequencing respectively).
The most abundant phylum among all the samples was Proteobacteria (Fig. 7.2),
representing 45% of all samples. In particular, Delta- and Gamma-, and
Epsilonproteobacteria – recently reclassified to a new phylum, Epsilonbacteraeota
(Waite et al. 2017) – were abundant in our samples. This abundance pattern
100%
Proteobacteria Thermotogae
δ
Actinobacteria Nitrospinae
90% Chloroflexi WS2
Bacteroidetes Aerophobetes
Epsilonbacteraeota Armatimonadetes
80% Acidobacteria Halanaerobiaeota
RELATIVE FREQUENCY
Atribacteria FBP
γ
Patescibacteria LCP-89
70% Gemmatimonadetes Fibrobacteres
Firmicutes BRC1
Planctomycetes Rokubacteria
60% α
Spirochaetes Hydrogenedentes
Caldiserica Lentisphaerae
ζ
Nitrospirae TA06
50% Cyanobacteria Tenericutes

Latescibacteria Fusobacteria
Chlamydiae Schekmanbacteria
Cloacimonetes FCPU426
40%
Aegiribacteria Poribacteria
Verrucomicrobia Modulibacteria
Kiritimatiellaeota Uncultured
30%
Entotheonellaeota Elusimicrobia
Acetothermia WOR-1
Dependentiae WPS-2
20% Synergistetes GN01
Zixibacteria Deinococcus-Thermus
Omnitrophicaeota Margulisbacteria
10% Marinimicrobia(SAR406clade) MAT-CR-M4-B07
Dadabacteria CK-2C2-2
Calditrichaeota BHI80-139
0%
AR_5
PB_1
PB_3
PB_8
PTF_2
PTF_4
SAMPLE
Fig. 7.2 Bacterial communities inhabiting the studied deep hydrothermal systems in the Gulf of
California. Histogram depicts phyla richness and relative frequency within samples collected from
Alarcón Rise (AR), Pescadero Basin (PB), and Pescadero Transform Fault (PTF)
(α = Alphaproteobacteria, γ = Gammaproteobacteria, δ = Deltaproteobacteria,
ζ = Zetaproteobacteria)
c oincided with previous studies reporting these taxa as a major component of pro-
karyotic communities in hydrothermal vents (Dick 2019), particularly, in similar
systems, such as the Guaymas Basin (Teske et al. 2002; Dombrowski et al. 2018).
Actinobacteria, Chloroflexi, and Bacteroidetes represented the second most
abundant phyla in our samples, again similar to bacterial community composition in
vent systems in Guaymas Basin and East Pacific Rise (Teske et al. 2002; Campbell
et al. 2013; He and Zhang 2016; Dombrowski et al. 2018). In particular,
Actinomarinales OTUs were observed to be a dominant component in all samples
(Table 7.1). This group of bacteria has been regarded as common marine bacteria
from the photic zone, being the smallest free-living cells among prokaryotes (Ghai
et al. 2013). Nonetheless, to the extent of our knowledge, this group has not been
reported from hydrothermal vents previously, reflecting the ample capacity of
marine microorganisms to adapt and colonize extreme environments.
The third most abundant phyla were Acidobacteria, Artribacteria, and
Patesibacteria that have also been detected in the neighboring vent system of
Guaymas Basin (Teske et al. 2002; Dombrowski et al. 2018), despite being
considered as a rare component of active hydrothermal vents (Zhang et al. 2016).
Table 7.1 List of shared OTUs within samples from hydrothermal vent systems in the southern Gulf of California
TAXA PTF_2 PTF_4 PB_1 PB_3 PB_8 AR_5 POTENTIAL ROLE / CHARACTERISTICS
Actinobacteria; Actinomarinales order (10 OTUs) 1.8 5.3 8 2.5 1.1 14.9 Smalest free living cells (Ghai et al. 2013)
Atribacteria; JS1 class (4 OTUs) 0 0 0 11 14.2 0 Fermentation products that support
methanogens (Carr et al. 2015)
Alphaproteobacteria; Methyloceanibacter genus (1 OTU) 0.4 1 0.6 0.03 0.05 0.03 Ch4 oxidation (Takeuchi et al. 2014)
Epsilonbacteraeota; Sulfurovum genus (2 OTUs) 2.6 0 0.05 18.2 10.3 0.06 Sulfur compounds oxidation / nitrogen cycle
(Mori et al. 2018)
Gammaproteobacteria; uncultured Thioalkalivibrio sp. (1 OTU) 0.1 0 0 0 0 5.9 Sulfur compounds oxidation (Cao et al. 2014)
Deltaproteobacteria; Desulfobacterales order (1 OTU) 0 0 0 3.6 17 0 Sulfur compounds reduction (Campbell et al.
2013)
Deltaproteobacteria; Desulfobacterales order; Sva0081 sediment 0.4 0.5 0.2 0.006 0 0 Sulfur compounds reduction (Campbell et al.
group genus (1 OTU) 2013)
Deltaproteobacteria; Desulfobacterales order; Desulfobulbus genus 0 0 0 0 0 1.8 Sulfur compounds reduction (Campbell et al.
(1 OTU) 2013)
Deltaproteobacteria; Syntrophobacterales order; 0.9 2.4 0.4 0.005 0.004 0.007 Sulfur compounds reduction (Kuever et al.
Syntrophobacteraceae family (1 OTU) 2005)
Synergistetes; Thermovirga genus (1 OTU) 0 0 0 1 0 0 Isolated from an oil well (Dahle and Birkeland
2006)
Cloacimonetes; MSBL8 family (1 OTU) 0 0 0 1.1 0.5 0 Long fatty chains degradation (Shakeri Yekta
et al. 2019)
Deltaproteobacteria; Deferrisoma genus (1 OTU) 0 0 0 0 0 0.1 Iron metabolism (Slobodkina et al. 2012)
Gammaproteobacteria; Acidiferrobacteraceae family (1 OTU) 0 0 0 0 0 0.08 Iron metabolism (Issotta et al. 2018)
Zetaproteobacteria; Mariprofundus genus (1 OTU) 0 0 0 0 0 0.3 Iron metabolism (Emerson et al. 2007)
Results reflect samples collected from Alarcón Rise (AR), Pescadero Basin (PB), and Pescadero Transform Fault (PTF). Values (occurring in more
than in a single site), abundant (≥1% reads) and iconic taxa (based on their reported metabolic capacities) within the overall OTUs database in per-
centage are categorized into three levels of color intensity (<0.09%; <0.9%; >1%)
L. Espinosa-Asuar et al.
Overall, these diversity patterns denote a marked similarity in terms of the dominant
component of high bacterial taxonomic ranks with neighboring vent systems, which
may suggest a significant effect of the environmental conditions in the assemblage
vent microbial communities.
At a local level, alpha diversity estimates showed that PTF sites and a single
sample from PB (PB_1) were the most diverse locations within the studied vent
systems (Table 7.2). Also, these sites showed high diversity Shannon values (9.3–10)
even when compared to hydrothermal vents in the East Pacific Rise and Guaymas
Basin (Campbell et al. 2013; He and Zhang 2016). The lowest diversity values were
obtained from PB samples (6.6, 7.6), and the AR showed intermediate values (8.3).
Interestingly, the opposite pattern has been reported for macrofauna (e.g., E. spicata
and L. barhami), since PTF harbors the lowest diversity levels in contrast to the PB
system (Goffredi et al. 2017). This result may be linked to the ability of microorgan-
isms to occupy micro-niches in relation to the macrofauna, contributing substan-
tially to ecosystem functions (Troussellier et al. 2017).
7.3.2 Beta Prokaryotic Diversity Patterns
Beta diversity distribution showed a strong association between the sample sources
(Fig. 7.3). This is related to each hydrothermal system exhibiting dissimilar physi-
cochemical conditions (Goffredi et al. 2017; Paduan et al. 2018; Salcedo et al.
2019). Consequently, samples from the same locality were clustered, except for
PB_1 that grouped to the PTF. Therefore, the Mantel test showed no significance for
the geographic distribution. These results suggest a strong effect of environmental
conditions in shaping microbial distribution, given the significant differences in
physicochemical, geological, and biotic settings among the three analyzed localities
(PB, PTF, and AR; Goffredi et al. 2017; Paduan et al. 2018; Salcedo et al. 2019).
To attain a better understanding of beta distribution patterns, diversity was
explored at the OTUs level (Fig. 7.4, Table 7.2). This approach demonstrated that
the majority of OTUs was unique to each sediment core (45.6% to 79.7% of OTUs,
Fig. 7.4), embodying a large number of rare species of microbial life known as the
Table 7.2 Observed OTUs and alpha diversity values (Faith phylogenetic diversity (PD) and
Shannon), within the six samples from hydrothermal vent systems in the southern Gulf of
California, including Alarcón Rise (AR), Pescadero Basin (PB), and Pescadero Transform Fault
(PTF)
Observed OTUs Faith PD Shannon
PTF_2 2262 89.9 10
PTF_4 1936 84.6 9.3
PB_1 1981 79.5 9.3
PB_3 1221 61.9 7.6
PB_8 646 45.9 6.6
AR_5 1453 68.8 8.3
Fig. 7.3 UPGMA cluster analysis based on the beta diversity Jaccard dissimilarity matrix.
Samples included six bacterial communities (Alarcón Rise (AR), Pescadero Basin (PB), and
Pescadero Transform Fault (PTF)) from hydrothermal vent systems in the southern Gulf of
California
rare biosphere (Sogin et al. 2006). This sizeable genetic pool contributes impor-
tantly to the metabolic potential of the community (Jousset et al. 2017) and is known
to increase in response to environmental changes. It is possible that in the analyzed
vent systems this rare biosphere plays a vital role in assuring the ecosystem func-
tions, acting as ecologically important keystone organisms, as they outnumber
abundant taxa (Kurm et al. 2019).
7.3.3 Microbial Ecological Roles (Metabolic Signatures)
Since abundant and shared species represent an important component of microbial

communities, the shared (occurring in more than in a single site), abundant (≥1%
reads), and “iconic” taxa (based on their reported metabolic capacities) within the
OTU database were explored. Firstly, the largest number of shared OTUs among
1569 (69.3%)
1500
1315 (66.3%)
1159 (79.7%)
1060 (54.7%)
OTUs NUMBER
1000
710 (58.1%)
500
295 (45.6%)
270
251
183179
62 60 59 58 57 46
36 34 30 27 26 26 26 24 22 19 18 15 15 14 14
12 10 9 9 8 6 6 5 5
0
PB_8
PB_3
AR_5
PTF_4
PB_1
PTF_2
2500 2000 1500 1000 500 0
OTUs PER SAMPLE
Fig. 7.4 Graphic representation of shared OTUs within the studied deep hydrothermal vent sys-
tems sites in the southern Gulf of California. Number of unique or shared OTUs (among samples)
is represented by black bars; for unique OTUs, a percentage value is shown and was calculated
with respect to the number of OTUs per site. Dots below bars denote the occurrence of particular
OTUs in a specific site, and lines indicate sites shared OTUs between sites. The number of OTUs
on each site is proportional to colored bars in the left of the main figure
sites, 179–270 (Fig. 7.4), reflected the clustering patterns as displayed by the beta
diversity analysis (Fig. 7.3), where PB_1, PTF_2, and PTF_4 diverged from PB_3
and PB_8 and formed another cluster. Moreover, the low number of shared OTUs
among all vent samples (58, 0.75% of the overall OTUs) highlighted again the
uniqueness of each microbial community.
The exploration of abundant OTUs in the samples (Table 7.1) allowed the infer-
ence on microbial “metabolic signatures” that could reveal the potential effect of
micro-niches driving the differentiation between microbial vent communities (Dick
2019), even in sites within the same system, since they are remarkably heteroge-
neous and exhibit sharp gradients. These “metabolic signatures” can reflect poten-
tial patterns for metabolic pathways associated with particular conditions, for
example, the presence and concentration of chemical compounds, such as sulfur
compounds, methane, petroleum, and other related hydrocarbons.
In hydrothermal vents, sulfur is a crucial element in sustaining microbial com-
munities (Dick 2019). The oxidation of sulfur compounds has been extensively
studied due to its importance in chemosynthetic pathways in hydrothermal fields
(Sievert et al. 2008); on the other hand, less is known about sulfur compounds
reduction (Cao et al. 2014). We found abundant hydrothermal vent sulfur/sulfide
oxidizing bacteria (SOB), such as Sulfurovum (Mori et al. 2018) in PB and PTF
samples (Table 7.1). These Epsilonbacteraeota are characteristic of sulfide-rich vent
fluids (Akerman et al. 2013), and basalt-hosted vent sites of 9°N East Pacific Rise
(Campbell et al. 2013). Moreover, the contribution of this taxon to the nitrogen
cycle has been reported as well (Li et al. 2018), highlighting its metabolic potential.
Furthermore, in AR we detected the sulfur compounds oxidizer abundant taxon
Thioalkalivibrio. These Gammaproteobacteria have been reported within hydro-
thermal chimneys on the Southwest Indian Ridge (Cao et al. 2014). With respect to
sulfur/sulfate reducing bacteria (SRB), we identified several Desulfobacterales
OTUs in all our samples (Table 7.1), including unique OTUs (data not shown). This
result agrees with previous work in Guaymas Basin, and the East Pacific Rise vent
systems (Teske et al. 2002; Dhillon et al. 2003; Campbell et al. 2013); particular
SRB taxa within Desulfobacterales, Desulfobulbus genus, were detected abundantly
for AR, and Syntrophobacteraceae family showed more abundance for PTF_4.
The presence of sulfur/sulfide-oxidizing and sulfur/sulfate-reducing bacterial
taxa (SOB and SRB) in all samples confirms the importance of sulfur compounds
on the maintenance of vent communities, but the differences could reveal the “meta-
bolic footprint” within each sample, such as particular taxa within each guild
(Table 7.1). For example, AR is presenting a distinct sulfur-related bacterial com-
munity including Thioalkalivibrio (SOB) and Desulfobulbus (SRB). Another exam-
ple is the lower levels of both SOB and SRB sulfur-related taxa in PB_1, pointing
that this site could have different environmental characteristics than the other two
PB samples. A third example is PB_4 showing high levels of SRB
Syntrophobacteraceae taxa, in contrast with low levels of SOB taxa; the lack of
observed sulfur compounds oxidizing prokaryotes leads us to hypothesize that
methane, and not sulfur compounds, in PB_4 site could be the essential source of
energy, based on the abundance of Methyloceanibacter, a methylotrophic taxon
(Takeuchi et al. 2014) in this sample (Table 7.1). Interestingly Goffredi et al. (2017)
reported the presence of methane in the end-member fluids of this area. Anaerobic
oxidation of methane provides a notable source of energy and biomass at hydrother-
mal vents, in addition to the better-known oxidation of sulfur compounds by bacte-
ria (Teske et al. 2002), and this metabolism could be coupled to the SRB (Muyzer
and Stams 2008; Cassarini 2017). A further possibility is that SO archaea (Sievert
et al. 2007; Nakagawa and Takai 2008) are filling this niche.
Sulfur and methane-related bacteria reflect the similarity among microbial taxa
of PTF samples and PB_1, where we found Syntrophobacteraceae and Sva0081
taxa (both SRB taxa; Kuever et al. 2005; Campbell et al. 2013), and
Methyloceanibacter (methane-related) in contrast to PB samples (PB_3 and PB_8)
that were characterized by Sulfurovum microorganisms (SOB), or Class JS1 from
Atribacteria, that are known to generate fermentation products that support metha-
nogens (Carr et al. 2015). Since the PB_1 is the only sample within PB hydrother-
mal system that is located easterly (Fig. 7.1), one may expect a low influence on the
sediments from venting fluids within this particular spot, triggering the observed
lower abundance of SOB and SRB taxa (Zinke et al. 2018).
Epsilonbacteriaceae (SOB) abundance within the PB_3 and PB_8 samples
(Fig. 7.2) coincides with the high levels of hydrogen reported for these sites
(Goffredi et al. 2017; Paduan et al. 2018), given the ability of the taxa to use
hydrogen for energy besides sulfur (Campbell et al. 2013). The abundance of these
taxa in PTF_2 sample (Fig. 7.2) could point to the presence of hydrogen in this site.
Whereas the reductive tricarboxylic acid cycle (rTCA), characteristic of
Epsilonproteobacteria (Hügler et al. 2005; Nakagawa and Takai 2008; Cerqueira
et al. 2015), could represent another “metabolic footprint” for PTF_2, PTF_4, and
PB_1, equally the Calvin–Benson–Bassham cycle for PB_2, PB_8, and AR, due to
the prevalence of Gammaproteobacteria (Hügler and Sievert 2011; Fig. 7.2).
The abundance of methane-related taxa (Table 7.1) demonstrated methane traces
in PFT samples (Salcedo et al. 2019), whereas the lowest abundance of these taxa at
AR confirmed the low concentrations of methane (Goffredi et al. 2017). Similarly,
the occurrence of OTUs related with the degradation of long fatty acids in PB,
including the MSBL8 family from Cloacimonetes, and Thermovirga, a genus iso-
lated from oil sediment samples; (Dahle and Birkeland 2006; Shakeri Yekta et al.
2019), agrees with reports of long fatty chains in these vents (Goffredi et al. 2017).
The AR basalt-hosted system presented ferric metabolism-related taxa (Table 7.2)
such as Deferrisoma, typically involved in iron reduction (Slobodkina et al. 2012),
Acidiferrobacteraceae and Mariprofundus, regarded as Fe-oxidators (Emerson et al.
2007; Issotta et al. 2018); the occurrence of these bacterial taxa reflects the basalt
high ferric concentration, which is characteristic of AR (Goffredi et al. 2017).
7.3.4 D
eep Hydrothermal Systems as an Approximation
to Understand Primeval Environments
To understand aqueous environments likely to have been present on the prebiotic

Earth, and to investigate whether they would be conductive to the origin of life, we
can learn from the different prebiotic analog sites that exist today in our planet, in
particular from the submarine hydrothermal vents (Deamer et al. 2019). Different
metabolic signatures found in this study between analyzed microbial communities
from southern Gulf of California hydrothermal vent systems give consideration to
diverse metabolic possibilities that reflect primordial life forms potentially originat-
ing in early Earth. Further detailed studies for these near (<100 km), but contrasting
environmental systems, should be done, as a frontier for fruitful exploration, discov-
ery, and research within this newly discovered Mexican ecosystem.
It has been proposed that in the Hadean (~ 4.6 to 4 billion years ago), in the
absence of oxygen, alkaline vents acted as electrochemical flow reactors, in which
alkaline fluids saturated in H2 mixed with relatively acidic ocean waters rich in CO2,
through a labyrinth of interconnected micropores with thin inorganic walls contain-
ing catalytic Fe(Ni)S minerals (Sojo et al. 2016). The difference in pH across these
thin barriers produced natural proton gradients with equivalent magnitude and
polarity to the proton-motive force required for carbon fixation in present bacteria
and archaea (Sojo et al. 2016).
Nowadays, alkaline vents form labyrinthine networks of interconnected micro-
pores bounded by thin inorganic walls, through which hydrothermal fluids (and
ocean waters) percolate. Such vents should have been even more common on the
early Earth, as the mantle was less differentiated from the crust. Hence, ultramafic
minerals could have been found across much of the ocean floor (Fyfe 1994; Shields
and Kasting 2007; Jaffrés et al. 2007).
Along with the Cuatro Ciénegas Basin, where microbial communities with
strong marine and hydrothermal vent resemblances (Souza et al. 2006) occur due to
the presence of an active fault (Souza et al. 2018), these extreme Mexican systems
(hydrothermal vents in the southern Gulf of California, and Cuatro Ciénegas hot
springs) pose ideal models to explore primordial environments (e.g., the origin of
life, early life, and astrobiology) where modern microorganisms metabolize ancient
forms of chemical energy (de Anda et al. 2018).
7.4 Conclusions and Perspectives
Herein, we described the rich prokaryotic diversity in hydrothermal systems of the

South of the Gulf of California represented by three different systems: PB, a carbon-
ated high-temperature vent, AR dominated by basalt high-temperature vents, and
PTF with low-temperature vents. All of which are characterized by particular sulfur
compounds oxidizing and reducing bacteria, among other taxa. Remarkably, dif-
ferentiated community structure assemblages among systems were observed, except
for PB_1 clustering with PTF samples. This pattern may be elucidated by the
detailed evaluation of the whole bacterial community.
Hydrothermal explorations pose future challenges and opportunities for science.
The abundance of apparently primordial life-forms originating and still evolving in
deep-sea vents and using rocky mineral walls in ocean-floor as power sources for
chemical reactions opens up a world of possibilities to further explore a several
hypotheses on the origin of life on Earth.
In this chapter, we provided evidence of the diverse chemotrophic microbial
communities inhabiting high-temperature and low-temperature hydrothermal vents
in the southern Gulf of California, and their characteristic “metabolic signatures”
that are strongly related to local environmental conditions. The presence of these
microbes confirms that life is possible in sea floor environments in hydrothermal
systems, which were perhaps present and active during most of the early Earth.
Acknowledgments This work was supported by SEP- Ciencia Básica CONACyT Grant 238245
and Pappit-DGPA grant IG200319. We want to thank Dr. Erika Aguirre Planter for technical sup-
port. We are grateful to the Monterey Bay Aquarium Research Institute and especially to
R. Vrijenhoek for his invaluable assistance in obtaining the samples herein analyzed, and for his
unyielding support. A special word of appreciation to the Western Flyer crew and the ROV Doc
Ricketts pilots for their invaluable help.
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Chapter 8
Andean Microbial Ecosystems: Traces
in Hypersaline Lakes About Life Origin
Luis A. Saona, Mariana Soria, Patricio G. Villafañe, Agustina I. Lencina,

Tatiana Stepanenko, and María E. Farías
Abstract High-altitude Andean lakes (HAALs) represent unique environments on

the Earth where one can study the biological chemistry of life in one of its most
extreme versions. The Atacama Desert, Argentine Puna, and Bolivian Altiplano har-
bor hypersaline lakes where polyextremophilic Andean Microbial Ecosystems
(AMEs) inhabit microbial mats, evaporitic mats, biofilms (BF), evaporites (EV),
and microbialites (Mi). These AMEs have two remarkable characteristics: (i) they
are the only ones in the world that inhabit areas ranging from 3100 to 4200 masl;
and (ii) they are excellent modern analogues of those which populated the primitive
Earth ~3 billion years ago. In this chapter, we will delve into the different kinds of
AMEs present in the HAAL, their formation, structure, and their adaptation to con-
ditions largely influenced by volcanic activity, UV radiation, arsenic content, high
salinity, low dissolved oxygen content, extreme daily temperature fluctuation, and
oligotrophic conditions. All of these physicochemical parameters recreate the early
Earth and even extraterrestrial conditions. The relevance of studying these ecosys-
tems does not lie only in scientific-descriptive and/or economic interest. The scien-
tific research community has a great responsibility to address climate change. In this
scenario, the AMEs could have played a key role, influencing changes that allowed
the origin of aerobic life and those who have faced the great climatic events of the
Earth.
8.1 Introduction
The hypersaline environments, ponds and lakes, are mainly dominated by NaCl (up
to saturation concentrations), and they are alkaline ecosystems (Risacher et al.
2003). They are widely distributed across the globe but are highly abundant in
L. A. Saona · M. Soria · P. G. Villafañe · A. I. Lencina · T. Stepanenko · M. E. Farías (*)

Laboratorio de Investigaciones Microbiológicas de Lagunas Andinas (LIMLA), Planta Piloto
de Procesos Industriales Microbiológicos (PROIMI), CCT, CONICET,
San Miguel de Tucumán, Argentina

168 L. A. Saona et al.
t errestrial biomes, such as deserts and steppes and in geologically interesting regions
including the East African Rift Valley. Other geographically well-known hypersa-
line environments are Great Salt Lake (Utah), the Dead Sea (Israel), and the alkaline
lakes of Wadi Natrun (Egypt) (Larsen 1980).
Environments, such as Puna (Argentina), Altiplano (Bolivia), or Atacama Desert
(Chile), exhibit unique geography, and they are a large reservoir of high-altitude
Andean lakes (HAAL). These lakes have wide environmental factors, such as UV
radiation, arsenic content, high salinity, low dissolved oxygen content, extreme
daily temperature fluctuation, and oligotrophic conditions, that represent a window
to the early Earth and even extraterrestrial conditions where currently we are look-
ing for life, like Mars (Farías et al. 2014; Albarracín et al. 2015).
Despite the extreme conditions, various substrates in these environments are
colonized by rich and diverse communities of phototrophic and heterotrophic
microorganisms, which are referred to as Andean Microbial Ecosystems (AME).
The AMEs are complex associations of microbes that in many cases influence or
induce mineral precipitation in lakes, hydrothermal vents, puquios, fumaroles of
volcanoes, and salt flats of the Central Andes Region (Southwest Bolivia, Northern
Argentina, and Chile) (Gomez et al. 2014). These ecosystems include:
• Biofilms, which are communities of microbial cells associated with a solid sur-
face and embedded in an exopolymeric substance (EPS) matrix (Marshall 1984).
• Microbial mats, which are benthic communities growing on a solid substrate
(e.g., sand, rock, or sediments) as vertical layers of functional groups of microbes
embedded in an mineral-organic matrix, more often exopolysaccharides, sili-
cates, and carbonates (Dupraz et al. 2009; Glunk et al. 2011; Albarracín et al.
2016a).
• Microbialites, which are microbial mats that present various degrees of mineral
precipitation and lithification (Dupraz et al. 2009; Glunk et al. 2011; Albarracín
et al. 2016a).
• Endoevaporitic domes, which are microbial mats associated with gypsum crusts
and halite.
Some of the Puna-Altiplano microbialites resemble those observed in the ancient
geological record, for example, stromatolites, which are the oldest evidence of life
on Earth (Allwood et al. 2006; Nutman et al. 2016, 2019). Thus, these systems,
inhabited by microorganisms known as “polyextremophiles,,” are excellent micro-
bial ecology models, and they provide us a unique opportunity to apply an inte-
grated geobiological approach to study the biochemist and molecular mechanisms
of the adaptation to extreme conditions like those that existed 3800 million years ago.
This chapter describes the HAALs, as a relatively unexplored natural laboratory,
where biofilms, microbial mats, endoevaporitic domes, and microbialites can be
found. These HAALs can be considered the modern counterparts of the ancient
microbial ecosystems. Analyzing all the physicochemical parameters that make the
arid Central Andean endorheic basins placed on Argentina (Puna), Chile (Atacama
Desert), and Bolivia (Altiplane) as one of the most extreme places on Earth. Lastly,
we will focus on the molecular and biochemist tools of AMEs to resist all these
8 Andean Microbial Ecosystems: Traces in Hypersaline Lakes About Life Origin 169
abiotic stress and even use it. All this information gives us interesting insights into
the ancient microbial life.
8.2 Life’s Origin
A long time ago, specifically in the Precambrian ocean, life arose (Mojzsis et al.
1996; Rosing 1999; Watanabe et al. 2000; Kempe and Kazmierczak 2002), and
from there it began its evolutionary path to the cells that inhabited different environ-
ments. The dating of these events is not entirely clear, and until a few years ago, the
consensus was that the oldest life records found to date corresponded to organo-
sedimentary structures, known as stromatolites, located in the Pilbara Block of
Western Australia dating back to 3.48 billion years back (Walter et al. 1980; Van
Kranendonk et al. 2008). These fossils and their dating suggested that the origin of
life occurred a few hundred million years before, between 3.7 and 3.8 billion years
ago (Watanabe et al. 2000; Kempe and Kazmierczak 2002). However, stromatolites
reported in the Southwest of Greenland in Isua Supracrustal Belt date back 3.7 bil-
lion years ago (Nutman et al. 2016, 2019) moving the date of the origin of life
beyond 4000 million years.
Four thousand million years ago, the Earth was in the period of time called
Archean; this eon is characterized by being between the first geological evidence
and the next 2500 million years. There, our last unique common ancestor (LUCA)
evolved and lived (Fox et al. 1980; Weiss et al. 2016), on a planet that, despite the
huge differences with today’s Earth was already habitable (Benedetto 2010).
The primitive Earth did not have the same physical-chemical composition as
today. The atmosphere of that time was formed gradually by the degasification of
the mantle and the gases contributed by the Great Meteorite Bombings. Although its
original composition is still a matter of speculation, there is a consensus on it. In the
atmospheric composition, gases, such as H2O, CO, CO2, H2, HCN, NH3, and CH4
initially predominated; and later the volcanic activity would have contributed gases
like N2, CO2, and H2O (Pla-García and Menor-Salván 1990; Holland 1994). The
first organisms to inhabit the Earth were prokaryotic microorganisms that carried
out anoxygenic photosynthesis, which is characterized for not releasing dioxygen as
a waste product (Kulp et al. 2008; Shih 2015). Later, cyanobacteria evolved and
developed oxygenic photosynthesis with which they began to release oxygen into
the atmosphere (Holland 1997; Summons et al. 1999; Kasting 2001). In this way, it
was not until 2.5 billion years ago when the level of oxygen in the atmosphere began
to increase in the event known as great oxygenation event (GOE) (Shih 2015). This
event allowed to anaerobic life began to breathe oxygen and could evolve giving
way to the great diversity of aerobic organisms that inhabit the Earth today.
In this metabolic context, the role of other elements that could have been a key
for the development of life has been widely studied. In this chapter, we will focus
on the role of the element arsenic. During the Achaean, the highest concentrations
of As were found in rock bodies called “green rock belts” (i.e., metamorphized into
green-amphibolite shale facies and intruded by granite bodies), because they con-
tain important massive sulfide deposits, thereby concentrating the As (Lahaye et al.
2001; Bundschuh et al. 2008; Benedetto 2010).
8.3 H
igh-Altitude Andean Lakes as Analogues to Primitive
Earth
8.3.1 Physicochemical Conditions in Andean Valleys
We already mentioned the physicochemical characteristics of the primitive Earth

where life originated; now let’s think about how that land looked like. Many images
exist that try to represent the landscapes of the ancient Earth of 4000 million years
ago; however, these are usually characterized by a mountainous cordon in the hori-
zon with reddish-brown colors and its active volcanoes. The water that joins that
horizon with the bay at the bottom of the image where the first life forms of which
there is register are outlined: stromatolites. It is possible to find very similar primi-
tive landscapes in the valleys of the Central Andes (Fig. 8.1).
The Central Andes region is home to a set of physicochemical, geological, and
mineralogical conditions very similar to those that are presumed to have existed on
the primitive Earth where life originated. This high-altitude region that encom-
passes Argentine, Chilean, and Bolivian territory is characterized by closed basins
where salt flats, volcanoes, hypersaline lakes, hydrothermal fountains, and deserts
abound. Due to its southern location and its altitude gradients, it is subjected to a set
of environmental conditions that transform it into one of the most extreme places on
the planet: low oxygen pressure, high UV radiation, constant volcanic eruptions,
high thermal fluctuation, and high aridity, among others (Vuille et al. 2000, 2004;
Garreaud et al. 2009; Albarracín et al. 2015). At this point, we will place special
emphasis on UV radiation, dryness, salinity, and the presence of toxic elements
(metals) for most life forms.
In terms of UV radiation, the Andean Valleys are one of the most irradiated areas
on the planet. The monthly average reaches 6.6 kW h m−2 d−1 with maximum irra-
diation of 310 W m−2 (Duffie and Beckman 2013). These high levels of radiation are
due in part to the latitude, which offers small midday solar zenith angles throughout
the year, in addition to clean atmospheres at an average height of 3600 m above sea
level (masl) with low aerosol content and low amount of clouds that reduces the
attenuation of solar radiation (Luccini et al. 2006).
This high radiation consequently affects the aridity of the region. Although from
January to April maximum rainfall is received, in a phenomenon called Bolivian
winter, there is low humidity (38.5%) and low precipitation (1.47 mm) in this region
(Albarracín et al. 2015). In the Atacama Desert, located from 19° S to 30° S in
northern Chile and extending from the Pacific coast to the Andes mountain range
(Davila et al. 2008; Contador et al. 2019), there are salt crusts present (halite and
plaster) that can be considered the last niche available for life in hyperarid environ-
Fig. 8.1 Overview of sampling sites. (A) Sampling in Pozo Bravo Lake in Salar de Antofalla,
Catamarca, Argentina. At the bottom of photo, the stromatolites are presents. In the photo samples
are being taken of microbial mats inhabiting the ground of lake. (B) Endoevaporitic systems in
Laguna 3 Quebradas, Catamarca, Argentina. (C) Microbialites present in Carachipampa,
Catamarca, Argentina. (D) Laguna La Brava, Antofagasta, Chile. In the bottom of the photo,
microbial mats can be observed. (E) Laguna Diamante, Argentina. This lake is the crater of Gálan
volcano. (F) Stromatolites of Laguna Socompa, Argentina
ments on Earth (Connon et al. 2007; Davila et al. 2008; Schulze-Makuch et al.
2018). These aridity conditions in the Atacama Desert date back 150 million years
ago, making it not only the driest on the planet but also one the oldest (Hartley et al.
2005; Clarke 2006).
Finally, due to the volcanic activity of the region (Smedley and Kinniburgh
2002), HAALs present high arsenic concentration (Demergasso et al. 2007;
Escudero et al. 2013; Rascovan et al. 2016; Kurth et al. 2017). The average concen-
tration varies between 0.8 mg L−1 and 11.8 mg L−1 (Albarracín et al. 2015). The
highest As concentration was found in Diamante Lake, located in the crater of Galán
Volcano, with an As concentration of 230 mg L−1 (Rascovan et al. 2016), which are

almost ten times higher than the concentration present in Mono Lake, a lake that has
been studied for its high As concentration (Oremland et al. 2009; Wolfe-Simon et al.
2011). New measurements made in the water column of this lake, report up to
300 mg L−1. This high concentration of As indisputably influences over the metabo-
lism of the microorganisms that inhabit these lakes.
In summary, the Andean Valleys region has climatic, geological, atmospheric,
and mineralogical conditions that make it the best analogue of the primitive Earth.
All these abiotic characteristics directly influence the selection, adaptation, and evo-
lution of the life that is a fundamental part of the landscapes of the Andes. In this
sense, both the HAALs and the AMEs that inhabit them represent a natural labora-
tory from which we can study and find more clues about the origin of life. Although
these ecosystems are modern and do not date from 3.7 million years ago, they are
the same type of life – referred to as biofilm, microbial mats, and/or organo-
sedimentary structures (microbialites) – that arose in the primitive Earth. In this
sense, during the last decade, these ecosystems have been studied from metage-
nomic, genomic, physiological, and biogeochemical approaches; areas in which our
research group has provided information specially referred to (i) abundance and
kind of ecosystems as well as the (ii) biological role of arsenic as part a primitive
metabolism for obtaining energy.
8.3.2 Andean Microbial Ecosystems (AMEs)
The Puna-Altiplano region has become an interesting region to study microbial

mineralization processes. The most widely studied types of microbial-induced
organomineralization are those of carbonate with calcium, as calcite and/or arago-
nite (Reid et al. 2000), calcium and magnesium (Vasconcelos and McKenzie 1997;
Glunk et al. 2011), and magnesium (Thompson and Ferris 1990; Sanz-montero and
Rodríguez-Aranda 2008).
Extreme changes in environmental factors, like salinity and temperature, together
with a favorable local geology and geomorphology, facilitate the formation of
closed lakes and groundwater springs, which are saturated in minerals, including
carbonates. Thus, a highly mineralizing setting together with physicochemical and
microbiological processes triggers carbonate precipitation and produces a set of
diverse organo-sedimentary structures, referred as AMEs (Burne and Moore 1987).
As previously stated, the AMEs are complex associations of microbes that gener-
ally are dominated by Proteobacteria, Bacteroidetes, and Firmicutes. Another par-
ticularity of these systems is the presence of Cyanobacteria, which thrive as primary
producers. Diatoms and fungi may also be present of these ecosystems (Farías et al.
2014; Rasuk et al. 2014; Fernandez et al. 2016).
Our research group worked in the analysis of the AMEs in the Puna-high Andes
region. In 2009, the first microbialite was reported in Laguna Socompa, followed by
Laguna Diamante in Argentina (Belluscio 2010) (Fig. 8.1). Since then, an extensive
surveying has been carried out to the salt flats, lagoons, geysers, and fumaroles in
the highlands of Bolivia, Argentina, and Chile. For 10 years, 80 environments were
examined, and 20% of them presented at least 1 type of these microbial ecosystems.
They include biofilms, microbial mats, microbialites, and endoevaporite inhabiting
gypsum domes.
All throughout the examination of the AMEs, some shared characteristics were
found, from a geological, physical, and chemical point of view: (i) active volcanic
incidence – all the microbial ecosystems found where in some way connected to
areas were active volcanoes are present. (ii) Underground water input. (iii) Mixed
zones with different salinities: underground low conductivity water and salar thalas-
sic water. (iv) Two main organo-sedimentary structures were found: oncolites
(Laguna Negra, Tres Quebradas, and Las Quinoas, among others) and microbial
mounds with a composite internal fabric of mesoclots and individual laminae. The
mounds facies consist of thrombolites (clotted fabrics), which commonly grade
upward and outward to stromatolites caps (laminae) (La Brava, Pozo Bravo, Ojo
Bravo, El Peinado). From the biological point of view: (v) Predominance of dia-
toms, the main component in all the studied systems. (vi) Predominance of anaero-
bic over aerobic photosynthetic microorganisms. (vii) Microbial rhodopsin as the
main system for producing ATP. (viii) Arsenic resistance and bioenergetic mecha-
nisms. (ix) Predominance of carbon fixation pathways alternative to the Calvin cycle.
The knowledge and recent research activities in these systems have served as the
basis for the declaration of protected areas in Laguna Socompa, Puquios de Mar,
Tolar Grande, Llamara, and Tebenquiche. Additionally, AMEs are now included in
baselines and environmental impact studies for lithium mining company projects in
Chile’s Salar de Atacama (Albemarle) and Catamarca, Argentina (Liex, Albemarle,
Ultra Lithium and Morena del Valle Minerals). Taken together, the preservation of
these unique ecosystems is a priority due to the benefits of AEMs to not only scien-
tific understanding of microbial adaptation to extreme environments, but also these
systems can be of great relevance to the economic development of local Andean
populations.
8.3.3 Early Metabolisms in Andean Valleys
As we detailed above, due to the poly-extreme conditions present in the HAALs and
in the Andean Valleys in general, the AMEs have selected specific genes that allow
them to have highly specialized metabolisms.
In the case of extreme UV radiation, microbial communities have efficient repair
mechanisms for accumulated DNA damage in the form of cyclobutane pyrimidine
dimer (Fernández Zenoff et al. 2006; Albarracín et al. 2012); express different pho-
tolyases to minimize the damage of direct exposure to solar radiation (Sancar 2003;
Albarracín et al. 2013); synthesize antioxidant enzymes to prevent damage from
oxidative stress (Albarracín et al. 2011; Di Capua et al. 2011); produce secondary
metabolites, such as pigments (Weber 2005; Kurth et al. 2015; Albarracín et al.
2016b); and form biofilms associated with minerals and rocks also to avoid direct
exposure to UV rays (Albarracín et al. 2016a; Rascovan et al. 2016).
AMEs must also be able to access water molecules in one of the driest deserts in
the world, which represents the last niche available for life in hyperarid environ-
ments (Davila et al. 2008). In this sense, the endolithic microbial communities of
the Atacama Desert, composed mainly of Cyanobacteria, inhabit evaporitic rocks of
sulfate and halite and appear to settle in the areas of maximum available humidity
(Rothschild 1990; Parnell et al. 2004; Dong et al. 2007). In addition, due to the
oxygen solubility decreases as a higher salt concentration, AMEs develop under
microaerophilic and anoxic conditions.
In the primitive Earth, around 3.8 billion years ago, the high exposure to UV
radiation, together with other conditions such as the alkaline pH of hydrothermal
sources, favored the biogenesis of macromolecules, either allowing the accumula-
tion of organic matter (avoiding acid hydrolysis) or contributing as source of energy
for the development of complex molecules relevant to life (Kempe and Kazmierczak
2002; Rapf and Vaida 2016). Similarly, it is presumed that high salinity was an
important factor in the formation of macromolecules from smaller organic mole-
cules, due to the low activity of water in these conditions (Knauth 1998). In addition
to these mechanisms, one of the metabolisms that we have deeply studied and that
is related to the reactions that were presumably present in the origin of life is the
arsenic metabolism.
Arsenic is a chemical element belonging to metalloids, although it is present
along the Earth’s crust, its relative abundance is very low and is considered an
extremely toxic element. An interesting aspect of arsenic in relation to its role in
biotic chemistry is that it is structurally similar to phosphorus; both have the same
oxidation state, atomic radius, and electronegativity. In its most characteristic oxi-
dized form (+5), arsenate and phosphate have a negative charge at physiological
pH. In fact, both molecules have similar speciation at different pH, as well as similar
pKa values (Anbar et al. 2009; Tawfik and Viola 2011) and thermochemical radii,
which differ by only 4% (Kish and Viola 1999).
As a result of the high volcanic activity, the As concentration in HAALs is very
high (Rascovan et al. 2016; Corenthal et al. 2016; Boutt et al. 2016). However,
despite their high toxicity, AMEs have specific genes that allow them to survive
under these conditions (Fig. 8.2). Here, we detail a brief summary of As biological
role studied in different AMEs.
8.3.3.1 Laguna Diamante
Laguna Diamante is placed at 4560 masl inside Volcano Gálan caldera at Argentinean
Puna (Fig. 8.1). This Lake presents extreme conditions of high conductivity
(180 mS), alkalinity (pH 10), and As concentrations reach 230 mg L−1. In this lake,
a red biofilm associated to gaylussite mineral and volcanic rock is present. This
microbial community is composed of more than 94% Haloarchaea. Previous
metagenomic analyses of red biofilm samples have revealed a high abundance of
Fig. 8.2 Scheme of arsenic metabolic cycle
genes used for arsenite oxidation (e.g., aioAB) (Fig. 8.2) and respiratory arsenate
reduction (e.g. arrAB) (Fig. 8.2) (Rascovan et al. 2016). The theorizing of this pro-
cess has shown that cells can breathe As and thus obtain energy from their oxidation
and reduction (Andres and Bertin 2016; Rascovan et al. 2016; Ordoñez et al. 2018).
This specific point creates a direct link to prebiotic chemistry: electrochemical
transfer through oxidation reactions by the aio gene is present before the divergence
between bacteria and archaea and even from times before the last universal common
ancestor (LUCA) (Lebrun et al. 2003; Kulp 2014). In other words, it is theorized
that even before the formation of the first cell, reactions between arsenic species
were already catalyzed by means of proto-enzymes.
Physiological assays with Halorubrum DM2 sp. (Laguna Diamante isolate)
where arsenic facilitated its growth suggested an important role of arsenic as a bio-
energetic component (Ordoñez et al. 2018). In this strain, an increment in the tran-
scriptional levels of the genes related to arsenic metabolism when arsenic was added
to the growth medium was observed by RT-qPCR (Stepanenko unpublished).
Although it is not yet possible to assign the underlying molecular mechanism, these
results suggest the idea that the biogeochemical cycle of arsenic is affected by
microbial metabolism and vice versa.
8.3.3.2 Laguna La Brava
La Brava placed in Salar de Atacama, Chile, at 2200 masl (Fig. 8.1) is drained with

groundwaters containing leached volcanic material (Corenthal et al. 2016; Boutt
et al. 2016), in which high concentrations of arsenic and sulfide are found. Hence,
the microbial metabolisms in the mats are possibly driven by anoxygenic photosyn-
thesis using reduced sulfur and arsenic compounds (Visscher et al., in prep).
Anaerobic processes could include fermentation, methanogenesis, sulfate, and
likely arsenate reduction.
In regard to As, due to widespread volcanism and geothermal activity character-
istic of the Precambrian, considerably more arsenic was accumulated on the Earth’s
surface than today (Cabral and Beaudoin 2007; Witt-Eickschen et al. 2009). Hence,
as modern counterpart of Precambrian system, a living microbial mat from Laguna
Brava has been investigated, using an array of conventional geochemical techniques
(Farias et al. 2017; Sancho-Tomás et al. 2018). This experimental approach allowed
to unravel the relationship between the microbial mat activity, mineral occurrence,
arsenic speciation, distribution of major and trace elements, and their relationship
with the mineralogy and the exopolymeric substances (EPS). In that way it was
shown that As was not linked to Ca or Si, and only moderately related to Fe, result-
ing from sorption onto an iron (oxy)hydroxide mineral. In that way, a number of
features that support the biological cycling of arsenic have being documented
(Sancho-Tomás et al. 2018). These include the incorporation of arsenic into exo-
polymeric substances, the identification of organic-rich globules containing abun-
dant arsenic with no other trace metals, and the heterogeneous distribution of As(III)
and As(V), either together or separately and at different scales. Although accurate
information on sulfur distribution and speciation are missing, the high concentra-
tion of both sulfur and arsenic in the water column supports the notion of a coupled
arsenic-sulfur cycle (Sancho-Tomás et al. 2018).
8.3.3.3 Laguna Socompa
Laguna Socompa is placed in Argentinean Puna at 3400 masl in the basis of the
homonym volcano (Fig. 8.1). As concentration in Socompa lake is 28 mgL−1
whereas in the microbialite the salinity rises to 32 mg L−1 (Farías et al. 2013). The
arsenic resistance mechanism developed by these microbial communities consisted
mainly on the reduction and extrusion of As (V) by diverse Acr3 efflux pumps,
associated to ArsC (thioredoxin type) reductases, which was also observed in strains
that were isolated from Socompa (Kurth et al. 2017; Ordoñez et al. 2018). In the
stromatolites, a complete set of arsenic metabolism genes was unraveled (Kurth
et al. 2017), including arsM methyltransferases from the most varied phyla, as well
as respiratory aioA oxidases, arrA reductases, and even novel arxA oxidases; there-
fore, the community is able to carry out a complete arsenic cycle, even using it as a
source of energy, allowing growth and adaptation of microorganisms to high con-
centration of arsenic.
8.4 Conclusions
Andean Microbial Ecosystems (AMEs) represent a unique reservoir of microbial

life adapted to extreme conditions; among them, arsenic resistance mechanisms and
bioenergetic pathways have developed in efficient strategies that allow these com-
munities to colonize all kinds of extreme ecosystems, from stromatolites developing
in volcanic influenced areas like Socompa lake (3400 masl) to gaylussite biofilms
evolving at 230 mg L−1 of As in an volcanic caldera at Volcano Gálan at 4560 masl.
The association of arsenic geochemical cycle associated to anoxygenic photosyn-
thesis and sulfur cycle is being deeply studied at this moment. These studies will
position AMEs as suitable extraterrestrial life search models.
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Chapter 9
Stromatolites in Crater-Lake Alchichica
and Bacalar Lagoon
Luisa I. Falcón, Patricia M. Valdespino-Castillo, Rocio J. Alcántara-

Hernández, Elizabeth S. Gómez-Acata, Alfredo Yanez-Montalvo,
and Bernardo Águila
Abstract Extant stromatolites have been considered ecological similes to their

ancient counterparts. We now know that these microbial assemblages are composed
of a great diversity of microbes, which couple and intertwine their metabolic capa-
bilities to create self-sustained microbial ecosystems. Presently, stromatolites thrive
in a vast diversity of aquatic environments including freshwater, hypersaline, coastal
lagoons, alkaline lakes, oligotrophic pools, abandoned pits, few marine systems,
and brackish waters. In this chapter, we will summarize the research that has been
done in two stromatolite-harboring sites in Mexico: the alkaline crater-lake
Alchichica and the oligotrophic karstic coastal lagoon of Bacalar. Alchichica is
located in the Transvolcanic belt in Central Mexico. It is a maar-alkaline crater lake
(salinity 8.5 gl−1, pH 9.5) with water chemistry determined by high contents of car-
bonates, sodium, a high Mg/Ca ratio, and particularly low Ca2+ concentrations
(~0.3 mM). Two main stromatolite-types, as defined by mineralogy, texture, and
microbial composition, develop along its periphery from surface to over 30 m in
depth. Alchichica is a modern environment that resembles Precambrian oceanic
conditions. Stromatolites from Alchichica have been dated radiometrically in
~1.1–2.8 ka BP. Bacalar is a coastal lagoon located in the Yucatan Peninsula, which
is a carbonate platform that emerged above sea level during the Oligocene. The
Bacalar lagoon has high carbonate concentrations as a result of the influx of ground-
L. I. Falcón (*) · E. S. Gómez-Acata · A. Yanez-Montalvo · B. Águila

Parque Científico y Tecnológico de Yucatán, Mérida, Yucatán, Mexico
e-mail: [email protected]; [email protected]; [email protected]
P. M. Valdespino-Castillo
MBIB, Lawrence Berkeley National Laboratory, Berkeley, USA
R. J. Alcántara-Hernández
Instituto de Geología, Universidad Nacional Autónoma de México, UNAM,
Ciudad Universitaria, Mexico City, Mexico

184 L. I. Falcón et al.
water (salinity 1.2 gl−1, pH 7.6–8.3). It holds the largest freshwater stromatolite
structures known, which have been radiometrically dated in ~6.8–9.2 ka BP. Their
mineralogy, shape, and texture are similar along the lagoon’s coast, but microbial
composition changes, possibly due to anthropogenic impact.
9.1 The Ecological Setting
9.1.1 Crater-Lake Alchichica
Alchichica is an oligotrophic, monomictic, saline, maar, soda, crater-lake located in

the Trans-Mexican Volcanic Belt in central Mexico (19° 24’ N, 97° 24’ W,
2300 m.a.s.l) (Fig. 9.1). It has a maximum depth of 60 m, contains alkaline water
(pH 8.7–9.2), and a high conductivity (13 dS m−1). The dominant anions are chlo-
rides and bicarbonates; whereas the dominant cations are sodium and magnesium
(Alcocer and Lugo 2003; Armienta et al. 2008; Mancilla Villa et al. 2014).
Kaźmierczak et al. (2011) described two distinct morphologic generations of stro-
matolites: (1) Columnar dome-like structures, rich in aragonite, forming near the
shore line, dated back to 1100 ybp and (2) Spongy cauliflower-like thrombolytic
structures, rich in hydromagnesite and aragonite, dominate the lake’s periphery
(Valdespino-Castillo et al. 2018) (Fig. 9.2). Developing inside the lake, these struc-
tures were dated back to 2800 ybp (Kaźmierczak et al. 2011). It is hypothesized that
these distinct morphologies and mineral compositions were the result of different
periods of drought and flood by groundwater influx to the lake.
9.1.2 Bacalar Lagoon
Bacalar is the largest freshwater karstic lake in the Yucatan Peninsula (Gamboa-
Pérez and Schmitter-Soto 1999) (Fig. 9.1). The lagoon is 40 km long, 1–2 km wide,
and 20 m at its deepest point (Gischler et al. 2008). It is located in the evaporitic
region of the Yucatan Peninsula, in an area defined as a “Coastal Transversal
Corridor,” due to its high hydrological connectivity (Perry et al. 2002; Hernández-
Arana et al. 2015). Bacalar lagoon has a high groundwater inflow and connections
with other water bodies – brackish lagoons, estuarine coastal lagoons, and reef
lagoons – especially during the rainy season (Perry et al. 2009; Hernández-Arana
et al. 2015). The physicochemical characteristics of the water are oligotrophic, alka-
line (7.5–8.3) with a temperature range of 28–31 °C (Gamboa-Pérez and Schmitter-
Soto 1999; Gischler et al. 2008; Beltrán et al. 2012; Centeno et al. 2012). The
hydrogeochemistry has been defined as Ca2+ > Mg2+ > Na+ > Sr2+ > K+. The abun-
dance of anions is SO42− >> HCO3− > Cl− > NO3− (Gischler et al. 2008; Castro-
Contreras et al. 2014; Velázquez et al. 2018). Sulfates show saturation throughout
9 Stromatolites in Crater-Lake Alchichica and Bacalar Lagoon 185
Fig. 9.1 Geographic location of stromatolites in Mexico. Crater-lake Alchichica (left) within the
central Mexican Transvolcanic Belt, and Bacalar lagoon (right) in southern Yucatan peninsula
Fig. 9.2 Mineral
composition of crater-lake
Alchichica stromatolite
types (AS, spongy; AC,
columnar) and Bacalar
lagoon stromatolites
(BAC). Modified from
Valdespino-Castillo
et al. 2018
the lagoon (Perry et al. 2009; Johnson et al. 2018). Calcium is higher in the northern
zone, with values similar to seawater (Gischler et al. 2008). A saturation of bicar-
bonate ions, with values greater than seawater, occurs in the southern part of the
lagoon, due to the presence of four sinkhole “cenotes.” In addition, it is considered
that the southern part of Bacalar lagoon has the greatest extent of freshwater stro-
matolites (~ 10 km) in the world (Gischler et al. 2008, 2011). Stromatolites in
Bacalar lagoon are distributed in reef patches near the shores throughout the lagoon.
These stromatolites have been radiometrically dated between 6790 and 9190 ybp
(Gischler et al. 2008). Their morphology varies with the depth of the shore (Gischler
et al. 2011). They have been described as forming crusts, heads, and ledges. The
internal texture of Bacalar stromatolites suggests that most of them are thrombo-
lites, but there are also stromatolites and oncolites present (Gischler et al. 2008,
2011; Castro-Contreras et al. 2014). Bacalar stromatolites are composed almost
entirely of calcite, with a mixture of plagioclase, kaolinite, and siderite (Valdespino-
Castillo et al. 2018; Yanez-Montalvo et al. 2020) (Fig. 9.2).
9.2 Molecular Approaches to the Study of Stromatolites
Stromatolites have been studied with mineral, biochemical, microscopic, and

genetic protocols. The study of their microbial diversity started with morphological
descriptions using different microscopic techniques (i.e., light microscopy, scan-
ning electron microscopy (SEM), transmission electron microscopy (TEM), and
laser confocal microscopy). Molecular biology and Next Generation Sequencing
techniques (NGS) have allowed the characterization of microbial communities
through gene amplification from environmental DNA (Centeno et al. 2012; Coman
et al. 2015; Lindsay et al. 2017; Valdespino-Castillo et al. 2018; Gómez-Acata
et al. 2019).
Studying stromatolites with molecular techniques started in this century by
Steppe et al. (2001), who were the first to extract DNA from microbial communi-
ties in stromatolites. Several different techniques have been used where the
principal objective is to obtain DNA of good quality and in great quantity for
downstream analysis such as metagenomic studies (e.g., Gómez-Acata et al.
2019). All of these methods have three principal steps: a breakage of both cell
wall and cell membranes with different approaches including mechanical disrup-
tion, physical, chemical, and enzymatic lysis; hydrophobic cell debris separation
(proteins and lipids are eliminated with organic solvents such as phenol:chloroform
and DNA precipitation using alcohols. Moreover, depending on the type of sam-
ple, some methods include other steps to eliminate any inhibitors produced by
microorganisms (i.e., exopolymeric substances (EPS) and carbonated minerals) at
the beginning of the DNA extraction method (pre-treatment) or after DNA pre-
cipitation (purification) (Gómez-Acata et al. 2019). Commercial kits (used for
environmental samples such as soil, plants, sediments, and biofilm) are a good
choice since these are efficient and quick DNA extraction methods (López-García
et al. 2005; Breitbart et al. 2009; Couradeau et al. 2011; Saghaï et al. 2015;
Lindsay et al. 2017). There is no best method for DNA extraction of stromatolites;
all are good and have success depending on the mineral composition and the
amount of EPS in the sample. Methods that could be inadequate for one sample
could be the best for another, and the method selection depends on the stromato-
lite degree of lithification.
Microbialites have also been studied through the analysis of RNA expression.
For this purpose, it is very important to isolate high-quality RNA, so elimination of
all nucleases in the environment is crucial for this purpose since nucleases degrade
RNA. Phenol is a very common reagent used for elimination of nucleases in RNA
extraction methods as well as β-mercaptoethanol and guanidine thiocyanate (Santos
et al. 2010; Mobberley et al. 2015; Louyakis et al. 2018). Furthermore, commercial
kits have been used with excellent results (Edgcomb et al. 2014; Alcántara-
Hernández et al. 2017; Valdespino-Castillo et al. 2014, 2017). When analyzing gene
expression patterns, sampling is fundamental, since enzymatic activity changes
with the time of the day; for this reason, the sample must be frozen immediately
with liquid nitrogen at the study site or when the study site is difficult to access,
some preservative solution could be used (i.e., RNA later) (Kruse et al. 2017).
Finally, the integrity of RNA must be determined through the RIN (RNA integrity
number) value and the sample must be free of any inhibitor. There are a few studies
of phages (viruses) in stromatolites (Desnues et al. 2008; White III et al. 2018), by
direct methods (isolation of viruses from samples purified with a cesium chloride
density gradient and formamide/CTAB extraction).
Alchichica crater-lake and Bacalar lagoon stromatolites have been studied to
understand their microbial community composition with genomic approaches. For
this purpose, DNA extractions have included phenol:chloroform with enzymatic
lysis, mechanical disruption, and chemical lysis (Centeno et al. 2012; Valdespino-
Castillo et al. 2014, 2017, 2018; Yanez-Montalvo et al. 2020) or using DNeasy
PowerSoil® (Couradeau et al. 2011) and Power Biofilm TM DNA isolation kits
(Qiagen, Carlsbad, CA, USA) (Ragón et al. 2014). In some studies, initial carbonate
dissolution was needed to obtain high-quality DNA (Table 9.1), suitable for down-
stream applications. Moreover, Alchichica crater-lake stromatolites have been
Table 9.1 DNA extraction techniques for microbial community studies in stromatolites from the
Bacalar lagoon and the Alchichica crater-lake
DNA
Lysis purification
Study site treatment Technique/commercial kit methoda Reference
Alchichica Carbonates (1) QuickPick™gDNA Kit None Couradeau
crater-lake dissolved with (Bio-Nobile, Parainen, Finland) et al. (2011)
HCl 33% and previous incubation for 3 h at
neutralized 56 °C with Proteinase K and
with PBS ViscozymeH
pH 7, 0.5 M (2) MoBioPowerSoil DNA kit
EDTA pH 9 (MoBio, Carlsbad, CA, USA)
previous incubation with
ViscozymeH (Sigma-Aldrich,
Buchs, Switzerland)
Bacalar None Chemistry lysis with CTAB and DNeasy Centeno et al.
lagoon and SDS, enzymatic lysis with Blood & (2012)
Alchichica lysozyme, freeze-thaw cycles, Tissue kit
crater-lake and mortar liquid nitrogen, (Qiagen,
extraction with Alameda, CA)
phenol:chloroform:isoamyl
alcohol, precipitation with
isopropanol
Alchichica None Power Biofilm ™ DNA Isolation None Ragon et al.
crater-lake Kit (MoBio, Carlsbad, CA, USA) (2014)
Alchichica None DNA was extracted as reported None Valdespino-
crater-lake by Centeno et al. (2012) Castillo et al.
(2014, 2017,
2018) and
Alcántara-
Hernández
et al. (2017)
Alchichica Carbonates Ground with mortar and pestle Power Saghaï et al.
crater-lake dissolved with and Power Biofilm ™ DNA Clean™ Pro (2015)
100 μL HCl Isolation Kit (MoBio, Carlsbad, DNA
33% then CA, USA) Clean-up Kit
neutralized (Mo Bio,
with 1 mL of Carlsbad, CA,
1:1 PBS pH 7 USA), to
and 0.5 M remove
EDTA pH 9 residual EPS
Methods that report an extra purification step after DNA extraction
a
studied with an RNA, cDNA approach (Valdespino-Castillo et al. 2017; Alcántara-

Hernández et al. 2017). The extraction was done with an RNA PowerSoil® Total
RNA Isolation kit (Mo Bio Laboratories, Carlsbad, CA). Then RNA was purified
with an RNeasy Mini kit (Qiagen, Venlo, the Netherlands) and DNA digestion with
DNAseI (Qiagen) was necessary.
9.3 Microbial Diversity
Stromatolite formation is due to mineral accretion, precipitation, or binding influ-

enced by microbial activity (Burne and Moore 1987). The main biological compo-
nents of stromatolites are bacteria, which represent over 90% of genetic diversity
(Centeno et al. 2012). Research suggests that stromatolites are formed by a micro-
bial succession, where microbial mats actively trap ions and detritus with mucous
EPS. This EPS forms nucleation centers, and when they become saturated, an extra-
cellular precipitation aided by microbial degradation occurs (Dupraz et al. 2009).
Although no concise bacterial metabolisms have been reported for microbialite gen-
esis, some metabolisms are most likely to influence precipitation, cementation, and
lithification of minerals (Riding 2011). For instance, the role of photosynthesis,
either oxygenic and/or anoxygenic, along with sulfur metabolism is key for mineral
precipitation (Arp et al. 2012). Photosynthesis elevates pH locally and increases the
saturation index of minerals in a microenvironment, thus forming a genesis or
growth by lamination (Gérard et al. 2013). Other metabolisms may be involved in
carbonate precipitation, including ureolysis, nitrification, and methane metabolism
(Zhu and Dittrich 2016). A commonality among stromatolites is the presence of
cyanobacteria, sulfate-reducing bacteria, and Proteobacteria. This bacterial compo-
sition seems to be key for the construction of stromatolites (Chagas et al. 2016).
9.3.1 M
icrobial Diversity of Alchichica
Crater-Lake Stromatolites
Studies in crater-lake Alchichica have described the microbial diversity of stromato-

lites in both stromatolite-types, around the lake’s periphery (Centeno et al. 2012)
and along depth gradients (Kaźmierczak et al. 2011; Couradeau et al. 2011, Águila
2018). Stromatolite colorations vary with depth; for instance, shallow microbialites
have a yellow-greenish coloration, while deeper microbialites exhibit brown-reddish
colorations, due to different photopigments (Kaźmierczak et al. 2011; Couradeau
et al. 2011; Águila 2018). Overall, both stromatolite types (spongy and columnar)
have a strong component of Cyanobacteria (Nostocales, Oscillatoriales, and
Synechococcales), Proteobacteria (mainly Alphaproteobacteria, followed by
Deltaproteobacteria), Bacteroidetes (mainly Cythophagia) and Actinobacteria
(Fig. 9.3) (Valdespino-Castillo et al. 2018).
9.3.1.1 Cyanobacteria of Alchichica Crater-Lake Stromatolites
Alchichica stromatolites contain a high diversity and abundance of cyanobacteria,

including several unique species (Tavera and Komárek 1996). A recent study of
cyanobacterial composition along a depth gradient (from 3 m to 30 m) found that
Fig. 9.3 Microbial composition based on 16SrDNA gene diversity associated with Alchichica
crater-lake (AC, columnar; AS, spongy) and Bacalar lagoon (BAC) stromatolites
Pleurocapsales, and particularly the Xenococcaceae family, are the main cyano-
bacteria present in deep stromatolites below 12 m (Águila 2018). These results
agree with previous studies (Couradeau et al. 2011; Kaźmierczak et al. 2011),
where a taxonomy/depth relationship was observed. For instance, Nostocales and
filamentous Synechococcales were found at shallow depths, while Pleurocapsales
and Chroococcales were associated with deeper stromatolites; no correlation was
found with cocci Synechococales. Interestingly, stromatolites from 30 m only
contained Chroococcidium gelatinosum and Xenococcus candelaria, suggesting
an important role in their formation (Águila 2018). The main cyanobacterial gen-
era described in Alchichica crater-lake stromatolites included: Rivularia,
Calothrix, Trichormus, Nodosilinea, Phormidesmis, Oculatella, Cyanobium,
Heteroleiblena, Chroococcus, Entophysalis, Chroococcopsis, Xenococcus,
Chroococcidium, Stanieria sp., Pseudanabaena, Synechococcus, and
Gloeomargarita along with a high diversity of unclassified cocci Synechococales
(Águila 2018) (Fig. 9.4).
Fig. 9.4 Representative images of the cyanobacterial diversity of crater-lake Alchichica. Several
classes of cyanobacteria are observed including Nostocales (a, c and f), Synechococcales (d and
e), Chroococcales (d, h, i and o), and Pleurocapsales (b, g, j, k, l, m and n). Scale bar is 5 μm
9.3.2 Microbial Diversity of Bacalar Lagoon
Bacalar stromatolites have shown a high content of Corg associated with a large cya-
nobacterial abundance, representing over 70% of their genetic diversity, followed
by Bacteroidetes and Proteobacteria (Valdespino-Castillo et al. 2018; Yanez-
Montalvo 2020) (Fig. 9.3). In addition to the above groups, Johnson et al. (2018)
described that reducing sulfate bacteria contribute significantly to the precipitation

of carbonates. Proteobacteria are represented primarily by classes Alpha, Beta, and
Deltaproteobacteria (Fig. 9.3).
9.3.2.1 Cyanobacteria of Bacalar Lagoon Stromatolites
Cyanobacteria in stromatolites from Bacalar lagoon are common and diverse at

the northern portion of the lagoon while the southern portion exhibits a low diver-
sity of cyanobacteria (Yanez-Montalvo et al. 2020). Main cyanobacteria dominat-
ing the stromatolites comprise Nostocales along with filamentous Synechococcales.
Main genera include Arthrospira, Nostoc, Rivularia, Calothrix, Mastigocladopsis,
Fischerella, Microcoleus, Phormidium, Aphanocapsa, Microcystis,
Synechococcus, Cyanobium, Nodosilinea, Chroococcidopsis, and Staneria
(Fig. 9.5).
9.4 Functional Ecology and Biogeochemical Cycling
As mentioned above, stromatolites harbor a rich and diverse microbial community

that also results in a diverse metabolic profile, including phototrophs, chemotrophs,
denitrifiers, sulfur oxidizers, sulfate-reducers, and P-cycling bacteria, among others.
These microbes are interrelated in highly complex metabolic networks within phys-
icochemical gradients at the microscale level in the stromatolite microstructure
(Wong et al. 2015). Metagenomic studies have confirmed this plethora of biogeo-
chemical pathways (Breitbart et al. 2009; Casaburi et al. 2016; Khodadad and Foster
2012; Kurth et al. 2017; Ruvindy et al. 2016; Warden et al. 2016), where the main
processes associated with elemental cycling (e.g., C, N, S, and P) are present, sug-
gesting thus functional redundancy. In addition, ecophysiological determinations
have also shown relevant metabolic features of microbialites (Beltrán et al. 2012;
Khodadad and Foster 2012). However, it seems that the environmental and geo-
chemical conditions of the surrounding media determine the main metabolic path-
ways used by these benthic communities (Breitbart et al. 2009; Kurth et al. 2017;
Ruvindy et al. 2016). Here, we will review the contributions regarding the N-cycle
and P-cycling processes in stromatolites from crater-lake Alchichica and Bacalar
lagoon, as stromatolites are known to often occur in oligotrophic environments
where microbial activity is largely restricted by nutrient availability, such as N or P
(Pepe-Ranney et al. 2012).
Fig. 9.5 Representative images of the cyanobacterial diversity of Bacalar lagoon stromatolites. In
Bacalar, observed classes of cyanobacteria include Nostocales (c, f, g and o) and Synechococcales
(a, b, d, e, h, i, j, k, l, m and n). Scale bar is 5 μm
9.4.1 N-Cycle
Nitrogen is an essential element for biomolecules and its obtainment is essential for
cellular development and growth. Consequently, this macroelement often limits
biomass activity, where fixed-N might have constrained primary production on geo-
logical timescales (Falkowski 1997). Nitrogen is used in both assimilatory and
dissimilatory pathways including organic N recycling reactions and redox transfor-

mations of dissolved inorganic nitrogen species (NH4+, NO3−, and NO2−); all of
them interrelated in assimilatory, energy-obtaining, and energy-dissipating pro-
cesses (Canfield et al. 2010).
Alchichica crater-lake and Bacalar lagoon are reported as meso-oligotrophic sys-
tems regarding the concentration of dissolved inorganic nitrogen (DIN) [Alchichica
~1.5 μM and Bacalar ~0.5 μM], where NO3− is the most representative dissolved
N-form in the oxic system. Their salinity varies in: (i) the origin of the ions
[Alchichica, athalassohaline inland soda lake and Bacalar, karstic inland-coastal
lagoon]; (ii) their type [Alchichica Na > Mg > K > Ca and Bacalar [Ca > Mg > Na];
and (iii) their concentration [Alchichica electrical conductivity 13 dS/m, Bacalar
2.2 dS/m]. Despite these differences, the stromatolites from both aquatic systems
can fix N2, mainly during daytime (Falcón et al. 2002; Beltrán et al. 2012) and count
with diazotrophic bacteria, such as Cyanobacteria and Alphaproteobacteria, as
revealed by 16S rRNA and nifH gene surveys (Beltrán et al. 2012; Centeno et al.
2012). N2 fixation has been found as a relevant feature in lithifying and non-
lithifying microbial mats (Beltrán et al. 2012), though stromatolites with excessive
NO3− availability can diminish N2-fixation rates due to the preference of nitrate
assimilation over N2 fixation (Breitbart et al. 2009). Metagenomic data suggest that
the assimilatory pathways are the most relevant in the stromatolite community,
including amino acid metabolism, allantoin degradation, nitrate assimilation, and
N2 fixation (Breitbart et al. 2009; Khodadad and Foster 2012; Ruvindy et al. 2016).
A study considering different types of stromatolites, where Bacalar lagoon and
Alchichica crater-lake samples were included, showed that the overall microbial
distribution patterns show a significant correlation with the N concentration in the
living tissue (Valdespino-Castillo et al. 2018), demonstrating thus, the relevance of
N in these microbial communities.
Denitrification is one of the most studied anaerobic respiration processes in stro-
matolites and microbial mats (Joye and Paerl 1994; Alcántara-Hernández et al.
2017), where nitrate (NO3−) is subsequently reduced to N2 (NO3−, NO2−, NO, N2O,
N2). This process is often used by microorganisms living in aerobic/anaerobic inter-
phases where O2 concentration fluctuates and an alternative electron acceptor is
needed. As a consequence, stromatolites harbor a rich denitrifying community,
especially when nitrate is available, as mentioned above. We surveyed the potential
nitrite respiring bacterial communities in stromatolites from Alchichica crater-lake
and Bacalar lagoon employing the genes encoding for nitrite reductases (nirK and
nirS) (Fig. 9.6). Our results showed that both microbial communities harbor diverse
phylotypes (at 5% nucleotide difference), with a main proportion of shared OTUs
(highlighted in green). Most of the nirK and nirS sequences were affiliated to
Alphaproteobacteria; however, gene nirS OTUs were related to Thioalkalivibrio
nitratireducens, a lithotrophic sulfur-oxidizing bacteria. These results suggest that
main phylotypes for nitrate respiration are present in both stromatolites, with differ-
ences in the less abundant sequences. Denitrification has been reported in other
metagenomic studies (Breitbart et al. 2009), and found as a shared feature among
stromatolites.
Fig. 9.6 Neighbor joining tree for genes involved in nitrite reduction in Alchichica lake and
Bacalar lagoon microbialites. (a) nirK gene (469 nucleotides) and (b) nirS gene (402 nucleotides).
Highlighted OTUs represent those with most clones (>5%) obtained in this study. Midpointed trees
with bootstrap values ≥50% (1000 replicates). Divergence is represented by each scale bar
The chemolithotrophic processes such as nitrification and anaerobic ammonia

oxidation (anammox) have showed differences among freshwater and marine stro-
matolites. In the Alchichica crater-lake, just a few phylotypes associated with aero-
bic and anaerobic ammonia oxidation were found, mainly related to Nitrosomonas
spp. (capable of growth in slightly saline conditions). This low diversity could be
related to bioenergetic constraints in this saline (alkaline) environment (Oren 2011),
where nitrification and anammox are limited. The stromatolites of Bacalar lagoon
have shown the presence of Thaumarchaeota, which likely drive ammonia oxidation
in this system (Centeno et al. 2012).
9.4.2 P-Cycling
Phosphorus is a structural element of cell membranes and it is also interrelated to

many other energetic processes in cellular function as well as an essential element
for DNA and RNA construction. Microbes play a fundamental role in acquisition
and remineralization of phosphorus within the ecosystems. In environmental sci-
ences, we are still in the process of understanding the identity of the microorgan-
isms harboring the genetic potential for the transformation of the different forms of
phosphorus. In the aquatic environment, dissolved organic phosphorus (DOP) is
usually the most abundant fraction (Kolowith et al. 2001) still largely uncharacter-
ized (Dyhrman et al. 2007). Using stromatolites and bacterioplankton as study mod-
els, we have analyzed their potential to use this phosphorus compartment.
Additionally, we compared stromatolites’ potential to that of bacterioplankton.
To utilize this fraction of the total P in aquatic environments, microbes harbor
diverse strategies, the better known are associated with pH-dependent (acid or alka-
line) phosphatases to release orthophosphate or polyphosphate chains by the degra-
dation of organic matter phosphate esters (Ruttenberg and Dyhrman 2005). Phytate
or phytic acid is another molecule that may be abundant in DOP, it is associated
overall with plant debris runoff to the aquatic environments (Suzumura and
Kamatani 1995). Some microbes can release orthophosphate from phytate using
specific enzymes, phytases. Following this logic, the microbial markers we used to
study microbial DOP utilization in stromatolites and bacterioplankton of the maar
lake Alchichica were alkaline phosphatases phoA and phoD, as well as beta propel-
ler phytases (3-phytase, BPP). Finally, we studied the transcription of these genetic
markers in seasonal and diel dynamics (Valdespino-Castillo et al. 2014, 2015, 2017).
Our results showed a large potential for DOP utilization using these specific
genetic markers, taxonomically related to calcium-dependent alkaline phosphatases
of Alpha-, Beta-, and Gamma-proteobacteria. A comparison with available datasets
(GenBank-2014) showed that the diversity of alkaline phosphatase phoX of
Alchichica was similar to the diversity of this particular marker in systems exhibit-
ing contrasting environmental characteristics (e.g., trophic state, phosphorus condi-
tion, concentration of Zn, Mg, and Ca, metal cofactors of these type of enzymes)
(Valdespino-Castillo et al. 2014). The diversity of BPP was taxonomically related to
Alpha- and Beta-proteobacteria, as well as to Bacteroidetes and Flavobacteriales.

Beta propeller phytases found in this alkaline lake were similar to other phytases
found in forest soils, glaciers, and lake sediments as well as from the gut of lake fish
(Huang et al. 2009). These comparisons intend to contribute to understand the diver-
sity of these bacterial metalloenzymes in natural waters; other markers should be
compared among different habitats as the databases for microbial functional genes
expand. A functional parallelism was found between stromatolites and bacterio-
plankton regarding DOP utilization where stromatolites concentrated most of the
genetic diversity associated with the markers studied (phoX, phoD, and BPP)
(Valdespino-Castillo et al. 2014). Alkaline phosphatase phoX was the marker among
DOP utilization enzymes that showed the highest ratio of unique sequences/total
sequences (Valdespino-Castillo et al. 2014).
Gene dynamics analyses showed a more stable pool of enzymes for DOP utiliza-
tion in stromatolites compared to bacterioplankton, in which DOP utilization poten-
tial was relevantly impacted by the hydrologic cycles of the lake (summer
stratification-winter circulation). The highest DOP utilization diversity of stromato-
lites together with this result suggests their resilience. The microbial communities
in the water column exhibited a faster response to environmental change (different
phylotypes were found in the intra-annual exploration (Valdespino-Castillo et al.
2014, 2017)). Together these results also suggest divergent strategies to face envi-
ronmental change between stromatolites and bacterioplankton. An environment
able to harbor high structural (and therefore functional) diversity might be a strategy
that allows stromatolites to persist after millions of years. In terms of phosphorus
utilization, stromatolite microstructure may offer a gradient of pH microenviron-
ments (i.e., gradients that may be stretched by redox activity, such as photosynthesis
and respiration), but the clarification of these gradients has been difficult to assess
due to their rocky nature.
More studies are still needed to comprehend the intricacies of stromatolites in
Alchichica crater-lake and Bacalar lagoon. These systems are unique and fragile. Both
of them are experimenting a detriment in their water quality, associated with lack of
wastewater treatment, and sustainable development. So far, we do not fully under-
stand the rate of damage to these ancient structures, hence increasing our concern
toward protecting these singular ecosystems. We now know how diverse and relevant
stromatolites are functioning as micro-ecosystems where all metabolisms needed for
their growth and maintenance exist, thanks to their great bacterial diversity.
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Chapter 10
The Origin and Early Evolution of Life
on Earth: A Laboratory in the School
of Science
José Alberto Campillo-Balderas and Arturo Becerra
Abstract The study and research in the origin of life in Mexico have deep roots in
the last century. However, the modern period of study of the origin and early evolu-
tion of life started in the early 1970s, when two groups were created to study the
topic at the Universidad Nacional Autónoma de México (UNAM). The first of these
groups began in 1974 in the School of Sciences and has continued to work since
then: analyzing comets and meteors as models of chemical events that occurred in
the early Earth; studying the central hypothesis of the “RNA world” and the role of
modified ribonucleotides on the early stages of evolution; using bioinformatics
tools and comparative genomics; and trying to infer very early stages of life. Here,
we present a brief description of the main lines of research of the first group and the
perspectives of the study of the origin and early evolution of life. This chapter
addresses a question that does not have a model site, since the environmental condi-
tions where it arose are still unknown. Nevertheless, many inferences can be made
out of experiments and comparative genomics.
10.1 T
he Study of the Origin and Early Evolution of Life
in Mexico
Study and research of the origin of life in Mexico have deep roots in the last century
since Alfonso L. Herrera, a remarkable naturalist and teacher, introduced the evolu-
tionary theory in basic education programs and proposed his theory of plasmogeny,
as a physicochemical process of the origin of life (Perezgasga et al. 2003; Ledesma-
Mateos and Barahona 2003). Herrera made an essential improvement in Mexican
education and life sciences. As Negrón-Mendoza said, “He is considered a corner-
J. A. Campillo-Balderas · A. Becerra (*)

Facultad de Ciencias, Universidad Nacional Autónoma de México, UNAM,
Mexico City, Mexico
e-mail: [email protected]; [email protected]

204 J. A. Campillo-Balderas and A. Becerra
stone of Biology in Mexico” (Negrón-Mendoza 1995). When Herrera had published

his hypotheses (Herrera 1904, 1905, 1919, 1924), Alexander I. Oparin was close to
announcing his foundational views about the heterotrophic origin of life, whose
conclusions were that living organisms are the historical result of a gradual transfor-
mation of lifeless matter (Lazcano 2016). First, in a book (Oparin 1924), which had
no international notice, since only a few Russian scientists read it, but then in 1938,
an English edition of Oparin’s book was published and became known worldwide
(Oparin 1938), where Oparin attempted to provide in detail a chemical process for
the origin of life (Miller et al. 1997).
In our view, the modern period of study of early evolution of life in Mexico could
have been started in the early 1970s, around the moment that Oparin visited our uni-
versity (Universidad Nacional Autónoma de México, UNAM), thanks to the academic
authorities of the university and the invitation of the young scientist Antonio Lazcano.
This event was not only an excellent opportunity to listen to the most crucial scientist
on the field, but also it was a critical moment that allowed the foundation of the mod-
ern view of the study of origins of life in our university and Mexico. A few years later,
were created in our university at least two new groups working in the origin of life:
one by Antonio Lazcano at the School of Sciences in 1974 and the other by the chem-
ist Alicia Negrón at the Institute of Nuclear Science, the laboratory of chemical evolu-
tion in 1976. In conjunction with research, Professor Lazcano founded the first course
about “Origin of Life” that has been imparted from 1974 until now as a requested and
successful program on the School of Science. Both laboratories were and are thriving
and vital groups that generate not only several and outstanding research but also a
good number of scientists who trained as students in both groups (Chap. 1). In this
chapter, we write about the main activities and investigations of the first one.
10.2 The Origin of Life Laboratory in the School of Science
10.2.1 Prebiotic Chemistry and RNA World
In the beginning, the laboratory in the School of Science at the UNAM was called
Laboratorio A. I. Oparin; it was founded in September 1974 by Antonio Lazcano.
Since this time, he had been working on the importance of extraterrestrial organic
compounds present in nuclei of comets and chondritic meteorites, next to Joan Oró,
Stanley L. Miller, and other prominent pioneers of the field. They analyzed comets
and meteors as models of chemical events that occurred in the solar system when
the Earth itself was forming, to explain the synthesis of organic molecules that
could provide the conditions of the primitive Earth (Oró et al. 1978, 1980; Bar-Nun
et al. 1981; Lazcano et al. 1983). Also, significant meetings occurred in the field on
those years including the second and third Mexican meetings of Origin of Life
(Fig. 10.1); the first as a tribute to Oparin in 1975; and the “Cosmochemistry and the
Origins of Life” where Joan Oró, Cyril Ponnamperuma, Lynn Margulis, George
Fox, and other founders of the field presented their results and research.
10 The Origin and Early Evolution of Life on Earth: A Laboratory in the School… 205
Fig. 10.1 The third Mexican meetings of Origin of Life. “Cosmochemistry and the Origins of
Life” where Joan Oró, Cyril Ponnamperuma, Lynn Margulis, George Fox presented their results
and research in 1979
The discovery of the catalytic activities of RNA molecules motivated Lazcano to

propose in 1986 the existence of what we now call the “RNA world,” a central
hypothesis in the modern study of the emergence of life, and which was suggested
the same year, independently, by Walter Gilbert and Bruce Alberts (Lazcano 1986).
Since that year until today, the group of “Origin of Life Laboratory” addressed the
study of this hypothesis, which has opened a line of research that seeks to under-
stand the origin of modified ribonucleotides (Fig. 10.2), such as coenzymes, alar-
mones, and histidine (Hernández-Morales et al. 2019; Vázquez-Salazar et al. 2018).
Nevertheless, how life originated on Earth is not fully knowable, the development
of catalytic RNAs and the diverse roles that RNA molecules and ribonucleotides
play in extant cells (Vázquez-Salazar and Lazcano 2018) has provided substantial
support to the possibility that this genetic polymer carried out a much more con-
spicuous role on the early stages of evolution, an epoch generally referred to as the
RNA world (Gilbert 1986).
Some of the main results in this subject and obtained by the founder, students,
and colleagues of the laboratory are (a) the demonstration of the catalytic activities
of pure peptides under prebiotic conditions (Shen et al. 1990); (b) the display that it
is possible to synthesize organic compounds of biochemical importance in reducing
and neutral atmospheres, which broke a paradigm that for the past 60 years indi-
cated otherwise (Bada et al. 2007; Cleaves et al. 2008); (c) the demonstration of the
similarity of formation of alkylated amino acids in chondritic meteorites and neutral
Fig. 10.2 The role of ribonucleotides, such as coenzymes, alarmones, and histidine, in the origin
and early evolution of life
and reducing atmospheres (Parker et al. 2011a, b); and (d) the analysis of the origi-
nal samples of the Miller experiment, which led to a research article that was pub-
lished in the journal Science (Johnson et al. 2008; Bada and Lazcano 2003).
10.2.2 Early Life and the Last Common Ancestor
Despite the unbeatable obstacles surrounding the understanding of early life, a key
question in astrobiology, there are two ways to approach it, especially to the evolu-
tionary transition between the non-living and the living, to the process that we call
the origin of life: (1) the synthetic approach or also called “bottom-up approach”
and (2) the reconstruction of early entities using the actual organisms or the “top-
down approach” (Fig. 10.3).
Our group has been using both approaches, mainly since the genomic era started
when the first two genomes were available and the comparison of them was plau-
sible. The first completely sequenced genome of a living organism was Haemophilus
influenzae (Fleischmann et al. 1995) then Mycoplasma genitalium a few months
Fig. 10.3 Approaches to early life. The synthetic approach or also called “bottom-up approach”
and the reconstruction of early entities using the actual organisms or the “top-down approach”
later (Fraser et al. 1995). At that moment we started to compare the genomes to infer
the gene complement and metabolic abilities of early stages, continuing a lineage of
research that began years before (Lazcano et al. 1988, 1992).
However, Mushegian and Koonin (1996) published the first inference of the
comparison of both genomes. They concluded that the information obtained sug-
gested a minimal gene set for cellular life and reflected some characteristics of the
last common ancestor of life, a cell that was a straightforward one that lacked a
DNA genome and complex metabolic pathways. Nevertheless, we propose that
polyphyletic gene losses can be bias the characterizations of the cenancestor
(Becerra et al. 1997). In our proposal, we insist that in the characterization of ances-
tral states, should be considered the biological aspects as well as the bioinformatic
or statistical data since we are doomed to have partial reconstructions and are not
exempt from false-negative results. Furthermore, a bioinformatics approach to the
origin of life is not feasible since all possible intermediates that may have once
existed have long since being lost.
Phylogenetic analyses based on comparative genomics provide essential clues on
the very early stages of biological evolution and diversification of life. Still, it is
hard to see how its applicability can be used beyond a time that corresponds to a
cellular evolution period in which protein biosynthesis was already working
(Becerra et al. 2007; Lazcano 2012).
In other words, phylogenetic analysis and comparative genomics cannot give us
direct information about the origin of life. Sensitive tools can help us infer the very
early stages of cellular evolution, as Woese and Fox discovered that, regarding the
similarities and differences in the small subunit of the ribosomal RNA molecule,
living beings are divided into three groups (Woese and Fox 1977a). They suggested
that there was a primitive entity in the division of these three lineages when the rela-
tion between genotype and phenotype had not yet evolved (Woese and Fox 1977b;
Delaye and Becerra 2012).
They named this ancestral entity a progenote, which implies a primitive state.
However, thanks to the development of DNA sequencing technology and the accu-
mulation of a vast amount and diversity of sequences and complete genomes in
databases, it was possible to start identifying the genes conserved in bacteria and
Archaea. Our group has a particular interest and has been working significantly in
this area. We have collaborated importantly in the reconstruction and characteriza-
tion of the cenancestor, also known as the last common ancestor (LCA), which
means the last common ancestor of currently living beings (Delaye et al. 2005;
Becerra and Delaye 2015).
Despite qualitative and quantitative differences in the methodology used to iden-
tify the gene complement of LCA, there is an overlap that reveals an impressive
level of conservation for a significant number of sequences involved in essential
biological processes. The results obtained include lists of repertoires of gene
sequences from incompletely represented basic biological processes, such as trans-
lation, transcription, biosynthesis of nucleotides and amino acids, energy metabo-
lism, sequences related to replication, and cellular transport (Muñoz-Velasco et al.
2018). Corroborating the proposal in 1992 by Lazcano, Fox, and Oró, that proposed
that the last common ancestor possessed genetic machinery similar to that of a mod-
ern bacterium (Lazcano et al. 1992; Delaye and Becerra 2012).
Nevertheless, the current descriptions of LCA are limited by the scant informa-
tion available, and the gene complement includes proteins that originated in differ-
ent epochs prior to LCA time. For instance, several highly conserved ribosomal
proteins may have originated during the RNA/protein world stage, whereas thymi-
dine kinase and thioredoxin reductase, which are involved in deoxyribonucleotide
biosynthesis, evolved at a later stage. Although we favor a bacterial-like ancestor, at
this moment, it is difficult to assess the metabolic pathways related to the LCA or
the environmental conditions in which it prospered (Becerra et al. 2007). Other
research groups propose that acetogenesis and methanogenesis are the oldest
metabolisms on Earth (Martin and Russell 2007; Sousa et al. 2013) and suggest that
both metabolic routes emerged in a hyperthermophilic environment, such as the
alkaline hydrothermal environment (Martin and Russell 2007), from an LCA
endowed with geochemically driven monocarbon-unit transformations (Sousa and
Martin 2014; Weiss et al. 2016). However, our results suggest that methanogenesis
is present only in the Archaea domain, whereas the acetyl-CoA synthesis from CO2,
or Wood-Ljungdahl pathway, is present in both bacteria and Archaea (Muñoz-
Velasco et al. 2018), but also we are interested in the metabolic adaptation to the
oxygen that increased its level in the primitive atmosphere (Alvarez-Carreño
et al. 2016).
10.2.3 Early Metabolic Evolution
Some authors suggest that the contemporary metabolic routes are descended from
prebiotic chemical pathways and differ by the intervention of enzymes (Degani
and Halmann 1967; Hartman 1975; de Duve 1991; Morowitz 1992). However, the
known prebiotic pathways are quite different from the present metabolic routes,
as Lazcano and Miller said: “the origin of metabolic pathways lies closer to the
origin of life than to the last common ancestor” (Lazcano and Miller 1999).
Therefore, to study the evolution and origin of the contemporary metabolic path-
ways, it gives limited or no knowledge about the process that occurred near the
beginning of life.
However, it is also essential to understand and inquire what kind of metabolism
was present in early life (close to LCA) and infer how was the process to assemble
the pathways and increase their number of enzymes. There are four main hypothe-
ses about the origin and assembly of metabolic pathways:
(a) The Horowitz hypothesis was the first attempt to explain this evolutionary pro-
cess and was proposed by Horowitz in 1945. It is also referred to as the retro-
grade hypothesis, which means the pathways were built up backward a step at a
time than in the forward direction using intermediates in the prebiotic condi-
tions (Horowitz 1945).
(b) The Granick hypothesis is the development of the biosynthetic pathway that
was assembled in the forward direction, where the prebiotic compounds do not
perform a role (Granick 1950, 1957).
(c) The Patchwork assembly hypothesis, an exciting idea proposed independently
by Ycas in 1974 and Jensen in 1976, according to which biosynthetic pathways
are the outcome of the serial recruitment of promiscuous enzymes endowed
with broad catalytic specificity that could react in different steps and routes.
(d) The semi-enzymatic origin of metabolic pathways, proposed by Lazcano and
Miller in 1999, suggests that the metabolic-like pathways produced crucial
components required by primitive living entities, and were originally non-
enzymatic or semi-enzymatic autocatalytic processes. That later became fine-
tuned by ribozyme- and protein-based metabolic routes (Lazcano and Miller
1999; Islas et al. 1998; Delaye and Lazcano 2005).
Unlike the origin of life, the early evolution of metabolic pathways has directly
benefited from the development of genomics and molecular evolution. Thanks to
the comparative analysis of complete sequences of prokaryotic and eukaryotic
genomes, it is possible to study the origin and evolution of metabolism. For instance,
the hypotheses mentioned above relate to a greater or lesser degree with gene dupli-
cation, which is a crucial mechanism of genome evolution that affects genomic
complexity, intrinsic genetic diversity (Innan and Kondrashov 2010), and is ulti-
mately a significant source of evolutionary innovation, as Lewis (1951) and Ohno
(1970) proposed it for the development of metabolic changes (Fani 2012). Our
group works on the subject since the bioinformatic tools are available, analyzing the
enzymatic substrate specificity and its implication in the early evolution of meta-
bolic pathways (Lazcano et al. 1994); the role of gene duplication in the evolution
of purine nucleotide salvage pathways (Becerra and Lazcano 1998); the evolution
of histidine biosynthesis (Fani et al. 1995, 1997, 1998; Alifano et al. 1996); the
evolution of autotrophy metabolism (Peretó et al. 1999; Becerra et al. 2014); and the
evolution of methanogenesis pathways (Muñoz-Velasco et al. 2018).
10.2.4 The Origin of Life and Viruses
In the first half of the twentieth century, there was a dichotomic explanation to the
origin of life that persists until now. For one side, the metabolism-first hypothesis is
based on the idea that life is “a dynamic, self-regulating, metabolizing system.”
Biochemists, like Oparin, promoted it. On the other side, the nucleocentric hypoth-
esis emphasizes that life is “a single molecule with the inherent capacity to undergo
such essential activities as self-duplication.” Geneticists, like Hermann Muller,
were the ones who supported the idea of an “autocatalytic enzyme/gene” as a pri-
mordial form of life (Podolsky 1996).
Precisely, in 1926, Muller proposed that viruses, as small primitive genes, self-
replicating, non-metabolizing agents, are the relics of a primordial form of life.
Even Jerome Alexander, Calvin Bridges, and, later, J.B.S. Haldane (then retracted)
reinforced this idea because they believed that viruses were the missing link between
nonlife and the primordial cells (Podolsky 1996; López-García 2012). With the dis-
covery of DNA in 1944 and its description as a molecule with genetic information
and evolutionary characteristics, the metabolism-first hypothesis lost strength.
Therefore, viruses seen as living fossils of a primitive living entity, the nucleocen-
tric became the virocentric hypothesis of the origin of life and viruses (Moreira and
López-García 2009) (Fig. 10.4a).
Later in the 1930s, Robert Green and Patrick Laidlaw (Green 1935; Laidlaw
1939) challenged the virocentric with their retrograde hypothesis of the origin of
viruses (Fig. 10.4b). According to them, all viruses descended from a cellular ances-
tor that degenerated and subsequently increased the level of parasitism. In 1945,
Burnet recognized the debate between those two hypotheses (virocentric and degen-
erate), but also he empirically proposed that viruses could be escaped fragments
from modern cells (Burnet 1945) (Fig. 10.4c). This hypothesis was also supported
later in 1970 with the discovery of the reverse transcriptase and the proposal of
RNA tumor viruses as mobile genetic elements of animal cells (Baltimore 1970;
Mizutani and Temin 1970; Temin 1970).
Fig. 10.4 The main hypothesis of the origin of virus. (a) The virocentric hypothesis of the origin
of life and viruses. (b) The retrograde hypothesis of the origin of viruses. (c) The escape
hypothesis
Virus research in the latter half of the twentieth century has allowed an increase
in the knowledge of the evolution of viruses. However, their origin is still a debate
because viruses are polyphyletic. With the sequencing of new viral genomes, the
free access to viral databases, and overall, with the expertise in the origin and
early evolution of life, our laboratory wanted to be part of the origin-of-viruses
debate in 2014. We realized that there were new refined versions of the three
hypotheses. With the findings of supposedly hallmark genes like RNA-dependent
RNA polymerase (replication) and capsid (structure) and the nature and size of
viral genomes, it has been proposed that RNA viruses originated in the prebiotic
RNA world: a kind of an “ancient virus world” or a virosphere that preceded the
contemporary biosphere (Koonin et al. 2006; Forterre 2006) (Fig. 10.4a). Later,
with the discovery of some double-stranded DNA megaviruses in 2001 and the
analyses of phylogenetic markers involved in DNA replication and repair, tran-
scription, and translation, viruses have been seen as a consequence of a degener-
ate origin of an ancestral cell and even proposed as a fourth domain of life (Boyer
et al. 2010; Claverie et al. 2006; Nasir et al. 2015) (Fig. 10.4b). Finally, with the
studies of horizontal gene transfer and blocks of genes as functional modules,
viruses could have a chimeric-escaped gene origin (Campbell 2001; Hendrix et al.
2000) (Fig. 10.4c).
We are aware of the highly divergent nature of viral genomes, which has compro-
mised the elucidation of deep phylogenetic relationships (Holmes 2009). Therefore,
we have based our work on the origin and early evolution of viruses in three strate-
gies: the study of all biological and ecological data, pangenomic analysis, and pro-
tein structure similarities for each viral family.
In a comparative data analysis of the chemical nature, genome size, and seg-
mentation, and host type of all viral references reported in GenBank, we have
shown that the viral genome size, like RNA viruses, does not correlate with the
evolutionary history of hosts (Campillo-Balderas et al. 2015). We found that RNA
viral families are well distributed in eukaryotic hosts. With the possibility of bias,
Cystoviridae and Leviviridae are the only two RNA viral families which infect
prokaryotes like Pseudomonas, Caulobacter, Acinetobacter, and pathogenic
Enterobacteriaceae. All of these hosts are found in the microbiome of many ani-
mals. There are no RNA viruses which infect Archaea. Accordingly, it appears
that RNA viruses have a recent origin closely related to the evolutionary history
of eukaryotes. It can thus be concluded that RNA viruses may be ancient, but not
primitive (Fig. 10.5).
Once we have designed the database with all the biological information, we have
been analyzing the pangenome of all families of DNA and RNA viruses at this
moment. We recruited the reference sequences of all proteins from GenBank,
searched for homologs, and clustered them into protein families and pangenomic
compartments (core, dispensable, and unique proteins) depending on their presence
and absence in each viral family. With this approach (Contreras-Moreira and
Vinuesa 2013), we expect to trace the biological history of all proteins that are
shared by each viral family and to determine whether they stem from cells or viruses
according to their distribution in phylogenies.
Fig. 10.5 RNA viral

families distributed in the
tree of life. RNA viruses
only infect the Eukarya
domain. There are no RNA
viruses in Archaea and
only two families that
infect Proteobacteria
(Pseudomonas,
Caulobacter,
Acinetobacter, and
pathogenic
Enterobacteriaceae). This
bacterial group coexist
with eukaryotes; therefore,
these viruses probably
evolved from the
eukaryotic viruses
We also want to analyze some tertiary structures like capsid proteins. A recent
study based on structural similarities has shown that capsid proteins probably
evolved from ancestral proteins of cells on multiple occasions (Krupovic and
Koonin 2017). In our laboratory, we have designed an approach for inferring phy-
logenies based on the construction of matrices of pairwise structural comparison
scores (Jácome et al. 2015). We expect to determine a deep phylogenetic relation-
ship of jelly-roll domain, the most prevalent fold of icosahedral capsid proteins,
based on this methodology.
With these preliminary results, we want to determine a consilience and discor-
dance of findings according to the biological and ecological information, the pange-
nomic analysis, and deep phylogenies of tertiary structures of all viral families.
With this, we expect to elaborate an origin and early evolution model, supporting
the idea that viruses are escaping genes from host genomes.
Finally, the physicochemical conditions in which life started cannot be deter-
mined unequivocally, and how life originated on Earth is unknowable. Nevertheless,
the development of cosmology, geology, and prebiotic chemistry allows more robust
inferences about this scenario. This synthetic approach together with the top-down
approach releases not only the reconstruction of patterns of early stages of life but
also an opportunity to learn and discover the process of evolution. Using prebiotic
chemistry, bioinformatics tools, comparative genomics, but above all, evolutionary
biology, students and professors from the Origin of Life laboratory in the School of
Science, we try to investigate one of the most exciting stages of life, the very early
stages of evolution.
Acknowledgments The authors would like to thank all the members of the Origen de la Vida
Laboratory at the Universidad Nacional Autónoma de México. We also acknowledge the help of
Dr. Ricardo Hernández-Morales and Dr. Alberto Vázquez-Salazar.
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Chapter 11
Cuatro Ciénegas as an Archaean
Astrobiology Park
Nahui Olin Medina-Chávez, Susana De la Torre-Zavala,
Alejandra E. Arreola-Triana, and Valeria Souza
“Exobiology is no more fantastic than the realization of space

travel itself, and we have a grave responsibility to explore its
implications for science and for human welfare with our best
scientific insights and knowledge.”
Joshua Lederberg 1960, Science
Abstract The Cuatro Ciénegas Basin (CCB), located in Coahuila, México, is con-
sidered a living laboratory for astrobiology. This site is an analogue for the Gale
Crater in Mars because of its gypsum-rich soils. Moreover, the “pozas” at the site
are extremely oligotrophic, which is characteristic of ancient oceans chemistry.
Despite the oligotrophic conditions, CCB harbors a very rich microbial diversity in
the form of stromatolites and microbial mats. Archaea data at CCB has been sparse
given that, in general, archaea do not represent more than 1% of the reads in a
metagenome. Here, we describe a new hyper-diverse site with an abundant core of
Archaea. This site, which we named “archaean domes,” recreates in their interior
the atmosphere of ancient Earth and Mars and can serve as a valuable resource for
astrobiological research.
N. O. Medina-Chávez · S. De la Torre-Zavala
Instituto de Biotecnología, Universidad Autónoma de Nuevo León, Facultad de Ciencias
Biológicas, San Nicolás de los Garza, NL, Mexico
A. E. Arreola-Triana
Departamento de Biología Celular y Genética, Universidad Autónoma de Nuevo León,
Facultad de Ciencias Biológicas, San Nicolás de los Garza, NL, Mexico
V. Souza (*)
Mexico City, Mexico

220 N. O. Medina-Chávez et al.
11.1 C
uatro Ciénegas Basin: A Living Laboratory
for Astrobiology
In 1976, the Viking landers performed experiments looking for life on Mars. These
experiments were designed to search for Class I organisms—those that live in rela-
tively moderate temperatures (> −33 °C) and high water activity (aw > 0.7), like
most of the organisms on Earth. The experiments yielded negative, but controver-
sial, results. Some of the controversy arose from the fact that these experiments
were not designed to look for organisms belonging to Classes II to IV, that is, organ-
isms that live in extreme conditions with low temperatures and high water activity,
high temperature and low water activity, or low temperature and low water activity
(Sagan and Lederberg 1976). More than 40 years ago, Sagan and Lederberg sug-
gested that future space crafts that landed on Mars should be equipped to detect the
full range of organisms that could potentially inhabit the planet. To more fully
understand the putative Martian life and the constraints it lives under, it is essential
to use analogue ecosystems on Earth.
Analogues mimic and hypothesize the origin and occurrence of life beyond
Earth. Astrobiologists on Earth work in analogue environments that are similar to
those found in space. Of particular interest are the extreme environments where life
could not be thought possible (Gupta et al. 2014; Jones et al. 2018). Soare et al.
(2001) describe three types of analogues. Analogues of the first order are based on
empirical evidence gathered from spacecraft, landers, and satellite images; on Earth,
features, such as impact craters, volcanoes, and aeolian dunes, serve as analogues of
the first order. Analogues of the second order are those based on indirect but highly
suggestive evidence, such as alluvial fans and gypsum veins, the latter of which
Steve Squyres called “the single most powerful evidence for liquid water on Mars”
(Szynkiewicz et al. 2010; Showstack 2011). Analogues of the third order are specu-
lative, unsupported by direct or indirect evidence—as long as there is no confirma-
tion of life existing elsewhere in the solar system, extremophile organisms are
analogues of the third order (Preston and Dartnell 2014).
The study of extremophiles has educated our search for life in other planets. Of
the terrestrial bodies in the Solar System, Mars is the most Earth-like, and features
of its geology and geography point to a warmer, wetter past, as evidenced by the
traces of gypsum identified by the OMEGA instrument on ESA’s Mars Express
orbiter (Bibring et al. 2005; Paige 2005; Preston and Dartnell 2014). On Earth, there
are over 30 sites that emulate the extreme conditions found on Mars, among which
are the Atacama Desert (Azua-Bustos et al. 2012), the Ka’u Desert (Seelos et al.
2010), the Mojave Desert (Salas et al. 2011), Rio Tinto (López-Archilla et al. 2001;
Amils et al. 2014), McMurdo Dry Valley (Chan-Yam et al. 2019), and permafrost
soils (Douglas and Mellon 2019). In Mexico there are two Mars analogues: the salt
flats of Guerrero Negro, Baja California Sur, and the Cuatro Ciénegas Basin,
Coahuila.
The Cuatro Ciénegas Basin (CCB) is an arid, gypsum-rich environment that is
considered an analogue of the Gale Crater and Olympia Undae on Mars (Szynkiewicz
11 Cuatro Ciénegas as an Archaean Astrobiology Park 221
et al. 2010; López-Lozano et al. 2012). The CCB is located at the bottom of a valley
in the northern Mexican state of Coahuila, at an altitude of 740 masl (Souza et al.
2012). The area receives approximately 200 mm of precipitation, and the ponds, or
“pozas,” are fed through extremely oligotrophic groundwater aquifers whose stoi-
chiometry resembles that of the early Earth. More than 200 pozas in the CCB are
considered hyper-diverse because of their heterogeneity and endemism found in the
microbial communities.
The geological description has been addressed previously (Meyer 1973;
Escalante et al. 2008; Souza et al. 2012, 2018a; Hipkin et al. 2013), but the main
picture exposes the CCB as an extreme scenery, due to its inner arid conditions that
allow high levels of oligotrophy, especially for phosphorus—the CCB reportedly
has the lowest levels of this element. Also, the high rate of radiation and the salty
emergence due to mineral and ion precipitation confers the CCB features that are
similar to the Martian landscape (López-Lozano et al. 2012; Souza et al. 2012).
Despite the oligotrophic state of the water in the CCB and the stoichiometric
levels of phosphorous that are below those needed to maintain DNA and protein
synthesis, the CCB has a remarkable level of biodiversity in all domains and taxa
and is one of the few sites in the world with living stromatolites (Elser et al. 2005;
Souza et al. 2006, 2008, 2012, 2018a; Alcaraz et al. 2008).
The research of these living fossil formations and the sediment rearrangements
(microbial mats) can give us a glimpse of how life could have looked on the early
stages this planet and others. Recently, CCB was described as a new evolutionary
model called a “lost world” (Souza et al. 2018b).
Multidisciplinary research teams are working at this place to demonstrate our
hypothesis: that a seed bank of ancestral lineages has been trapped inside the deep
aquifer of a mountain with a magmatic pouch under its sediments; when water from
the aquifer rises to the surface and refills the pozas, it brings with it the deep bio-
sphere from the seed bank. This mountain, named San Marcos y Pinos, has been
giving shelter and isolation to these relictual communities for thousands or millions
of years and allowing speciation and divergence to take place (Souza et al. 2018b).
A particular hydrological system within CCB, the Churince, possesses a large
diversity of microorganisms from the Bacteria and Archaea domains, specifically
5167 operational taxonomic units (OTUs) with a 97% identity from 16s rRNA gene
sequencing (Souza et al. 2018b). These identifications suggest that CCB has a
marine ancestry with certain phyla like Actinobacteria, Bacteroidetes, and
Proteobacteria (Souza et al. 2018b).
These extreme physicochemical and environmental conditions, along with the
ancient microbial communities found at this site, make the CCB a model for the
Earth’s earliest ecosystems—a Precambrian Park—and a perfect analogue for the
gypsum-rich Martian soils (López-Lozano et al. 2012; Souza et al. 2012).
CCB shows that life can not only hide and survive under the sediments but also
transform these extreme environments through their metabolisms. We hypothesize
that the study of the CCB can gleam information about the ancient Martian sea that
left its imprint in the sediments of the central mount of the Gale Crater and help
missions, such as the Mars Science Laboratory, trace the biosignatures left by
changes in chemical stoichiometry through the Martian eras. If life exists or has
existed in the Gale Crater, it is very possible that it left a biosignature imprint in the
gypsum crystals (Edwards et al. 2006). In this case, a firm understanding of an
analogous region such as the CCB would be invaluable to the search for life on Mars
(Newsom et al. 2001; Szynkiewicz et al. 2010; López-Lozano et al. 2012).
11.2 S
tromatolites and Microbial Mats in CCB: Revelations
of the History of Life
Stromatolites are the legacy of early life on Earth, representing a treasure trove for
studies of paleoecology and evolution. These formations give an insight into micro-
bial interactions, their role in the appearance of Eukaryotes, and how changes in the
atmosphere and climate have affected this and other planets with the “ideal” condi-
tions for life (McNamara and Awramik 1992; Breitbart et al. 2009). More informa-
tion about stromatolites can be found in Chap. 4 of this book.
The formation of stromatolites has decreased since the appearance of plants and
metazoans (Awramik 1971), and living stromatolites can be found in a few localities
including Hamelin Pool, Shark Bay, Australia (Reid et al. 2003; Burns et al. 2004;
Papineau et al. 2005), Yellowstone National Park, United States (Berelson et al.
2011), and in the Cuatro Ciénegas Basin (CCB) (García-Oliva et al. 2018; Souza
et al. 2018b). The bacterial communities in the CCB stromatolites rely on the avail-
ability of light, water depth, current flow, and desiccation (Paterson et al. 2008).
A vast number of studies have proved that stromatolites and microbial mats in
the CCB have a “marine signature,” implying that the whole ecosystem may be
considered relics, coming from an ancient marine community (Souza et al. 2006;
Desnues et al. 2008).
In 2008, Desnues et al. (2008) performed a comparative metagenomic analysis
of viral communities from the freshwater stromatolites in Pozas Azules II (PAII)
and Rio Mesquites (RM) within the CCB and the marine stromatolites from
Highborne Cay, Bahamas. They compared their sequences with nonredundant
Genbank and SEED databases and found that the viral communities in the stromato-
lites were unrelated, with 98.8% unique sequences for Highborne Cay, 99.3% for
PAII, and 97.7% for Rio Mesquites. Assembly of contigs shows that freshwater
stromatolites (PAII and RM) did not produce any cross-contigs with marine micro-
bialites (Highborne Cay), suggesting that none of the viruses was shared between
them (Desnues et al. 2008).
One year later, Breitbart et al. (2009) conducted metagenomics and isotopic
analyses of the stromatolites in PAII and RM. They found that both samples had
Bacteria dominance with an 87% in PAII and 95% in RM samples. Interestingly, a
comparison against SEED database distinguished 1061 (PAII) and 203 (RM)
archaeal sequences, and more than 86% belonged to Euryarchaeota phylum. Their
findings also showed that although the stromatolites at these sites are different in
function and taxa, the genes involved in ammonia assimilation, nitrogen fixation,
assimilatory nitrate reduction, and phosphorus metabolism genes were present in
both metagenomes, demonstrating a great adaptation to the CCB thanks to poly-
phosphate accumulations that serve a reservoir. It has been proposed that stromato-
lites are present in the CCB because of its stoichiometric nutrient constraints (Elser
et al. 2005), which cause the coordinated metabolic activity from the whole micro-
bial community to create microenvironments that favor carbonate precipitation
(Breitbart et al. 2009).
Besides stromatolites, CCB also harbors microbial mats, another extraordinary
well-organized biological structure. A microbial mat is a vertically stratified micro-
bial community, defined by physicochemical gradients that support most biogeo-
chemical cycles in aquatic environments, making these structures self-sufficient
(Bolhuis et al. 2004). In 2012, Peimbert and collaborators (2012) did a metage-
nomic comparison of two aquatic microbial mats (red and green mats). They found
that the red mat lacked phosphorus and was dominated with Pseudomonas, whereas
the green mat was nitrogen-limited with Cyanothece as the most abundant genus,
making these mats metabolically different and diverse, although heterotrophic taxa
was shared between the two mats.
For the reasons described above, the stromatolites and microbial mats in the
CCB are perfect astrobiology analogues of the third order. These successful and
efficient microbial communities are capable of cycling the few nutrients available to
them, just like their counterparts did during the Archean Eon.
11.3 T
he Archaean Domes in Cuatro Ciénegas Basin: A Lost
World Enclosing a Secret of the Tough Archaean Life
The exploration of the CCB in March 2016 led by Dr. Valeria Souza identified
“Archaean Domes” (AD) located near the Poza Azul II, within the Pozas Azules
Ranch. Archaean Domes are a very particular site that under dry conditions, which
span most of the year, looks just like another salty place. However, after a heavy
rain, the water dissolves the salty crust allowing not only photosynthesis to be less
constricted but, more importantly, establishing a communication between the deep
biosphere and the surface. Under wet conditions, a complex system of dome-like
structures forms in this unique, shallow, clay-rich pond. Inside, these domes are
dominated by methane-rich gases that simulate the conditions of the late Archean
Eon (Medina-Chávez et al. 2019), whereas the outer shell presents all the photosyn-
thetic lineages as well as several fungi and protozoa (Espinosa-Asuar, to be submit-
ted). During the dry season in the CCB, the AD area dries up, leaving a crystalline
salty crust on the top layer of the mat (Medina-Chávez et al. 2019).
Microbial activity can be limited by desiccation stress and low substrate diffu-
sion (Schimel et al. 2007; Aanderud et al. 2015), causing dormancy events; micro-
bial metabolism is reactivated once the soils are soaked again (Fierer and Schimel
2003; Aanderud et al. 2015). These dormancy processes driven by harsh conditions
could be key players in these biosphere fluctuations, leading to microbial commu-
nity maintenance. Similar conditions of stress may be happening in other planets,
and dormancy periods like the ones described could make detection of life more
difficult.
Results revealed that AD mats comprise an abundant, hyper-diverse, and cohe-
sive archaeal core community that maintains its abundance and diversity through
time and despite environmental fluctuations. Members of five phyla (Euryarchaeota,
Crenarchaeota, and the unexpected Thaumarchaeota, Korarchaeota, and
Nanoarchaeota) displayed 230 species (OTU’s at 97% cutoff) corresponding to a
5% of archaeal relative abundance from a whole-genome SGS analysis (Medina-
Chávez et al. 2019), which is an uncommon value even for other hypersaline micro-
bial mats (Fernandez et al. 2016; Babilonia et al. 2018). It is worth noting that a
study of genetic diversity of archaea among stromatolites in several regions in
Mexico, including the CCB, showed a low abundance (up to 1.76% of the commu-
nity) of Archaea (Centeno et al. 2012). Similar results were obtained from the analy-
sis of two aquatic microbial mats from the CCB ponds in 2012, where Archaea
abundance reached only 2% of the community in a green mat (Peimbert et al. 2012).
Taking into account that the Archaean Domes have not been extensively explored
and sampled as other archaeal-rich sites (Wong et al. 2016), the AD in CCB undoubt-
edly represent a model to study how archaeal communities thrive in conditions simi-
lar to the expected to be found on exoplanets.
The AD site in CCB is extremely saline, reaching up to 53% during the wet sea-
son and staying under saturation the rest of the year. The site is surrounded by a
carbonate layer and poor vegetation. Because the microbial mats can be found only
beneath the salt and not the carbonate, it is likely that salinity is an important abiotic
factor for the community below. In the AD, fluctuations in the relative abundances
of rare taxa through time seem to be related to precipitation; the behavior of the
archaeal community is consistent with the findings of a study on community net-
works in a (non-hypersaline) microbial mat within the Churince Pond in the CCB,
which demonstrates that halotolerant taxa increases during dry conditions (De Anda
et al. 2018). Chloride and sulfates are characteristic minerals on Mars, suggesting
that brines and other hypersaline environments may have been a part of Martian
history (Martínez and Renno 2013; Smith et al. 2014). If this were the case, then
halophilic microorganisms could be part of the “rare taxa” (Lynch and Neufeld
2015) in the biota of another planet.
NASA’s Curiosity rover found different levels and concentrations of methane
throughout its mission. In June 2019, it detected the highest levels of methane
(Greicius 2019), a relevant finding considering that there are only two possible
sources for the production of this gas: the interaction given by rocks and water (in
this case, frozen water) and microbial communities (Sherwood Lollar et al. 2007;
Etiope and Sherwood Lollar 2013; Dean et al. 2018). If the methane found on Mars
is biogenically produced, it is likely to be the product of organisms similar to
Euryarchaeota, which we found in high abundance throughout the seasons in the
hypersaline, desiccated AD.
Polyextremophilia is another phenomenon expected and studied for Mars ana-

logues. Halophilia and alkalophilia are often found together, for example,
Yellowstone and Rio Tinto are both hypersaline and alkaline (White et al. 2015). If
there are microbes on Mars, then we can hypothesize that Archaea or other Bacteria
of an extreme nature would be the most likely candidates. These domains contain
methanogenic bacteria and polyextremophiles that are adapted to survive not one
but several extreme conditions at once (Mesbah and Wiegel 2008; Seckbach 2013).
Salinity is not the only abiotic factor leading the presence of extremophilic micro-
organisms—temperature, pressure, oxygen availability, and pH also play a major
role on the composition and structure of microbial communities. The AD of CCB,
besides its extreme salinity, reaches pH values of 10 during wet seasons (data not
shown), whereas the pH at the Poza Azul II at a distance of 50 m from the AD
reaches 7.9 (Centeno et al. 2012; Espinosa-Asuar et al. 2015).
11.4 Conclusions
Ever since its discovery, the Cuatro Ciénegas Basin has not ceased to amaze us. The
site harbors a richness of endemic macro and microbiota, and its cohesive microbial
community thrives in extreme environments, building niches that seem relict from
an ancient sea and adapting to the feedback and biological input of the Anthropocene.
The oligotrophic, gypsum-rich, and poly-extreme environments in CCB have
recently been described as an invaluable ecological and functional model for the
study of adaptations and evolution in harsh, fluctuating environments. This puts the
domain Archaea, for the first time, as a core component of the CCB microbiome,
with a fundamental role in the maintenance of the hyper-diversity of this particular
site. The study of the ecology and the evolution of the Archaean Domes in the CCB
can take us deeper into a “lost world” that will continue to help us understand the
cycles and biosignatures that life, past or present, may leave on Martian or other
habitable extraterrestrial soils.
Acknowledgments We want to thank Dr. Laura Espinosa-Asuar and Erika Aguirre Planter for
their technical help. To Pronatura Noreste for their help in logistics and getting access to their
ranch and APFF Cuatrociénegas for their support .This research was funded by PAPIIT- DGAPA,
UNAM grant IG200319 to VS and Luis E. Eguiarte.
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Souza V, Eguiarte LE, Elser JJ, Travisano M, Olmedo-Álvarez G (2018a) A microbial Saga: how
to study an unexpected hot spot of microbial biodiversity from scratch? In: Souza V, Olmedo-
Álvarez G, Eguiarte LE (eds) Cuatro Ciénegas ecology, natural history and microbiology.
Springer, Cham, pp 1–20
Souza V, Moreno-Letelier A, Travisano M, Alcaraz LD, Olmedo G, Eguiarte LE (2018b) The lost
world of Cuatro Ciénegas Basin, a relictual bacterial niche in a desert oasis. eLife 7:e38278
Szynkiewicz A, Ewing RC, Moore CH, Glamoclija M, Bustos D, Pratt LM (2010) Origin of
terrestrial gypsum dunes-implications for Martian gypsum-rich dunes of Olympia Undae.
Geomorphology 121:69–83
White RA III, Power IM, Dipple GM, Southam G, Suttle CA (2015) Metagenomic analysis reveals
that modern microbialites and polar microbial mats have similar taxonomic and functional
potential. Front Microbiol 6:966
Wong H, Ahmed-Cox A, Burns B (2016) Molecular ecology of hypersaline microbial mats: cur-
rent insights and new directions. Microorganisms 4(1):6
Index
A astrological research, 35
Abiotic model, 112 habitable and signature of life, 64–67
Actinobacteria, 118 origin of life, 32, 33
Aerosol collector and pyrolizer (ACP), 61 planets, stars, 62–64
Alphaproteobacteria, 114 plasmogenesis, 32
Andean microbial ecosystems (AME) rock
arsenic geochemical cycle, 177 micrometeoroid, 37
early earth, 168 research, 45
HAAL (see High-altitude Andean solar system, 42, 43
lakes (HAAL)) society, 34
hypersaline environments, 168 Astrobiology and Planetaryicy
life’s origin, 169–170 satellites, 53–54
Archaea, 224, 225
Archaeobacteria, 76
Astrobiology B
biosignature (see Biosignature) Bacterial communities, 155, 158, 160, 162
CHONPS, 3 Biosignatures, 21, 23, 24, 26, 90–92
definition, 2
earth’s history, 2
extraterrestrial life, 2 C
historical-collective phenomenon, 2–3 Calcium and aluminum inclusions (CAIs), 42
minimal living systems, 2 Calvin cycle, 173
solar system (see Solar System) Calvin–Benson–Bassham cycle, 161
Astrobiology and Planetary Enceladus, Cassini Plasma Spectrometer (CPS), 61
53–54, 56, 57 Centro de Investigaciones Químicas (CIQ), 59
Astrobiology and Planetary Europa, 52, 54, 56 Chloride-carbonate (Cl-CO3), 57
Astrobiology and Planetary habitability, 56 Cuatro Ciénegas, 122
Astrobiology and Planetary Mars Cuatro Ciénegas Basin (CCB), 35, 142
curiosity, 51 Archaean Domes (AD), 223–225
habitability, 51 astrobiology, 220–222
search for organics, 49–51 extremophiles, 220, 225
Astrobiology and Planetary sciences Mars analogues, 220, 225
astrobiological research, 38–41 stromatolites and microbial mats, 222–223

https://doi.org/10.1007/978-3-030-46087-7
230 Index
D Horizontal gene transfer (HGT), 76–78

Darwinian evolution, 3 Hydrogen cyanide (HCN), 60
Deep hydrothermal systems Hydrothermal vent systems, 151
alpha prokaryotic diversity
patterns, 154–157
beta prokaryotic diversity patterns, 157–158 I
community structure, 152, 162 Instituto de Ciencias Nucleares (ICN), 54, 60
deep-sea communities, 150
DNA isolation, 153
Gulf of California, 152 J
microbial ecological roles, 158–161 Jupiter Galilean system, 52
origin of life, 151, 161, 162 Jupiter system, 15
primeval environments, 161–162
prokaryotic diversity (see Prokaryotic
diversity) L
sediment samples, 153 Laboratoire Interuniversitaire des Systèmes
16S rRNA amplicon data, 154 Atmosphériques (LISA), 59
16S rRNA gene, 153 Laser confocal microscopy, 186
vent communities, 150 Laser-induced plasma (LIP), 60
Drake Equation, 26 Last archaeal common ancestor (LACA), 79
Last bacterial common ancestor (LBCA), 79
Last universal common ancestor (LUCA), 139,
E 144, 175
East African Rift Valley, 168 ancestral potein reconstruction, 81–83
Eukaryotes, 119 Earth-Moon system, 83
Exogenous and endogenous energy sources, 58 molecular sequences, 75–78
Exoplanets, 34 nature and existence, 78–79
Exopolymeric substance (EPS), 93, 168, 176 reverse gyrase, 79–81
Extrasolar planets/exoplanets, 18 solar system, 83
Extremophiles, 5–7 temperature, 84
Light microscopy, 186
Lost City hydrothermal field (LCHF), 151
F
Firmicutes, 118
M
Martian meteorites, 13
G Mat-building filamentous cyanobacteria, 116
Galileo Europa Mission (GEM), 52 Metabolic signatures, 158–161
Gas chromatograph and mass spectrometer Metallic meteorites, 43
(GC-MS), 61 Meta-omic approaches, 98
Meteorites
achondrites, 43
H carbonaceous, 42
Habitable zone, 19 chondrites, 42
High-altitude Andean lakes (HAAL), 168 chondrules, 42
AMEs, 172–173 classification, 42
early metabolisms iron meteorite Yanhuitlán, 43, 44
Andean Valleys, 173 metallic, 43
arsenic metabolism, 174 multidiscilinary group, 45, 46
Laguna Diamante, 174, 175 Methanogenesis, 21
Laguna La Brava, 175, 176 Microbial diversity
Laguna Socompa, 176 Alchichica crater-lake
water molecules, 174 stromatolites, 189–191
physicochemical conditions, 170–172 Bacalar lagoon stromatolites, 191–193
Highborne Cay microbialites, 121, 122 stromatolites, 189
Index 231
Microbialite Planetary collisions

abiotic (spectror hypothesis), 112 Citlalin Tlamina, 49
Actinobacteria, 118, 119 crusts, 46
Alphaproteobacteria, 114 design and construction, 49
biotic driven hypothesis, 112, 113 surfaces, 46
classification, 107 Planktonic Cyanobacteria, 114
Cyanobacteria, 114 Polycyclic aromatic hydrocarbons (PAH), 13
definition, 107 Polyextremophiles, 168
environments, 111 Pozas Azules II (PAII), 222
Eukaryotes, 119 Prokaryotic diversity, 152, 162
Firmicutes, 118
forming ecosystem
Clinton Creek, 126, 127 R
Cuatro Ciénegas, 122 Rare biosphere
Highborne Cay, 121, 122 after LUCA, 144
Kelly Lake, 125, 126 astrobiology, 142–144
Pavilion Lake, 123, 125 common vs. rare, 139–140
Shark Bay, 121 ecological niches warrant
forming ecosystems, 110, 111 investigation, 143
fossils, 111 extremophiles, 140–142
global distribution, 110, 111 life on Earth, 140, 143, 144
hydromagnesite forms, 111 16S rRNA gene, 143
mat-building filamentous cyanobacteria, shotgun metagenomics, 143
115, 116 Remotely operated vehicle (ROV), 153
Oceania, 111 Rio Mesquites (RM), 222
perssistent ecosystems, 109
SOBs, 116
SRBs, 117 S
stromatolite, 108 Scanning electron microscopy (SEM), 186
viruses, 120 Scientific Event Alert Network (SEAN), 44
Molecular biology, 186 Search for Extraterrestrial Intelligence
Molecular clouds, 4 (SETI), 26
Shark Bay microbialites, 121
Small subunit rRNA (SSU rRNA)
N molecule, 76
Nanodiamonds, 5 Sociedad Mexicana de Astrobiología
Next Generation Sequencing techniques (SOMA), 34
(NGS), 186 Solar System, 2, 5–12, 15
Nucleoside diphosphate kinase (NDK), 81 Callisto, 15
Enceladus, 16
Europa, 15, 16
O habitable planets, 18, 19, 26
Origin and early evolution of life icy satellites, 15
Mexico, 203–204 intelligent life, 25, 26
Origin of life life in exoplanets, 22–25
last common ancestor (LCA), 206–209 Mars, 12–14
prebiotic chemistry and RNA, Proxima Centauri, 20
204–206 signatures of life, 21, 22
and viruses, 210–213 Titan, 16–18
Venus, 9, 11
Stratospheric Observatory for Infrared
P Astronomy (SOFIA), 52
Pescadero Basin (PB) system, 152 Stromatolites, 108
Pescadero Transform Fault (PTF), 152 astrobiology, 90
Photosynthesis, 23 Bacalar lagoon, 184–186
232 Index
Stromatolites (cont.) T
biosignatures Theory of Life, 3
mineral, 95–97 Thrombolite, 109
morphology and fabrics, 91–95 Titan
organic molecules, 97–99 Cassini-Huygens, 61
constraints of habitability, 100 Exogenous and endogenous energy
crater-lake Alchichica, 184 sources, 58
Mexico, 184, 185 HCN, 60
microstructure, 192 LIP, 60
molecular approaches, 186–188 LISA, 59
N-cycle, 192–196 Transmission electron microscopy
P-cycling, 192, 196–197 (TEM), 186
Sulfate-reducing bacteria (SRBs), 117
Sulfur/sulfate reducing bacteria (SRB), 160
Sulfur-oxidizing bacteria (SOBs), 116 U
Synechococcus sp, 114 Unidentified flying objects (UFOs), 1
Systems biology approaches, 98 Universal biology, 3

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Cuatro Ciénegas Basin: An Endangered Hyperdiverse Oasis

More information about this series at http://www.springer.com/series/15841

Astrobiology and Cuatro

ISSN 2523-7284 ISSN 2523-7292 (electronic)

© Springer Nature Switzerland AG 2020

Mexico City, Mexico Valeria Souza

1 What Is Astrobiology? ���������������������������������������������������������������������������� 1

7 Bacterial Communities from Deep Hydrothermal Systems:

Abstract Astrobiology is an inherently multidisciplinary field that is focused on

1.1 Aliens Everywhere

© Springer Nature Switzerland AG 2020 1

1.2 What Astrobiologists Are Looking for?

phenomenon”; we should thus conceptualize life on Earth as a single individual, a

1.3 The Limits of Life

Table 1.1 Classification of extremophiles as defined by their environment

­ ultiple stresses, and they are recognized as polyextremophiles; common examples

1.4 Extreme Sites on Earth

1.5 Where Are Astrobiologists Looking for Life?

1.6.1 Venus, Earth’s Hot Twin

(1978)b, Vega exploration Galileo

1.6.2 Mars, the Martians, and the Emergence of Astrobiology

1.6.2.1 Martians on the Rocks

1.6.2.2 Discovering Mars

1.6.3 Life in the Cold: The Icy Satellites

1.6.3.1 Europa, Ganymede, Callisto, and Enceladus

organics provided by cometary impacts has been proposed as another source of

chemistry developing on its substantial atmosphere. Cassini-Huygens, one of the

1.7 Habitable Planets Around Other Stars

which is limited by pressure and temperature conditions, both provided by the

1.8 Signatures of Life

microbial mats, as a probable consequence of methanogens tolerating different

1.8.1 Fingerprints of Life in Exoplanets

1.9 What About Intelligent Life?

1.10 Our Habitable Planet

Hays L et al. (2015) NASA astrobiology strategy

Seager S et al (2012) An astrophysical view of earth-based metabolic biosignature gases.

Abstract A small community of scientists in Mexico has been contributing to the

All authors contributed equally to the text, listed in alphabetical order.

© Springer Nature Switzerland AG 2020 31

2.1 Astrobiology and Planetary Sciences in Mexico

The exploration of other worlds is a young endeavor, telescopes and meteorites

2.1.1 Plasmogenesis: A Hypothesis for the Origins of Life

life is a physicochemical phenomenon that can happen on other planets. Today

2.1.2 Origins in the Lab

2.1.3 The Mexican Society of Astrobiology

2.2 The Rocks That Fell from the Sky

Earth. In the following section, we present the general classification of meteorites

2.2.1 The History of the Solar System Written in Rocks

2.2.3 Understanding Alien Rocks: Research in México

On October 4, 1990, a group of researchers of the UNAM participated in the

2.2.3.1 A Multidisciplinary Group to Study Meteorites

on how chondrules were formed. Our approach is to create chondrules in the

2.3 Planetary Collisions

2.3.1 Impact Studies from Mexico

At UNAM, we have studied several of the previous phenomena, in particular, impact

2.4 Mars: The History of the Search for Organics

an atmosphere composed by carbon dioxide and nitrogen. On Earth, life emerged

2.4.1 Mars on Earth: Clues on the Search for Organic Matter

2.4.2 Curiosity Is What Is Needed

Mexico City, Mexico Valeria Souza

1 What Is Astrobiology? �� 1

7 Bacterial Communities from Deep Hydrothermal Systems:

1.1 Aliens Everywhere

1.2 What Astrobiologists Are Looking for?

1.3 The Limits of Life

ultiple stresses, and they are recognized as polyextremophiles; common examples

1.4 Extreme Sites on Earth

1.5 Where Are Astrobiologists Looking for Life?

1.6.1 Venus, Earth’s Hot Twin

1.6.2 Mars, the Martians, and the Emergence of Astrobiology

1.6.2.1 Martians on the Rocks

1.6.2.2 Discovering Mars

1.6.3 Life in the Cold: The Icy Satellites

1.6.3.1 Europa, Ganymede, Callisto, and Enceladus

1.7 Habitable Planets Around Other Stars

1.8 Signatures of Life

1.8.1 Fingerprints of Life in Exoplanets

1.9 What About Intelligent Life?

1.10 Our Habitable Planet

2.1 Astrobiology and Planetary Sciences in Mexico

2.1.1 Plasmogenesis: A Hypothesis for the Origins of Life

2.1.2 Origins in the Lab

2.1.3 The Mexican Society of Astrobiology

2.2 The Rocks That Fell from the Sky

2.2.1 The History of the Solar System Written in Rocks

2.2.3 Understanding Alien Rocks: Research in México

2.2.3.1 A Multidisciplinary Group to Study Meteorites

2.3 Planetary Collisions

2.3.1 Impact Studies from Mexico

2.4 Mars: The History of the Search for Organics

2.4.1 Mars on Earth: Clues on the Search for Organic Matter

2.4.2 Curiosity Is What Is Needed

2.5 Icy Worlds: Europa and Enceladus

2.6.1 The Voyager and Ground-Based Observations Era

2.6.2 The Cassini-Huygens Era

2.6.3 The Future for Titan Exploration

2.7 Planets Around Other Stars

2.8 Habitable Worlds and Signatures of Life

3.1 Early Life as Revealed by Molecular Sequences

3.2 The Nature of LUCA and Its Conditions of Existence

3.5 When Did LUCA Live?

3.6 Conclusions and Perspectives

4.2 Long Live the Stromatolites and Their Biosignatures

4.4 Stromatolite Products as Biosignatures: Minerals

5.1 Physical Classification and Brief History

5.2 Microbialites as the Oldest Persistent Ecosystems

5.2.1 Global Distribution of Microbialites

5.2.2 Proposed Microbialite Formation Hypotheses

5.2.2.1 Abiotic (Spectator Hypothesis)

5.2.2.2 Biotic-Driven Hypothesis

5.2.3 Major Members of the Microbialite Microbiome

Alphaproteobacteria are abundant in marine, freshwater, and artificial marine-

5.2.3.2 Planktonic Cyanobacteria

5.2.3.3 Filamentous Cyanobacteria, Mat Builders

5.2.3.4 Sulfur-Oxidizing Bacteria (SOBs)

5.2.3.5 Sulfate-Reducing Bacteria (SRBs)

Representatives of the phylum Actinobacteria appear to be rare within microbialite-

5.2.4 Previously Studied Microbialite-Forming Ecosystems

5.2.4.1 Shark Bay Microbialites

5.2.4.2 Highborne Cay Microbialites