Full Text

Title
Molecular microbial ecology of Mars-like environments on earth,

for application in astrobiology
Advisor(s)
Pointing, SB; Aitchison, JC
Author(s)
Chan, Wai, Olivia.; .
Citation
Issued Date
URL
Rights
2012
http://hdl.handle.net/10722/173841
The author retains all proprietary rights, (such as patent rights)

and the right to use in future works.
Abstract of thesis entitled
MOLECULAR MICROBIAL ECOLOGY OF

EXTREME ENVIRONMENTS ON EARTH, FOR
APPLICATION IN ASTROBIOLOGY
Submitted by
Wai Olivia Chan

for the degree of Doctor of Philosophy
at The University of Hong Kong in February 2012
Astrobiology is a multidisciplinary topic that addresses the origin,

distribution and evolution of life in the universe. One of the key questions relates
to whether life could have evolved on other planetary bodies, and Mars has been
the major focus. Biologists contribute to this question by studying the ecology of
extreme environments on Earth that share closest analogy to Mars past or
present environment. In this thesis, molecular-level interrogations were used to
address some aspects of microbial biodiversity, ecology and stress tolerance in
two such extreme environments. The high-altitude cold and intense UV
irradiance of central Tibet was selected as an analogue for Mars surface today,
whilst cold alkaline high-carbonate freshwater lakes were chosen as an analogue

for Mars previous late wet phase.
Biological soil crusts from central Tibet supported a diverse
microflora and these were variously bacteria or eukarya dominated. The
relatively well-developed eukarya-dominated crusts were characterized and
showed they comprised of Stichococcus bacillaris, plus alphaproteobacteria,
betaproteobacteria, bacteroidetes and gemmatimonadetes. In order to evaluate
the diversity of radiation-tolerant taxa in these soils, samples were exposed to
ionizing radiation and viability, physiology and phylogenetic identity determined.
The most radio-tolerant taxa isolated and characterized were from the radiation
tolerant phylum Deinococci (15kGy), whilst a relatively diverse range of
Actinobacteria, Bacilli, Cyanobacteria and Proteobacteria were also recovered
after exposure to doses up to 10kGy.
This implies the high-radiation
environment has selected for tolerance among diverse phyla, with tolerances that
far exceed environmental exposure. It is not known at this stage if they all
employ similar protective strategies.
Microbial reefs that have developed in cold alkaline lakes in British
Columbia were studied as analogues for a late-wet Mars environment.
Molecular ecological analysis revealed that communities consisted largely of of
Proteobacteria (alpha), Cyanobacteria (Leptolyngbya) and Acidobacteria, with
similarities in community assembly to marine stromatolites. Microbial diversity
varied spatially and temporally within microbialites, and indicated that
geographically proximal structures can develop with different communities.
Significant changes also occur between summer and winter when the lake
surface is frozen. Investigation of other nearby lakes with similar geochemistry
but not supporting microbialites revealed extensive microbial mats. These

developed in the presence of relatively high concentrations of methane or sulfate,
and their biodiversity reflected this with several putative methanotrophic and
sulphate utilizing taxa identified.
No obvious cues that inhibit or promote
microbialite formation were observed in this study.
MOLECULAR MICROBIAL ECOLOGY

OF MARS-LIKE ENVIRONMENTS ON
EARTH, FOR APPLICATION IN
ASTROBIOLOGY
by
Wai Olivia Chan

B.Sc. (Dist.) Iowa State University
M.Sc. Iowa State University
A thesis submitted in partial fulfillment of the requirements for the

Degree of Doctor of Philosophy at The University of Hong Kong
September 2012
Declaration
I declare that this thesis represents my own work, except

where due acknowledgement is made, and that it has no been
previously included in a thesis, dissertation or report submitted
to this University or any other institution for a degree, diploma
or other qualification.
Signed.
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Acknowledgements
My deepest gratitude goes to my supervisor Dr. Stephen
Pointing. Thank you for your support throughout the research process.
Your guidance, inspiration and encouragement are deeply appreciated.
Special acknowledgment to Dr. Christopher McKay and Dr.
Darlene Lim from the NASA Ames Research Center. Thank you for
introducing me to the Pavilion Lake Research Project group and making
my stay in NASA Ames a great learning experience. Thank you for
believing in me and giving me the opportunity in furthering my research in
Ames. Special mention to Dr. Sharmila Battacharya for sharing her
laboratory, and Dr. Oana Marcu, Max Sanchez and Matt Lera for their
kind assistance in the laboratory.
I would like to express my sincere thanks to everyone in the
Pavilion Lake Research Project group. It was my great pleasure working
with the top research group in North America. Special thanks to Donnie
Reid and Mike Delaney for their assistance during the lake diving
operation. Thank you for keeping me alive under water.
My sincere thanks to Dr. Esther Ruiz, Dr. Alberto GonzalesFairen, Dr. Alfonso F. Davila, Jhony Zavaleta, Dr. Giuseppe Marzo and
Dr. Azzurra De Luca. Working in Ames was one of the most memorable
and enjoyable moments in my life. Thank you for treating me like family. I
appreciate all the love and the gift of friendship.
I would like to thank Professor Jonathan Aitchison from the
Earth Science Department from the University of Hong Kong Tibet
Research Group for organizing an unforgettable field trip to Tibet.
"#!
Many thanks to Dr Maggie Lau, Dr Donnabella Lacap, Kelly

Lau, Fiona Wong, Yuki Chan, Anthony Ha, Anthony Woo, Miko Ng,
Charmaine Yung and Subramanya from the Extremophiles Research
Group. Thank you for your assistance in the laboratory and most
importantly your constant encouragement and support. Life would be
miserable without you all.
I would like to thank my mother, my husband and my brother. I
would not be able to complete this without your love. Thank you for your
patience and understanding through the daunting moments. To my
beloved cat Ah Mol who seems to understand all my moods and takes
special interest in my research papers, thank you.
Lastly, I would like to express my appreciation to all my friends
in Hong Kong, US and Canada for their kindness.
Namaste.
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TABLE OF CONTENTS
DECLARATION
ACKNOWLEDGMENTS
vi
TABLE OF CONTENTS
viii
LIST OF FIGURES
xv
LIST OF TABLES
CHAPTER 1
xxiv
ASTROBIOLOGY
Introduction of astrobiology
What is astrobiology
The beginning of astrobiology
Possible life in space

Studies of Mars-like environments
4
10
of Earth
CHAPTER 2
Mars-like terrestrial environments
12
Mars-like aquatic environments
16
Molecular approaches in astrobiology
18
Project Scope
22
MOLECULAR DIVERSITY AND
24
ECOLOGICAL SIGNIFICANCE OF
SOIL MICROBIAL COMMUNITIES IN
THE HIGH ALTITUDE DESERTS OF
TIBET
"###!
Introduction
Biological soil crusts (BSCs) in
24
24
ecosystems
Biological Soil Crusts in hyperarid
24
deserts
Hyperarid deserts in Tibet, China
26
Objectives
27
Materials and Methods
28
Site description
28
Sample collection
28
DNA extraction and polymerase
29
chain reaction (PCR) amplification

Real-time quantitative PCR (q-PCR)
30
analysis
Clone library construction
30
Statistical analysis
32
Results
34
Samples description
34
Real-time quantitative PCR analysis
35
Bacterial and archaeal diversity in
38
high altitude deserts
Bacterial community diversity
42
Eukaryal community diversity
42
Discussion
49
Conclusion
56
"#!
CHAPTER 3
ISOLATION OF IONIZING
57
RADIATION TOLERANT BACTERIA

FROM HIGH ALTITUDE DESERTS
OF TIBET PLATEAU, CHINA
Introduction
Objectives
57
63
64
Site description
64
Sample collection
64
Total organic content and radiation
65
test
CHAPTER 4
Culture and Isolation
65
Analytical Profile Index (API)
66
Cloning
66
Sequence assembly and alignment
66
Phylogenetic analysis
67
Results
68
Discussion
82
Conclusion
86
MOLECULAR ECOLOGY OF
87
FRESHWATER MICROBIALITE
STRUTURES IN A CARBONATERICH LAKE IN BRITISH COLUMBIA,
"!
CANADA
Introduction
87
Microbialites as a living fossil
87
Microbialites in Pavilion Lake
89
Objectives
89
90
Site location
90
Sample collection
92
Microscopy
94
DNA extraction and polymerase
94

95
analysis
Terminal restriction fragment length
95
polymorphism (t-RFLP)
Clone library restriction
96
96
96
Results
97
Sites and samples morphology
97
description
Real-time quantitative PCR analysis
102
Terminal restriction fragment length
105
polymorphism
Bacterial diversity of microbialites at
"#!
107
Pavilion Lake
Willow Point vs. Three Poles
113
Summer season vs. budding
132
season
CHAPTER 5
Discussion
148
Conclusion
158
MICROBIAL DIVERSITY OF
160
CARBONATE-RICH, MICROBIAL
MATS IN SALINE, ALKALINE LAKES
OF THE CARIBOO PLATEAU,
BRITISH COLUMBIA
Introduction
160
Microbial mats in ecosystems
160
Hypersaline lakes as extreme
163
environment
Hypersaline lakes on Cariboo
165
Plateau
Objectives
168
Site Description
168
Sampling Collection
168
Water Chemistry
169
DNA extraction and Polymerase
169
166
"##!
170
analysis
Clone library construction
170
170
170
Results
171
Water Chemistry
171
171
analysis
Bacterial and archaeal diversity in
174
hypersaline lakes
Bacterial communities of
180
Goodenough and Deer Lakes

Archaeal communities of
191
Goodenough and Deer Lakes

Discussion
196
Conclusion
203
APPENDIX
CHAPTER 6
204
SYNTHESIS
207
Thesis Summary
207
Synthesis of the findings
212
High radiation arid environments
212
Carbonate-rich lakes
212
Implications for astrobiology
214
"###!
Limitations of the study and future
216
works
REFERENCES CITED
218
"#$!
LIST OF FIGURES
Figure 1.1
Image of the planet Mars
Figure 1.2a
Image Hematite pebbles on Mars surface taken by 9

Mars Rover Opportunity
Figure 1.2b
Image of disappearing ice on Mars subsurface
Figure 1.3
Mars surface with river bed feature
10
Figure 1.4
Schematic drawing of the molecular methods used 21

in this study
Figure 2.1
Sandstorm in the Gansu Province of China in 2006
26
Figure 2.2
The satellite image of 2 sampling sites
28
Figure 2.3
Soil sampling at Site 5520m
29
Figure 2.4
Photo of sample collection at N1 (5240m)
35
Figure 2.5
Samples collected from H (4661m) site
35
Figure 2.6
The absolute abundance of archaeal, bacterial and
37
eukaryal communities by real-time quantitative

PCR (q-PCR) analysis.
Figure 2.7
The relative abundance of archaeal, bacterial and
38
eukaryal communities by real-time quantitative

PCR (q-PCR) analysis
Figure 2.8
Plots of rarefaction curves for bacterial clone
40
library of site N1
Figure 2.9
Plots estimated OTUs richness for bacterial clone
40
library of site N1
Figure 2.10
Plots of rarefaction curves for eukaryal clone

library of site N1
"#!
41
Figure 2.11
Plots estimated OTUs richness for eukaryal clone
41
library of site N1
Figure 2.12
The relative abundance of microbial structure of at
43
5240m (Site N1)

Figure 2.13
Relative
abundance
of
bacterial
phylotypes 48
obtained from N1 clone library

Figure 2.14
Relative
abundance
of
eukaryal
phylotypes 48
obtained from N1 clone library

Figure 3.1
Map of sampling sites
65
Figure 3.2
Averaged total organic content of samples
68
collected from different altitudes

Figure 3.3
Survival of bacteria in soil after exposed to various 69

level of gamma radiations
Figure 3.4
Retrieved Deinococcus from 4810m soils exposed 70

to 15kGy gamma radiation
Figure 3.5
Phylogenetic tree of the survived samples from
74
Deinococcus-Thermus group based upon

Maximum Likelihood analysis of full 16S rRNA
gene sequence data
Figure 3.6
Phylogenetic tree of the survived samples from 75

Proteobacteria
Likelihood
group
analysis
of
based
full
upon
16S
Maximum
rRNA
gene
sequence data
Figure 3.7

Cyanobacteria
group
"#$!
based
upon
Maximum
Likelihood
analysis
of
full
16S
rRNA
gene
sequence data
Figure 3.8

Firmicutes group based upon Maximum Likelihood
analysis of full 16S rRNA gene sequence data
Figure 3.9

Actinobacteria
Likelihood
group
analysis
based
of
full
upon
16S
Maximum
rRNA
gene
sequence data
Figure 4.1a
Cross-sectional view of an ancient stromatolite in
88
Medicine Bow National Forest, Laramie, WY

Figure 4.1b
Modern stromatolites in Shark Bay, Western
88
Australia and example of surface stromatolite

sample
Figure 4.2
Pavilion Lake and the map showing the location of
90
Pavilion Lake in British Columbia, Canada

Figure 4.3a
Picture of Pavilion Lake surrounded by Marble
91
Canyon
Figure 4.3b
Diving operation in Pavilion Lake
91
Figure 4.4
A schematic cross-section of Pavilion Lake from
94
Willow Point to Three Poles

Figure 4.5a
Pavilion Lake shallow-intermediate facies
99
Figure 4.5b
Pavilion Lake intermediate facies
99
Figure 4.5c
Cross-section of intermediate turret structure from
99
Willow Point
"#$$!
Figure 4.5d
Deep facies from 33m depth
99
Figure 4.6a
Close up picture of shallow to intermediate facies
100
Figure 4.6b
Close up picture of microbialite
100
Figure 4.6c
Macroscopic picture of Green pigmented cell grew
100
up to 1.5cm in diameter
Figure 4.6d
Macroscopic picture of purple-pigmented cell grew
100
up to 1.2cm in diameter
Figure 4.7a
Light microscopy of oscillatorian cyanobacterial
101
morphotypes collected from microbialites

Figure 4.7b
Light microscopy of coccoid cyanobacterial
101
morphotypes collected from microbialites

Figure 4.8a-b
The absolute abundance and relative abundance
104
of archaeal, bacteria and eukaryal communities in

microbialites by real-time quantitative PCR
analysis
Figure 4.9a-b
Nonmetric multidimensional scaling plot of Bray
106
Curtis similarities for bacteria rRNA gene

recovered from Willow Point and Three Poles, and
summer and budding seasons
Figure 4.10
109
library of Willow Point

Figure 4.11
Plots of estimated OTUs richness for bacterial
109
clone library of Willow Point

Figure 4.12

library of Three Poles
"#$$$!
110
Figure 4.13
110
clone library of Three Poles

Figure 4.14
111
library of Three Poles in summer season

Figure 4.15
111
clone library of Three Poles in summer season

Figure 4.16
112
library of Three Poles in budding season

Figure 4.17
112
clone library of Three Poles in budding seasons

Figure 4.18
Relative abundance of bacterial phylotypes
130
obtained from Willow Point clone library

Figure 4.19
Relative abundance of bacterial phylotypes
131
obtained from Three Poles clone library

Figure 4.20
Phylogenetic
relationship
among 138
Alphaproteobacteria from Willow Point and Three

Poles and based upon Maximum Likelihood
Figure 4.21
Phylogenetic
relationship
among 139
Betaproteobacteria from Willow Point and Three

Figure 4.22
Phylogenetic
relationship
among 140
Deltaproteobacteria from Willow Point and Three
"#"!

Figure 4.23
Phylogenetic
relationship
among 141
Gammaproteobacteria from Willow Point and

Three Poles and based upon Maximum Likelihood
Figure 4.24
Phylogenetic relationship among Acidobacteria 142

from Willow Point and Three Poles and based
upon Maximum Likelihood analysis of full 16S
rRNA gene sequence data
Figure 4.25
Phylogenetic relationship among Bacteroidetes 143

Figure 4.26
Phylogenetic relationship among Chloroflexi from 144

Willow Point and Three Poles and based upon
gene sequence data
Figure 4.27
Phylogenetic relationship among Cyanobacteria 145

""!
Figure 4.28
Phylogenetic relationship among Gemmatimonas 146

Figure 4.29
Phylogenetic relationship among Nitrospira from 146

Willow Point and Three Poles and based upon
gene sequence data
Figure 4.30
Phylogenetic relationship among Planctomycete 147

and uncultured bacteria from Willow Point and
Three Poles and based upon Maximum Likelihood
Figure 5.1
A conceptual model of biogeochemical cycling in 161

microbial mats
Figure 5.2
Stratified structures in hypersaline microbial mats 164

at Guerrero Negro, Baja California
Figure 5.3
Location
and
geological
setting
of
southern 165
Cariboo Plateau lakes

Figure 5.4
The absolute abundance of archaeal, bacterial and
173
eukaryal communities in Goodenough Lake and

Deer Lake by real-time quantitative PCR analysis
Figure 5.5
The relative abundance of archaeal, bacterial and

eukaryal communities in Goodenough Lake and
""#!
173
Deer Lake by real-time quantitative PCR analysis

Figure 5.6
Rarefaction curves for bacterial clone library from 176

Good Enough Lakes mat
Figure 5.7
Plots of estimated OTU richness of bacterial clone 176

library from Good Enough Lakes mat
Figure 5.8
Rarefaction curves for bacterial clone library from 177

Deer Lakes mat
Figure 5.9
Plots of estimated OTU richness for bacterial clone 177

library from Deer Lakes mat
Figure 5.10
Rarefaction curves for archaeal clone library from
178
from Good Enough Lakes mat

Figure 5.11
Plots of estimated OTU richness for archaeal clone 178

library from Good Enough Lakes mat
Figure 5.12
Rarefaction curves for archaeal clone library from
179
from Deer Lakes mat. All curves were averaged

over 1000 simulations
Figure 5.13
Plots of estimated OTU richness for archaeal clone
179
library from Deer Lakes mat

Figure 5.14
Phylogenetic relationship among bacteria from
188
Goodenough and Deer Lakes based upon

gene sequence data
Figure 5.15
Relative abundance of bacteria in Goodenough 190

Lake microbial mat.
Figure 5.16
Relative abundance of bacteria in Deer Lake
""##!
190
microbial mat
Figure 5.17
Phylogentic relationship among archaea from
194
Goodeough Lake and Deer Lake based upon

gene sequence data
Figure 5.18
Relative abundance of
archaeal diversity of 195
Goodenough Lake and Deer Lake.
""###!
LIST OF TABLES
Table 1.1
Comparison of bulk parameters on Earth and Mars
Table 1.2
Comparison between Mars surface and deserts 12

environments on Earth
Table 2.1
Table of sampling sites altitudes and GPS 28

locations
Table 2.2
Absolute and relative abundance of bacteria, 37

archaea and eukarya quantified by qPCR and their
paired rations in soil crusts from Tibet high altitude
deserts
Table 2.3
Summary of bacterial and eukaryal clone libraries 39

for Site N1
Table 2.4
Summary of community structure at 5240m (Site
43
N1)
Table 2.5a-d
Identities of 16S rRNA gene sequences obtained 44

from site N1 (5240m)
Table 2.6

from site N1 (5240m)
61
Table 3.1
List of radiation tolerant organisms
Table 3.2
Identities of clones recovered after various gamma 71

radiations
Table 3.3
Morphology description of selected clones
72
Table 3.4
Chemical properties of selected clones
73
Table 4.1
Locations of Willow Point and Three Poles
91
Table 4.2
The list of samples that was included in this study
93
Table 4.3
List of selected microbialites samples for q-PCR 102

analysis
Table 4.4
Results from real-time quantitative PCR analysis. 103

Results were averaged from at least three runs
Table 4.5
Coverage summary of clone libraries constructed 108

for microbialites in Pavilion Lake
Table 4.6a-j

from site Willow Point
Table 4.7a-e

from site Three Poles
Table 4.8
Summary of recovery phyla from microbialites at 132

Willow Point, Three Poles and stromatolites at
Shark Bay
Table 4.9

from Three Poles during summer season
Table 4.10a-
from Three Poles during budding season
Table 4.11
Summary of bacterial assemblages isolated from 137

seasonal libraries of Pavilion Lake microbialites
Table 5.1
Locations and characters of lakes on Cariboo 168

Lakes
Table 5.2
Microbial abundance of Goodenough Lake and 172

Deer
Lake
from
real-time
analysis
""!
quantitative
PCR
Table 5.3
Coverage summary of clone libraries constructed 175

for microbial mats in Goodenough Deer Lakes
Table 5.4a-d
Identities of 16S rRNA (bacterial) gene sequences
181
obtained from Goodenough Lake

Table 5.5a-c
Identities of 16S rRNA (bacterial) gene sequences
185
obtained from Deer Lake

Table 5.6
Identities of 18S rRNA (archaeal) gene sequences
192
obtained from Goodenough Lake

Table 5.7
Identities of 18S rRNA (archaeal) gene sequences

obtained from Deer Lake
"""!
193
Chapter 1. Astrobiology.
1.1 Introduction to Astrobiology

1.1.1 What is astrobiology?
Astrobiology is a multidisciplinary study of the origin, distribution
and evolution of life. The fundamental questions in Astrobiology are (NASA
Astrobiology Institute):
(1) How does life begin and evolve? Is there life beyond Earth?
and, if so,
(2) How can we detect it?
(3) What is the future of life on Earth and in the universe?
For the past few decades, it was named as exobiology, which

focused on searching life forms beyond earth (Dick and Strick 2004). In
1996, NASA expanded the scope of exobiology and redefined exobiology
into Astrobiology. Exobiology and astrobiology share the same concerns of
the origins of life and the search of life beyond earth; But both placed life in
the context of its planetary history encompassing the search for planetary
systems, the study of biosignatures, and the past, present, and future of life
(NASA Astrobiology Institute). Astrobiology is raised to a multidisciplinary
level, which includes chemistry, physics, biology and geology, with added
new techniques and concepts. Compared with other sciences that are
becoming more and more specialized, Astrobiology has increasingly become

more generalized, making use of many specialties to study various questions
(Dick and Strick 2004).
1.1.2The beginning of Astrobiology
Are we alone? Is there life in space?
According to an ancient Chinese mythology, Chinese believed

there was a beautiful lady, Change (), who was separated from her
husband and lived on the moon by herself. It was because she accidentally
swallowed an immortality pill, and then started to float off to the moon due to
an overdose. However, she was not alone on the moon, she was
accompanied by a jade rabbit, who was making herbal medicine on the
moon. In early 17th century, Galileo Galilei created the first astronomical
telescope for lunar observation (Drake 1957). Mountains and craters were
observed on the moon surface. Nonetheless, the curiosity of human being
about space has never ceased. The ideas of Man in space and Man on the
moon had been raised and triggered the later space program.
In 1957, the first earth-orbiting artificial satellite, Sputnik, was
launched by the Soviet Union. This ignited the Space Race between the
Soviet Union and the United States of America during the Cold War (Dick
and Strick 2004). Even though it was largely a strategic event between two
countries, it accelerated the early development in space missions and

astrobiology. On July 29, 1958 the National Aeronautics and Space
Administration (NASA) was established. NASA is a government agency of
the United States and responsible for the nations public space program.
Their mission is to pioneer the future in space exploration, scientific
discovery and aeronautic research. Over the past 50 years, NASA has
conducted numerous successful missions, which have brought back
valuable images of the moon and over 400 kilograms of lunar samples. After
the fall of the Soviet Union, NASA remains and plays a leading role in both
space exploration and astrobiology.
1.2 Possible life in space

Follow the water to life. ------ slogan of Abscicon 2002 (Irion
2002)
It is possible to imagine that life biochemistries and forms in outer

space are very different to that on Earth. However, in terms of searching for
life in extraterrestrial environments, scientists are limited to the life systems
as we know on Earth (Gale 2009). They cannot survive individually but exist
in groups of different life forms (Ruiz-Mirazo et al. 2004). There are three
important ingredients that scientists believe are necessary for life: (1)
chemical elements such as carbon, hydrogen, oxygen and nitrogen that
formed the building blocks of living things, (2) an energy source that living
organism can used, and (3) liquid water (Squyres 2004). Among these three
basic requirements, searching for liquid water is the main strategy for search
for life (Irion 2002). This is because simple molecules will not react among
themselves and form a complex molecule; the unique physiochemical
characteristics of water make it a perfect solvent that brings them together
(Gale 2009). Liquid water also forms structural components in living cell and
regulate biological processes. (Pohorille and Wilson 1995). For earth life,
liquid water covers 75% of earth surface and is the most effective medium
that can stay stable over a wide temperature that mediates important
geochemical, probiotic and biological reactions (Mancinelli 2005; Gale 2009).
Some scientists believe liquid water is only an aspect of the environment that
life has evolved with, whereas life can exist without water (Walter 1999).
Lines of evidence suggest that methane could be an alternative solvent and
energy source for life (Chan 2005). However, up to date, no life has ever
been
detected
in
methane
lakes
on
Titan
or
other
planets
(http://www.esa.int/SPECIALS/Cassini-Huygens/index.html). Earth is the

only planet in the universe that we know with certainly has flourished with life
and liquid water remains the prime criteria in the search of extraterrestrial
life.
Most locations in the universe are not suitable for life, only a small
region in solar systems is considered habitable (Gonzalez et al. 2001a, b;
Gale 2009). They are far away from extreme lethal radiation events and the
surface temperature would allow liquid water to exist for long periods of time.
Among all the planets within the habitable zone in solar systems, Mars
attracts most of the scientists attention. Mars, the red planet, is always the
top priority in terms of searching life in space. Apart from it is the closest
planet from earth; this planet shares very similar physical conditions with the
Earth (table 1.1). Mars is a terrestrial planet covered with a thin layer of
atmosphere, which consist of carbon dioxide predominately (95%), with a
mix of nitrogen, argon, oxygen and traces gases. Very much like the earth,
Mars has similar four seasons, with a daily temperature ranging from about 60C to +30C at equator in summer and -130C at the poles in winter
(Leovy 2001; Squyres 2004; Williams 2007)
Figure 1.1. Image of the planet Mars. Image credit: NASA/JPL/Malin Space
Science Systems.
From the old images captured by Viking missions, Mars was
depicted a dry, lifeless planet, and mostly covered by hyper-arid land (Klein
1979; Mancinelli 1998). The soil is depleted in organic material (Biemann et
al. 1977; Navarro-Gonzlez et al. 2003) and indicated the presence of one or
more reactive oxidants (Oyama and Berdahl 1977; Navarro-Gonzlez et al.
2003). Due to the low atmospheric pressure and low surface temperature
(table 1.1), liquid water is very unstable on Mars surface. Water ice at polar
areas converted to vapor directly. Moreover, the planets surface is highly
oxidizing (Gmez-Silva et al. 2008), because the high ultraviolent and
cosmic radiation cannot be diverted by the thin ozone layer and the weak
magnetic field on Mars. Organic compounds can be destroyed under such
radiation.
6
Table 1.1. Comparison of bulk parameters on Earth and Mars. (Leovy 2001;
Squyres 2004; Williams 2007; Gale 2009)
Earth
Mars
Planet
from 3rd
position
4th
Sun
Average distance from 1.49 x108
2.27x108
Sun (km)
Equatorial radius (km)
6378
3396
Mass (kg)
5.97x1024
0.64
Density (kg/m )
5515
Gravitational force
9.80
3.71
(ms-2)
Surface
temperature -90 to +58
-170 to -8
(C)
Average
surface 15
-60
temperature (C)
Moon
Length of day
24 hr
24.6 hr
Length of year
365.25 days
678 earth days
Water availability
75% of the planet are No

covered
with
liquid
water
liquid possibly
but
some
water, ice covered at subsurface permafrost.

two poles
Atmospheric
density 1.217
~0.020
(kg/m3)
Major
Atmospheric 78.08%
composition
Nitrogen, 95.32% Carbon
(by 20.95% Oxygen
volume, dry air)
Dioxide, 2.7% Nitrogen,

1.6%
Argon,
0.13%
Oxygen, 0.08% Carbon

Monoxide
Description
Flourished with life
No life ever detected
However, Mars is not as dry as initially thought. Mars Orbital

Camera (MOC) and Mars Odyssey Orbiter brought back high-resolution
Mars images showing polar ice cap, subsurface water-ice in northern arctic
plain and the gullies at mid-latitude give strong evidences that water once
flowed
on
Mars
(http://www.nasa.gov/mission_pages/mars/news/mgs-
20061206.html). In 2004, Two mobile robots, Spirit and Opportunity Mars

Rovers were sent to look for any evidences could prove Mars were formerly
wet enough for life. They discovered evidences of salt water and pebble-like
hematite embedded on Mars outcrop rocks (NASA Facts: Mars Exploration
Rover). Hematite is a mineral that requires water to form on Earth. Mar. In
2008, Phoenix Mars Lander targets the North Arctic Region. Frozen water
was found in the soil sample from a trench approximately 2 inches deep
(http://phoenix.lpl.arizona.edu/06_20_pr.php). Life supporting niches found
at subsurface might support microbial life on Mars (Gale 2009). Moreover,
strong evidence proved that Mars was a warmer and wetter planet (Walter
1999). Geological features such as out-flow channel, valleys and lahars were
abundant since early Mars history (Baker and Milton 1974; Carr 1981;
Squyres 1984; Squyres and Carr 1986; Christianson 1989). By comparing to
earth surface morphology, planetary geologists hypothesized that liquid
water once flowed across the martian surface. (Lewis 1972; Baker and
Milton 1974; Anders and Owen 1977; McElroy et al. 1977; Clark and Baird
1979; Pollack and Black 1979; Pieri 1980; Carr 1981, 1986; Squyres 1984;
Squyres and Carr 1986; Christianson 1989). Even though there is no

unambiguous indication of residual life discovered on Mars, it is reasonable
to assume that life could have possibly originated and evolved on Mars
(Mancinelli 2005a, b).
Figure 1.2a (left). Image Hematite pebbles on Mars surface taken by Mars
Rover Opportunity. Image credit: Mars Exploration Rover Mission, JPL,
NASA. Figure 1.2b (right). Image of disappearing ice on Mars subsurface.
Image credit: NASA, JPL-Caltech, University of Arizona and Texas A&M
University.
Figure 1.3. Mars surface with riverbed feature. Image credit: NASA, JPL,
MGS Project.
1.2.2 Studies of Mars-like environments on Earth
Analogue means two similar things that are comparable. In
astrobiology, comparing Earth and Mars would be very valuable in terms of
searching for life of Mars. Analogues have been used since the very
beginning of space exploration. For example, similar morphology structures
on Earth were used to interpret images from Mars. Since Mars is an
unknown planet to us, there are many possible areas for us to explore on
Mars. There are several principal habitats that life could be present, i.e.
surface, subsurface and atmosphere. However, due to the limited financial
resources and technology, it is impossible to explore every single inch of
area on Mars. Alternatively, there are Mars-like environments on Earth
defined for this purpose.
10
Extreme Mars-like environments on Earth play a role for Mars

exploration, because many of these extreme environments resemble the
conditions that exist on Mars and other planets (Gross 1997; Cleaves and
Chalmers 2004; Seckback 2006). Extremophiles, organisms living in
environments beyond what we consider ambient level of thermal, moisture,
radiation and/or other stresses, can survive, adapt and benefit at these
unusual conditions (Dion 2008). There are no absolute definition of extreme
environments, their conditions can vary but not limited to extreme low to high
temperature, low to high pH or high salinity. Highly selective physiochemical
properties in environments can serve as model systems for exploring
fundamental ecological principles such as relationships between the diversity
of microorganism and extreme condition environments (Smith et al. 2006).
Understanding microbial adaptation mechanism in these extreme conditions
will lead to limiting factors for life on Earth and therefore provide clues for
where we can pinpoint the area or substance that we should focus on.
Microbiology of extreme environments is very important in astrobiology.
11
1.2.2.1 Mars-like terrestrial environments

Table 1.2. Comparison between Mars surface and desert environments on
Earth. (http://pubs.usgs.gov/gip/deserts/; Leovy 2001; Squyres 2004)
Earths hot desert Earths cold desert Mars surface
Averaged
20-25 (max. up to
Temperature
49C)
< 10
-60
(C)
Water
< 250mm
< 250mm
Possibly some
availability
precipitation per
precipitation per
subsurface
year
year with ice
permafrost and
deposits
CO2 ice at two

poles
Example
Features
Atacama, Gobi
Arctic and
Desert
Antarctic
Sand, sand dune
Ice, snow dune
Whole planet
Arid land, sand,
loosen rocks
Radiation level
Very low
Very low
Extreme UV and
background
background
ionizing radiation
radiation
radiation with
due to less
more intensive UV
protection
radiation (due to
reflection of ice)
Description
Intensive
Intensive freeze-
Extreme radiation
weathering
thaw action
level with no life

form detected
It is because we are living on Earths surface, we would assume

that it is a more favorable habitat for life. In fact, life on Mars surface is much
12
more challenging due to its exposure to environmental stresses, such as

temperature, wind and radiation.
Radiation is one of the factors that can limit life, as they interact
directly with DNA and proteins and cause damages to cell (Evans 2001).
The Sun provides energy for heating and photosynthesis;
however, excessive solar radiation would also cause damage to living cell
(Ehling-Schulz and Scherer 1999). Fortunately, the majority of harmful
radiation is greatly reduced by a thick atmospheric layer and the strong
magnetic field before it reaches earth surface (Gale 2009). Conversely, this
protection is absent on Mars (Leovy 2001; Squyres 2004). If life is present
on Mars, they are required to tolerate high radiation levels by repairing such
damages (Mattimore and Battista 1996; Zahradka et al. 2006) or avoid over
exposure to radiation. Life could also take refuges to protect them from high
UV radiation. Even modest covering of rocks and soils are enough to prevent
organism loss their viability in Martian environments (Cockell et al. 2006).
Previously, it was discussed that water was one limiting factor for
life (Horowitz et al. 1972). Consequently, deserts that characterized low
moisture, low nutrients and high UV radiation were considered one of the
major Mars analogues on Earth (e.g. Atacama deserts). If microbial life does
exist in extreme arid environments, it is possible that they could exist on
Mars. In this study, high altitude deserts were focused because of their
enormous level of UV radiation compared to the other areas on Earth.
13
There are three characteristics that define these areas as Marslike environments. Firstly, there are very low levels of organic material and
the present organics are refractory. Secondly, there are very low levels of
soils bacteria; these are nearly undetectable at some of the locations
(Navarro-Gonzalez et al. 2003). Lastly, the soil contains an oxidizing agent
with the ability to oxidize at equal rate L- and D- amino acids, as well as Land D-sugars. Despite all the unfavorable conditions, highly diverse
microorganisms were found in driest deserts on Earth (Navarro-Gonzalez et
al. 2003; Cockell and Stokes 2004). Extreme soil systems are complicated
with high heterogeneity. Therefore, it is more likely that Mars is a very large
planet with microbial niches, microbial life is dispersed and difficult to detect
(Warren-Rhodes et al. 2006). Microorganisms can be hidden by lifeless
surroundings (Crawford and Newcombe 2008). Extreme arid environments
not only offer the opportunity to investigate where and what to search for
extraterrestrial life (Torsvik and vres 2008), They also provide a physical
platform to test the methods and technologies for future Mars exploration.
Permafrost is another terrestrial environment on Earth that is
studied as Mars analogue. Permafrost or permafrost soil refers to ground,
comprised of soil, sediment, and includes ice and organic matter that remain
below freezing point (0C) for at least two consecutive years (van
Everdingen 2005). Permafrost can be found at high latitudes regions (Arctic
and Antartic regions), West Siberia, Alaska, Mackenzie Delta and Eurasian
Northeast. Despite the fact that permafrost covers a significant area of the
14
polar region (more than 25% of the polar land surface and part of the costal
sea shelves) (Romanovshii et al. 2005), it is also a common phenomenon
found in our solar system. In astrobiology, the Arctic and Antarctic
permafrost environments are studied as an analogue for extraterrestrial
permafrost environments.
Permafrost extreme environment is characterized by permanent
subzero temperature, low oxygen concentration, absence of free water, and
high salt concentrations in thin water films. Permafrost can extend up to
1000m into the subsurface (Williams and Smith 1989) and is subdivided into
three layers by their different temperature and living condition. Due to the
seasonal freezing and thawing, it was thought permafrost is hostile for life
and not even microorganisms could survive under such harsh environment.
However, in addition to life from three domains (Bacteria, Archaea and
Eukarya) were isolated from there; permafrost acted as a giant freezer that
preserved the oldest form of life on Earth. Significant amount of viable
ancient microorganism was isolated from two cores from the two Polar
Regions. The cells collected from arctic were dated to ~3million years and
~5millions for cells from Antarctic. Permafrost provides a unique stable
physicochemical
complex
that
able
to
retain
viability
of
these
microorganisms that no other habitats could maintain (Gilichinsky et al.

2005).
Scientists interests on permafrosts microbial communities can be
dated back since 1911 (Omelyansky 1911). A number of investigations were
15
than carried out to study on revealing microbial diversity, abundance and

physiology within the active layer and perennially frozen ground of
permafrost or other circumarctic environments
(Kris 1940; James and
Sutherland 1942; Boyd 1958; Boyd and Boyd 1964; Zvyagintsev et al. 1985;
Khlebnikova et al. 1990; Rivkina et al. 2000; Kobabe et al. 2004; Gilichinsky
et al. 2005; Zak and Kling 2006; Liebner and Wagner 2007).
A consortium
of microorganism was found in permafrost extreme environment. In addition,

the detection of methane in Martian surface and atmosphere indicated the
methanogenic archaea at permafrost extreme would be the potential
candidate for life on Mars (Wagner et al. 2001; Morozova et al. 2007).
1.2.2.2 Mars-like aquatic environments

Lines of evidences proved that Mars contained abundant liquid on
its surface at some time in the past (Lewis 1972; Baker and Milton 1974;
Anders and Owen 1977; McElroy et al. 1977; Clark and Baird 1979; Pollack
and Black 1979; Pieri 1980; Carr 1981, 1986; Squyres 1984; Squyres and
Carr 1986; Christianson 1989); however, some unknown events occurred
and resulted in losing its atmosphere. Therefore this water body started to
evaporat and eventually was lost due to the temperature change. As water
started to evaporate, this large water body formed individual small pockets of
brine lakes. These evaporative systems were concentrated with dissolved
minerals (eg. carbonate, sulfate). Several recent studies indicated Mars is a
salty planet, possible life were suggested based on these evaporites and the
16
liquid water present on early Mars (Rothschild 1990; Banin and Mancinelli
1995). Therefore, organisms that can survive in these Mars evaporative
systems provide us clues to how life could have existed on Mars during that
period of time.
Halophile, organisms that can adapt in high salt environments,
was suggested to be present and evolved on Mars. Halophiles can be
recovered from any environments with salt, including cold, hot, dry, wet
alkaline and neutral conditions. They represent one of the most diversity
group microorganisms. They can be bacteria, eukarya or archaea. Great
diversity in halophiles suggested that life in high salt concentration is easy to
evolve.
Stromatolites are another life form that maybe useful analogs for
Mars within these brine systems. They are carbonate structures formed by
microorganism (Burne and Moore 1987; Pierson 1992). Stromatolites are the
oldest record of life available on Earth, they were once the most abundant
life structure on the planet over three billions ago (Hoehler et al. 2001). They
occupy the hypersaline environments that are comparable to the aquatic
environments on early Mars. Therefore, they not only play a significant role
on understanding the early evolution of life, they also provide information of
how life may originate in early moist Mars and their evolution (McNamara
and Awramik 1992).
17
1.3
Molecular approaches in Astrobiology
The history of life could be preserved in the rocks and stored as

genetic information in every organism. Therefore, the development of
molecular approaches, especially in microbiology, has a huge impact on the
understanding
life
in
the
universe
and
their
evolution.
Molecular
phylogenetics studies can map out extraordinarily complex history of

evolutionary changes in life. This may answer the question of how complex
organism human intelligence evolved from a simple unicellular organism and
the relationship between the impacts of environmental changes to the
evolution of in life.
Microbial diversity and community structures are a study to
describe different aspects of complexity and variation microbial population
and communities (Torsvik and vres 2008). Many Microbial communities in
soil and aquatic environments on Earth are complex systems with highly
functional and phylogenical diversity. Therefore, sophisticated molecular
approaches are critical to describe the Mars-like environments on Earth and
in the development for astrobiology.
On the early Earth, life was more likely to be in microscopic form.
However, microorganisms are relatively small in size and with non-distinctive
morphological characters, which can be a limitation as basis for their
classification (Ward et al. 1998). Moreover, studies of microorganism have
long been restricted to the microscopic description of samples and the
isolation of a limited number of microorganisms. These traditional
18
microbiology techniques are selective and not quantitative (Vestal and

White, 1989; White et al. 1997); most microorganisms in the environment are
viable but not culturable (Amann et al. 1995; McCarthy and Murray 1996;
Pace 1996; White et al. 1997). Viable counts of bacteria in environmental
samples determined with classical methods represent only a small fraction
(0.1% to 1%) of the active microbial community (Olsen and Bakken, 1987;
White et al. 1997). Therefore, culture independent techniques were
developed to overcome the selective bias of the traditional cultivation
techniques.
Metagenomics is recovering genetic information directly from
environmental samples. This overcomes the limitation in cultivation as well
as PCR biases in molecular studies. Metagenomics can be used to generate
information on the potential functioning of individual microbial species and
their roles in the ecosystem (Rondon et al. 2000; Tringe et al. 2005), but it is
challenging (to isolates and extract DNA with high molecular weight and high
purity.
The most common approach for assessing microbial diversity is
using small sub-unit ribosomal RNA recovered from samples. rRNA genes
from bulk DNA were then amplified by Polymerase chain reaction (PCR) and
are cloned. Consequently, the clones can be identified by DNA sequencing
analysis (Torsvik and vres 2008). Since it is the most common approach,
results are ready for comparative studies to other researches. The
disadvantages of these approaches are that they are time consuming and
19
labor intensive for routine analysis. Especially when it comes to complex

communities like soil or microbialites ecosystems, our understanding of the
extent microbial diversity of many extreme environments are very limited.
Therefore, community-fingerprinting techniques such as denaturing gradient
gel electrophoresis (DGGE) and terminal restriction fragment length
polymorphism (T-RFLP) are frequently used to screen large number of
samples variation in spatial and temporal scale. Real-time polymerase chain
reaction provides detection and yet quantification of targeted DNA molecule.
These approaches offer high solution in describing microbial diversity and
composition, and therefore led to the discovery of third domain of life
Archaea. All these methods, including culture dependant and culture
independent technique, have their own advantages and limitation. Choice of
particular methods is based on expertise and laboratories; therefore, in
recent years polyphasic studies required multi molecular techniques were
applied to address our understanding of microbial diversity in Mars-like
environments (Pointing et al. 2009).
Furthermore, The importance of this diversity data is that
scientists could apply them to understand the function and ability of these
microorganisms in extreme environments. Billi et al. (2000) and Cockell et al.
(2005)
isolated
Chroococcidiopsis
from
extreme
environments
and
investigated their physiological change after simulated it with radiation in

laboratory. The demonstration of radiation resistance in Chroococcidiopsis
explained the repair mechanisms and therefore hint the strategy that enable
20
these organisms to live and survive in some of the most extreme

environments on Earth (Billi et al. 2000).
Figure 1.4. Schematic drawing of the molecular methods used in this study.
21
1.4 Project Scope

The general objective of this study was to conduct a molecular
level study of microbial community diversity for previously unstudied Marslike terrestrial and aquatic environments. They include high-altitude deserts
in Tibet, and carbonate-rich and hypersaline lakes in British Columbia.
This current chapter provides a brief history of astrobiology as
well as Mars. The possibility of life on Mars and the implication of Mars-like
environments on Earth have been presented. In addition, the molecular
approaches in study Mars-like environments were discussed.
In chapter two, a previously unstudied hyperarid soil environment
on Tibet Plateau was studied. A multi-domain survey, included Q-PCR
analysis, was conducted for the biological soil crusts collected from varies
altitude levels. Bacteria and eukaryal clone libraries of selected site were
constructed to provide an insight view of microbial diversity and composition
of high altitude deserts.
Since dehydration and desiccation produce same type of DNA
damage, microbes were isolated from high altitude Tibet soil and then
exposed to high doses of ionizing radiation. Results revealed in chapter
three expands our knowledge of radiation and desiccation tolerant bacteria,
as well as the distribution of these bacteria in high altitude Tibet deserts.
In chapter four, the focus was switched from hyperarid to aquatic
Mars-like environments. Microbial communities of microbialites collected
from a freshwater carbonate rich lake were studied. Molecular approaches
22
included Q-PCR, TRFLP and clone library construction were used. In

addition, comparisons were made to show the spatial and temporal variation
of these freshwater microbialites.
The final experimental chapter presents a comparison of
microbial mats communities of two hypersaline lakes with different water
chemistry. Bacterial and archaeal clone libraries were constructed to look at
their prokaryote communities.
General conclusions based upon results are made in the final
chapter, including an identification of limitations to the study and therefore
suggestion for future study.
23
Chapter 2. Molecular diversity and ecological significance of

soil microbial communities in the high altitude deserts of Tibet.
2.1 Introduction
2.1.1 Biological soil crusts (BSCs) in ecosystems
Biological soil crusts are complex ecosystems that consist of
Cyanobacteria, algae, microfungi, lichens and bryophytes in different
combinations and abundance. They are located on the topsoil level and
restricted to the area where higher plants are limited and therefore can
receive the maximum sunlight. Biological soil crusts act like a barrier
against soil erosion (Campbell 1979; Belnap and Gardner 1993) and
control the nutrient intake within soil system (Evans and Johansen 1999;
Belnap 2002). They are found in many soil environments on Earth,
varying from the tropics to polar region, as well as temperate climates to
extreme arid deserts (Starks et al. 1979; de Winder 1990).
2.1.2 Biological Soil Crusts in hyperarid deserts

Biological soil crusts are commonly found in arid environments.
For example, Sonoran Deserts, Great Basin, Colorado Plateau,
Chihuahuan Deserts (Redfield et al. 2002; Nagy et al. 2005; Gundlapally
and Garcial-Pichel 2006; Soule et al. 2009). Hyperarid desert areas are
characterized by their low moisture, low nutrient, extreme temperature,
and high UV environments. However, a wide diversity of microorganism
communities were found under such unfavorable conditions (GarciaPichel et al. 2001; Chanal et al. 2005). Biological soil crusts in hyperarid
24
deserts are vital soil inhabitant for microorganism (Belnap and Lange
2001, Garcia-Pichel et al. 2001); they play important roles in the
biogeochemistry and geomorphology of desert areas (Eldridge and
Greene 1994; Evans and Johansen 1999; Garcia-Pichel et al. 2001).
These assemblages of microorganism structures are dominated by
Cyanobacteria and microalgae; they are the primary producers in deserts
and are largely responsible for both carbon and nitrogen inputs (Evans
and Johansen 1999; Belnap 2002). They also reduce soil erosion with
their ability of extracellular polymeric secretions (Eldridge and Greene
1994), and also pave the way for further succession by higher plant.
The composition of these microbial desert crusts at various
localities have been studied intensively. Research has emphasized on
the cyanabacterial or algal component of these communities (Killian and
Fehr 1935; Vogel 1955; Shielfs 1957; Shields and Durrell 1964;
Friedmann and Galun 1974; Garcia-Pichel et al. 2001). For the past
decade, molecular approaches have been applying to study the microbial
diversity of phototrophic and non-phototrophic bacteria of BSCs (Kuske et
al. 2002; Yeager et al. 2004, 2007; Nagy et al. 2005; Gundlapally and
Garcia-Pichel 2006), as well as the eukaryal and archaeal communities
(Nagy et al. 2005; Bate and Garcia-Pichel 2009; Soule et al. 2009).
However, recent studies only emphasise an individual domain, with little
attention paid to the overall microbial biodiversity across three domains of
life (Pointing et al. 2009). Understanding these microorganisms that
survive in the most extreme arid conditions help us determine the limits to
life on Earth, recognize the interactions between biotic and abiotic
25
components of the ecosystem, and perhaps serve as models for potential

life on Mars. Several publications [Science (2003) 302:1018-1021; Nature
(2004) 431: 414] have identified such communities in the Antarctic and
Atacama deserts and highlighted their scientific significance, and
proposed
that
further
research
is
needed
to
determine
the
microorganisms dynamics dealing with desiccation stress, ionizing, and

UV radiation at their substrate and tolerant levels.
2.1.3. Hyperarid deserts in Tibet, China
Figure 2.1. Sandstorm in the Gansu Province of China in 2006 (Copyright

to National Geographic).
Dryland environment cover more than 35% of the global
terrestrial land mass and are expanding rapidly due to anthropologic
effect (Bhatnagar and Bhatnagar 2005; Housman et al. 2005). Despite
the fact that desert soil is one of the major reservoirs of bioavailable
nitrogen (Walvoord et al. 2003), understanding desert ecosystems can
26
contribute to future solutions for desertification and changes in

microclimate. In China, approximately 28% of the land mass are
characterized as desert and this number is growing rapidly in recent
decades due to environment degradation. The recent sandstorms in
Beijing, Ningxia, Shaanxi and Xinjiang provinces (April 2006) (fig 2.1)
confirmed that these effects are not only catastrophic at the ecological
level but also significantly impact Chinas environment and economic
prosperity. Prior studies have been carried out extensively in many hot
and cold arid environments, however, due to political reasons, the
Tibetan deserts have been inaccessible and thus ignored in the past.
2.1.4 Objectives
In this study, a culture-independent survey of multi-domain
microbial biodiversity in two biological soil crusts from high altitude soils
on Tibet Plateau was conducted. The study targeted eukaryal dominated
soil crust types (based on visual morphology). The study aimed to test the
following hypothesis:
1) Biological soil crusts at high altitudes support multidomain microbial diversity.
2) Different
crust
types
(visual
different microbial composition.
27
morphology),
reflects
2.2 Materials and methods

2.2.1 Site description
This experiment included 2 sites at different altitudes of the
Tibetan Plateau. They were 4661m and 5240m. Their GPS locations
were listed in table 2.1 and their relative positions were shown by satellite
image in figure 2.2 provided by Google Earth.
Table 2.1. Table of sampling sites altitudes and GPS locations.
!"#$! %&#"#'($)*+,!
-./0#".1!
"!
#$$%!
&'(!)*%+##,-!.#(!/*))+0',1!
-%!
)'#0!
&%(&&*)/+&0,-!.)(%%*2+#.,1!
Figure 2.2. The satellite image of 2 sampling sites (GOOGLE Map).

2.2.2 Sample collection
Soil Crust samples were collected from Tibet desert during the
summer (May) season of 2006. Samples were scooped using an alcoholsterilized (Isopropyl alcohol was used, since ethanol was not permitted to
be transported to Tibet.) spatula and stored in sterile-petri-dishes.
Samples were then stored in darkness until they were processed.
28
Figure 2.3. Soil sampling at Site 5520m.

2.2.3 DNA extraction and polymerase chain reaction (PCR) amplification
DNA extraction was done using a MO BIO UltraClean Soil
DNA Isolation Kit (http://www.mobio.com/) following the manufacturers
instructions. Final product was diluted in 50!l distilled water and kept in
4C. To visualize the extracted DNA, gel electrophoresis (1% agarose)
with ethidium bromide (EtBr) staining was run at 90 volts for 20 mins.
The 16S rRNA genes were amplified by polymerase chain
reaction using three sets of primers (Universal bacterial primers: 8F (5AGA GTT TGA TCC TGG CTC AG-3) and 1391R (5-CCG TCA ATT
CMT TTG AGT TT-3); eukaryal primers NS1 (5'-GTA GTC ATA TGC
TTG TCT C-3') and 1391R (5-CCG TCA ATT CMT TTG AGT TT-3); and
archaeal primers Farch9 (5-CTG GTT GAT CCT GCC AG-3) and 1391R
(5-CCG TCA ATT CMT TTG AGT TT-3). A 0.01 to 1.0 !l of DNA
template was used in a 50!l polymerase chain reaction (PCR) mixture
(1.5mM MgCl2, 0.2 mM of each deoxynucleoside triphosphate (dNTP),
29
0.3 !l of each primer pair and 1.0U of taq DNA polymerase). Thermal
cycling conditions were as follows: denaturation at 94"C for 1 min, 55"
C for 1 min, 72"C for 1 min, followed by an extension of 72"C for 10
mins. Gel electrophoresis (1% agarose) with EtBr staining under UV light
confirmed the purity of the amplified product.
2.2.4 Real-time quantitative PCR (q-PCR) analysis

PCR amplification was quantified in real-time (Applied
Biosystems prism 7000, California) by flourometric monitoring with SYBR
Green 1 dye (Invitrogen, California). All standard curves were constructed
using plasmids from cloned rRNA genes (Qiagen, California) separately
for archaea, bacteria and eukarya. The number of copies in standards
was
calculated
using
the
Zbio.net
online
converter
(http://www.molbiol.ru). Slopes of the standard curves generated were 3.03, -3.21, and -3.28 for Archaea, Bacteria, and Eukarya respectively. All
three standard curves achieved a high correlation coefficient (>0.99).
Quantification of genes in each sample was performed in triplicate.
Dissociation curves were studied for each run to ensure the threshold
cycle (Ct) was given by efficient and specific amplification. Absolute copy
number of genes was obtained by interpolation with the respective
standard curves generated.
2.2.5 Clone library construction

Site N1 was selected for clone library construction (total 3
libraries, 1 archaeal libraries discarded) based upon the q-PCR results
30
and t-RFLP profiles. Each PCR products were gel-purified using the GE
Heathcare illustra GFX PCR DNA and Gel Band Purification kit
(http://www.gelifesciences.com) prior to cloning. The amplicons were
ligated into a pDrive-cloning vector using Invitrogen TOPO TA Cloning
Kit
(http://www.invitrogen.com)
following
the
product
instructions.
Plasmids were extracted from positive transformants (Mini-MTM Plasmid

DNA extraction system, Viogene, Taiwan). 100 plasmids were selected
for bacterial library and 50 plasmids were selected for eukayra library. All
clones were sequenced using the BigDye Terminator Cycle Sequencing
kit (Applied Biosystems, California) and performed by Applied Biosystems
3730 Genetic Analyzer. Sequences were manually refined by BioEdit
(http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and then aligned using
the ClustalW.
Sequences alignments retrieved from clones were used to
construct a DNAdist (DOTUR package) DNA distance matrix. Phylotypes
were delineated on the basis of 97% sequence similarity with the aid of a
free algorithm DOTUR (Schloss and Handelsman, 2005). All sequences
generated by this study have been deposited in the NCBI GenBank
database. Approximate phylogentic affiliations were then determined by
BLAST
searches
of
the
NCBI
GenBank
database
(http://www.ncbi.nlm.nic.gov). Estimates of clone library sampling effort

were made using the freeware EstimateS (Colwell 2006). Sampling effort
was assessed by calculation of Coverage and Rarefaction curves,
estimates of library richness were made using the non-parametric
31
estimators ACE and Chao 1. All O.T.U delineation was made on the basis
of sequenced phylotypes.
2.2.6 Statistical analysis

Diversity indices were calculated following Hunt et al., 2004 to
estimate the abundance and diversity of taxa present in the samples
during the sampling periods.
To determine how well the sample size reflects the apparent
diversity within the clone library, library coverage (C) was calculated
based on the proportion of a clone library to the infinite size that was
sampled (Hunt et al. 2004).
C = 1 (n1 / N)
Where n1 is the number of clones that occurred once and N is
the total number of clones examined.
Rarefaction curve was calculated using EstimateS (Colwell
2006). Rarefaction curves were used as a reference to show the
goodness of sampling whlist the non-parametric estimator ACE and
classic CHAO 1 were used for estimating OTU (Operational Taxonomic
Unit) richness. It was tested whether perusal difference in the curves
resulting from different number of randomizations existed. All analyses in
this study were based upon 1000 randomizations, in which each OTU
sequence was treated as a separate sample.
Non-metric multi-dimensional scaling ordinations (NMDS) were
used to visualize Bray Curtis Similarities (diversity data) and Euclidean
Distances (environmental data).
In BEST analyses The BIO-ENV
32
procedure was used to maximize the rank correlation between biotic and
environmental data, thereby establishing a ranking (!w) for the effects of
environmental variables on diversity. All analyses were performed using
Primer v6.1.6 (Clarke and Gorley 2006). All results stated as significant
have a confidence level of P <0.05 unless stated otherwise.
33
2.3 Results
2.3.1 Samples description
Soil samples based on visual identification of biological soil
crusts were collected from two different altitudes in the arid Tibet tundra.
Soils are typically of low moisture level and nutrient status (Thomas
1997). No higher plants were observed and the area was covered by bare
soil with loose rocks. The collected soil crust samples were very dry and
crumbly, but somehow they were cohesive and stayed flat after collection.
(fig.
2.5).
Microscopic
observation
revealed
mixed
microbial
morphologies, however specific identification beyond phylum based upon

observation was not possible.
34
Figure 2.4. Photo of sample collection at N1 (5240m)
Figures 2.5. Samples collected from H (4661m) site. Flat soil crusts were
cohesive surface structures and stored in a petri-dish.
2.3.2 Real-time quantitative PCR analysis
Soil samples were analyzed by Real-time quantitative PCR to
compare their communities distribution of the three domains, archaea,
bacteria and eukarya. Results were shown in Table 2.2 and the graphical
illustrations of absolute and relative abundance of the communities were
shown in Figure 2.6 and 2.7.
Tibet deserts were dominated by bacteria and eukarya, with
very low abundance of archaea. Two types of community pattern were
identified among four sampling sites, but they showed no relevance to the
35
sampling altitudes. The total biomass abundance in sites 4661m (H) and
5240m (N1) ranges from 2.5-6.3x105 copies per gram (fig. 2.6). At the
sites 4661m (H) and 5240m (N1), the abundance of eukarya were 88%
and 80% respectively (fig. 2.7).
36
Table 2.2. Absolute and relative abundance of bacteria, archaea and

eukarya quantified by qPCR and their paired rations in soil crusts from
Tibet high altitude deserts.
H
N1
4661m
5240m
Average abundance (SSU rRNA copy no.
per gram)
Archaea
2.2x103
2.2x103
4
Bacteria
2.7x10
1.3x105
Eukarya
2.2x105
5.0x105
Relative abundance
Archaea
0.9%
0.4%
Bacteria
10.7%
21.0%
Eukarya
88.5%
78.7%
Stantard Deviation (SSU rRNA copy no. per
gram)
2
Archaea
1.5x10
1.6 x102
Bacteria
6.3x102
4.7x104
4
Eukarya
3.7x10
8.6x103
Ratio
Archaea/
8.27 x10-2
1.69 x10-2
Bacteria
Bacteria/Eukarya
1.21 x10-1
2.67x10-1
'(&!!"
!"#$%&'()*"&+,*+-().-$/0(#12%3)
'(%!!"
'($!!"
'(#!!"
'(!!!"
-./0120"
30456170"
&!!"
8149060"
%!!"
$!!"
#!!"
!"
)"$%%'*"
+'",#$!*"
Figure 2.6. The absolute abundance of archaeal, bacterial and eukaryal

communities by real-time quantitative PCR (q-PCR) analysis.
37
$!!"#
!"#$%&'"()*+,-$,."(/01(
,!"#
+!"#
*!"#
)!"#
0123453#
(!"#
637894:3#
'!"#
;47<393#
&!"#
%!"#
$!"#
!"#
-#'))$.#
/$#(%'!.#
Figure 2.7. The relative abundance of archaeal, bacterial and eukaryal

communities by real-time quantitative PCR (q-PCR) analysis.
2.3.3 Bacterial and archaeal diversity in high altitude deserts

Based on the qPCR result. Site N1 (5240m) was selected for
clone libraries construction for a throughout microbial communities
description. All three archeael, bacterial and eukaryal libraries were
constructed. However, the retrieved clones from archaeal library have the
closest match with eukaryotes. Other sets of archaeal specific primers
were attempted with same negative result. This may due to the low
abundance of archaea that revealed in qPCR analysis; therefore that
particular library was discarded.
The cloning sizes of bacterial and eukaryal libraries were 100
and 52 clones respectively (Table 2.3). Further analyses were made
under the OTU definition of 97% nucleotide similarity. Rarefaction curves
of 2 clone libraries were shown in Figure 2.8 and 2.10. Rarefaction curves
38
could not been used for concluding their difference in richness, it is

because they do not address the precision of the observed species
richness (Hughes et al. 2001). However, it could show if sampling effort
made is comparable among libraries. None of these curves has reached
an asymptote, but both bacterial libraries were not linear. Eukaryal curve
showed a strong approximation towards the asymptote and bacterial
curve also showed the tendency to curve downward under the given
sampling effort. This indicated that the sizes of clone libraries, although
unequal, were satisfactory to allow the estimation of total OTUs richness.
Both non-parametric richness estimators ACE and Chao1
estimations resulted in similar trends for both lakes (fig. 2.9 and 2.11).
The estimated curves by Chao1 were more stable and had better
concave downward shapes; therefore they were used in estimating the
richness of OTUs.
Table 2.3. Summary of bacterial and eukaryal clone libraries for Site N1.
Bacteria
Eukarya
# of clones analyzed
100
52
# of OTUs (97% cutoff)
62
Chao 1 richness
74
3.6
Coverage, %
Average similarity to
phylotypes using BLAST
18
98
95
97
39
#!"
!"#$%&'()'*+,-'($-%&.%/'
'#"
'!"
&#"
&!"
%#"
%!"
$#"
$!"
#"
!"
!"
%!"
'!"
(!"
)!"
$!!"
$%!"
!"#$%&'()'01(2%-'-3#41%/'
Figure 2.8. Plots of rarefaction curves for bacterial clone library of site N1.
All curves were averaged over 1000 simulations.
$!!"
!"#$%&'()'*+,-'($-%&.%/'
+!"
)!"
*!"
(!"
#!"
,-."
'!"
-/01"$"
&!"
%!"
$!"
!"
!"
%!"
'!"
(!"
)!"
$!!"
$%!"
!"#$%&'5)'01(2%-'-3#41%/'
Figure 2.9. Plots estimated OTUs richness for bacterial clone library of
site N1. All curves were averaged over 1000 simulations.
40
(#$"
!"#$%&'()'*+,-'($-%&.%/'
("
'#$"
'"
&#$"
&"
%#$"
%"
!#$"
!"
!"
%!"
&!"
'!"
(!"
$!"
)!"
!"#$%&'0)'12(3%-'-4#52%/'
Figure 2.10. Plots of rarefaction curves for eukaryal clone library of site
N1. All curves were averaged over 1000 simulations.
("
!"#$%&'()'*+,-'($-%&.%/'
'#$"
'"
&#$"
&"
*+,"
%#$"
+-./"%"
%"
!#$"
!"
!"
%!"
&!"
'!"
(!"
$!"
)!"
!"#$%&'()'12(3%-'-4#52%/'
Figure 2.11. Plots estimated OTUs richness for eukaryal clone library of
site N1. All curves were averaged over 1000 simulations.
41
2.3.4.1 Bacterial assemblage diversity

All 62 unique phylotypes were sequenced. They included
alpha-,
beta-,
delta-
and
gamma-Proteobacteria,
Acidobacteria,
Actinobacteria, Bacteroidetes, Cyanobacteria, Gammatimonadetes and

Planctomycetes. Both phototrophic (Cyanobacteria) and heterotrophic
(Proteobacteria) communities were present within the environments.
Common soil bacteria groups were detected, such as Acidobacteria and
Bacteroidetes, however no Deinococcus-Thermus was dectcted in the
clone library. Detailed descriptions of the closest blast match of the
sequences were listed in Table 2.5a-d. The most abundant bacterial
group was Bacteroidetes, and the most abundant bacterial sequence in
both sampling sites was beta-Proteobacteria, which affiliated to
Naxibacter sp. BUZ1 (FJ657440). The relative abundance of bacterial
assemblage was shown in Figure 2.13.
2.3.4.2 Eukaryal assemblage diversity

All 4 unique phylotypes were sequenced and the detailed
descriptions of the closest blast were shown in Table 2.6. The closest
blast match of the dominant clone is a terrestrial microalgae Stichococcus
bacillaris (88%). The graphical illustration of relative abundance of
eukaryal assemblage was shown in Figure 2.14.
Interpolation of clone library sequence data with qPCR data
allowed an estimation of relative abundance for all phylotypes across all
domains in the community. Detail description of the abundance was
shown in Table 2.4 and Figure 2.12.
42
Table 2.4. Summary of community structure at 5240m (Site N1).

Domain (%)
Phylum
Absolute Abundance
(%)
0.4
Archaea (0.4)
Archaea
Bacteria (21)
Cyanobacteria
1.5
Alphaproteobacteria
3.4
Betaproteobacteria
2.3
Deltaproteobacteria
0.2
Gammaproteobacteria
0.6
Acidobacteria
1.0
Actinobacteria
1.5
Bacteroidetes
4.4
Gemmatimonadetes
3.8
Planctomycetes
0.2
Unknown
2.1
Viridiplantae-Stichococcus
69.6
Rhizaria-Protaspis
4.6
Paecilomyces
4.5
Iodophanus carneus
1.5
Eukarya (78.7)
!"#$%&%'
()*+'
,%#-&".%'
/0)(+'
123%"4%'
56)7+'
Figure 2.12. The relative abundance of microbial structure of at 5240m

(Site N1).
43
Table 2.5a. Identities of 16S rRNA gene sequences obtained from site N1 (5240m).
Code
Genbank
accession
number
Relative
abundance
(%)
Putation Phylum
Closest Genebank Match

Identity
Accession
Number
Percent
Source
similarity
N1TB_01
HQ144091
Alphaproteobacteria
Brevundimonas sp. AKB-2008-VA7
AM989006
99
drinking water
N1TB_02
HQ144092
Alphaproteobacteria
Kaistobacter sp. Gsoil 634
AB245347
97
soil
N1TB_03
HQ144093
Alphaproteobacteria
Kaistobacter sp. IMER-A2-13
FJ436424
98
soil
N1TB_04
HQ144094
Alphaproteobacteria
Roseococcus sp. LW5
FM956480
95
water
N1TB_05
HQ144095
Alphaproteobacteria
Roseomonas gilardii
AY150045
94
culture
N1TB_06
HQ144096
Alphaproteobacteria
Roseomonas gilardii
AY150045
92
culture
N1TB_07
HQ144097
Alphaproteobacteria
Sphingomonadaceae bacterium PB125
AB220112
98
culture
N1TB_08
HQ144098
Alphaproteobacteria
Sphingomonas melonis
AB334774
99
sea urchin
N1TB_09
HQ144099
Alphaproteobacteria
Sphingomonas sp. YC6722
EU707560
96
culture
N1TB_10
HQ144100
Betaproteobacteria
uncultured Curvibacter sp.
EF663074
98
cropland
N1TB_11
HQ144101
Betaproteobacteria
Naxibacter sp. BUZ1
FJ657440
99
culture
N1TB_12
HQ144102
Betaproteobacteria
Naxibacter sp. BUZ1
FJ657440
94
culture
N1TB_13
HQ144103
Deltaproteobacteria
uncultured delta proteobacterium
EF073932
94
pasture
N1TB_14
HQ144104
Gammaproteobacteria
Pseudomonas sp. MY1106
EU082808
97
glacier
N1TB_15
HQ144105
Gammaproteobacteria
Stenotrophomonas maltophilia
FJ765513
99
waste soil
N1TB_16
HQ144106
Acidobacteria
uncultured Acidobacteria bacterium
EF447036
96
volcanic environment
N1TB_17
HQ144107
Acidobacteria
AY571796
96
soil
N1TB_18
HQ144108
Acidobacteria
EU751349
97
sandstone
44
Table 2.5.b (cont.). Identities of 16S rRNA gene sequences obtained from site N1 (5240m).
Code
Genbank
accession
number
Relative
abundance
(%)
Putation Phylum

Identity
Accession
Number
Percent
Source
similarity
N1TB_19
HQ144109
Acidobacteria
AY571790
98
soil
N1TB_20
HQ144110
Acidobacteria
AY571792
97
soil
N1TB_21
HQ144111
Actinobacteria
actinobacterium PB90-5
AJ229241
98
culture
N1TB_22
HQ144112
Actinobacteria
Rhodococcus sp. djl-6-2
DQ185597
100
culture
N1TB_23
HQ144113
Actinobacteria
uncultured actinobacterium
EF016798
96
desert Soils
N1TB_24
HQ144114
Actinobacteria
uncultured actinobacterium
AY250866
95
cryptoendolithic
community
N1TB_25
HQ144115
Bacteroidetes
Hymenobacter roseosalivarius
Y18834
97
culture
N1TB_26
HQ144116
Bacteroidetes
Y18834
97
culture
N1TB_27
HQ144117
Bacteroidetes
Y18834
95
culture
N1TB_28
HQ144118
Bacteroidetes
Rhodocytophaga aerolata
EU004198
86
culture
N1TB_29
HQ144119
Bacteroidetes
Rhodocytophaga aerolata
EU004198
95
culture
N1TB_30
HQ144120
Bacteroidetes
uncultured Segetibacter sp.
EU223960
93
soil
N1TB_31
HQ144121
Bacteroidetes
Spirosoma rigui
EF507901
93
culture
N1TB_32
HQ144122
Bacteroidetes
Spirosoma panaciterrae
EU370956
87
soil
N1TB_33
HQ144123
Bacteroidetes
uncultured Bacteroidetes bacterium
EF522294
95
sandstone
N1TB_34
HQ144124
Bacteroidetes
EF683046
97
atmosphere
N1TB_35
HQ144125
Bacteroidetes
EF522294
94
endolithic sandstone
N1TB_36
HQ144126
Bacteroidetes
EF683046
98
atmosphere
45
Table 2.5c (cont.). Identities of 16S rRNA gene sequences obtained from site N1 (5240m).
Code
Genbank
accession
number
Relative
abundance
(%)
Putation Phylum

Identity
Accession
Number
Percent
Source
similarity
N1TB_37
HQ144127
Bacteroidetes
AY921801
97
farm soil
N1TB_38
HQ144128
Cyanobacteria
Microcoleus vaginatus CSU-U-KK1
EF667962
99
culture
N1TB_41
HQ144129
Cyanobacteria
Phormidium sp. 195-A12
EU282429
98
permafrost
N1TB_42
HQ144130
Gemmatimonadetes
uncultured Gemmatimonadetes bacterium
AY921899
97
farm soil
N1TB_43
HQ144131
Gemmatimonadetes
EF072223
93
Georgia
N1TB_44
HQ144132
Gemmatimonadetes
AY921912
94
farm soil
N1TB_45
HQ144133
Gemmatimonadetes
AY921912
93
farm soil
N1TB_46
HQ144134
Gemmatimonadetes
AY921705
88
farm soil
N1TB_47
HQ144135
Gemmatimonadetes
AY921912
93
farm soil
N1TB_48
HQ144136
Gemmatimonadetes
AY921894
89
farm soil
N1TB_49
HQ144137
Gemmatimonadetes
AY922065
92
farm soil
N1TB_50
HQ144138
Gemmatimonadetes
AY921939
95
farm soil
N1TB_51
HQ144139
Gemmatimonadetes
EF072223
94
Georgia
N1TB_52
HQ144140
Gemmatimonadetes
AY921939
92
farm soil
N1TB_53
HQ144141
Planctomycetes
uncultured planctomycete
EU979098
92
soil
N1TB_54
HQ144142
unidentified clone
uncultured bacterium
AY274139
96
gold mine
N1TB_55
HQ144143
unidentified clone
uncultured endolithic bacterium
AB374366
96
subsurface of rock
N1TB_56
HQ144144
unidentified clone
AB374381
89
below the rock surface
46
Table 2.5d (cont.). Identities of 16S rRNA gene sequences obtained from site N1 (5240m).
Code
Genbank
accession
number
Relative
abundance
(%)
Putation Phylum

Identity
Accession
Number
Percent
Source
similarity
N1TB_57
HQ144145
unidentified clone
AB374370
91
subsurface of rock
N1TB_58
HQ144146
unidentified clone
AB473917
89
N1TB_59
HQ144147
unidentified clone
AB473896
97
N1TB_60
HQ144148
unidentified clone
uncultured soil bacterium
DQ378239
96
soil
N1TB_61
HQ144149
unidentified clone
AF507699
89
forest soil
N1TB_62
HQ144150
unidentified clone
EU861961
96
soil
Table 2.6. Identities of 18S rRNA gene sequences obtained from site N1 (5240m).
Code
Genbank
accession
number
Relative
abundance
(%)
Putation Phylum

Identity
Accession
Number
Percent
Source
similarity
N1TE_01
HQ143746
88.46
Viridiplantae
Stichococcus bacillaris
AB055865
99
culture
N1TE_02
HQ143747
3.85
Rhizaria
Protaspis sp. CC-2009b
FJ824125
94
marine environment
N1TE_03
HQ143748
5.77
Fungi
Paecilomyces sp. 080834
DQ401104
99
mangrove
N1TE_04
HQ143749
1.92
Fungi
Iodophanus carneus
U53380
96
culture
47
?"%8+('4=+)():.0-
@8A8'B8C0!"#$%#&'()'*%+()&,%-
./0-
3)44%(,4'8%6)():-
1)(%#&'()'*%+()&,%-
.>0-
..02)"(%#&'()'*%+()&,%-
<=%8'*%+()&,%90-
.03%44%#&'()'*%+()&,%-
50!+,6'*%+()&,%70-
1%+()&',6)():;;0-
!+(,8'*%+()&,%90-
Figure 2.13. Relative abundance of bacterial phylotypes obtained from N1

clone library.
.+6$#,&78$6()(/0)
4949:;)
.-&"+(/#()(/0) /01123445*D0-
<&=&/%+>'()
$+->6'(;0-
!"#$%&$&$$'()
*+$#,,+-#(>>0-
Figure 2.14. Relative abundance of eukaryal phylotypes obtained from N1

clone library.
48
2.4 Discussion
Even though arid and hyperarid environments appear harsh for
living organisms, complex prokaryotic communities were present in different
desert environments on Earth (Rainey et al. 2005; Chanal et al. 2005;
Garcia-Pichel et al. 2001). These microorganisms formed a biotic desert
crust that played a critical role that affects the structure, function and
productivity of arid lands, including soil stabilization, water retention, carbon
and nitrogen fixation and soil fertility (Belnap and Lange 2001; Redfield et al.
2002). In this study, an algal-dominated biological soil crust was
characterized using a multidomain approach.
This study surveyed two soil crusts samples collected from two
different altitudes. No observation related to geographic location or altitude
was recognized. Previous studies on biological soil crusts in arid
environments have revealed bacteria dominated communities (Bates and
Garcia-Pichel 2009) with low abundance of archaea and eukarya. Unlike
other hot and cold deserts, my results indicated eukaryal-dominated crusts
were identified on Tibet Plateau. High abundance of green algae in Tibet
suggested algae is an important member of high altitude deserts. It may due
to their high tolerance to both desiccation and photophysiology (Cardon et al.
2008). In this study, we focused in these eukarya-dominated soil crusts.
Detailed sequence-based analysis of highest sampling site N1 was carried
out to fully characterize microbial diversity.
49
Tibetan deserts present a unique high altitude desert ecosystem,

due to the high level of UV radiation observed (Blumthaler et al. 1997). High
abundance Cyanobacteria and photosynthetic eukarya were recovered from
Site N1. It is because these microorganisms contain sunscreen pigments
that could protect them from of ultraviolet radiation (Garcia-Pichel and
Castenholz
1991;
Bdel
et
al.
1997).
In
particular,
extracellular
polysaccharides that important substances in binding soil in biological soil

crust, would also offer UV protection (Ehling-Schulz and Scherer 1999). In
addition, Deinococcus-Thermus phyla, which could tolerate high UV
radiation by carrying out DNA repair mechanism (Moseley 1983; Smith et al.
1992; Minton 1994; Mattimore and Battista 1996). They were found
abundant in extreme soil environments (Nagy et al. 2005; Pointing et al.
2009), but no Deinococcus were retrieved from this study. It is not certain if it
is absence from the Tibet high altitude environment or high competition
within the soil ecosystem, further experiments in chapter three are required
to address this observation.
Bacteria comprised 21% of the community with 62 unique
sequences spanned in 10 phyla. Previous study has indicated that
distribution of soil crust bacteria are related to local factors such as soil
chemistry (Dequiedt et al. 2009). Spatial and temporal variation may also
lead to changes in biodiversity (van der Gast et al. 2008). Primary production
is important for any ecosystem since it is the basis from which others gain
nutrients for growth (Begon et al. 1996). When it came to Tibet high altitude
50
deserts with an extreme climatic condition, an area that was sparsely

vegetation with limited higher plants, photoautotrophic communities play an
essential role for ecosystem. At site N1 (5240m), the communities were
dominated by phototrophs comprised by Stichococcus, Cyanobacteria and
Choroflexi. The autotrophs community was dominated by a green soil alge,
Stichococcus bacillaris, which is an opportunistic species that is only
occasionally found in abundance in nature (Round 1965). Stichococcus sp.
was previously identified Negev Desert in Isarel (Lange et al. 1992; Kidron
1995; Karnieli et al. 1999; Veste et al. 2001 a,b; Galun and Garty 2001) The
environment represented during sampling time may favour the growth of this
species.
Other than Stichococcus, Choroflexi and Cyanobacteria were
present as phototrophs in the community in Tibet deserts. Cyanobacteria
show a widespread distribution in moist and arid soil environments (Palmer
and Friedmann 1990; de Chazal et al. 1992; Whitton and Potts 1999; Miller
and Bebout 2004; Smith et al. 2006). Cyanobacteria, can tolerate
desiccation and high doses of ionizing radiation (Potts 1999; Ehling-Schulz
and Scherer 2009). In particular, Microcoleus vaginatus recovered in this
study could synthesis a biopolymer, extracellular polysaccharides, that could
regulate the water content within the cell and protect the cell wall from
damage during swelling and shrinkage (Grilli Caiola et al. 1993, 1996), as
well as form sheaths that could bind soil particles together (Belnap and
Gardner 1993). Exceptionally low abundance of Cyanobacterial phylotypes
51
recovered from Tibet supported the observation from polar region (Pointing
et al. 2009). This may indicate the competition for water between eukarya
and Cyanobacteria in the soil; Cyanobacteria abundance are highly localized
and more abundant at hypolithic strata (Cockell 2004; Pointing et al. 2009).
Interestingly, Chloroflexi, which were not common to arid environments,
were obtained from Tibet and other deserts (Nagy et al. 2005; Pointing et al.
2009).
Unlike other hot desert environment, heterotrophs were the
majority
group
present
Alphaproteobacteria,
at
Site
N1
Betaproteobacteria
(5240m).
They
Acidobacteria,
comprised
of
Actinobacteria,
Bacteroidetes and Gammatimonadetes. This combination of bacteria was

commonly found in soils, as well as extreme hyperarid soils (Connon et al.
2007). Bacteriodetes was the largest bacteria group in Site N1, it has been
previous reported from other BSCs from arid environments (Garcia-Pichel et
al. 2003; Hughes and Lawley 2003; Smith et al. 2004; Nagy et al. 2005;
Gundlapally and Garcia-Pichel 2006), but low abundance agricultural soil
(Boyer et al. 2001). High abundance of proteobacteria was detected from
Tibet deserts and other desert area (Garcia-Pichel et al. 2003). It was
proposed proteobacteria and Bacteriodetes were also able to produce EPS
(Gundlapally and Garcia-Pichel 2006) that could help in BSCs formation.
Alphaproteobacteria
are
common
soil
component,
whereas
Betaproteobacteria reflects the presence of desert plants (Nagy et al. 2005).

Gemmatimonadetes were thought to be minor component of BSCs, but large
52
amounts
were
isolated
from
Tibet.
Moreover,
Actinobacteria
are
heterotrophs that play a significant role in the carbon cycle in soil, as well as
arid environments (Nagy et al. 2005; Gundlapally and Garcia-Pichel 2006).
Although the archaea are common in soils worldwide (Rutz and
Kieft 2004; Chanal et al. 2005; Fierer et al. 2005; Nagy et al. 2005; Soule et
al. 2009), they appear to have revealed their biological limit in extreme arid
deserts, they have been recorded as absent in deserts such as the Atacama
(Warren-Rhodes et al. 2006) and polar deserts (Pointing et al. 2009) In Tibet
deserts, failure in retrieving archaeal clone from library construction and the
extreme low ratio of archaea to bacteria abundance (0.002 to 0.08 in Tibet
whereas 0.05 from arid lands in North America) in Site N1 indicated that the
population of archaea was absent or present at a very low level, and there
were no recoverable archaeal phylotypes in Tibet high altitude deserts. It is
suggested archaea are unable to tolerate the extreme environmental stress
in Tibet high altitude deserts.
The study of the unique algal dominated soil crusts in Tibet is very
important for several ecological reasons: (1) Many studies worldwide have
shown that the biological soil crusts can be critical factor in soil stability in
deserts area. They reduce water and wind erosion (Belnep 2001; Belnap
and Lange 2001; Warren 2001). In Tibetan high altitude deserts, the
polysaccharides extruded by Cyanobacteria and algae bind soil particles and
linked them together (Tisdale and Oades 1982; Schulten 1985; Belnap and
Gardner 1993). They protect the top soil fine particles, and hence, stabilize
53
the soil and have huge influence on preventing desertification and

sandstorms in northern China (Goudie 1978; Kovda 1980; Tsoar and Pye
1987); (2) They also can maintain site productivity and conserve soil
moisture (Garcia-Pichel and Belnap 1996; Warren 2002), because they
protect the plant essential nutrients that are often bound to these top soil
particles (Danin and Ganor 1991; Verrecchia et al. 1995; Belnap 2001).
Biological soil crusts serve as major primary producer and nitrogen-fixing
communities in arid environments (Belnap and Lange 2001). They also
promote the development of vascular plants (Belnap and Lange 2001); (3)
Green algal is closely related to water sensitive higher green plant, but still
could survive under extreme desiccated environments. Molecular studies of
algal crusts provide useful information of photoprotection of higher plant
under environmental stress and protection against the effects of extreme
dehydration (Cardon et al. 2008).
A highly diverse community of Cyanobacteria, alga, heterophic
bacteria and individual fungi in Tibet deserts proposed that a high trophic
complexity existed. Individual species may rely on each other for
sustenance, as well as nutrients recycling. Indeed, a detail survey of
microbial distribution in relation to environmental stress and physical factors
are required to understand whether the data presented here is a truly stable
structure of Tibet deserts. Seasonal variation may influence soil chemistry
soil water content soil humidity and atmospheric humidity. Is the eukaryadominant crust often present in Tibet? Or was it resulted from a recent
54
stochastic rain? However, due to the inaccessibility of Tibet deserts in winter.

Physical constraints were a major limit for sampling in this study. Repeated
sampling throughout the year is impossible to attend these questions.
Tibet high altitude deserts are characterized by low moisture and
low organic content condition. Long, and extremely cold winters in Tibet
inhabit the population of high plants and animals, and so any available water
content is usually frozen. The conditions are very similar to the Martian
surface (Gmez-Silva et al. 2008). Mars is a cold dry planet, with a harsh
and hostile surface condition. Due to the lack of magnetic field and a thick,
dense atmosphere, Mars surface is exposed to extreme ultraviolet and
space radiation (as low as 200nm wavelength) and somehow is very
comparable to the conditions on early Earth. Even thought the UV flux
observed from current Earth is 3 orders magnitude lower than Mars surface
(Cockell et al. 2000; Crdoba-Jabonero et al. 2003; Patel et al. 2003), Tibet
is the tallest and largest arid region on Earth. Tibet may not present the UV
condition on Mars surface but it is similar to the condition of Mars subsurface
or Mars cave where UV radiation and temperature are greatly reduced and
liquid water is more likely to present. Therefore, it is suggested Tibet high
altitude deserts could serve as Mars analogue, microbes that survive in Tibet
could provide a proxy for scientists to focus for searching life on Mars and
also provide information of how life was evolved from the early Earth.
55
2.5 Conclusion
Biological soil crusts from high altitude locations in Tibet were
characterized by molecular approaches.
1.
Eukaryal phylotypes dominated biological soil crusts,

followed by bacteria and very low or zero abundance of
archaeal phylotypes.
2.
Phylogenetic analysis revealed that 10 bacterial phyla were

detected
and
proteobacteria,
Bacteroidetes,
gemmatimonadetes were the dominant groups. On the

other hand, Stichococcus was the prevalent eukaryal
phylotype in the community.
56
Chapter 3. Isolation of ionizing radiation tolerant bacteria from

high altitude deserts of Tibet plateau, China.
3.1 Introduction
Radiation is a process of energetic particles passing from one
object to another object. It can be divided into ionizing and non-ionizing
radiation based on their frequency and wavelength. Ionizing radiation is
more harmful with their shorter wavelength and higher frequency. It is
because ionizing radiation is a higher energy kind of radiation that is able
to ionize an atom or molecule. In general, doses as low as 0.1-1.0 Gy of
gamma radiation can cause lethal damage for some organisms (Upton
1982; Sutherland et al. 2000) (table 3.1). Therefore, some types of
radiation are essential tools for sterilization in food and medical industry
(Silliker 1980; Frazier 1988; Pointing 1995; Pointing et al. 1996). Ionizing
radiation has always been a part of our environment, but the natural
sources of ionizing radiation emitted on Earth terrestrial environment and
from the cosmic are very low. The average worldwide background dose is
2.4 millisievert (mSv) per year, which is not detectable in human cell
(http://www.who.int/ionizing_radiation/en/).
Gamma radiation (denoted as ! ) is one of three natural
radiations formed by radioactive atoms. Unstable atomic nucleus
transform to a more stable atom by emitting ionizing particles. Unlike
alpha and beta radiation, gamma radiation is penetrating, causing diffuse
damage throughout living cell (http://www.who.int/ionizing_radiation/en/).
57
Their ionizing power can producing free radicals, breaking chemical

bonds, producing new chemical bonds and cross-linkage between
marcomolecules, and damaging molecules that regulate vital cell
processes
(Upton
1982;
http://web.princeton.edu/sites/ehs/osradtraining/biologicaleffects/page.ht
m). At low doses, cells can undergo cellular repair processes rapidly.
Conversely, at extreme high doses, when cells cannot be replaced
immediately, ionizing radiation will lead to cell death and therefore tissue
failure. Furthermore, it can also cause chronic damage, such as mutation
in
cells
that
may
lead
to
cancer
(http://web.princeton.edu/sites/ehs/osradtraining/biologicaleffects/page.ht
m; http://www.who.int/ionizing_radiation/en/).
Although the background doses of ionizing radiation on Earth
is very low; there are organisms from bacteria and archaea domains that
can tolerate extreme level of ionizing radiation (Rainey et al. 2005) (Table
3.1). Their tolerance level for radiation varies between species (Battista
and Rainey 2001; Rainey et al. 2005). In 2005, Soil samples from the
Sonoran Desert were collected and exposed to various doses of gamma
radiation between 0kGy and 30kGy (Rainey et al.). Results showed that
several strains of Deincoccus, Geodermatophilus and Hymenobacter
were isolated as the dose of gamma radiation to which the soils were
exposed increased. Ionizing radiation tolerant organisms are not only
limited to arid environments, they have been recovered from a wide range
of non-arid environments including sewage (Ito et al. 1983), dried food
(Lewis 1973; Maxcy and Rowley 1978), textiles (Kristensen and
58
Christensen 1981), high level of nuclear waste (Philips et al. 2002;

Fredrickson et al. 2004), thermally polluted water (Carreto et al. 1996). It
is interesting to note that many of these environments were considered
dry or desiccated. On the other hand, since damage induced by ionizing
radiation is similar to the one induced by desiccation, DNA repair capacity
of a radiotolerant species affects the capability of surviving dehydration
as well (Maxcy and Rowley 1978). Since the background ionizing
radiation on Earth is low, it has been postulated that radioresistant quality
is not a result of evolutionary adaptation to radiation (Mattimore and
Battista 1996), but rather to compensate for desiccation. Consequently, it
is assumed that desiccation tolerant species are also ionizing radiation
tolerant species.
Ionizing radiation and desiccation can break the double strand
in
DNA
(Dose
et
al.
1992;
Mattimore
and
Battista
1996).
Chroococcidiopsis and Deinococcus are the few species that were able to
repair such damages. The resistance ability of Chroococcidiopsis is not
comparable to Deinococcus, and therefore, Deinococcus were studied
intensively for their DNA repair mechanism (Zahradka et al. 2006). They
can carry out a two-stage DNA repair process. This process involved
extended synthesis-dependant stand annealing (ESDSA), followed and
completed by crossovers. Chromosomal fragments with overlapping
homologies are used both as primer and a template for massive
synthesis of complementary single strands, as occurs in a single-round
multiplex polymerase chain reaction. Products were then anneal
contiguous
DNA
fragments
into
59
long,
linear,
double
stranded
intermediates and further mature into circular chromosomes (Zahradka et

al. 2006).
Apart from DNA repair mechanism, some microorganisms can
produce sunscreen to protect them from high UV level. For example,
mycosporine-like amino acids in all Cyanobacteria, fungi, and lichens
(Evans et al. 2001); scytonemin in Cyanobacteria (Garcia-Pichel and
Castenholz 1991; Bdel et al. 1997); Melanins in black fungi (Prota
1999); phenolic compounds in lichens (Rikkinen 1995) and anthocyanins
in mosses (Post 1990). Extracelllular polysaccharides may also offer UV
protection (Ehling-Schulz and Scherer 1999). These substances were
concentrated on the parts that expose to UV, while shaded part contains
less pigmented cell (Bdel et al. 1997).
Behavioral and morphological adaptations are also important
in avoiding UV exposure (Evans et al. 2001). Some microbes can even
work as group to avoiding UV. For Cyanobacteria, heavy pigments
species (e.g. scytonema, Nostoc, Calothrix) often layered on top and
relatively less pigmented species (e.g. Microcoleus) are sheltered, and
glide to the surface when water is available (Evans et al. 2001). Mosses
and lichens often roll up sensitive surface when dry (Evans et al. 2001).
60
Table 3.1. List of radiation tolerant organisms.

Species
Phylum
!-radiation
tolerance
level (kGy)
Bacteria
Actinobacteria
9
Brooks and Murray 1981;

Rainey et al. 1997, 2005
!-Proteobacteria
15
Green and Bousfield 1983;

Ito and Iizuka 1971; Rainey
et al. 2005
"-Proteobacteria
<1
Nishimura et al. 1994
"-Proteobacteria
0.1
DiRuggiero et al. 1997
Actinobacteria
25
Ferreira et al. 1999
Actinobacteria
15
Ferreira et al. 1999
Cyanobacteria
15
Billi et al. 2000
1.75
Collins et al. 2000
Not specify
Nicholson et al. 2000
Kocuria rosea
Methylobacterium
radiotolerans
Acinetobacter
radioresistens
E. colli
Rubrobacter
xylanophilus
Rubrobacter
radiotolerans
Chroococcidiopsis
sp.
Hymenobacter
actinosclerus
Bacillus sp.
Deinococcus
radiodurans
Deinococcus
proteolyticus
Deinococcus
radiophilus
Deinococcus
radiopugnans
Deinococcus grandis
Deinococcus
geothemalis
Deinococcus murrayi
Kineococcus
radiotolerans
Deinococcus
hohokamensis
Deinococcus
navajonenis
Deinococcus
hopiensis
Deinococcus
apachensis
Deinococcus
maricopensis
Deinococcus
pimensis
Deinococcus
yavapaienssis
Deinococcus
papapogonensis
Deinococcus
sonorensis
Reference
FlexibacterCytophagaBacteroides
Firmicutes
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
Actinobacteria
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
Battista and Rainey 2001

Not specify
Not specify
Not specify
Not specify
Not specify
Not specify
Philips et al. 2002
30
Rainey et al. 2005
25
Rainey et al. 2005
25
Rainey et al. 2005
17
Rainey et al. 2005
13
Rainey et al. 2005
30
Rainey et al. 2005
30
Rainey et al. 2005
30
Rainey et al. 2005
25
Rainey et al. 2005
61
Arthrobacter sp.
Friedmanniella sp.
Geodermatophilus
sp.
Nocardioides sp.
Bosea sp.
Chelatococcus sp.
Corbulabacter sp.
Planococcus sp.
Sphingomonadacea
e sp.
Spirosoma sp.
Deinococcus
radiomollis
Deinococcus
claudionis
Deinococcus
altitudinis
Deinococcus
alpinitundrae
Truepera radiovictrix
Actinobacteria
Actinobacteria
0-3
0-3
Rainey et al. 2005

Rainey et al. 2005
Actinobacteria
0-30
Rainey et al. 2005
Actinobacteria
!-Proteobacteria
!-Proteobacteria
!-Proteobacteria
Firmicutes
0-3
5-9
5-15
5-15
5-9
Rainey et al. 2005

Rainey et al. 2005
Rainey et al. 2005
Rainey et al. 2005
Rainey et al. 2005
!-Proteobacteria
5-15
Rainey et al. 2005
5-9
Rainey et al. 2005
Not specify
Callegan et al. 2008
Not specify
Not specify
Not specify
Albuquerque et al. 2005
Bacteroidetes/Chl
orobi
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
DeinococcusThermus
Archaea
Desulfurococcus
amylolyticus
Thermococcus
stetteri
Pyrococcus furiosus
Pyrococcus abyssi
Thermococcus
gammtolerans
Thermococcus
radiotolerans
Thermococcus
marinus
Crenarchaeota
1.2-2.5
Kopylov et al. 1993
Euryarchaeota
1.2-2.5
Kopylov et al. 1993
Euryarchaeota
Euryarchaeota
2.5
2.5
Jolivet et al. 2003a

DiRuggiero et al. 1997
Euryarchaeota
30
Jolivet et al. 2003b
Euryarchaeota
30
Jolivet et al. 2004
Euryarchaeota
20
Jolivet et al. 2004
Eukarya
Corollospora
maritima
Zallerion maritimum
Aspergillus niger
Aureobasidium
pullulans
Chaetomium
globosum
Letographium
procerum
Eutardigrade
Richtersius coronifer
Human
Fungi
3.1
Pointing et al. 1996
Fungi
Fungi
3.1
6

Fungi
13.05
Fungi
Fungi
10.1
Animal
<1
Jnsson et al. 2005
Animal
01-1 Gy
62
Sutherland et al. 2000
The Tibetan high altitude deserts are characterized by their low

moisture and extreme UV radiation. Even though the ionzing radiation
level on Mars are magnitude higher than Earth, Tibetan deserts serve as
the closest analogs of Mars surface. Understanding the abundance and
phylogenetic diversity of ionizing radiation bacteria in Tibet arid
environment would provide useful insight for the modern microbial
ecology and maybe correlate the possible link between ionizing radiation
resistances and desiccation resistances.. In chapter 2, result shows that a
wide
range
of
bacteria,
including
Cyanobacteria
proteobacteria,
Bacteroidetes and Gammatimonadetes, would survive in Tibet and not

necessarily has to be Deinococcus. It is interesting to see whether these
microbs can survive after expose to high gamma radiation. This provides
the wider implication of identifying microorganism that is potentially able
to tolerant extreme conditions of aridity and UV radiation, on Mars surface
has value to astrobiology research.
Objectives
In this study, I set out to isolate ionizing radiation resistant
bacteria from Tibet desert at different altitudes, where the highest level of
UV radiation could be observed on Earth. This study provided further
insight into the diversity of ionizing tolerant organisms from high altitude
arid soil and their relationship to varies radiation level. This research also
describes the new taxa that were not previously reported as ionizing
tolerant organisms and possibly new species of the genus Deinococcus.
63

3.2.1 Site description
The Tibet plateau is located west of Mainland China and north
of Nepal. As a result of two continental plates collided together, Tibet has
some of the tallest mountains, including Mount Everest, they formed the
tallest and largest region in the world today and in all geologic history.
The atmosphere in Tibet plateau is severely dry, due to the
blocking of ocean moisture by surrounding mountains. The annual
precipitation is only 400-500 mm (rainfall and snowfall) with a huge
temperature differences within a day (Chang 1981). With the high altitude
level, UV radiation received from the sun is enormously intense
compared to other areas on Earth. The UV level can be up to 2.53
mW/cm2 (UV-A) and 1.27 W/cm2 (UV-B) in Tibet area (Wong et al. 2010).
As UV radiation is correlated to altitude level, with every 1000m increase
in altitude, UV radiation level increased 10-15% (Blumthaler et al. 1997).
Therefore, the radiation level of highest sampling point in this study is
about 201% more than sea level.

Surface (upper 2 cm) soil samples were collected using a
sterile scoop in a sparsely vegetated area at 4 different altitude levels;
they include 4638m, 4810m, 5240m and 5520m. The soil was stored
aseptically at ambient temperature until they were processed. A map of
sampling sites was shown in Figure 3.1.
64
Figure 3.1. Map of sampling sites.

3.2.3 Total organic content and radiation test
2g of soil samples were weighted and then combusted in
furnace 450C for two hours. The total organic content was revealed by
the weight differences after combustion.
Another portion of 2g samples were gamma irradiated at a
commercial sterilization facility (Isotron plc, Swindon, UK), using a
60
Co
source, whist maintaining aseptic technique in style transfer throughout.
3.2.4 Culture and Isolation

After exposed to various levels of radiation, the samples were
serially diluted and plated on nutrient agar (Oxoid Ltd., Basingstoke,
Hampshire, England). Plates were incubated at 5C and 20C for 5 days.
The number of CFU per g soil was determined after 5 days of incubation.
65
Very few Colonies were observed from the 5C plates. However, plates
were discarded due to the slow growth rate, Selected colonies from 20C
purified phylogenetic analysis.
3.2.5 Analytical Profile Index (API)

API 20 (NE system) (Analytab Products, Inc., Planview, N.Y.)
was used for testing biochemistry of the surviving cultures. They included
tests for reduction of nitrate, indoles production, fermentation, Arginine
DiHydrolase, UREase, hydrolysis of esculin, hydrolysis of gelatin, !galactosidase and oxidase, as well as 12 asimmilation tests (glucose,
arabinose, manose, manitol, N-Acetyl-Glucosamine, maltose, potassium
gluconate, capric acid, adipic acid, malate, trisodium citrate and
phenyiacetic acid). Manufactured protocol was followed.
3.2.6 Cloning
Sequences were obtained from clone libraries that described in
Section 2.2.5.
3.2.7 Sequence assembly and alignment

All clones were sequenced using the BigDye Terminator Cycle
Sequencing kit (Applied Biosystems, California) and performed by
Applied Biosystems 3730 Genetic Analyzer. Sequences were manually
refined by BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and
then aligned using the Clustal W.
66
All aligned sequences generated by this study have been

deposited in the NCBI GenBank database. Approximate phylogentic
affiliations were then determined by BLAST searches of the NCBI
GenBank database (http://www.ncbi.nim.nic.gov).
3.2.8 Phylogenetic analysis

Phylogenetic analysis were conducted using PAUP* 4.0b8
(Swofford 2001) (model selection GTR+I+G). Model sequence of
evolution that best fit the data by hierarchical likelihood ratio test (hLRTs)
was first generated using MODELTEST v.3.06 program (Posada and
Crandall 1998). Based on this model, maximum likelihood trees were
generated with a starting tree obtained by stepwise addition, sequence
addition taken as-is and tree bisection-reconnection (TBR) branch
swapping. Gaps were treated as missing data. Nonparametric bootstrap
support (Felsentein 1985; Sanderson 1989) was calculated for each
internal node with a simple stepwise sequence addition, TBR branch
swapping and 1000 replicates. Baysenian posterior probablities were also
calculated. Phylogenetic trees were drawn using TREEVIEW (Page
1996).
67
3.3 Results
Four sets of soil samples were collected from Tibet plateau at
different altitudes (4638m, 4810m, 5240m and 5520m). The mean total
organic contents of the samples showed a negative trend correlate to
altitude.
&$"!#
89$"()9&5",'#)#9,$%,$)0:6)
'"!!#
&!"!#
&"$!#
%$"!#
&"!!#
%"$!#
%!"!#
%"!!#
$"!#
!"$!#
!"!!#
!"#$%&'"()"*+,-",#%)./,)01234567)
'"$!#
!"!#
()'*+#
(*%!+#
$&(!+#
$$&!+#
;($'$+-%)
Figure 3.2. Averaged total organic content and bacterial abundance of

samples collected from different altitudes.
Similarly, a negative trend was observed for the amount of cell

counts (fig. 3.2). A set of non-irritated samples was used as a control. For
un-irradiated samples, about the same value of CFU/g were observed at
different altitudes. They ranged from 1.2x108 to 2.32x 109 CFU/g There
was a decrease in the number of CFU/g recovered from the soil samples
with increasing doses of gamma irradiation, and this was observed all
altitudes, except for 4810m. At 15kGy level of radiation, samples from
68
4638m, 5240m and 5520m were failed to produce colonies, but soil from
4810m maintained the CFU/g to 7.21x104.
&$"!#
'()$*#
'+%!*#
$&'!*#
$$&!*#
!"#$%&'()*#
&!"!#
%$"!#
%!"!#
$"!#
!"!#
!#
$#
%!#
+,--,#.,/0,102"#345267.3#$8+9*#
%$#
Figure 3.3. Survival of bacteria in soil after exposed to various level of

gamma radiations.
A total of 19 ionizing radiation tolerant isolates were cultivated
from soil samples. Partial 16S rRNA gene sequences were determined
for all isolates recovered from the samples. Comparison of these 16S
rRNA gene sequences to the public databases using the BLAST (blastn)
facility (www.ncbi.nlm.nih.gov/BLAST/) enabled us to assign each isolate
to a taxonomic group at the family or genus level and in some cases
species level. All 19 isolates fell into 6 different taxonomic groups
(Actinobacteria,
Alphaproteobacteria,
Cyanobacteria,
Deinococcus-
Thermus, Firmicutes, Gammaproteobacteria) based on their closest

relatives (Table 3.2). 13 of the isolates were recovered from samples of
69
4638m, 4810m and 5240m that exposed to 5kGy doses. 3 isolates were
recovered from 4810m samples that exposed to 10kGy doses. And only 3
isolates that affiliated with the Deinococci recovered from 4810m samples
that exposed to 15-kGy dose of gamma radiation.
The morphological and chemical properties of selective clones
are further described in Table 3.3 and 3.4.
Figure 3.4. Retrieved Deinococcus from 4810m soils exposed to 15 kGy

gamma radiation.
70
Table 3.2. Identities of clones recovered after various gamma radiations.

Genbank
accession
number
Altitude
(m)
R24
HQ144151
4638
R25
HQ144152
4638
R07
HQ144153
R16-1
Code
Gamma
radiation
level (kGy) Putation Phylum

Identity
Accession
Number
Firmicutes
Bacillus sp. A1(2008)
FJ535468
99
mine soil
Actinobacteria
Arthrobacter sp. Z63zhy
AM412214
99
deep-sea sediment
4810
Deinococcus-Thermus
Deinococcus ficus
AY941086
98
culture
HQ144154
5240
Cyanobacteria
cf. Leptolyngbya sp. Greenland_9
DQ431004
88
hot spring
R16-2
HQ144155
5240
Actinobacteria
Micrococcus sp. MOLA 4
AM990780
99
sea water
R16-3
HQ144156
5240
Cyanobacteria
DQ431004
88
hot spring
R16-4
HQ144157
5240
Actinobacteria
Micrococcus sp. TPR14
EU373424
99
root
R16-5
HQ144158
5240
Actinobacteria
Micrococcus sp. MOLA 4
AM990780
99
sea water
R18-1
HQ144159
5240
Alphaproteobacteria
Sphingomonas sp. SKJH-30
AY749436
99
culture
R18-2
HQ144160
5240
Alphaproteobacteria
AY749436
99
culture
R18-4
HQ144161
5240
Alphaproteobacteria
AY749436
99
culture
R18-5
HQ144162
5240
Gammaproteobacteria
Pseudomonas sp. PCRP7(2)
EU255304
99
soil
R30
HQ144163
5520
Actinobacteria
Micrococcus sp. G541
EU086818
98
culture
R09
HQ144164
4810
10
Firmicutes
Planomicrobium sp. ISL-41
FJ265708
97
culture
R10
HQ144165
4810
10
Deinococcus-Thermus
Deinococcus ficus
AY941086
98
culture
R11
HQ144166
4810
10
Firmicutes
Planococcus sp. Tibet-IX21
DQ177487
97
permafrost
R12
HQ144167
4810
15
Deinococcus-Thermus
Deinococcus ficus
AY941086
97
culture
R13
HQ144168
4810
15
Deinococcus-Thermus
Deinococcus ficus
AY941086
98
culture
R14
HQ144169
4810
15
Deinococcus-Thermus
Deinococcus ficus
AY941086
99
culture
71
Percent
Source
similarity
Table 3.3. Morphology description of selected clones.
72
Table 3.4. Chemical properties of selected clones. (NA= not available)
73
Figure 3.5. Phylogenetic tree of the survived samples from DeinococcusThermus group based upon Maximum Likelihood analysis of full 16S
rRNA gene sequence data. Sequence codes refer to those given in Table
3.1 Rooted tree supported by bootstrap values for 1000 replicates (first
number) and Bayesian posterior probabilities (second number). Scale bar
represent nucleotide change per position.
18 out of 19 recovered sequences were then further analyzed
phylogeneticlly. 5 different phylogenetic trees were presented here to
revealed five distinct groups: they were Deinococcus-Thermus (fig. 3.5),
Proteobacteria (fig. 3.6), Cyanobacteria (fig. 3.7), Firmicutes (fig. 3.8) and
Actinobacteria (fig. 3.9). Out of the 18 sequences, 5 formed a closely
related to Deincoccus ficus sp. that was isolated from rhizosphere of ficus
tree (Lai et al. 2006).
74
Figure 3.6. Phylogenetic tree of the survived samples from Proteobacteria

group based upon Maximum Likelihood analysis of full 16S rRNA gene
sequence data. Sequence codes refer to those given in Table 3.1 Rooted
tree supported by bootstrap values for 1000 replicates (first number) and
Bayesian posterior probabilities (second number). Scale bar represent
nucleotide change per position.
Figure 3.7. Phylogenetic tree of the survived samples from Cyanobacteria

75
Figure 3.8. Phylogenetic tree of the survived samples from Firmicutes

Figure 3.9. Phylogenetic tree of the survived samples from Actinobacteria

Actinobacteria
Actinobacteria is a high GC, gram-positive bacteria group that
is commonly found in soils (Kuske et al. 1997; Dunbar et al. 1999;
Macrae et al. 2000), it is because they play an important role in carbon
cycling in soil ecosystem (Nagy et al. 2005; Gundlapally and GarciaPichel 2006).
Actinobacteria were also found as a major component in

76
other extreme desert environment. (Atacama Desert soils and Antarctica

soil) (Connon et al. 2007, Niederberger et al. 2008). Four clones were
isolated from 4638m, 5240m and 5520m soil samples that exposed to 5
kGy of gamma radiation. The 16S rRNA gene analysis showed that they
were closely affiliated to Arthrobacter sp. and Micrococcus sp..
Many strains of Arthrobacter were described in several studies
about their resistance to gamma radiation (Yoshinaka et al. 1973; Suzuki
et al. 1988; Rainey et al. 2005) and therefore some strains were
reclassified to new genus Rubrobacter. This taxa was abundant in many
arid soil (Chanel et al. 2005), They can survive under 0-5 kGy of gamma
radiation but not in high doses.
Micrococcus can survive with little water and it was previously
recovered from non-arid soil that exposed to ionizing radiation between 3
and 13 kGy (Rainey et al. 2005). In this experiment, it is the only
cultivated microorganism collected from the highest altitude sample site
(5520m).
Firmicutes
Firmicutes were commonly found in soil and arid soil
environments (Chanel et al. 2005). Furthermore, they could produce
endospores, which are resistant to desiccation, and survive under
extreme conditions. Three clones of Firmcutes were isolated from site
4638m and 4810m. Sample R-9 was omitted from the phylogenetic
analysis due to the poor quality in sequencing. Sample R-09 and R-11
were closely affiliated with Planomicrobium/Planococcus sp.. Sample R-
77
24 is closely affiliated with Bacillus sp.. They all were previously isolated
from extreme environments, such as permafrost and mine soil.
Bacillus class was well documented that they could tolerate
extreme level of UV radiation (Newcombe et al. 2005). They can form
resistant spore to survive the most extreme terrestrial environment
(Nicholson et al. 2000). They were isolated from arid and non-arid
environments (Chanal et al. 2005), spacecraft and their associated
assembly facilities (Newcombe et al. 2005).
In this study, clones that phylogenically closed to Planococcus
and Planomicrobium could survive up to 10kGy. Planococcus was first
described as radiation tolerant in 2005 (Rainey et al.), whereas
Planomicrobium was never reported as radiation tolerated.
Proteobacteria
Only Alpha- and Gamma- sub-phyla of proteobacteria clones
from 5240 samples survived after being exposed to 5 kGy of gamma
radiation.
Previous study indicated proteobacteria was the most
prevalent group in extreme desert environments (Connon et al. 2007).

Several strains were discovered their radiation tolerant ability.
For example, Methylobacterium and Chelatococcus (Rainey et al. 2005).
Sphingomonas sp. SKJH-30 is the only phylotype of Alphaproteobacteria
retrieved from the soils. Sphingomonas sp. is a gram-negative, rod
shape, chemohetetropic bacterium that was also found in extreme deepsea hydrothermal vent environment (Naganuma et al. 2007). Their family
78
Sphngomonadaceae was reportedly resistant to high doses of radiation of

up to 15 kGy (Rainey et al. 2005).
The Gammaproteobacteria clone is closely affiliated with
Pseudomonas sp. PCRP7 (2). It is also a gram-negative rod shape
bacteria found in high altitude soil (Selvakumar et al. 2009).
Pseudomonas was very susceptible to low-intensity ionizing radiation (Ito
and Iizuka 1971).
Cyanobacteria
Cyanobacteria are important components in desert soil
ecosystem. It could carry out photosynthesis and secrete extracellular
polysaccharides, which would help mount the bare soil together and
formed desert biotic soil crust (Eldridge and Greene 1994). Their abilities
in producing EPS were discussed to be related to their capacity of
resisting desiccation (Billi and Potts 2000; Potts 1996). They could
regulate the water content within the cell and protect the cell wall from
damage during swelling and shrinkage (Grilli Caiola et al. 1993, 1996).
They dominated the most extreme arid habitats in hot and cold deserts
(Palmer and Friedmann 1990; de Chazal et al. 1992; Nienow and
Friedmann 1993).
Cyanobacteria can tolerate desiccation as well as high doses
of ionizing radiation (Potts 1999; Billi et al. 2000). Cyanobacteria also
have been studied extensively for the ability to tolerate UV radiation
(Garcia-Pichel and Castenholz 1999; Ehling-Schulz and Scherer 2009).
The limit for gamma radiation Chroococcidiopsis was tested in previous
79
study (Billi et al. 2000) Two Cyanobacteria clones were recovered from
5240m sample that exposed to 5kGy of radiation. They are both closely
affiliated to an oscillatoriales cyanobacterium --- cf. Leptolyngbya sp.
Greenland_9.. This is the first report of Leptolyngbya!s radiation
tolerance.
Deinococcus-Thermus
The 16S rRNA gene analysis clearly showed that the isolates
that could survive highest doses (15 kGy) of gamma radiation belong to
the Deinococcus Thermus group. The Deinococci are a small family of
non-spore-forming bacteria that exhibit a unique characteristic which is
being able to tolerant high level of either gamma (greater than 25kGy), or
UV radiation, or both (Murray 1992; Battista 1997; Ferreira et al. 1997,
2000; Fredrickson et al. 2004; Suresh et al. 2004). They were intensively
studied for their two-stage process of DNA repair mechanism (Zahradka
et al. 2006) for desiccation and radiation. So far, at least 29 species were
recognized. D. alpinitundrae, D. altitudinis, D. apachensis, D.claudionis,
D. frigens, D. geothemalis, D. grandis, D. hohokamensis, D. hopiensis,
D. indicus, D. marmoris, D. maricopensis, D. murrayi, D. navajonenis, D.
papapogonensis, D. pimensis, D. proteolyticus, D. radiodurans, D.
radiomollis, D. radiophilus, D. radiopugnans, D.saxicola, D. sonorensis,
D.
yavapaienssis,
and
Truepera
radiovictrix
isolated
from
the
environments have been identified ionizing radiation tolerate (Brooks and

Murray 1981; Oyaizu et al. 1987; Ferrira et al. 1997; Hirsch et al. 2004;
Suresh et al. 2004; Albuquerque et al. 2005; Rainey et al. 2005; Callegan
80
et al. 2008). Particularly D. radiomollis, D.claudionis, D. altitudinis, D.

alpinitundrae were isolated from alpine soil from 3500 to 5060m
(Callegan et al. 2008). The Deinococcus clones cultivated from this study
are another high altitude species of Deinococcus that was reported
radiation sensitive.
5 strains were isolated from 4810m soils were shown mostly
related to species of the genus Deinococcus. These Deinococcus from
this study survived through several levels gamma radiations from 5 to 15
kGy doses. They are Gram positive, aerobic and oxidase-positive, except
for R-10. Colonies on nutrient agar were smooth, pale pinkish, circular
with entire edges, except for R-10 gave a pale yellowish color.
Deinococcus Sample R-12 was selected for further biochemical
description. The following compounds were utilized as sole carbon
sources for sample R-12 (which produce positive results in the API test):
D-glucose, L-arabinose, D-mannitol, N-acetyl-glucosamine, D-maltose,
potassium gluconate, adipic acid, malic acid and trisodium citrate.
According to the gene sequence similarity calculations, the most closely
related strain was Deinococcus ficus (97 to 99%), which was previously
isolated from rhizosphere in temperate region.
81
3.4 Discussion
The desert of Tibet is a high altitude arid environment
characterized by an extreme aridness, large temperature variation and low
organic content in soil. This study expanded our knowledge of the diversity
of ionizing radiation tolerant bacteria in high altitude desert soils. A novel
consortium of extremophiles that tolerated relatively high ionizing radiation
exposure was revealed in this chapter.
Diverse collections of bacteria were cultivated after exposing
samples under varies level of gamma radiation. Several taxa such as,
Leptolyngbya and Planomicrobium were never reported as radiation tolerant
organism. This indicates there maybe many organisms that are able to
tolerate ionizing radiation, but have not yet been identified. Many of them
only can survive relatively low level of gamma radiation (Ito and Iizuka 1971;
Grant and Patterson 1989; DiRuggiero et al. 1997; Jolivet et al. 2003, 2004;
Phillips et al. 2002); however the doses are considered vital to most
organisms (Upton 1982; Sutherland et al. 2000). For this experiment, only
Deinococcus has been to survive extreme doses of ionizing radiation up to
15 kGy.
The total carbon content of the soil samples provides a rough
estimation of available organic nutrients. As altitudes go up, the ultraviolet
radiation from the sun is more intense (Blumthaler et al. 1997). It is assumed
higher altitude environment is harsher than the lower altitude environments
and therefore population of bacterial assemblage is lower at high altitude.
82
Results agreed that high altitude level higher altitude is less habitable than
the lower altitude environments. However, survival of soil bacteria is not
correlated to altitude or organic content level, but related to the presence of
Deincoccus. Where site at 4810m has a more complex of radiation tolerate
community was present. This is suggested Deinococcus is a key genus of
ionizing tolerant community from the environments. Furthermore, the
majority of the culturable population was lost by exposing it to the gamma
radiation. This implied that culturable microorganisms are very sensitive to
radiation. Culturable-independent molecular approaches are important to
microbial diversity study of intense UV environments, as well as
extraterrestrial exploration for life.
In this chapter, wide range of bacteria was isolated, including
Actinobacteria, Cyanobacteria, Deinococcus, Firmicutes and proteobacteria
(alpha and gamma). This assemblage is common to soil environments and
other arid environments (Chanel et al. 2005). After exposing these microbes
to gamma radiation, further approved that radiotolerant bacteria are not
limited to Deinococcus. In 2005, similar study was done in Sonoran Desert
(Rainey et al.), only Actinobacteria, Alphaproteobacteria, Bacteriodetes and
Firmicutes were survived after exposed to gamma radiation. A more diverse
radiotolerant bacteria assemblage in Tibetan Plateau indicate high altitude
deserts are unique from other arid environments. It is expected a more
complex and yet radiation tolerant communities were needed to support the
ecosystem in high altitude arid environments. Moreover, it is interesting to
83
note that no Deinococcus phylotypes were recovered from the bacterial

clone library constructed from previous chapter, but several isolates were
discovered in this study. this may indicate that Deinococcus taxa are
particular low abundance in soil and thus PCR recovery bias affects their
recovery in clone libraries.
The Tibet high altitude desert is one of the oldest desert
environments on Earth. It is characterized with its extreme aridity and
tremendously high level of UV radiation. In such extreme condition,
vegetation in Tibet plateau is limited and majority of landscape are covered
by deserts soil. On the other hand, current knowledge of Mars brought back
by Phoenix Mars Mission (http://phoenix.lpl.arizona.edu) presented a similar
environments observed in Mars. Extremely arid environment bombarded
with high level of UV radiation that are similar to early Earth situation.
Therefore, the Tibet high altitude deserts provide an analogue to explore the
limit of life under Mars-like conditions. Radiation and desiccation tolerant
species Deinococcus isolated from this study may suggest the possible type
of organism scientist could expect from Mars. Their DNA repair mechanism
implied the possible life strategy on Mars as well as on early Earth. These
mechanisms also suggested future medical solution of human disease
regarding increasing UV radiation level on Earth. The isolates of
Cyanobacteria Leptolyngbya expanded our knowledge of radiation tolerance
of phototrophs, which is fundamental indication for origin of life and evolution
of higher plant evolution from such microorganism. Whilst the strategies for
84
radiation tolerance and repair are well documented and investigated for the
Deinococci (Mattimore and Battista 1996; Battista 1997), and to a lesser
extent for the Cyanobacterial genus Chroococcidiopsis (Potts 1999; Billi et
al. 2000), relatively little is known about the tolerance and repair status for
other bacterial phyla. Some investigators have suggested that Deinococci
and Chroococidiopsis share a similar pathway for repair (Billi et al. 2000),
and of particular interest would be to investigate taxa such as Rubrobacter
which have also been isolated form other desert environments (Rainey et al.
2005), to see if they share this pathway. If so, this may offer insight into
possible
lateral
gene
transfer
involvement
radiation/desiccation repair pathway.
85
in
aquisition
of
the
3.5 Conclusion
In this study, ionizing resistant bacteria of high altitude deserts on
the Tibet Plateau were isolated and characterized.
A total 19 isolates spanning 6 phyla were detected from the irradiated
soils. Many of them were previously reported as ionizing radiation tolerant,
whereas Planomicrobium and Leptolyngbya were first described as ionizing
tolerant in this experiment.
The Cyanobacteria and proteobacterial taxa tolerated doses of up to 5
kGy, whereas higher doses up to 15 kGy were tolerated only by
Deinococcus isolates.
86
Chapter 4. Molecular
Ecology
of
Freshwater
Microbialite
Structures in a Carbonate-rich Lake in British Columbia,

Canada.
4.1 Introduction
4.1.1 Microbialites as a living fossil
Microbialites are organosedimentary carbonate structures
formed in shallow water by trapping binding of sediment and/or the net
carbonate-precipitating activities of benthic microbial communities (Burne
and Moore 1987; Pierson 1992). Some evidences indicate abiotic
processes may also contribute to the formation of microbialites (Walter
1976; Riding 1990; Grotzinger and Rothman 1996; Sunmer and
Grotzinger 1996, 2000; Riding and Awramik 2000; Storrie-Lombardi et al.
2004),
but
the
exact
evolutionary
mechanism
remains
largely
uncharacterized.
Stromatolites are a laminated form of microbialites which were
the most abundant life form will origins some 3.5~3.8 billion years ago
(fig. 4.1a). They thrived for almost 85% of the Earths history and played a
major role in evolutionary history. They were instrumental in consuming
the carbon dioxide in the early Earth atmosphere and gives out free
oxygen and hydrogen gases (Hoehler et al. 2001). Microbialites consist of
a diverse and complex community of bacteria and algae, which are
dominated by Cyanobacteria.
Stromatolites populations decreased dramatically by the late
Neoprotezrozic (Awramik 1981, Walter et al. 1992), and are relatively rare
87
nowadays (fig.4.1b). Nevertheless, modern living microbialites are still

active in several biogeochemical cycles (Stal 2000). They involved active
recycling of carbon, nitrogen, oxygen and sulfur (Pinckney et al. 1995).
Studying these microbial mediated precipitation and dissolution of
calcium carbonate may improve our ability to interpret the modern
environmental
issues
such
as
global
warming.
These
modern
microbialites structures provide information to interpret their fossil

counterparts and for understanding microbial lithification processes
(Riding 2000). They are not only the proxies for ancient ecosystems; they
also provide potential aid for searching possible extraterrestrial life on
Mars.
Figure 4.1a (Top). Cross-sectional view of an ancient stromatolite in

Medicine Bow National Forest, Laramie, WY. Photo credit of Kerri
Lathrop. Figure 4.1b (bottom). Modern stromatolites in Shark Bay,
Western Australia. Scale bar (bottom right) approximately 1 m. Inset:
example of surface stromatolite sample (Burns et al. 2009).
88
4.1.2. Microbialites in Pavilion Lake

Microbialites are mostly found in shallow marine areas and
hypersaline lakes. Extensive studies were carried out in Shark Bay West
Australia (fig. 4.1b) (Burns et al. 2009; Goh et al. 2009) and Highborne
Cay, Bahamas (Foster et al. 2009). These environments normally were
defined as extreme for most living organisms. Recently, a unique
freshwater habitat, a high carbonate depositing lakes in British Columbia,
has been shown to support microbialites. This introduces interesting
application in astrobiology since carbonate-rich water is thought to have
persisted during Mars late water phase (Squyres et al. 2004).
4.1.3. Objectives
A large multidisciplinary study has set out to understand the
biogenesis, ecology and geobiological interaction of microbialite reefs in
Pavilion Lake. However, this was restricted to traditional microscopic,
mineralogical and biogeochemical analyses (Lavel et al. 2000). Very little
molecular analysis has been carried out for Pavilion Lake Research
Project, although this technique has become standard in microbial
ecology. Therefore, in this study, the microbial communities of the
microbialites from Pavilion Lake were characterized by 16S ribosomal
RNA genes amplified from environmental DNA directly retrieved by
samples. Sequenced based methods have yielded both quantitative and
qualitative information to resolve the community structure, function and
spatio-temporal
variation
within
89
these
microbialites.

4.2.1 Site location
Pavilion Lake was first described and studied by Lavel et al.
(2000). It is located at south of British Columbia, approximately 420km
northeast of Vancouver (+505157.00, -121 4420.00) (fig. 4.2).
Pavilion Lake is a small, deep and elongated (5.7 x 0.8 km) lake nestled
in a steep-walled limestone valley known as Marble Canyon. It is
dominated by karst hydrology with no surface stream entrance. Pavilion
Lake water is very clear with a 15m average Secchi depth and with
slightly alkaline water chemistry (pH 8).
Figure 4.2. The drawing of Pavilion Lake and the map showing the
location of Pavilion Lake in British Columbia, Canada (Lavel et al. 2000).
The Inset, side-scan sonar image showing that unique assemblages of
microbialites occur along the edges of the basins in finger-like fields
orientated roughly perpendicular to the shoreline. (Lavel et al. 2000)
90
Table 4.1 Locations of Willow Point and Three Poles.

Transect
Latitude
Longitude
Willow Point
5051'54.75"N
12144'47.48"W
Three Poles
5052'12.06"N
12144'23.15"W
Figure 4.3a (top). Picture of Pavilion Lake surrounded by Marble Canyon.

Figure 4.3b (bottom). Diving operation in Pavilion Lake.
91

Sampling trips were undertaken in July 2007, April 2008
and August 2008. Diver-recovered samples were taken along two
transects, at two opposite sides of the lake (Willow Point and Three
Poles) (Table 4.1), and microbialites were collected from each type of
structure. Microbialites in Willow Point were not included in the seasonal
study since the site was still covered by floating ice during the sampling
trip in April 2008.
Samples (Table 4.2) were dissected and stored in an
Eppendoff (New York, USA) 1.5ml microcentrifuge tubes with RNAlater
solution (Ambion, California, USA). Samples were then kept at -20C in
darkness and shipped to The University of Hong Kong by FedEx Next
Day Delivery for further analyses.
92
Table 4.2. The list of samples that was included in this study.
93
Figure 4.4. A schematic cross-section of Pavilion Lake from Willow Point

to Three Poles.
4.2.3 Microscopy
Microscopic examination of the mat was carried out using
either
Olympus
SZH10
stereomicroscope
or
BX
50
compound
microscope with differential interference contrast optics, both fitted with

an Olympus DP11 digital camera. Cyanobacterial morphotypes were
identified to familial level with reference to Bergeys Manual of Systematic
Bacteriology
(http://www.archive.org/stream/bergeysmanualofd1957amer#page/n5/mo
de/2up).
4.2.4 DNA extraction and polymerase chain reaction (PCR) amplification

DNA extraction was achieved using the MO BIO Powersoil
DNA Isolation Kit (http://www.mobio.com/) following the manufacturers
94
instructions. Final product was eluded in 100!l distilled water and kept in
4C. To visualize the extracted DNA, gel electrophoresis (1% agarose)
with ethidium bromide (EtBr) staining was run at 90 volts for 20 mins.
The 16S rRNA genes were amplified by polymerase chain
reaction using a pair of universal bacterial primers (8F (5-AGA GTT TGA
TCC TGG CTC AG-3) and 1391R (5-CCG TCA ATT CMT TTG AGT TT3)). A 0.01 to 1.0 !l of DNA template was used in a 50!l polymerase
chain reaction (PCR) mixture (1.5mM MgCl2, 0.2 mM of each
deoxynucleoside triphosphate (dNTP), 0.3 !l of each primer pair and
1.0U of taq DNA polymerase). Thermal cycling conditions were as
follows: denaturation at 94"C for 1 min, 55"C for 1 min, 72"C for 1
min, followed by an extension of 72"C for 10 mins. Gel electrophoresis
(1% agarose) with EtBr staining under UV light confirmed the purity of the
amplified product.

Please refer to Section 2.2.4.
4.2.6 Terminal restriction fragment length polymorphism (t-RFLP)

All PCR reactions of samples from three sampling seasons
and their replicates were carried out using a FAM-labeled forward primer
and primer pairs as described in previous section. Gel-purified amplicons
were digested using two restriction enzymes (Cfo1 and Msp1) and the
most informative selected for further analysis (Msp 1 for 16S/18S rRNA,
Hae III for ITS). Fragment analysis was achieved by capillary
95
electrophoresis (Applied Biosystems 3730 Genetic Analyzer), using a

GeneScan ROX-labeled GS500 internal size standard. t-RFLP patterns
and quality were analyzed using the freeware Peak ScannerTM Software
v1.0 provided by Applied Biosystems (http://www.appliedbiosystems.com)
and a data matrix comprised mainly the fragment size and abundance
was generated. The software based on scripts Perl and R were then used
to identify true peaks and bin fragments of similar size according to Abdo
et al. (2006). The relative abundance of a true terminal restriction
fragment within a given t-RFLP pattern was generated as a ratio of the
respective peak area. All t-RFLP GeneScan reads were repeated in
triplicate.

Four bacterial clone libraries were constructed to obtained
phylogenetic identities of the communities; samples for cloning were
selected based upon similarity analysis of t-RFLP data. For Willow Point
and Three Poles clone libraries, 3 representative samples were selected
and pooled based on the relative distance shown in Nonmetric
multidimensional scaling plot of Bray Curtis Similarities for community tRFLP profiles. For the seasonal study, one single sample was picked
from the cluster. Protocols please refer to Section 2.2.5.
96
4.3 Results
4.3.1 Sites and samples morphology description
Site description has been documented over several years by
the Pavilion Lake Research Project team, and my study is part of this
larger research project. The microbialites in Pavilion Lake occur at depths
from 10m to 55m. They have been characterized into shallow to
intermediate (~10-15m), intermediate (~20m), deep (~20-30m) facies
according to distinct changes in their morphologies and internal
microstructures (Lavel et al. 2000). In 2005, a new facies structure was
observed at 46-55m and they are morphologically distinct from the
previously described microbialites. Both porosity and friability of
microbialites decreased with depth.
The shallow to intermediate facies consisted of microbialite
mounds (fig. 4.5a). Their surfaces were dominated by green- and purplepigmented Cyanobacteria (fig. 4.6a). Heterotrophs and diatoms were also
observed on surfaces (fig. 4.6a). Larger (up to 3m tall) and less friable
microbialite domes were found at intermediate depth. The intermediate
facies consist of turret structures on top, with or without a leaf structure at
the base. The turret microbialites often displayed an internal channel up
to 1cm diameter filled with fine grain sediments (fig. 4.5b and 4.5c). It was
suggested that this structure was responsible for groundwater seepage
into the lake (Lavel et al. 2000). The deep facies are extremely dense and
display unique dentritic microstructures (fig. 4.5d). They were proposed to
be analogues for ancient carbonate structures Epiphyton and Girvanella.
Seasonal variations were also observed from microbialites morphology,
97
surface green-pigmented buds grew up to 1-cm in diameter during

budding season (fig. 4.6b).
98
Figure 4.5a. (upper left). Pavilion Lake shallow-intermediate facies

(18m depth). The shallow to intermediate facies consisted of microbialite
mounds. Figure 4.5b. (upper right). Pavilion Lake intermediate facies
(22m depth). The intermediate facies consist of turret structures on top
with or without a leaf structure at the base. Figure 4.5c. (lower left)
Cross-section of intermediate turret structure from Willow Point. A
hollow internal conduit was onserved. Figure 4.5d. (lower right) Deep
facies from 33m depth. Relatively dense calcite leaves from deep watermicrobialite.
99
Figure 4.6a. (top left). Close up picture of shallow to intermediate

facies. Their surfaces were dominated by green and purple-pigmented
Cyanobacteria. Heterotrophs and diatoms were also observed on
surfaces. Figure 4.6b. (top right). Close up picture of microbialite.
Green pigmented cells collected from budding season grew up to 1cm in
diameter. Figure 4.6c. (bottom left). Macroscopic picture of Green
pigmented cell grew up to 1.5cm in diameter. Figure 4.6d. (bottom
right). Macroscopic picture of purple-pigmented cell grew up to
1.2cm in diameter.
100
Figure 4.7a. Light microscopy oscillatorian Cyanobacterial morphotypes

collected from microbialites. (Scale bar 10!m)
Figure 4.7b. Light microscopy of coccoid Cyanobacterial morphotypes

collected from microbialites. (Scale bar 5!m)
101
4.3.2 Real-time quantitative PCR analysis

Real-time quantitative PCR (q-PCR) analysis of selected
samples (table 4.3) was carried out to enumerate abundance of the
various phyla present within the microbialites at different depths.
Numerical data was shown in Table 4.4 and the representative diagrams
were shown in figure 4.8a and 4.8b. The q-PCR data revealed that rDNA
signatures were an order of magnitude higher in young microbialite
exposed in the water column compared to older structures on the lake
bed. The q-PCR data also indicated that bacteria dominate the
community in both locations, typically comprising over 90% of
recoverable rDNA signatures.
Eukarya and archaea accounted for
comparatively low abundance of rDNA signatures in young microbialites.

An interesting observation was that the basal parts of the microbialite
structures at the sediment-water interface supported markedly higher
abundance of archaeal signatures compared to those in the water
column.
Table 4.3. List of selected microbialites samples for q-PCR analysis

Location
Depth(m)
Structure
TP 35F (top)
Three Pole
10.7
Shallow
TP 35F (bottom)
Three Pole
10.7
Shallow
TP 85F (top)
Three Pole
26.0
Deep
TP 85F (bottom)
Three Pole
26.0
Deep
WP 71F (top)
Willow Point
21.6
Intermediate
WP 71F (bottom)
Willow Point
21.6
Intermediate
WP 90F (top)
Willow Point
27.0
Deep
WP 90F (bottom)
Willow Point
27.0
Deep
102
Table 4.4. Results from real-time quantitative PCR analysis. Results were averaged from at least three runs (n/a= not available).
103
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)*+,-.,"
&%!!!"
/,012-3,"
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:!6"
9!6"
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7!6"
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4-05,2,"
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!6"
;<"'#=" ;<"'#=" ;<"9#=" ;<"9#=" D<"8$=" D<"8$=" D<":!=" D<":!="
>1?@A" >B?11?CA" >1?@A" >B?11?CA" >1?@A" >B?11?CA" >1?@A" >B?11?CA"
Figure 4.8a (Top) and Figure 4.8b (bottom). Results from real-time
quantitative PCR (q-PCR) analysis. The absolute abundance of archaeal,
bacteria and eukaryal communities in microbialites (top) and the relative
abundance of archaeal, bacteria and eukaryal communities in
microbialites (bottom).
104
4.3.3 Terminal restriction fragment length polymorphism (tRFLP)

A large number of samples from Three Poles and Willow Point
were collected and screened for bacterial diversity/abundance using
Terminal restriction fragment polymorphism analysis (tRFLP) of the 16S
rRNA gene. Non-metric multidimensional scaling and cluster analysis
were used to visualize relationships between samples.
In Figure 4.9a, the relationship between Three Poles and
Willow Point microbialites at different depths were shown. Two distinct
clusters were grouped according to sample location, indicating a small
but significant difference (P value) between locations. Furthermore,
temporal variation at Three Poles was shown in Figure 4.9b. Samples
from summer season (2008 Aug) and budding season (2008 April) were
analyzed and a clear shift of the microbial communities were observed
from the NMDs plot.
Therefore, representative samples were then
chosen to focus cloning effort in order to obtain a sequenced-based

dataset. Four bacteria clone libraries were constructed to further
characterize the taxa responsible for the spatial and temporal variability.
In view of the low percentage abundance of eukarya and archaea as
determined using q-PCR, all subsequent work focused on bacteria only.
105
Figure 4.9a (top), 4.9b (bottom), Nonmetric multidimensional scaling plot

of Bray Curtis similarities for bacteria rRNA gene recovered from (4.9a)
Willow Point and Three Poles, and (4.9b) summer and budding seasons.
106
4.3.4 Bacterial diversity of microbialites at Pavilion Lake

Clone
library
construction
was
conducted
on
total
community DNA from the microbialites at both sampling locations.

Universal bacterial primers were used for amplifying 16S rDNA.
Representative clones based on 97% sequence similarity cutoff were
sequenced and compared with known sequences by BLAST analysis.
For Willow Point and Three Poles libraries, the size of
bacterial libraries were 246 and 245 clones respectively. 164 and 72
unique phylotype groups were identified from both transects. Another set
of clone libraries were conducted for the temporal study at Three Poles. A
total of 107 clones were obtained for the summer season library, with
another 124 clones were obtained from budding season. 11 distinctive
phylotypes were identified from summer season whereas 30 were
obtained from the budding season (Table 4.5).
The coverage of the clone libraries was assessed by
rarefaction. None of the rarefaction curves for bacterial clone libraries has
reached an asymptote and shown in Figure 4.10 and 4.12 for Willow
Point and Three Poles libraries. These indicated that very diverse
communities were present in both locations. Due to financial and time
constraints, the cloning effort was limited to 250 clones per sample. In
the seasonal study, two library rarefaction curves showed the tendency to
curve downward under the given sampling effort (fig. 4.14 and 4.16). This
indicated that the sizes of clone libraries, although unequal, were
satisfactory to allow the estimation of total OTU richness.
107
Both non-parametric richness estimators ACE and Chao 1

estimations resulted in similar trends for 4 libraries (fig. 4.11, 4.13, 4.15
and 4.17). Since richness curves estimated by Chao 1 were generally
more stable and had better concave-downward shapes, the estimated
richness OTUs was therefore evaluated based on this statistic.
Table 4.5. Coverage summary of clone libraries constructed for
microbialites in Pavilion Lake.
Willow Point
Three Poles
246
245
164
72
Chao 1 richness
162
75
Coverage, %
49
77
93
94
Summer (August)
Budding (April)
107
124
11
30
11.5
34
96
83
97
97
Chao 1 richness
Coverage, %
108
'&!"
!"#$%&'()'*+,-'($-%&.%/'
'%!"
'$!"
'#!"
'!!"
&!"
%!"
$!"
#!"
!"
!"
(!"
'!!"
'(!"
#!!"
#(!"
)!!"
!"#$%&'()'01(2%-'-3#41%/'
Figure 4.10. Plots of rarefaction curves for bacterial clone library of Willow
Point. All curves were averaged over 1000 simulations.
#!!"
!"#$%&'()'*+,-'($-%&.%/'
'&!"
'%!"
'$!"
'#!"
'!!"
*+,"
&!"
+-./"'"
%!"
$!"
#!"
!"
!"
(!"
'!!"
'(!"
#!!"
#(!"
)!!"
!"#$%&'()'01(2%-'-3#41%/'
Figure 4.11. Plots of estimated OTUs richness for bacterial clone library
of Willow Point. All curves were averaged over 1000 simulations.
109
*!"
!"#$%&'()'*+,-'($-%&.%/'
)!"
(!"
'!"
&!"
%!"
$!"
#!"
!"
!"
'!"
#!!"
#'!"
$!!"
$'!"
%!!"
!"#$%&'()'01(2%-'-3#41%/'
Figure 4.12. Plots of rarefaction curves for bacterial clone library of Three
Poles. All curves were averaged over 1000 simulations.
#!!"
!"#$%&'()'*+,-'($-%&.%/'
+!"
*!"
)!"
(!"
'!"
,-."
&!"
-/01"#"
%!"
$!"
#!"
!"
!"
'!"
#!!"
#'!"
$!!"
$'!"
%!!"
!"#$%&'()'01(2%-'-3#41%/'
of Three Poles. All curves were averaged over 1000 simulations.
110
!"#$%&'()'*+,-'($-%&.%/'
'#"
'!"
&"
%"
$"
#"
!"
!"
#!"
$!"
%!"
&!"
'!!"
'#!"
!"#$%&'()'01(2%-'-3#41%/'
Poles in summer season. All curves were averaged over 1000
simulations.
'$"
!"#$%&'()'*+,-'($-%&.%/'
'#"
'!"
&"
()*"
%"
)+,-"'"
$"
#"
!"
!"
#!"
$!"
%!"
&!"
'!!"
'#!"
!"#$%&'()'01(2%-'-3#41%/'
of Three Poles in summer season. All curves were averaged over 1000
simulations.
111
&#"
!"#$%&'()'*+,-'($-%&.%/'
&!"
%#"
%!"
$#"
$!"
#"
!"
!"
#!"
$!"
%!"
&!"
'!!"
'#!"
'$!"
!"#$%&'()'01(2%-'-3#41%/'
Poles in budding season. All curves were averaged over 1000
simulations.
'!"
!"#$%&'()'*+,-'($-%&.%/'
&#"
&!"
%#"
%!"
()*"
$#"
)+,-"$"
$!"
#"
!"
!"
#!"
$!"
%!"
&!"
'!!"
'#!"
'$!"
!"#$%&'()'01(2%-'-3#41%/'
of Three Poles in budding season. All curves were averaged over 1000
simulations.
112
4.3.4.1 Willow Point vs. Three Poles

Detailed descriptions of the closest blast match of sequences
obtained from Willow Point and Three Poles showed in Table 4.6 a-j and
4.7 a-e. Willow Point clone library comprised Proteobacteria (Beta, Delta
and Gamma), Actinobacteria, Bacteroidetes, Chloroflexi, Elusimicrobia,
Firmicutes,
Fusobacteria,
Gemmatimonadetes,
Nitrospirae,
Plantomycetes and Verrucomicrobia. The most abundant phyla were

Alphaproteobacteria (21.5%), Acidobacteria (11.4%) and Cyanobacteria
(13.8%). Three Poles had a less diverse bacterial community. They
included Proteobacteria (Alpha, Beta, Delta and Gamma) Acidobacteria,
Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes,
Nitrospirae, Plantomycetes and Verrucomicrobia. The community was
dominated by Betaproteobacteria (63.1%), in particular, a common
freshwater bactera --- Delftia lacustris. A number of photosynthetic
bacteria (Cyanobacteria, Chloroflexi and Firmicutes) were recorded from
both locations. Pseudoanabaena sp. Oscillatoria sp. and Calothrix sp.,
which have been reported by morphological observations (Lavel et al.
2000) were also recovered from libraries. The graphical illustrations of
relative abundance of Willow Point and Three Poles communities are
shown in Figure 4.18 and 4.19 (inset).
A surprisingly high abundance for the Betaproteobacterium,
Delftia lacustris, was detected from a sample used in clone library
construction from Three Poles. It was concluded that this may have
represented an anomalous sample with contamination from water column
bacteria, therefore, an additional illustration of its community was
113
constructed for comparison. Figure 4.19 is shows the community

structure of Three Poles after Delftia lacustris was omitted. It is interesting
to note that the community structure of Three Poles is fairly similar to the
one in Willow Point after omitting Delftia lacustris. Three Poles was also
dominated by Alphaproteobacteria and Acidobacteria, and then with
comparable percentage of other phyla. As a result, it could be concluded
that the community composition at the phylum level was similar for both
locations, however at the phylogenetically-defined species level, Willow
Point was more diverse than Three Poles.
Phylogenetic trees of all bacteria clones were generated to
further resolve the relationships among bacteria (fig. 4.20- 4.30).
114
Table 4.6a. Identities of 16S rRNA gene sequences obtained from site Willow Point. (ND= Not determined)
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Identity
Acession
Number
Percent
similarity
Source
WP0807B_001
HQ143750
0.8
Alphaproteobacteria
Erythromicrobium ramosum
AB013355
99
ND
WP0807B_002
HQ143751
0.4
Alphaproteobacteria
Hyphomonas sp. MOLA 55
AM990830
99
sea water
WP0807B_003
HQ143752
2.0
Alphaproteobacteria
Rhodobacter sp. HTCC515
AY584573
97
ND
WP0807B_004
HQ143753
0.8
Alphaproteobacteria
Rhodobacter sp. Jip03
AB122032
97
rotten rice straw
WP0807B_005
HQ143754
2.0
Alphaproteobacteria
Sphingomonas sp. BAC310
EU131001
99
ND
WP0807B_006
HQ143755
0.4
Alphaproteobacteria
Sphingomonas sp. V29
AB534590
98
grassland soil
WP0807B_007
HQ143756
0.4
Alphaproteobacteria
Sphingopyxis sp. SM105
EF424407
97
oil-contaminated site
WP0807B_008
HQ143757
0.4
Alphaproteobacteria
alpha proteobacterium J05
FM211709
96
ND
WP0807B_009
HQ143758
0.8
Alphaproteobacteria
CU925753
98
anaerobic digester
WP0807B_010
HQ143759
0.8
Alphaproteobacteria
AM934956
97
WP0807B_011
HQ143760
0.4
Alphaproteobacteria
AM936241
92
WP0807B_012
HQ143761
0.4
Alphaproteobacteria
FM209172
98
hydrocarboncontaminated soil
WP0807B_013
HQ143762
0.4
Alphaproteobacteria
EU361382
89
ocean water
WP0807B_014
HQ143763
0.4
Alphaproteobacteria
DQ070828
93
basalt glass
WP0807B_015
HQ143764
0.4
Alphaproteobacteria
AF445724
94
hot spring
WP0807B_016
HQ143765
0.4
Alphaproteobacteria
AB425062
100
Biofilm
WP0807B_017
HQ143766
0.4
Alphaproteobacteria
uncultured alpha proteobacteria bacterium

clone QEDN7CD03
uncultured alpha proteobacterium clone
AMLD3
CM3H04
delph2D3
HF200_27P07
JdFBGBact_41
SM2D12
uncultured alpha proteobacterium clone 13-90ArvAB
uncultured Green Bay ferromanganous
micronodule bacterium MND8
AF292999
89
freshwater
115
Table 4.6b(cont). Identities of 16S rRNA gene sequences obtained from site Willow Point. (NA= possible putative chimeric sequences.)
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Acession
Number
Percent
similarity
uncultured Hyphomicrobiaceae bacterium

clone D25_45
clone Plot17-2D11
clone TDNP_Wbc97_245_1_118
EU266918
95
tar-oil contaminated
aquifer sediments
EU440683
99
agricultural soil
FJ517066
95
water
Identity
Source
WP0807B_018
HQ143767
0.4
Alphaproteobacteria
WP0807B_019
HQ143768
0.4
Alphaproteobacteria
WP0807B_020
HQ143769
0.4
Alphaproteobacteria
WP0807B_021
HQ143770
0.8
Alphaproteobacteria
uncultured Nitratireductor sp. clone CL3.D14
FM175760
97
rivulet
WP0807B_022
HQ143771
0.4
Alphaproteobacteria
uncultured Nitratireductor sp. clone CL3.D14
FM175760
91
rivulet
WP0807B_023
NA
0.4
Alphaproteobacteria
uncultured Rhizobiales bacterium clone AMIB9 AM935452
100
WP0807B_024
HQ143772
0.4
Alphaproteobacteria
uncultured Rhodobacter sp. clone CL4.E41
FM175921
97
rivulet
WP0807B_025
NA
0.4
Alphaproteobacteria
uncultured Rhodobacter sp. clone SHBZ498
EU639007
89
thermophilic microbial
WP0807B_026
HQ143773
0.4
Alphaproteobacteria
FJ516819
96
biofilm
WP0807B_027
HQ143774
1.2
Alphaproteobacteria
FM209100
97
WP0807B_028
HQ143775
0.4
Alphaproteobacteria
EF018173
89
rhizosphere
WP0807B_029
HQ143776
1.2
Alphaproteobacteria
AM935177
94
WP0807B_030
HQ143777
0.4
Alphaproteobacteria
AM936041
89
WP0807B_031
HQ143778
0.8
Alphaproteobacteria
AM934842
96
WP0807B_032
HQ143779
0.8
Alphaproteobacteria
uncultured Rhodobacteraceae bacterium clone

TDNP_Bbc97_72_2_135
uncultured Rhodobacterales bacterium clone
delph1C11
uncultured Rhodospirillaceae bacterium clone
Amb_16S_515
AMOC11
CMPA12
uncultured Rhodospirillales bacterium clone
AMMG11
Plot17-2E11
EU440692
93
agricultural soil
WP0807B_033
HQ143780
0.4
Alphaproteobacteria
uncultured Sphingomonadaceae bacterium
AB478689
99
biofilm mats
WP0807B_034
HQ143781
0.4
Alphaproteobacteria
uncultured Sphingomonadaceae bacterium

clone Elev_16S_598
EF019373
92
rhizosphere
116
Table 4.6c(cont). Identities of 16S rRNA gene sequences obtained from site Willow Point. (NA= possible putative chimeric sequences.)
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Identity
Acession
Number
Percent
similarity
Source
WP0807B_035
HQ143782
0.4
Betaproteobacteria
Dechloromonas hortensis strain MA-1
AY277621
94
culture
WP0807B_036
HQ143783
0.4
Betaproteobacteria
Leptothrix sp. S1.1
DQ241397
96
culture
WP0807B_037
HQ143784
0.4
Betaproteobacteria
Methylibium aquaticum
DQ664244
96
freshwater pond
WP0807B_038
HQ143785
0.4
Betaproteobacteria
Methylibium fulvum strain: Gsoil 328
AB245357
96
soil
WP0807B_039
HQ143786
0.4
Betaproteobacteria
uncultured beta proteobacterium clone 2630
EF447071
88
cave environment
WP0807B_040
HQ143787
0.4
Betaproteobacteria
uncultured beta proteobacterium clone A3
AJ867896
92
lake water
WP0807B_041
HQ143788
0.4
Betaproteobacteria
uncultured beta proteobacterium clone AMHF9 AM935541
95
WP0807B_042
HQ143789
0.4
Betaproteobacteria
uncultured beta proteobacterium clone D3A02
EU753673
90
dry stromatolite
WP0807B_043
HQ143790
1.2
Betaproteobacteria
GQ302539
97
cold spring
WP0807B_044
HQ143791
0.8
Betaproteobacteria
FJ517011
96
water
WP0807B_045
HQ143792
0.4
Betaproteobacteria
FJ946595
97
arctic snow
WP0807B_046
HQ143793
0.4
Betaproteobacteria
EF074054
92
pasture
WP0807B_047
HQ143794
0.4
Betaproteobacteria
EF018502
95
trembling aspen
rhizosphere
WP0807B_048
HQ143795
0.4
Betaproteobacteria
EF018556
92
rhizosphere
WP0807B_049
HQ143796
1.6
Betaproteobacteria
EF020008
96
trembling aspen
rhizosphere
WP0807B_050
NA
0.4
Betaproteobacteria
EF019806
88
rhizosphere
WP0807B_051
NA
0.4
Betaproteobacteria
uncultured beta proteobacterium clone swxj275

uncultured beta proteobacterium clone
TDNP_Wbc97_131_1_38
uncultured Hydrogenophaga sp. clone ESSE11
uncultured Ideonella sp. clone GASPWB2S3_E07
uncultured Nitrosomonadaceae bacterium
clone Amb_16S_1138
clone Amb_16S_819
clone Elev_16S_1398
clone Elev_16S_997
uncultured Rhodocyclaceae bacterium clone
Elev_16S_975
EF019789
90
rhizosphere
117
Table 4.6d(cont). Identities of 16S rRNA gene sequences obtained from site Willow Point. (NA= possible putative chimeric sequences.)
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Identity
Acession
Number
Percent
similarity
Source
WP0807B_052
HQ143797
0.4
Deltaproteobacteria
Anaeromyxobacter dehalogenans strain R
EU331403
86
garden soil
WP0807B_053
HQ143798
0.8
Deltaproteobacteria
uncultured delta proteobacterium clone AMEB4 AM935293
98
WP0807B_054
HQ143799
0.4
Deltaproteobacteria
uncultured delta proteobacterium clone TH1-8
AM690812
92
freshwater lake
WP0807B_055
HQ143800
0.4
Deltaproteobacteria
DQ395065
92
harbor sediment
WP0807B_056
HQ143801
0.8
Deltaproteobacteria
AM935618
99
WP0807B_057
HQ143802
0.4
Deltaproteobacteria
AM935395
81
WP0807B_058
HQ143803
0.4
Deltaproteobacteria
AM935632
90
WP0807B_059
NA
0.4
Deltaproteobacteria
EU440668
92
agricultural soil
WP0807B_060
HQ143804
0.4
Deltaproteobacteria
AM936195
91
WP0807B_061
HQ143805
0.4
Deltaproteobacteria
uncultured delta proteobacterium clone VHSB5-73

uncultured Desulfuromonadaceae bacterium
clone AMDF7
uncultured Desulfuromonadales bacterium
clone AMGD9
uncultured Myxococcales bacterium clone
AMDH12
uncultured Myxococcales bacterium clone
Plot17-2B09
uncultured Syntrophobacterales bacterium
clone CMIF2
uncultured Syntrophobacterales bacterium
clone CMPG5
AM936099
94
WP0807B_062
HQ143806
0.4
Gammaproteobacteria sulfur-oxidizing bacterium OAII2
AF170423
88
hydrothermal vent
WP0807B_063
NA
0.4
Gammaproteobacteria uncultured Beggiatoa sp. clone CL5.H29
FM176297
90
rivulet
WP0807B_064
HQ143807
0.4
Gammaproteobacteria uncultured Coxiella sp. clone Ax29_G2
EF092201
91
Axinella corrugata
WP0807B_065
HQ143808
0.4
Gammaproteobacteria uncultured Francisella sp. clone DS058
DQ234142
83
mangrove
WP0807B_066
NA
0.8
Gammaproteobacteria uncultured gamma proteobacterium clone 11
FJ024321
92
marine basalts
WP0807B_067
HQ143809
1.2
AY921861
97
farm soil
WP0807B_068
HQ143810
0.4
Gammaproteobacteria uncultured gamma proteobacterium clone

AKYG629
uncultured
gamma proteobacterium clone
Gammaproteobacteria
AKYG629
AY921861
95
farm soil
118
Table 4.6e(cont). Identities of 16S rRNA gene sequences obtained from site Willow Point. (NA= possible putative chimeric sequences.)
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Acession
Number
Percent
similarity

AKYG629
TDNP_LSbc97_50_2_129
AY921861
95
farm soil
FJ516866
98
sediment
Identity
Source
WP0807B_069
HQ143811
0.4
WP0807B_070
HQ143812
0.4
WP0807B_071
HQ143813
0.4
Gammaproteobacteria uncultured type I methanotroph clone site1-54
EF101327
94
sediment
WP0807B_072
HQ143814
0.4
Proteobacteria
EF587740
97
sediment
WP0807B_073
NA
0.4
Proteobacteria
EF020010
88
rhizosphere
WP0807B_074
HQ143815
0.4
Proteobacteria
EU297091
89
cropland
WP0807B_075
HQ143816
2.4
Acidobacteria
FN428746
90
sediment
WP0807B_076
HQ143817
1.6
Acidobacteria
AY921896
97
farm soil
WP0807B_077
HQ143818
0.8
Acidobacteria
AY921966
96
farm soil
WP0807B_078
HQ143819
0.8
Acidobacteria
AY922028
96
farm soil
WP0807B_079
NA
0.4
Acidobacteria
AM935777
87
WP0807B_080
NA
0.4
Acidobacteria
AM935576
86
WP0807B_081
HQ143820
0.4
Acidobacteria
AM936585
96
WP0807B_082
HQ143821
0.4
Acidobacteria
EF032752
87
cyanobacterial mat
WP0807B_083
HQ143822
0.4
Acidobacteria
DQ648914
93
PCB contaminated soil
WP0807B_084
HQ143823
0.8
Acidobacteria
FJ538144
98
paddy field soil
WP0807B_085
HQ143824
0.4
Acidobacteria
methanotrophic proteobacterium clone

ProfundalSite3-TypeI-1
uncultured proteobacterium clone
Elev_16S_1400
uncultured proteobacterium clone GASPKA1S3_H12
uncultured Acidobacteria bacterium clone
1C57
AKYG1558
AKYG1643
AKYG502
AMAA1
AMDB7
ARN6
HAVOmat15
lhad15
MBNT13
uncultured Acidobacteria bacterium clone OSC128
EF612358
95
soil
119
Table 4.6f(cont). Identities of 16S rRNA gene sequences obtained from site Willow Point.
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Acession
Number
Identity
Percent
similarity
Source

AY211077
VC47
EF061944
XME31
uncultured Acidobacteriaceae bacterium clone FJ475356
AhedenS12
96
anaerobic VCdegrading enrichment
95
mangrove sediment
94
forest soil
Acidobacteria
uncultured Acidobacterium sp. clone sw-xj104
GQ302574
95
cold spring
0.4
Acidobacteria
GQ302574
96
cold spring
HQ143830
0.4
Acidobacteria
GQ302570
98
cold spring
WP0807B_092
HQ143831
1.2
Actinobacteria
Kineococcus-like bacterium AS2988
AF060671
94
culture
WP0807B_093
HQ143832
0.4
Actinobacteria
EF020143
98
rhizosphere
WP0807B_094
HQ143833
0.4
Actinobacteria
uncultured Actinobacterium clone

Elev_16S_1570
uncultured Actinobacterium clone GASPMB1W2_A11
EF664832
84
forest
WP0807B_095
HQ143834
0.8
Antarctic
Antarctic bacterium strain L04
EU636059
93
Collins glacier
WP0807B_096
HQ143835
0.4
Bacteroidetes
uncultured Bacteroidetes bacterium clone

AKYG1020
AY921733
95
farm soil
WP0807B_097
HQ143836
0.8
Bacteroidetes
uncultured Bacteroidetes bacterium clone B2
EF125949
93
biofilm reactor
WP0807B_098
HQ143837
0.4
Bacteroidetes
EF020032
95
WP0807B_099
HQ143838
0.4
Bacteroidetes
EF020032
95
WP0807B_100
HQ143839
0.4
Bacteroidetes

Elev_16S_1427
Elev_16S_1427
Elev_16S_443
EF019261
96
trembling aspen
rhizosphere
trembling aspen
rhizosphere
trembling aspen
rhizosphere
WP0807B_101
HQ143840
0.4
Bacteroidetes
uncultured Cytophaga sp. clone 025
GU269399
94
soil
WP0807B_102
HQ143841
0.4
Bacteroidetes
uncultured Petrimonas sp. clone CL5.H19
FM176290
98
rivulet
WP0807B_086
HQ143825
0.4
Acidobacteria
WP0807B_087
HQ143826
0.4
Acidobacteria
WP0807B_088
HQ143827
0.4
Acidobacteria
WP0807B_089
HQ143828
0.4
WP0807B_090
HQ143829
WP0807B_091
120
Table 4.6g(cont). Identities of 16S rRNA gene sequences obtained from site Willow Point. (ND= Not determined)
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Acession
Number
Identity
Percent
similarity
Source
uncultured Saprospiraceae bacterium clone

FJ517043
TDNP_Wbc97_197_1_75
uncultured Sphingobacteriales bacterium clone FJ536929
ENR22
85
water
96
sludge
Chloroflexi
Sphaerobacter thermophilus DSM 20745
AJ420142
84
ND
0.4
Chloroflexi
93
rivulet
HQ143846
0.4
Chloroflexi
94
sediment
WP0807B_108
HQ143847
0.4
Chloroflexi
uncultured Caldilineaceae bacterium clone

FM175755
CL3.D9
uncultured Chloroflexi bacterium clone 16 T9d- FM242343
oil
uncultured Chloroflexi bacterium clone
AY922012
AKYG434
85
farm soil
WP0807B_109
HQ143848
0.4
Chloroflexi
uncultured Chloroflexi bacterium clone AMNC2 AM934722
96
WP0807B_110
HQ143849
0.4
Chloroflexi
uncultured Chloroflexi bacterium clone I2C
FJ205348
83
deep marine sediments
WP0807B_111
HQ143850
0.4
Chloroflexi
AB448826
90
deep subseafloor
sediments
WP0807B_112
HQ143851
0.8
Chloroflexi
uncultured Chloroflexi bacterium clone:

IODP1324B1H2.39
uncultured Chloroflexus sp. clone: 20-91ArvAB
AB425067
89
Biofilm
WP0807B_113
HQ143852
0.4
Cyanobacteria
Calothrix sp. BECID33
AM230683
93
rock surface
WP0807B_114
HQ143853
0.4
Cyanobacteria
Chroococcidiopsis sp. CC1
DQ914863
88
quartz hypoliths
WP0807B_115
HQ143854
1.2
Cyanobacteria
Chroococcidiopsis sp. CC1
DQ914863
95
quartz hypoliths
WP0807B_116
HQ143855
0.4
Cyanobacteria
Cylindrospermum stagnale PCC 7417
AJ133163
91
culture
WP0807B_117
HQ143856
2.4
Cyanobacteria
Gloeotrichia echinulata URA3
AM230705
97
culture
WP0807B_118
HQ143857
0.4
Cyanobacteria
DQ431005
89
Arctic hot springs
WP0807B_119
HQ143858
1.2
Cyanobacteria
DQ431004
91
hot spring
WP0807B_103
HQ143842
0.4
Bacteroidetes
WP0807B_104
HQ143843
0.4
Bacteroidetes
WP0807B_105
HQ143844
0.4
WP0807B_106
HQ143845
WP0807B_107
121
Table 4.6h(cont). Identities of 16S rRNA gene sequences obtained from site Willow Point. (ND= Not determined; NA= possible putative chimeric sequences.)
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Identity
Acession
Number
Percent
similarity
Source
WP0807B_120
HQ143859
0.8
Cyanobacteria
Leptolyngbya frigida ANT.LH53B.2
AY493576
90
culture
WP0807B_121
HQ143860
1.6
Cyanobacteria
Leptolyngbya sp. FYG
FJ933259
93
cone forming mats
WP0807B_122
HQ143861
0.4
Cyanobacteria
Leptolyngbya sp. HBC1
EU249120
89
marine stromatolite
WP0807B_123
HQ143862
1.6
Cyanobacteria
Leptolyngbya sp. LLi18
DQ786166
91
hot stream
cyanobacterial mat
WP0807B_124
HQ143863
0.8
Cyanobacteria
Pseudanabaena sp. 63-1
EF110976
90
cyanobacterial mat
WP0807B_125
HQ143864
2.0
Cyanobacteria
uncultured cyanobacterium clone 1DP2-N20
EU780335
98
coral
WP0807B_126
HQ143865
0.4
Elusimicrobia
Elusimicrobium minutum Pei191
CP001055
80
ND
WP0807B_127
NA
0.4
Firmicutes
uncultured Brevibacillus sp. clone 075
GU245921
91
soil
WP0807B_128
HQ143866
0.4
Firmicutes
EF651093
82
cropland
WP0807B_129
HQ143867
0.4
Fusobacteria
uncultured Firmicutes bacterium clone

AUVE_04A01
uncultured Fusobacteria bacterium clone
D15_35
EU266872
85
tar-oil contaminated
aquifer sediments
WP0807B_130
HQ143868
0.4
Gemmatimonadetes
DQ431899
95
sediment
WP0807B_131
HQ143869
0.4
Gemmatimonadetes
AY921980
91
farm soil
WP0807B_132
HQ143870
0.4
Gemmatimonadetes
AM935382
94
WP0807B_133
HQ143871
0.4
Gemmatimonadetes
EF220497
93
soil
WP0807B_134
HQ143872
0.4
Gemmatimonadetes
EF612387
93
soil
WP0807B_135
HQ143873
0.4
Nitrospirae

clone AKYG1742
clone AMGC8
clone Fl-1F_D03
clone OS-C40
micronodule bacterium MNF8
AF293012
97
freshwater
WP0807B_136
HQ143874
0.4
Nitrospirae
uncultured Nitrospira sp. clone Amb_16S_847 EF018579
97
trembling aspen
rhizosphere
122
Table 4.6i(cont). Identities of 16S rRNA gene sequences obtained from site Willow Point.
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Identity
Acession
Number
Percent
similarity
Source
WP0807B_137
HQ143875
0.4
Nitrospirae
uncultured Nitrospirae bacterium clone: 356
AB252945
95
iron-oxidation biofilm
WP0807B_138
HQ143876
4.9
Nitrospirae
uncultured Nitrospirales bacterium clone

3PJM46
FJ535101
99
cave wall biofilm
WP0807B_139
HQ143877
0.4
Planctomycetes
uncultured Pirellula sp. clone CL5.H34
FM176302
94
rivulet
WP0807B_140
HQ143878
0.4
Planctomycetes
AM935707
89
WP0807B_141
HQ143879
0.4
Planctomycetes
uncultured Planctomycetacia bacterium clone

AMBH12
uncultured Planctomycetales bacterium clone
delph1B8
FM209076
93
WP0807B_142
HQ143880
0.4
Planctomycetes
uncultured planctomycete clone D1E01
EU753675
88
dry stromatolite
WP0807B_143
HQ143881
0.4
Planctomycetes
uncultured planctomycete clone DEL34
AJ616275
97
river biofilm
WP0807B_144
HQ143882
0.4
Planctomycetes
uncultured planctomycete clone DEL75
AJ616291
91
river biofilm
WP0807B_145
HQ143883
0.4
Planctomycetes
uncultured planctomycete clone g10
EU979019
95
soil
WP0807B_146
HQ143884
0.4
Planctomycetes
uncultured planctomycete clone GASPMB1W1_C11
EF664756
95
forest
WP0807B_147
HQ143885
0.4
Planctomycetes
uncultured planctomycete clone LC1-32
DQ289903
90
Sediment
WP0807B_148
HQ143886
0.4
Planctomycetes
uncultured planctomycete clone MVS-107
DQ676396
90
suboxic freshwaterpond sediment
WP0807B_149
HQ143887
0.4
Planctomycetes
uncultured planctomycete clone: COM-32
AB451755
95
turfgrass
WP0807B_150
HQ143888
0.4
Planctomycetes
uncultured planctomycete clone: COM-33
AB451756
93
turfgrass
WP0807B_151
HQ143889
0.4
Planctomycetes
AB433105
88
deep subseafloor
sediments
WP0807B_152
HQ143890
0.8
Verrucomicrobia
AY874111
96
tufa
WP0807B_153
HQ143891
0.4
Bacteria
uncultured planctomycete clone:

IODP1319B109.42
uncultured Verrucomicrobia bacterium clone
TRK26
iron-reducing bacterium enrichment culture
clone HN-HFO140
FJ269108
83
paddy soil from mining

area
123
Table 4.6j(cont). Identities of 16S rRNA gene sequences obtained from site Willow Point.
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Identity
Acession
Number
Percent
similarity
Source
WP0807B_154
HQ143892
0.4
Bacteria
uncultured anaerobic bacterium clone C-4
DQ018787
84
anaerobic swine lagoon
WP0807B_155
HQ143893
0.4
Bacteria
uncultured bacterium clone 661191
DQ404914
98
contaminated sediment
WP0807B_156
HQ143894
0.4
Bacteria
uncultured bacterium clone DS3-53
DQ463232
90
River sediment
WP0807B_157
HQ143895
0.8
Bacteria
uncultured bacterium clone FFCH16215
EU134352
92
soil
WP0807B_158
HQ143896
1.2
Bacteria
uncultured bacterium clone MBNT5
FJ538136
95
paddy field soil
WP0807B_159
HQ143897
0.4
Bacteria
uncultured hydrocarbon seep bacterium

BPC023
AF154087
88
hydrocarbon seep
WP0807B_160
HQ143898
0.4
Bacteria
uncultured marine bacterium clone AntCL1D1
DQ906720
97
Antarctic sea water
WP0807B_161
HQ143899
0.4
Bacteria
uncultured marine bacterium clone AntCL1H5
DQ906739
89
Antarctic sea water

collected from 20 m
WP0807B_162
HQ143900
0.4
Bacteria
uncultured soil bacterium clone 1_H12 16S
EU589283
97
rice paddy field soil
WP0807B_163
HQ143901
0.4
Bacteria
uncultured soil bacterium clone

FACE.R2.EC.F04
FJ621051
90
sweetgum plantation
soil
WP0807B_164
HQ143902
0.4
Bacteria
uncultured soil bacterium clone L1A.10E07
AY989265
98
soil
124
Table 4.7a. Identities of 16S rRNA gene sequences obtained from site Three Poles. (NA= possible putative chimeric sequences.)
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Acession
Number
Identity
Percent
similarity
Source
TP0807B_001 HQ143903
0.4
Alphaproteobacteria
Nordella oligomobilis
AF370880
96
culture
TP0807B_002 HQ143904
0.4
Alphaproteobacteria
alpha proteobacterium Shinshu-th1
AB121772
98
soil
0.4
Alphaproteobacteria
AY921897
89
soil
TP0807B_004 HQ143905
0.4
Alphaproteobacteria
AY921677
96
farm soil
TP0807B_005 HQ143906
0.4
Alphaproteobacteria

AKYG421
AKYH996
delph2D3
FM209172
99
TP0807B_006 HQ143907
0.4
Alphaproteobacteria
uncultured alpha proteobacterium clone G06-2 FM253673
96
biofilm
TP0807B_007 HQ143908
0.8
Alphaproteobacteria
AF293000
92
freshwater
TP0807B_008 HQ143909
0.4
Alphaproteobacteria
AM934764
89
TP0807B_009 HQ143910
0.4
Alphaproteobacteria
EU440683
99
agricultural soil
TP0807B_010 HQ143911
0.8
Alphaproteobacteria
AM935308
97
TP0807B_011 HQ143912
1.7
Alphaproteobacteria
EF019251
99
trembling aspen
rhizosphere
TP0807B_012 HQ143913
0.4
Alphaproteobacteria
FJ516923
94
upper sediment
TP0807B_013 HQ143914
2.1
Alphaproteobacteria
AM934842
96
TP0807B_014 HQ143915
0.4
Alphaproteobacteria

micronodule bacterium MNG3
clone AMNG7
clone Plot17-2D11
uncultured Hyphomicrobium sp. clone
AMEC10
Elev_16S_430
TDNP_USbc97_210_1_88
AMMG11
Plot17-2E11
EU440692
93
agricultural soil
TP0807B_015 HQ143916
0.4
Betaproteobacteria
Delftia acidovorans isolate CI11
DQ530080
85
soybean rhizosphere
TP0807B_016 HQ143917
61.8
Betaproteobacteria
Delftia lacustris strain 332
EU888308
99
culture
TP0807B_017 HQ143918
0.4
Betaproteobacteria
uncultured beta proteobacterium clone A05-1
FM253568
97
biofilm
TP0807B_003
NA
125
Table 4.7b (cont). Identities of 16S rRNA gene sequences obtained from site Three Poles.
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Acession
Number
Identity
Percent
similarity
Source

clone Amb_16S_819
uncultured Rhodocyclaceae bacterium clone
Elev_16S_1785
EF018556
92
EF020265
96
uncultured delta proteobacterium clone g66
EU979075
95
soil
EF663509
97
grassland
FJ516992
94
water
AB451791
89
AM935650
91
AM934915
96
AM936521
97
AM934884
96
turfgrass-degrading
composting process
Acidobacteria
uncultured Acidobacteria bacterium clone 1947 EF188318
98
Altamira Cave
0.4
Acidobacteria
AY281355
98
soil
TP0807B_030 HQ143931
0.4
Acidobacteria
AY921884
97
farm soil
TP0807B_031 HQ143932
0.4
Acidobacteria
AY921984
97
farm soil
TP0807B_032 HQ143933
0.8
Acidobacteria
AM935084
98
TP0807B_033 HQ143934
0.4
Acidobacteria
FM877550
98
uranium mill tailings
TP0807B_034 HQ143935
0.4
Acidobacteria

39p18
AKYG1538
AKYG1760
AMPB1
BuhD-250
GASP-45KB-168-H09
EU044408
95
dune
TP0807B_018 HQ143919
0.4
Betaproteobacteria
TP0807B_019 HQ143920
2.1
Betaproteobacteria
TP0807B_020 HQ143921
0.4
Deltaproteobacteria
TP0807B_021 HQ143922
0.8
Deltaproteobacteria
TP0807B_022 HQ143923
0.4
TP0807B_023 HQ143924
0.4
TP0807B_024 HQ143925
0.4
TP0807B_025 HQ143926
0.4
TP0807B_026 HQ143927
0.8
TP0807B_027 HQ143928
0.4
TP0807B_028 HQ143929
0.8
TP0807B_029 HQ143930
uncultured delta proteobacterium clone GASPMA3S2_D07

uncultured
delta proteobacterium clone
Deltaproteobacteria
TDNP_Wbc97_103_1_10
uncultured delta proteobacterium clone: COMDeltaproteobacteria
68
Deltaproteobacteria
clone AM935650
Deltaproteobacteria
clone AMJG9
CM41C5
AMJD7
126
trembling aspen
rhizosphere
trembling aspen
rhizosphere
Table 4.7c (cont). Identities of 16S rRNA gene sequences obtained from site Three Poles.
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Acession
Number
Identity
Percent
similarity
Source
TP0807B_035 HQ143936
0.4
Acidobacteria
uncultured Acidobacteria bacterium clone MVS- DQ676412

66
98
suboxic freshwaterpond sediment
TP0807B_036 HQ143937
0.8
Acidobacteria
uncultured Acidobacterium sp. clone BuhC-16
FM866291
98
TP0807B_037 HQ143938
0.8
Acidobacteria
uncultured Acidobacterium sp. clone BuhC-67
FM866294
98
TP0807B_038 HQ143939
0.4
Acidobacteria
GQ302567
98
cold spring
TP0807B_039 HQ143940
0.4
Actinobacteria
AM935506
96
TP0807B_040 HQ143941
0.4
Chloroflexi
uncultured Actinomycetales bacterium clone

AMHB12
uncultured Anaerolineae bacterium clone
AMBA6
AM935711
96
TP0807B_041 HQ143942
0.4
Chloroflexi
uncultured Chloroflexi bacterium clone AD008
EF076118
93
TP0807B_042 HQ143943
0.4
Chloroflexi
96
farm soil
TP0807B_043 HQ143944
0.8
Chloroflexi
83
soil
TP0807B_044 HQ143945
0.4
Chloroflexi

AY921805
AKYG617
AY922170
AKYH910
uncultured Chloroflexi bacterium clone g68-MR- EU979101
68
marine sponge Agelas
dilatata
92
soil
TP0807B_045 HQ143946
0.4
Chloroflexi
uncultured Chloroflexi bacterium clone LC1-24 DQ289898
88
Sediment
TP0807B_046 HQ143947
0.4
Chloroflexi
uncultured Chloroflexi bacterium clone: HAuD- AB113601

MB19
89
geothermal water
TP0807B_047 HQ143948
0.8
Cyanobacteria
Cyanothece sp. PCC 7425
CP001344
93
culture
TP0807B_048 HQ143949
0.4
Cyanobacteria
Microcoleus paludosus SAG 1449-1a
EF654090
95
culture
TP0807B_049 HQ143950
0.4
Cyanobacteria
Microcoleus sp. HTT-U-KK5
EF654070
94
culture
TP0807B_050 HQ143951
0.8
Firmicutes
EF651373
91
cropland
TP0807B_051 HQ143952
0.4
Firmicutes
uncultured Firmicutes bacterium clone

AUVE_10E05
uncultured Firmicutes bacterium clone GASPMB3S2_A06
EF665637
91
forest
127
Table 4.7d (cont). Identities of 16S rRNA gene sequences obtained from site Three Poles.
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Acession
Number
Identity
Percent
similarity
Source
TP0807B_052 HQ143953
0.4
Gemmatimonadetes

clone AMFC3
AM935228
96
TP0807B_053 HQ143954
2.5
Nitrospirae
uncultured Nitrospira sp. clone CL3.D11
FM175757
98
rivulet
TP0807B_054 HQ143955
0.4
Nitrospirae
uncultured Nitrospiraceae bacterium clone

D10_16
EU266788
99
aquifer sediments
TP0807B_055 HQ143956
0.4
Nitrospirae
uncultured Nitrospirae bacterium clone 2646
EF447064
98
cave environment
TP0807B_056 HQ143957
0.4
Planctomycetes
Planctomycetacia bacterium WSF3-27
FJ405890
92
bamboo grove soil
TP0807B_057 HQ143958
0.4
Planctomycetes
Planctomycetales bacterium Ellin7224
AY673390
90
soil
TP0807B_058 HQ143959
0.4
Planctomycetes
FJ475365
91
forest soil
TP0807B_059 HQ143960
0.4
Planctomycetes
uncultured Planctomycetaceae bacterium

clone AhedenS28
uncultured Planctomycetacia bacterium clone
GASP-MA2W3_D06a
EF663320
93
cropland
TP0807B_060 HQ143961
0.4
Planctomycetes
uncultured planctomycete clone AKYG587
AY921932
93
soil
TP0807B_061 HQ143962
0.4
Planctomycetes
uncultured planctomycete clone DPC183
DQ269110
90
surface of marine macroalga
TP0807B_062 HQ143963
0.4
Planctomycetes
uncultured planctomycete clone I8A
FJ205363
91
deep marine sediments
TP0807B_063 HQ143964
0.4
Planctomycetes
CU925984
96
mesophilic anaerobic
digester
TP0807B_064 HQ143965
0.4
Verrucomicrobia
FJ205241
84
hydrothermal
TP0807B_065 HQ143966
0.4
Verrucomicrobia
uncultured Planctomycetes bacterium clone

QEDN4AG12
B6H
TRK26
AY874111
97
tufa
TP0807B_066 HQ143967
0.4
Bacteria
uncultured bacterium clone AUVE_04F02
EF651153
92
cropland
TP0807B_067 HQ143968
0.4
Bacteria
uncultured bacterium clone BacC-s_051
EU335160
98
soil
TP0807B_068 HQ143969
0.4
Bacteria
uncultured soil bacterium clone 2-1
AY326516
90
soil
128
Table 4.7e (cont). Identities of 16S rRNA gene sequences obtained from site Three Poles.
code
Genbank
accession
number
Relative
abundance
Putative Phylum
(%)

Acession
Number
Identity
Percent
similarity
Source
TP0807B_069 HQ143970
0.4
Bacteria
uncultured soil bacterium clone F6-139
EF688389
93
soil
TP0807B_070 HQ143971
0.4
Bacteria
uncultured soil bacterium clone F6-139
EF688389
93
soil
TP0807B_071 HQ143972
0.4
Bacteria
uncultured soil bacterium clone S042
AF507707
90
forest soil
TP0807B_072 HQ143973
0.4
Bacteria
uncultured soil bacterium clone S102
AY037620
89
soil
129
5*66%)-6(8%;*)*9.
H*''=,(6-,'(+-%.
<0.
1%,)*'-%.
F0.
G"%8,)(6A,*)*9.
40.
/0.
D=9(+%,)*'-%.
C0.
D-'6-,=)*9.
<0.
E-)'(9#-'%*.
F0.
!"#$%&
#'()*(+%,)*'-%.
//0.
B"=9-6-,'(+-%.
C0.
1*)%&
#'()*(+%,)*'-%.
20.
?A%8(+%,)*'-%..
<>0.
3*")%&
#'()*(+%,)*'-%.
40.
?$"('(:"*@-.
>0.
1%,)*'(-;*)*9.
>0.
!,-;(+%,)*'-%.
<<0.
!8)%',)-,.
+%,)*'-=6. !,)-8(+%,)*'-%.
<0.
/0.
5%66%&
#'()*(+%,)*'-%.
40.
78,"%99-:-*;.
#'()*(+%,)*'-%.
<0.
Figure 4.18 Relative abundance of bacterial phylotypes obtained from

Willow Point clone library.
130
F*''B,(7-,'(+-%.
/1.
2%,)*'-%.
51.
!"#$%&
#'()*(+%,)*'-%.
/01.
E"%<,)(7@,*)*C.
31.
D-)'(C#-'%*.
31.
6*77%)-7(<%9*)*C.
2*)%&
#'()*(+%,)*'-%.
31.
:1.
A-'7-,B)*C.
81.
=@%<(+%,)*'-%.
01.
=$"('(>"*?-.
31.
4*")%&
#'()*(+%,)*'-%.
51.
6%77%&
#'()*(+%,)*'-%.
81.
!,-9(+%,)*'-%.
:;1.
!,)-<(+%,)*'-%.
:1.
D-)'(C#-'%*.
81.
6*77%)-7(<%9*)*C.
E"%<,)(7@,*)*C.
81.
:1. A-'7-,B)*C.
:1.
=@%<(+%,)*'-%.
=$"('(>"*?-.
/1.
81.
!,)-<(+%,)*'-%.
:1.
!,-9(+%,)*'-%.
6%77%&
;1.
#'()*(+%,)*'-%.
:1.
4*")%&
#'()*(+%,)*'-%.
81.
F*''B,(7-,'(+%.
:1.
2%,)*'-%.
81.
!"#$%&
#'()*(+%,)*'-%.
G1.
2*)%&
#'()*(+%,)*'-%.
;81.
Figure 4.19. Revised chart (after omitting Delftia lacustris) of relative

abundance of bacterial phylotypes obtained from Three Poles clone
library. Smaller pie, original bacterial distribution of Three Poles clone
library
131
Table 4.8. Summary of recovery phyla from microbialites at Willow Point,

Three Poles (revised), and stromatolites at Shark Bay (Goh et al. 2009).
Three
Phylum abundance (%)
Williow Point
Shark Bay
Poles
Alphaproteobacteria
22
24
29
Betaproteobacteria
9
8
Deltaproteobacteria
5
7
4
Gammaproteobacteria
5
3
29
Unclassified proteobacteria
1
Acidobacteria
11
16
Actinobacteria
2
1
11
Antarctic bacteria
1
Bacteroidetes
4
8
Chloroflexi
4
8
Cyanobacteria
14
4
11
Elusimicrobia
0.4
Firmicutes
1
3
Fusobacteria
0.4
Gammatimonadetes
2
1
Nitrospirae
6
8
Planctomycetes
5
8
5
Verrucomicrobia
1
2
Unknown
6
7
1
Deinococcus
1
Cadidate divison OP11
2
4.3.4.2. Summer season vs. budding season

The results of this study were deemed to require further
support since during the investigation a high degree of heterogeneity was
encountered when sequencing clone libraries.
This was regrettably
outside the scope of this thesis. For the summer season in 2008, the
highest portion of sequences was related to Alphaproteobacteria (97.2%),
whereas the majority of clones in the budding season library had
sequence identities related to an uncultured bacterium (29.8%),
Gammaproteobacteria
(26.6%),
Alphaproteobacteria
132
(14.5%)
and
Betaproteobacteria (12.9%) (table 4.11). By comparing the clone libraries

obtained from Three Poles in 2007 summer and 2008 summer, high
abundance of Gammaproteobacteria was obtained in 2008 but not in
2007. Their difference may due the possibly large variation between year,
and the samples for 2008 summer were collected by others and change
of custody may have affected results (e.g. Gammaproteobacteria are
other indication of contamination). Detail descriptions of the closet blast
matches from seasonal clone libraries were shown in Table 4.9 and Table
4.10 a-b.
133
Table 4.9. Identities of 16S rRNA gene sequences obtained from Three Poles during summer season.
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Identity
Acession
Number
Percent
similarity
Source
TP0708B_001
HQ233606
0.9
Alphaproteobacteria
Phyllobacterium sp. AKB2008VA5
AM989040
99
treated drinking water
TP0708B_002
HQ233607
0.9
Deltaproteobacteria
EF221515
92
soil (Antarctic)
TP0708B_003
HQ233608
17.8
Gammaproteobacteria Enhydrobacter sp. KB3-12
FN377702
99
sediment (Arctic)
TP0708B_004
HQ233609
5.6
FN377702
98
sediment (Arctic)
TP0708B_005
HQ233610
1.9
FN377702
98
sediment (Arctic)
TP0708B_006
HQ233611
34.6
Gammaproteobacteria uncultured Enhydrobacter sp.
EU305591
99
wastewater plant
TP0708B_007
HQ233612
9.3
EU305591
99
wastewater plant
TP0708B_008
HQ233613
1.9
EU305591
96
wastewater plant
TP0708B_009
HQ233614
25.2
Gammaproteobacteria uncultured gamma proteobacterium FM994667
99
digestive tract of deep-sea limpet
TP0708B_010
HQ233615
0.9
Gammaproteobacteria uncultured gamma proteobacterium FM994667
97
digestive tract of deep-sea limpet
TP0708B_011
HQ233616
0.9
Chloroflexi
94
hydrocarbon-contaminated soil
uncultured Chloroflexi bacterium
134
AM935790
Table 4.10a. Identities of 16S rRNA gene sequences obtained from Three Poles during budding season. (NA= possible putative chimeric sequences).
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Identity
Acession
Number
Percent
similarity
Source
TP0408B_001 HQ233617
0.8
Alphaproteobacteria
Chelatococcus sp. ES-SL-4
FJ529045
99
excess sludge of oilfield
TP0408B_002 HQ233618
5.6
Alphaproteobacteria
Methylocaterium sp. AKB-208-ES6
AM989020
99
TP0408B_003 HQ233619
8.1
Alphaproteobacteria
Phyllobacterium sp. AKB-2008-VAS
AM989040
99
TP0408B_004 HQ233620
10.5
Betaproteobacteria
Delftia acidovorans SPH-1
CP000884
100
culture
TP0408B_005 HQ233621
0.8
Betaproteobacteria
Mitsuaria sp. H29L1B
EU714912
99
mixed hay soils
TP0408B_006 HQ233622
1.6
Betaproteobacteria
AY569280
98
pink microbial mat
TP0408B_007 HQ233623
0.8
FJ358880
90
marine reef sandy sediment
TP0408B_008 HQ233624
0.8
FJ816058
99
rhizosphere soil
TP0408B_009 HQ233625
0.8
EU352764
98
culture
TP0408B_010 HQ233626
0.8
uncultured Aquabacterium sp. clone

YJQ-2
Deltaproteobacteria
clone OX43
Acinetobacter calcoaceticus strain
Gammaproteobacteria
PUCM1011
Acinetobacter haemolyticus strain NK
Gammaproteobacteria
2.BH-7
Gammaproteobacteria Acinetobacter sp. PND-5
EF494200
99
culture
TP0408B_011 HQ233627
0.8
Gammaproteobacteria Acinetobacter sp. SS-2
AM396494
99
deep sea sediment
TP0408B_012 HQ233628
11.3
Gammaproteobacteria E. coli
EU723821
99
hemp retting water
0.8
Gammaproteobacteria E. coli
CP001637
99
culture
TP0408B_014 HQ233629
0.8
Gammaproteobacteria Halomonas sp. gy6
FJ514813
99
farmland soil
TP0408B_015 HQ233630
5.6
Gammaproteobacteria Pseudomonas sp. G3DM-15
EU037276
100
chromium contaminated soil
TP0408B_016 HQ233631
4.0
Gammaproteobacteria Stenotrophomonas maltophilia
FJ405363
99
sweet potato plants
TP0408B_017 HQ233632
0.8
Gammaproteobacteria
FM176309
97
rivulet
TP0408B_018 HQ233633
0.8
Acidobacteria
EF417748
92
soil
TP0408B_013
NA
uncultured Thiorhodospira sp. clone

CL5.H46
135
Table 4.10b (cont). Identities of 16S rRNA gene sequences obtained from Three Poles during budding season. (NA= possible putative chimeric sequence).
code
Genbank
accession
number
Relative
abundance
(%)
Putative Phylum

Identity
Percent
similarity
GU184127
99
culture
AY360611
88
oxic rice field soil
AB113601
88
geothermal water
EF110976
87
cyanobacterial mat
94
mangrove soil
97
freshwater lake
98
lake
TP0408B_018 HQ233634
6.5
Actinobacteria
TP0408B_019 HQ233635
0.8
Chloroflexi
TP0408B_020 HQ233636
0.8
Chloroflexi
TP0408B_021 HQ233637
0.8
Cyanobacteria
TP0408B_022 HQ233638
0.8
Planctomycetes
TP0408B_023 HQ233639
0.8
Fibrobacteres
TP0408B_024 HQ233640
0.8
Bacteria
uncultured planctomycete clone MSBDQ811901

4A3
uncultured Fibrobacteres bacterium
AM690985
clone TH3-98
aquatic bacterium R1-G8
AB195775
TP0408B_025 HQ233641
0.8
Bacteria
uncultured bacterium clone 5-21
TP0408B_026 HQ233642
0.8
Bacteria
TP0408B_027 HQ233643
0.8
Bacteria
0.8
Bacteria
29.8
Bacteria
TP0408B_028
NA
TP0408B_029 HQ233644
Rhodococcus sp. SY095
Acession
Number

clone M10Ba21
clone: HAuD-MB19
uncultured bacterium clone BACuB7C11

uncultured bacterium clone
LaC15L69
uncultured bacterium clone RUGL1266
uncultured bacterium clone W2-16
136
Source
sediment of Guanting
Reservoir
soil
DQ833466
98
GQ128110
88
EF667818
98
GQ421122
99
uncultivated river sediment

bacterium
soil
FJ545601
99
seawater
Table 4.11. Summary of bacteria isolated from seasonal libraries of

Pavilion Lake microbialites.
Putative Phylum
Summer
(August 2008)
(%)
Budding
(April 2008)
(%)
Alphaproteobacteria
0.9
14.5
Betaproteobacteria
12.9
Deltaproteobacteria
0.9
0.8
Gammaproteobacteria
97.2
26.6
Acidobacteria
0.8
Actinobacteria
6.5
Chloroflxi
0.9
1.6
Cyanobacteria
0.8
Fibrobacteres
0.8
Planctomycetes
0.8
Uncultured bacteria
33.9
137
Alp h a p ro teobacteria
Gammaproteobacteria
TP0807B_004 (HQ143905)
TP0408B_026 (HQ233641)
U n c u ltu re d a lp h a p ro teobacterium (A Y 9 2 1 6 7 7 )
TP0807B_006 (HQ143907)
74/87 U n c u ltu re d a lp h a p ro tebacterium (F M 2 0 9 1 7 2 )
TP0807B_005 (HQ143906)
100/97 WP0807B_012 (HQ143761)
T P 0 7 0 8 B _0 0 1 (HQ233606)
100/100 P h y llo b a c te riu m s p . EBBLQ01 (F J 1 7 8 7 8 5 )
TP0408B_003 (HQ233619)
U n c u ltu re d H y p h o m ic robiaceae (A M 9 3 4 7 6 4 )
100/
A g ro b a c te riu m tu m e fa ciens (A Y 6 2 6 3 8 7 )
WP0807B_003 (HQ143752)
63/54
100/97 R h o d o b a c te ra c e a e b a c terium (D Q 6 2 8 9 6 4 )
WP0807B_024 (HQ143772)
WP0807B_026 (HQ143773)
100/81
WP0807B_009 (HQ143758)
/54
U n c u ltu re d R h o d o b a c ter sp. (E U 6 3 9 0 0 7 )
WP0807B_004 (HQ143753)
WP0807B_025
U n c u ltu re d R h iz o b ia les (A M 9 3 5 4 5 2 )
100/96
WP0807B_027 (HQ143774)
100/64
WP0807B_023
WP0807B_002 (HQ143751)
100/100 H y p h o m o n a s s p . M O L A 55 (A M 9 9 0 8 3 0 )
100/98 TP0408B_002 (HQ233618)
99/61
M e th y lo b a c te riu m sp. V3 (A F 3 2 4 2 0 1 )
TP0408B_001 (HQ233617)
94/77
TP0807B_002 (HQ143904)
A lp h a p ro te o b a c te riu m (A B 1 2 1 7 7 2 )
100/97
U n c u ltu re d H y p h o m ic robiaceae (E U 4 4 0 6 8 3 )
100/88 TP0807B_009 (HQ143910)
90/69
WP0807B_019 (HQ143768)
TP0807B_003
96/79
U n c u ltu re d a lp h a p ro teobacterium (A Y 9 2 1 8 9 7 )
U n c u ltu re d alp h a p ro teobacterium (A M 9 3 4 9 5 6 )
98/66
WP0807B_010 (HQ143759)
100/76
N o rd e lla o lig o m o b ilis (A F 3 7 0 8 8 0 )
TP0807B_001 (HQ143903)
/87
U n c u ltu re d N itra tire ductor sp. (F M 1 7 5 7 6 0 )
WP0807B_021 (HQ143770)
WP0807B_034 (HQ143781)
WP0807B_022 (HQ143771)
100/92
U n c u ltu re d H y p h o m ic robium sp. (A M 9 3 5 3 0 8 )
88/62
TP0807B_010 (HQ143911)
WP0807B_018 (HQ143767)
56/
WP0807B_020 (HQ143769)
U n c u ltu re d H y p h o m ic robiaceae (F J 5 1 7 0 6 6 )
98/93 U n c u ltu re d a lp h a p ro teobaterium (A B 4 2 5 0 6 2 )
93/85
WP0807B_016 (HQ143765)
WP0807B_006 (HQ143755)
90/
100/ WP0807B_008 (HQ143757)
WP0807B_033 (HQ143780)
87/
0 .1
WP0807B_005 (HQ143754)
54/88
E ry th ro m ic ro b iu m ra m osum (A B 0 1 3 3 5 5 )
81/96
WP0807B_001 (HQ143750)
WP0807B_007 (HQ143756)
100/
U n c u ltu re d S p h in g o m o nadaceae (E F 0 1 9 3 7 3 )
100/94
TP0807B_007 (HQ143908)
U
n
c
u ltu re d micronodule bacterium (A F 2 9 3 0 0 0 )
100/
WP0807B_161 (HQ143899)
TP0807B_008 (HQ143909)
100/97
WP0807B_015 (HQ143764)
66/
U n c u ltu re d a lp h a p ro teobacterium (A F 4 4 5 7 2 4 )
WP0807B_014 (HQ143763)
WP0807B_017 (HQ143766)
WP0807B_073
WP0807B_029 (HQ143776)
99/85
100/
TP0807B_012 (HQ143913)
U n c u ltu re d R h o d o s p irillaceae (A M 9 3 5 1 7 7 )
100/84 TP0807B_011 (HQ143912)
U n c u ltu re d micronodule bacterium (A F 2 9 2 9 9 9 )
83/
U n c u ltu re d R h o d o s p irillaceae (A M 9 3 6 0 4 1 )
99/67
WP0807B_013 (HQ143762)
U n c u ltu re d a lp h a p ro teobacterium (E U 3 6 1 3 8 2 )
59/ U n c u ltu re d alp h a p ro teobacterium (A M 9 3 6 2 4 1 )
TP0408B_028 (HQ233643)
78/
U n c u ltu re d R h o d o s p irillales (A M 9 3 4 8 4 2 )
100/73
TP0807B_013 (HQ143914)
WP0807B_031 (HQ143778)
100/92
WP0807B_011 (HQ143760)
100/
TP0807B_014 (HQ143915)
/93
WP0807B_032 (HQ143779)
U n c u ltu re d R h o d o s p irillales (E U 4 4 0 6 9 2 )
92/86
100/98
100/100
Figure 4.20. Phylogenetic relationship among Alphaproteobacteria from

Willow Point and Three Poles and based upon Maximum Likelihood
analysis of full 16S rRNA gene sequence data. Sequence codes refer to
those given in Table 4.6 a-j, 4.7 a-e, 4.9 & 4.10 a-b. Rooted tree
supported by bootstrap values for 1000 replicates (first number) and
138
Betaproteobacteria
TP0408B_004 (HQ233620)
92/67
100/83
100/69
Delftia lacustris strain 332 (EU888308)

TP0807B_016 (HQ143917)
Delftia acidovorans isolate CI11 (DQ 530080)
WP0807B_045 (HQ143792)
79/
WP0807B_036 (HQ143783)
100/85 Uncultured beta proteobactreium clone 187 (AB252908)
73/
Uncultured Ideonella sp. clone GASP-WB2S3 (EF074054)

WP0807B_037 (HQ143784)
77/
TP0408B_005 (HQ233621)
TP0408B_006 (HQ233622)
100/63
WP0807B_046
(HQ143793)*
WP0807B_038 (HQ143785)
96/
68/
50/
Uncultured beta proteobacterium clone A05-1 (FM 253568)

TP0807B_017 (HQ143918)*
WP0807B_035 (HQ143782)
95/70
92/64
Dechloromonas hortensis (AY277621)
WP0807B_041 (HQ143788)
Uncultured beta proteobacterium clone AMHF9 (AM 935541)
100/88
100/64
88/
WP0807B_048 (HQ143795)
TP0807B_018 (HQ143919)
Uncultured Nitrosomonadaceae bacterium clone Amb819 (EF018556)
99/
51/
100/
WP0807B_042 (HQ143789)
Uncultured beta proteobacterium clone D3A02 (EU753673)
Uncultured beta proteobacterium clone TDNP Wbc97 (FJ517011)
100/100
99/
WP0807B_044 (HQ143791)
WP0807B_047 (HQ143794)
Uncultured Nitrosomonadaceae bacterium clone Elev997 (EF019806)

100/ 96/
100/
90/
100/54
Uncultured Rhodocyclaceae bacterium clone Elev1785 (EF020265)

Uncultured betaproteobacterium clone 2630 (EF447071)
WP0807B_043 (HQ143790)
96/
99/
WP0807B_049 (HQ143796)
TP0807B_019 (HQ143920)
Uncultured Rhodocyclaceae bacterium clone Elev975 (EF019789)
WP0807B_051
WP0807B_050
0.1
Figure 4.21. Phylogenetic relationship among Betaproteobacteria from

139
Deltaproteobacteria
Alphaproteobacteria
WP0807B_056 (HQ143801)
U ncultured D esulfuromonadaceae (AM 935618)
U ncultured deltaproteobacterium COM-68 (AB451791)
100/98
77/
TP0807B_023 (HQ143924)
77/
Iron reducing bacterium HN-HFO140 (FJ269108)
U ncultured deltaproteobacterium TH1-8 (AM 690812)
53/
69/
WP0807B_057 (HQ143802)
WP0807B_153 (HQ143891)
U ncultured M yxococcales AMDH12 (AM 935632)
/73
WP0807B_058 (HQ143803)
/70
TP0408B_007 (HQ233623)
TP0807B_024 (HQ143925)
100/84
100/
U ncultured D esulfuromonadales AMCB1 (AM 935650)
U ncultured deltaproteobacterium VHS-B5-73 (D Q 395065)
100/98
WP0807B_055 (HQ143800)
U ncultured deltaproteobacterium SI-2M_A09 (EF221515)
100/96
TP0708B_002 (HQ233607)
/82
TP0807B_020 (HQ143921)
78/82
U ncultured deltaproteobacterium g66 (EU 979075)
94/
WP0807B_052 (HQ143797)
100/69
Anaeromyxobacter dehalogenans (EU 331403)
TP0807B_021 (HQ143922)
100/99
53/
U ncultured deltaproteobacterium GASP-MA3S2 (EF663509)
100/67
TP0807B_022 (HQ143923)
U ncultured deltaproteobacterium TDNP Wbc97 (FJ516992)
WP0807B_059
73/
U ncultured D esulfuromonadales AMGD9 (AM 935395)
100/73
U ncultured D esulfuromonadales AMJG9 (AM 934915)
TP0807B_025 (HQ143926)
100/100
0.1
Figure 4.22. Phylogenetic relationship among Deltaproteobacteria from

140
Gammaproteobacteria
100/54
Betaproteobacteria
TP0708B_005 (HQ233610)
TP0708B_010 (HQ233615)
57/52
TP0708B_008 (HQ233613)
62/68
TP0708B_004 (HQ233609)
TP0708B_003 (HQ233608)
T P 0 7 0 8 B _0 0 7 (HQ233612)
T P 0 7 0 8 B _0 0 6 (HQ233611)
100/100
M o ra xe lla sp . E ve re st-gws-63 (E U 5 8 4 5 2 5 )
U n cu ltu re d g a m m a p ro teobacterium BG4 (F M 9 9 4 6 6 7 )
M o ra xe lla o slo e n sis PCWCW3 (G Q 2 8 4 4 7 2 )
51/51
T P 0 7 0 8 B _0 0 9 (HQ233614)
100/82
T P 0 4 0 8 B _0 2 5 (HQ233640)
100/66
92/68
T P 0 4 0 8 B _0 1 1 (HQ233627)
100/68
T P 0 4 0 8 B _0 0 9 (HQ233625)
100/98
T P 0 4 0 8 B _0 0 8 (HQ233624)
T P 0 4 0 8 B _0 1 0 (HQ233626)
T P 0 4 0 8 B _0 1 4 (HQ233629)
100/100 E scherichia co li (J0 1 8 5 9 )
100/95
T P 0 4 0 8 B _0 1 2 (HQ233628)
A eromonas so b ria A T C C 4 3 9 7 9 T (X 7 4 6 8 3 )
U n cu ltu re d m a rin e b a cterium AntCL1D1 (D Q 9 0 6 7 2 0 )
100/78
WP0807B_160 (HQ143898)
86/
T P 0 4 0 8 B _0 1 5 (HQ233630)
100/99
T P 0 4 0 8 B _0 3 0 (HQ233644)
T h e rm o p h ilic m e th a n o troph HB (U 8 9 2 9 9 )
WP0807B_068 (HQ143810)
64/
WP0807B_069 (HQ143811)
TP0807B_026 (HQ143927)
U n cu ltu re d ga m m a p ro teobacterium (A M 9 3 6 5 2 1 )
100/76
76/64 WP0807B_067 (HQ143809)
U n cu ltu re d B e g g ia to a sp. (F M 1 7 6 2 9 7 )
WP0807B_063
/56
WP0807B_066
TP0807B_027 (HQ143928)
100/96
WP0807B_072 (HQ143814)
100/100
93/
U n cu ltu re d m e th a n o trophic proteobacterium (E F 5 8 7 7 4 0 )
WP0807B_159 (HQ143897)
WP0807B_062 (HQ143806)
T P 0 4 0 8 B _0 1 7 (HQ233632)
WP0807B_070 (HQ143812)
100/94
U n cu ltu re d g a m m a p ro teobacterium (F J5 1 6 8 6 6 )
U n cu ltu re d C o xie lla sp. Ax29 (E F 0 9 2 2 0 1 )
S u lfu r-o xid izing bacterium OAII2 (A F 1 7 0 4 2 3 )
WP0807B_071 (HQ143813)
100/
/75
WP0807B_065 (HQ143808)
U n cu ltu re d F ra n cise lla sp. DS058 (D Q 2 3 4 1 4 2 )
U n cu ltu re d h yd ro ca rb on seep bacterium BPC023 (A F 1 5 4 0 8 7 )
T P 0 4 0 8 B _0 1 6 (HQ233631)
WP0807B_030 (HQ143777)
WP0807B_028 (HQ143775)
100/95
100/100
0.1
Figure 4.23. Phylogenetic relationship among Gammaproteobacteria from

141
Nitrospira
Acidobacteria
P0807B_135 (HQ143873)
TP0807B_008 (HQ143909)
TP0807B_053 (HQ143954)
UCyanobacteria
n cu ltu re d Nitro sp ira sp. CL3 (F M 1 7 5 7 5 7 )
WP0807B_138
(HQ143876)
WP0807B_091 (HQ143830)
99/
100/65
TP0807B_055
(HQ143956)
TP0807B_028
(HQ143929)
/55
WP0807B_077
(HQ143818)
WP0807B_136
(HQ143874)
100/100
WP0807B_086 (HQ143825)
/62
100/95
/100
U n cu ltu re d acid o b a cterium (A Y 9 2 1 9 6 6 )

TP0807B_029 (HQ143930)
TP0807B_036 (HQ143937)
/51
TP0807B_033 (HQ143934)
/78
U n cu ltu re d acid o b a cterium HAVOmat (E F 0 3 2 7 5 2 )
WP0807B_082 (HQ143821)
Gemmatimonadetes
WP0807B_087 (HQ143826)
100/100
100/97
U n cu ltu re d acid o b a cterium XME31 (E F 0 6 1 9 4 4 )
WP0807B_131 (HQ143869)
100/96(HQ143820)
/98
WP0807B_081
100/87
U n cu ltu re d G e m m a tim o nas sp. A1816 (E U 2 8 3 5
WP0807B_078
(HQ143819)
100/90
U n/98
cu ltu re d acid o b a cterium
BuhC-67 (F M 8 6(HQ143871)
6294)
WP0807B_133
TP0807B_037 (HQ143938)
100/58
U n cu
U n cu ltu re d100/98
b a cte riu m D A008
(Y 1ltu
2 5 9re
7 )d G e m m a tim o nadetes OS-C40 (E F 6 1 2 3
TP0807B_038 (HQ143939) WP0807B_134 (HQ143872)
/60 100/63
U n cu ltu re d acid o b a cterium AMAA1 (A M 9 3 5 7 7 7 )
U n cu ltu re d G e m m a tim o nadetes AMGC8 (A M 9 3 5 3 8
100/91
100/
/65
WP0807B_080
U n cu ltu re d100/97
acid o b a cteriumWP0807B_132
AMDB7 (A M 9 3 5 5 7 6 )(HQ143870)
TP0807B_034 (HQ143935)
WP0807B_130 (HQ143868)
/79
100/100 WP0807B_084 (HQ143823)
/60
U n cu ltu re d acid o bTP0807B_052
a cterium MBNT13 (F(HQ143953)
J5 3 8 1 4 4 )
A cid o b a cte riu m ca p su la tum (D 2 6 1 7 1 )
TP0807B_032 (HQ143933)
100/98
80/58
0.1
Nitrospira
P0807B_135 (HQ143873)
Figure 4.24. Phylogenetic

relationship
among Acidobacteria from Willow
/62 TP0807B_008
(HQ143909)
TP0807B_053
(HQ143954)
Point and Three Poles and based upon Maximum Likelihood analysis of
U n cu ltu re d Nitro sp ira sp. CL3 (F M 1 7 5 7 5 7 )
full 16S rRNA gene/100sequence
data.
Sequence codes refer to those given
WP0807B_138
(HQ143876)
TP0807B_055
(HQ143956)
in Table 4.6 a-j, 4.7 a-e, 4.9 & 4.10 a-b. Rooted tree supported by
WP0807B_136 (HQ143874)
bootstrap values for 1000 replicates (first number) and Bayesian posterior
probabilities (second number). Scale bar represent nucleotide change per
position.
Gemmatimonadetes
WP0807B_131 (HQ143869)
U n cu ltu re d G e m m a tim o nas sp. A1816 (E U 2 8 3 5 6 4 )
WP0807B_133 (HQ143871)
U n cu ltu re d G e m m a tim o nadetes OS-C40 (E F 6 1 2 3 8 7 )
WP0807B_134 (HQ143872)
U n cu ltu re d G e m m a tim o nadetes AMGC8 (A M 9 3 5 3 8 2 )
WP0807B_132 (HQ143870)
WP0807B_130 (HQ143868)
TP0807B_052 (HQ143953)
100/96
100/87
/98
100/98
100/63
100/91
100/97
0.1
142
Bacteroidetes
78/78
WP0807B_104 (HQ143843)
WP0807B_096 (HQ143835)
WP0807B_098 (HQ143837)
/86 /80
WP0807B_099 (HQ143838)
WP0807B_100 (HQ143839)
/100
/92
Uncultured Bacteroidetes AS38 (EU283366)
Uncultured bacterium clone 661191 (DQ404914)

100/54
/69
100/59
100/79
WP0807B_155 (HQ143893)
Uncultured Saprospiraceae TDNP Wbc97 (FJ517043)
Uncultured Cytophaga sp. (AB015265)
100/99
WP0807B_103 (HQ143842)
100/
Uniden Cytophagales OPB73 (AF027008)

Flexibacter ruber IFO16677 (AB078065)
WP0807B_097 (HQ143836)
79/78
WP0807B_101 (HQ143840)
100/99
52/63
100/94
100/85
Uncultured bacterium R1-18 (AB280282)

Uncultured bacterium MBNTS (FJ538136)
100/89
100/74
100/97
100/90
WP0807B_158 (HQ143896)
WP0807B_162 (HQ143900)
Uncultured soil bacterium 1_H12 (EU589283)
Arcocella aquatica NO-502T (AJ535729)
TP0807B_015 (HQ143916)
TP0408B_024 (HQ233639)
51/100
Uncultured Petrimonas sp. (FM176290)

WP0807B_102 (HQ143841)
Rhodothermus marinus (AF217494)
Uncultured Cytophaga sp. BD2-15 (AB015543)

Prosthecochloris aestuarii CHP 3401 (AJ291826)
Caldothrix abyssi LF13T (AJ430587)
0.1
Figure 4.25. Phylogenetic relationship among Bacteroidetes from Willow

full 16S rRNA gene sequence data. Sequence codes refer to those given
position.
143
Gammaproteobacteria
100/54
Betaproteobacteria
TP0708B_005 (HQ233610)
TP0708B_010 (HQ233615)
57/52
TP0708B_008 (HQ233613)
62/68
TP0708B_004 (HQ233609)
TP0708B_003 (HQ233608)
T P 0 7 0 8 B _0 0 7 (HQ233612)
T P 0 7 0 8 B _0 0 6 (HQ233611)
100/100
M o ra xe lla sp . E ve re st-gws-63 (E U 5 8 4 5 2 5 )
U n cu ltu re d g a m m a p ro teobacterium BG4 (F M 9 9 4 6 6 7 )
M o ra xe lla o slo e n sis PCWCW3 (G Q 2 8 4 4 7 2 )
51/51
T P 0 7 0 8 B _0 0 9 (HQ233614)
100/82
T P 0 4 0 8 B _0 2 5 (HQ233640)
100/66
92/68
T P 0 4 0 8 B _0 1 1 (HQ233627)
100/68
T P 0 4 0 8 B _0 0 9 (HQ233625)
100/98
T P 0 4 0 8 B _0 0 8 (HQ233624)
T P 0 4 0 8 B _0 1 0 (HQ233626)
T P 0 4 0 8 B _0 1 4 (HQ233629)
100/100 E scherichia co li (J0 1 8 5 9 )
100/95
T P 0 4 0 8 B _0 1 2 (HQ233628)
A eromonas so b ria A T C C 4 3 9 7 9 T (X 7 4 6 8 3 )
U n cu ltu re d m a rin e b a cterium AntCL1D1 (D Q 9 0 6 7 2 0 )
100/78
WP0807B_160 (HQ143898)
86/
T P 0 4 0 8 B _0 1 5 (HQ233630)
100/99
T P 0 4 0 8 B _0 3 0 (HQ233644)
T h e rm o p h ilic m e th a n o troph HB (U 8 9 2 9 9 )
WP0807B_068 (HQ143810)
64/
WP0807B_069 (HQ143811)
TP0807B_026 (HQ143927)
100/76
76/64 WP0807B_067 (HQ143809)
U n cu ltu re d B e g g ia to a sp. (F M 1 7 6 2 9 7 )
WP0807B_063
/56
WP0807B_066
TP0807B_027 (HQ143928)
100/96
WP0807B_072 (HQ143814)
100/100
93/
U n cu ltu re d m e th a n o trophic proteobacterium (E F 5 8 7 7 4 0 )
WP0807B_159 (HQ143897)
WP0807B_062 (HQ143806)
T P 0 4 0 8 B _0 1 7 (HQ233632)
WP0807B_070 (HQ143812)
100/94
U n cu ltu re d C o xie lla sp. Ax29 (E F 0 9 2 2 0 1 )
S u lfu r-o xid izing bacterium OAII2 (A F 1 7 0 4 2 3 )
WP0807B_071 (HQ143813)
100/
/75
WP0807B_065 (HQ143808)
U n cu ltu re d F ra n cise lla sp. DS058 (D Q 2 3 4 1 4 2 )
U n cu ltu re d h yd ro ca rb on seep bacterium BPC023 (A F 1 5 4 0 8 7 )
T P 0 4 0 8 B _0 1 6 (HQ233631)
WP0807B_030 (HQ143777)
WP0807B_028 (HQ143775)
100/95
100/100
0.1
Figure 4.26. Phylogenetic relationship among Chloroflexi from Willow

full 16S rRNA gene sequence data. Sequence codes refer to those given
position.
144
Lyngbya majuscula NAC8 47 (GU724198)

/58
Lyngbya sordida NAC8-49 (GU724201)
Lyngbya sordida NAC8 53 (GU724206)
Lyngbya bouillonii PAL08 16 (GU111927)
Uncultured Prochloron sp. clone HA 97 (DQ357965)
/56
73/99
Prochloron sp. (X63141)
Uncultured Prochloron sp. clone AO 101 (DQ357968)
Uncultured Prochloron sp. clone AO 105 (DQ357958)
98/70 Microcystis novacekii isolate TAC65 (AB012337)
Microcystis novacekii isolate TAC20 (AB012336)
72/55 Microcystis aeruginosa isolate TAC170 (AB012340)
Microcystis ichthyoblabe isolate TAC48 (AB012338)
Microcystis wesenbergii isolate TAC52 (AB012335)
100/98
74/65
Microcystis sp. KND9506 (U66194)
Microcystis sp. isolate T17 1 (AB012330)
100/100 Chroococcus sp. VP2 02 (FR798932)
100/99
Chroococcus sp. 9E 05 (FR798922)
Chroococcus sp. strain JJCM (AM710384)
100/100 Gloeothece sp. PCC 69091 (EU499305)
Gloeothece membranacea PCC 6501(X78680)
Radiocystis sp. strain JJ30 3 (AM710389)
100/100
73/91
100/58
100/
98/56
Snowella litoralis 0TU35S07 (AJ781039)

100/86
100/100
Snowella rosea 1LM40S01 (AJ781042)
100/98
Snowella litoralis 1LT47S05 (AJ781041)
Woronichinia naegeliana 0LE35S01 (AJ781043)
Synechocystis salina LEGE 06155 (HQ832911)
Synechocystis sp. PCC 6805 (AB041938)
Synechocystis sp. MMG 8 (FJ839359)
Synechocystis aquatilis 1LT32S04 (FM177503)
Synechocystis sp. CCALA_700 (GQ375043)
Merismopedia glauca 0BB39S01 (AJ781044)
Synechococcus sp. strain PCC7003 (AB015059)
100/99
Synechococcus sp. strain PCC 7002 (AJ000716)
Merismopedia punctata PMC26006 (GQ859638)
61/74
100/96
100/59
96/71
93/78
53/
Aphanocapsa sp. HBC6 (EU249123)
/54
53/68
51/78
100/96
Crocosphaera watsonii WH 8501 (AY620237)

Uncultured cyanobacterium clone A115-7 (AY323171)
Aphanothece sacrum (AB119259)
Cyanothece sp. PCC 8801 (AF296873)
90/
80/
100/100
Oscillatoria sancta SAG 7479 (EU196639)

100/85
Hydrocoleum lyngbyaceum HBC7 (EU249124)
Lyngbya sp. BAN TS30 (HQ419199)
Trichodesmium havanum str F34_5 (AF518770)
Trichodesmium sp. (X70767)
Hydrocoleum sp. GV58 (DQ883636)
Oscillatoria margaritifera NAC8 54 (GU724207)
Oscillatoria sp. NAC8 18 (GU724195)
Oscillatoria sp. PAB 21 (EU253967)
Stromatolite
(Bahamas)
61/67
100/
100/81
86/
Coral
Trichodesmium hildebrandtii (AF091322)
/61
53/68 Uncultured bacterium clone StromD08 (EU917988)

53/99
Uncultured bacterium clone StromC08 (EU917975)
Uncultured bacterium clone StromH08 (EU918027)
Uncultured cyanobacterium clone BBD 216-32 (DQ446127)
53/93
53/51
51/52 Tychonema tenue SAG 482 (GQ324973)

100/96
Tychonema sp. K27 (GQ324965)
Tychonema bourrellyi HAB663 (FJ184385)
Microcoleus vaginatus PCC 9802 (AF284803)
Arthrospira maxima (AF260509)
Arthrospira platensis EEW4 (HQ008227)
53/68 Arthrospira platensis MMG 9 (FJ839360)
100/97
Spirulina sp. EEW5 (HQ008228)
Arthrospira sp. PCC 9901 (EU183352)
Lyngbya majuscula CCAP 14464 (HQ419207)
(Montastraea sp.)
(Florida & Bahamas)

Stromatolite
(Australia & others)
100/99
76/67
Planktothrix pseudagardhii HAB639 (GQ457311)

Phormidium sp. Ant Brack 3 (AF263321)
/75
/57
/100
Chroococcidiopsis sp. Gob 91 19 (AY301042)

Chroococcidiopsis sp. PCC 7431 (AB074506)
/99
Chroococcidiopsis thermalis (AB039005)
/100
Chroococcidiopsis sp. 9E 07 (FR798923)
Chroococcidiopsis sp. MMG 6 (FJ839357)
Chroococcidiopsis sp. BB792 SAG 2023 (AJ344552)
Plectonema wollei JW 2010c (HQ419202)
Plectonema wollei NO99 (HQ419204)
85/82 Brasilonema sp. CENA114 (EF117246)

Brasilonema octagenarum UFV OR1 (EF1508552)
100/95
Brasilonema terrestre CENA116 (EF490447)
100/68
Brasilonema bromeliae SPC 951 (DQ486055)
100/100 Crinalium epipsammum SAG 2289 (AB115964)
Crinalium magnum SAG 3487 (AB115965)
Microcoleus chthonoplastes WW10 (EF654058)
Microcoleus chthonoplastes CCY9608 (GQ402024)
Microcoleus chthonoplastes WW11 (EF654059)
Uncultured bacterium clone TP0807B 049 (HQ143950)
/89
Uncultured bacterium clone TP0807B 048 (HQ143949)
Phormidium murrayii ANTLPE2 (AY493598)
100/100
Phormidium aerugineo caeruleum strain 9N 01 (FR798943)
72/98
73/94
I-III
Coral
Pseudanabaena mucicola PMC26906 (GQ859643)
53/
Pseudanabaena sp. strain 1a 03 (FR798944)
Pseudanabaena PCC7403 (AB039019)

53/65
53/75
100/99
53/
(Siderastrea sp.)
Uncultured bacterium clone WP0807 (HQ143853)

Uncultured bacterium clone WP0807B 124 (HQ143863)
Pseudanabaena sp. 63 1 (EF110976)
Filamentous cyanobacterium 72-1 (EU196365)
Uncultured Gloeobacter sp. clone HAVOmat17 (EF032784)
(Bahamas)
Symploca atlantica CCY9617 (GQ402026)

80/70
100/80
Symploca sp. CCY0030 (GQ402025)
Lyngbya sp. UTEX LB 2516 (HQ419209)
Symploca sp. HBC5 (EU249122)
Symploca sp. VP642b (AY032933)
100/69
100/84
Uncultured Cylindrospermopsis sp. clone TH16S99 (FJ847397)

Raphidiopsis mediterranea FSS1 1501 (HQ730899)
53/99
Raphidiopsis sp. PMC0005 (GQ859597)
Cylindrospermopsis raciborskii form 1 (AF067818)
/98
Cylindrospermopsis raciborskii strain 09A (AF516728)
/97 Sphaerospermopsis aphanizomenoides PMC64110 (HQ157698)
/56
Aphanizomenon aphanizomenoides ANA280B (FJ234886)
Anabaena aphanizomenoides CENA188 (FJ830569)
Cuspidothrix issatschenkoi PMC21003 (GQ859624)
88/99
Aphanizomenon issatschenkoi 473 (FJ424566)
Dolichospermum flos aquae ANA311E (HQ157685)
53/
/59
Anabaena smithii TAC431 (AY701554)
100/97
Anabaena mucosa strain 1tu35s5 (AJ630425)
100/98
Dolichospermum planctonicum PMC20003 (GQ859619)
Aphanizomenon sp. 326 (AJ293121)
/99
/53
Anabaena cf cylindrica PMC9705 (AJ293119)
89/53
Aphanizomenon gracile PMC64910 (HQ157700)
58/78 Dolichospermum heterosporum (AB558957)
Anabaena lemmermannii 1tu32s11 (AJ630424)
Trichormus variabilis strain KCTC AG10269 (DQ234833)
99/68
Anabaena sp. PCC 7108 (AF317629)
Wollea saccata ACCS 045 (GU434226)
Aphanizomenon ovalisporum UAM289 (HQ259629)
Calothrix sp. PMC23404 (GQ859627)
Cylindrospermum sp. PMC23804 (GQ859607)
Trichormus doliolum (AJ630455)
Gloeotrichia echinulata PYH14 (AM230704)
69/89
Gloeotrichia echinulata URA3 (AM230705)
Nostoc_linckia (AB074503)
Gloeotrichia sp. PMC23604 (GQ859628)
99/70 Uncultured bacterium clone WP0807B 117 (HQ143856)
Coleodesmium sp. ANTL52B5 (AY493596)
68/
Tolypothrix distorta clone 163 5B1638 (AF334694)
100/66
/51
Spirirestis rafaelensis clone 143 2A1591 (AF334690)
70/
Coleodesmium wrangelii clone 144 2B1592 (AF334702)
Nostoc flagelliforme str Sunitezuoqi (GU810186)
100/97
63/
Nostoc sp. ANTL52B7 (AY493623)
Nostoc verrucosum (AB511947)
90/
Mojavia pulchra JT2 VF2 (AY577534)
Nostoc sp. 1b 05 (FR798942)
Nodularia harveyana strain UTEX B2093 (AF268021)
Nodularia sp. PCC 9350 (AY038034)
Nodularia sp. isolate BCNOD9427 (AJ224447)
Nodularia spumigena GSL023 (FJ546713)
51/
53/78
Nodularia sphaerocarpa (AF268018)
Nodularia sp. PCC 731041 (AJ133184)
51/
Tolypothrix sp. TOL328 (AM230706)
100/74
Tolypothrix sp. PCC 7504 (AM230669)
Calothrix elenkinii RPC1 (GU292083)
Nostoc sp. PCC_7120 (AF317631)
Anabaena iyengarii RPAN6 clone 1 (GQ466520)
Anabaena variabilis RPAN16 clone 1 (GQ466514)
Anabaena laxa RPAN14 clone 1 (GQ466510)
Anabaena spiroides RPAN58 clone 1 (GQ466495)
100/69
Anabaena variabilis (AB074502)
Trichormus azollae Kom BAI1983 (AJ630454)
Anabaena sp. BHUAR002 (HM235817)
97/96
Microcoleus sp.
Coral
(Florida & others)
Pseudoanabaena sp.
Microbialite
(Canada)
Coleodesmium sp.
Westiellopsis sp. 1590-2 (AJ544089)

Westiellopsis sp. 89-7854 (AJ544222)
Fischerella muscicola strain SAG2027 (AJ544077)
Westiellopsis sp. AKM9 (HM573464)
Nostochopsis lobatus 921 (AJ544080)
Westiellopsis prolifica SAG 1693 (AJ544086)
Fischerella sp. CENA161 (EU840724)
Nostochopsis sp. 89-45 (AJ544081)
Fischerella sp. IAM M 263 (AB093491)
Mastigocladus laminosus Greenland 8 (DQ431003)
70/53
100/95
Fischerella muscicola PCC 7414 (AB075986)
99/65
Mastigocladus laminosus SAG 484 (EU116035)
Fischerella sp. MV9 (DQ786169)
Fischerella sp. JSC 11 (HM636645)
51/58
Mastigocladus laminosus Kovacik 19877B (EU116034)
100/52
94/82
Fischerella_major NIES 592 (AB093487)
Uncultured Fischerella sp. clone YL099 (HM856465)
100/100 Chlorogloeopsis sp. Greenland 5 (DQ431000)
Chlorogloeopsis sp (X68780)
100/97
100/99 Chlorogloeopsis fritschii PCC 6912 (AB093489)
Chlorogloeopsis sp. PCC 9212 (AB075982)
Stigonema ocellatum SAG 4890 (AJ544082)
100/89
Stigonema ocellatum SAMA 35 (GQ354275)
Scytonema sp. UCFS10 (HM629428)
Uncultured cyanobacterium clone D1F06 (EU753647)
/80
Uncultured cyanobacterium clone D3B06 (EU753653)
Uncultured cyanobacterium clone D2H03 (EU753652)
53/57
100/54
Uncultured cyanobacterium clone D1E07 (EU753645)
53/75
Uncultured cyanobacterium clone D2D02 (EU753650)
91/88 Calothrix sp. 2T08 (FR798918)
Calothrix parietina 2T10 (FR798917)
/80
Calothrix sp. PCC 7103 (AM230700)
/98
Calothrix brevissima AKM12 (HM573459)
Rivularia sp. MU24 UAM 305 (EU009149)
/89
Rivularia sp. VP4 08 (FR798919)
/58
Uncultured Chroococcidiopsis sp. clone_QB91 (FJ790640)
95/94
87/
58/
99/70
69/76
IV-V
Cyanobium sp. 1LT47S01 (FM177491)

90/ Cyanobium sp. PCC 6904 (AF216944)
98/64
Chroococcales cyanobacterium BN23 (HM346184)
Aphanothece sp. CENA118 (HQ380799)
Aphanothece minutissima 2LT34S03 (FM177488)
Cyanodictyon sp. strain_JJCD (AM710382)
Uncultured Chroococcales cyanobacterium clone AP107 (AY943947)
Aphanothece sp. 0BB21S01 (AJ639901)
Arctic cyanobacterium 49S1 (DQ851567)
95/
Uncultured Synechococcus sp. clone XZZLH7 (EU703495)
Merismopedia tenuissima 0BB46S01 (AJ639891)
Merismopedia sp. CENA106 (EF088332)
Cyanobium sp. LEGE 06137 (HQ832914)
Prochlorococcus sp. GP2 (AF001472)
53/82
Prochlorococcus sp. MIT9215 (AF115271)
Uncultured Prochlorococcus sp. clone JL WNPG T5 (AY664105)
/64
Candidatus Synechococcus spongiarum P129SC1 (EF656451)
98/
53/
Prochlorococcus sp. NATL2A (AF001467)
Prochlorococcus marinus (X63140)
Prochlorococcus sp. MIT9303 (AF001469)
Uncultured cyanobacterium clone BBD216b-20f (EF123579)
/62
Uncultured Synechococcus sp. clone PmeaH2OA6 (EU249939)
Uncultured cyanobacterium clone WA-71f (EF123581)
Synechococcus WH8101 (AF001480)
Synechococcus sp. CB0205 contig00003 (NZ_ADXM01000002)
53/
Microbialite
(Spain)
53/
Calothrix sp.
Chroococcidiopsis sp.
/86
Uncultured cyanobacterium clone 1DP1-N8 (EU780371)

Uncultured cyanobacterium clone MD3-8 (FJ425596)
Leptolyngbya margaretheana strain 1T12 (FR798934)
Leptolyngbya PCC7104 (AB039012)
Spirulina laxissima strain SAG 25680 (DQ393278)
Uncultured cyanobacterium clone CD207B07 (DQ200576)
99/78 Phormidium persicinum SAG 80.79 (EF654085)
88/77
Leptolyngbya sp. PCC7375 (AF132786)
Leptolyngbya PCC7375 (AB039011)
53/57
Leptolyngbya sp. P2b-2 (EF372581)
100/100
100/94
100/92
Leptolyngbya sp. FLKBBD1 (EF110975)
Leptolyngbya sp. HBC2 (EU249128)
100/71
Uncultured cyanobacterium clone 4DP1-A17 (EU780373)
53/
Filamentous cyanobacterium FLK9 (EU196364)
Cyanobacterium 62-2 (EU223007)
Filamentous cyanobacterium 73-2 (EU196366)
Halomicronema sp. TFEP1 (AF320093)
100/99
Halomicronema sp. SCyano39 (DQ058860)
Halomicronema sp. PCyano40 (DQ058890)
Uncultured cyanobacterium clone ss2206-8 (HM748584)
Pseudanabaena sp. LEGE-07190 (HQ832919)
100/96
Phormidium priestleyi ANTLACV51 (AY493586)
Uncultured cyanobacterium clone 1HP1-B24 (EU780310)
100/69
Uncultured cyanobacterium clone 3MP1-D1 (EU780279)
100/97 Limnothrix sp. B15 (GQ848190)
95/91
Limnothrix sp. B14 (HM151382)
100/100
Geitlerinema cf. lemmermannii P-SA (EU196642)
100/100 Limnothrix sp. CENA111 (EF088336)
Limnothrix redekei 2LT25S01 (FM177493)
Acaryochloris sp. HICR111A (EU873540)
Acaryochloris marina strain MBIC-11017 (AY163573)
Acaryochloris sp. HICR111A (EU873540)
Uncultured Acaryochloris sp. clone HICR111A2-9 (GQ370819)
100/100
Uncultured Acaryochloris sp. clone HICR111A2-9 (GQ370819)
Uncultured Acaryochloris sp. isolate SRODG092 (FM995185)
Uncultured bacterium clone TP0807B-047 (HQ143948)
Uncultured Thermosynechococcus sp. clone DTB123 (EF205530)
/87
/94
Uncultured Thermosynechococcus sp. clone TP111 (EF205554)
Uncultured Thermosynechococcus sp. clone DTB140 (EF205542)
Microbialite
(Canada)
100/82
Coral
(Hawaii, Carribean
& others)
70/63
100/99
53/
58/
77/
Outgroup
100/
100/85
80/58
53/
53/
87/58
100/80
Leptolyngbya sp.
Coral
(Porites sp.)
(Australia)
Microbialite
(Canada)
Acaryochloris sp.
Acaryochloris sp. JJ8A6 (AM710387)

Uncultured bacterium clone WP0807B-121 (HQ143860)
/75
I, III

63/89
/62
Uncultured cyanobacterium clone D3C04 (EU753642)
Uncultured cyanobacterium clone D2A05 (EU753635)
Uncultured cyanobacterium clone D1G07 (EU753633)

Uncultured cyanobacterium clone D1A07 (U753623)
75/64
Leptolyngbya sp. 1T12c (FR798935)
53/
53/55
67/78
53/
Uncultured cyanobacterium clone D2C04 (EU753638)
Microbialite
(Spain & Canada)
Plectolyngbya hodgsonii ANTLG21 (AY493615)

100/99
Plectolyngbya hodgsonii ANTLPR22 (AY493583)
/98
Plectolyngbya hodgsonii ANTLG22 (AY493618)
Leptolyngbya foveolarum VP1-08 (FR798945)
Uncultured Gloeothece sp. clone TRK02 (AY874086)
100/94
Leptolyngbya sp.
53/61
53/
53/
53/
52/79
Uncultured cyanobacterium clone 1DP2-H24 (EU780334)

Uncultured cyanobacterium clone 1DP2-B8 (EU780332)
Uncultured bacterium clone WP0807B_125 (HQ143864)
53/
Uncultured cyanobacterium clone 2DP1-F24 (EU780387)
53/
Uncultured cyanobacterium clone 1DP2-M1 (EU780354)
Uncultured cyanobacterium clone 1DP2 P7 (EU780346)
Uncultured cyanobacterium clone 2DP1 I24 (EU780372)
68/
53/
53/
53/
/57
Coral
53/
0.2
53/86
53/69
53/87
53/54
53/95
89/88
Uncultured cyanobacterium clone 1DP2 B3 (EU780349)

Uncultured cyanobacterium clone 2DP1 C8 (EU780364)
Uncultured cyanobacterium clone pItb vmat-79 (AB294971)
Uncultured cyanobacterium clone MD3 43 (FJ425631)
/97
Uncultured cyanobacterium clone MD3-14 (FJ425602)
Uncultured cyanobacterium clone 1DP1-L11 (EU780385)
Uncultured cyanobacterium clone 2DP1-E9 (EU780386)
(Turbinaria sp.)
(Australia)
Microbialite
(Canada)
Uncultured Brasilonema sp. clone NGP-11 (EU037931)

Clostridium perfringens (Y12669)
0.2
Figure 4.27. Phylogenetic relationship among Cyanobacteria from Willow Point and
Three Poles and based upon Maximum Likelihood analysis of full 16S rRNA gene
sequence data. Pink * refers to clone given in Table 4.6 a-j, 4.7 a-e, 4.9 & 4.10 a-b. Blue
branches refer to samples from marine environments and Green samples refer to clone
from freshwater environments. (I:Chroococcales; II:Pleuricaapsales; III: Oscillatoriales;
IV:Nostocales; V:Stigonematales) Rooted tree supported by bootstrap values for 1000
replicates (first number) and Bayesian posterior probabilities (second number). Scale
bar represent nucleotide change per position. (PDF file with complete sequence codes
are available on request).
145
TP0807B_034
(HQ143935) U n cu ltu re d acid o b a cterium (A Y 9 2 1 9 6 6 )
100/98
WP0807B_138
(HQ143876)
TP0807B_029
WP0807B_084
(HQ143823) (HQ143930)
80/58
100/100
TP0807B_055 (HQ143956)
/60
TP0807B_036
(HQ143937)
U
n
cu
ltu
re
d
acid o b a cterium
MBNT13 (F J5 3 8 1 4 4 )
Gemmatimonadetes
WP0807B_136 (HQ143874)
/51
TP0807B_033
(HQ143934)
A cid o b a cte riu m ca p su la tum (D 2 6 1 7 1 )
/78
WP0807B_131
(HQ143869)
U n cu ltu100/96
re d(HQ143933)
acid
o b a cterium HAVOmat
(E F 0 3 2 7 5 2 )
TP0807B_032
100/87
U
n
cu
ltu
re
d
G
e
m
m
a
tim
o nas sp. A1816 (E U 2 8 3 5
WP0807B_082 (HQ143821)
/98
WP0807B_133
(HQ143871)
WP0807B_087 (HQ143826)
100/100
Gemmatimonadetes
n cu
G eom
a tim o nadetes
OS-C40
100/97 100/98
UU
n cu
ltultu
rere
d dacid
b amcterium
XME31 (E
F 0 6 1 9(E
4F
4 )6 1 2 3
WP0807B_131
(HQ143869)
100/96
/98
WP0807B_134
(HQ143872)
Nitrospira 100/63
100/87
UWP0807B_081
n cu ltu re d G e m m(HQ143820)
a tim o nas sp. A1816 (E U 2 8 3 5 6 4 )
U
n
cu
ltu
re
d
G
e
m m a tim o nadetes AMGC8 (A M 9 3 5 3 8
100/91
/98
WP0807B_078
(HQ143819)
WP0807B_133 (HQ143871)
100/90
U
n
cu
ltu
re
d
G
e
m
m
a
tim
o
nadetes
OS-C40 (E(F
F6
WP0807B_132
(HQ143870)
100/98
U n 100/97
cu ltu re d acid
o b a cterium
BuhC-67
M182 368672) 9 4 )
P0807B_135
(HQ143873)
WP0807B_134
(HQ143872)
100/63
WP0807B_130
(HQ143868)
TP0807B_037
(HQ143938)
/62 TP0807B_008
(HQ143909)
U n cu ltu re d G
e m m a tim o nadetes
AMGC8 (A M 9 3 5 3 8 2 )
100/91
100/58
TP0807B_052
(HQ143953)
U
n
cu
ltu
re
d
b
a
cte
riu
m
D
A008
(Y
1
2597)
TP0807B_053
(HQ143954)
WP0807B_132
(HQ143870)
100/97
TP0807B_038
(HQ143939)
WP0807B_130
/60
U n(HQ143868)
cu ltu re d Nitro sp ira sp. CL3 (F M 1 7 5 7 5 7 )
U n cu ltu reTP0807B_052
d acidWP0807B_138
o b a(HQ143953)
cterium AMAA1
(A M 9 3 5 7 7 7 )
(HQ143876)
/100
100/
/65
WP0807B_080
TP0807B_055 (HQ143956)
0.1
U n cu ltu re d acid oWP0807B_136
b a cterium AMDB7
(A M 9 3 5 5 7 6 )
(HQ143874)
0.1
Figure 4.28.
Phylogenetic
relationship among Gemmatimonas from
TP0807B_034
(HQ143935)
WP0807B_084
and Three
Poles and(HQ143823)
based upon Maximum Likelihood
/79 Willow Point 100/100
/60
U
n
cu
ltu
re
d
acid
a cterium
MBNT13
(F J5
3 8 1to
44)
analysis of full 16S rRNA gene sequenceo bdata.
Sequence
codes
refer
cida-e,
o b a cte
ca p su
la tum
(D 2 6tree
171)
those given in Table 4.6 a-j, A
4.7
4.9riu&m4.10
a-b.
Rooted
TP0807B_032
(HQ143933)
Gemmatimonadetes
WP0807B_131 (HQ143869)
100/96
100/87
U n cu ltu re d G e m m a tim o nas sp. A1816 (E U 2 8 3 5
Nitrospira
/98
WP0807B_133 (HQ143871)
U n cu ltu re d G e m m a tim o nadetes OS-C40 (E F 6 1 2 3
100/98
WP0807B_134
(HQ143872)
P0807B_135
(HQ143873)
100/63
/62 TP0807B_008
U n cu(HQ143909)
ltu re d G e m m a tim o nadetes AMGC8 (A M 9 3 5 3 8
100/91
TP0807B_053
(HQ143954)
WP0807B_132
(HQ143870)
100/97
U n cu ltu re
d Nitro sp ira sp.
CL3 (F M 1 7 5 7 5 7 )
WP0807B_130
(HQ143868)
WP0807B_138
(HQ143876) (HQ143953)
TP0807B_052
/100
TP0807B_055 (HQ143956)
WP0807B_136 (HQ143874)
/79
/100
0.1
Figure 4.29. Phylogenetic relationship among Nitrospira from Willow Point

Gemmatimonadetes
and Three Poles and based upon Maximum Likelihood analysis of full
16S rRNA gene sequence
data.
Sequence codes
refer to those given in
WP0807B_131
(HQ143869)
100/96
100/87
Table 4.6 a-j, 4.7 a-e, 4.9 & 4.10
Rooted
supported
bootstrap
U na-b.
cu ltu
re d Gtree
emm
a tim o nasbysp.
A1816 (E U 2 8 3 5 6 4 )
/98
values for 1000
replicates WP0807B_133
(first number)(HQ143871)
and Bayesian posterior
U nScale
cu ltu re
G e m m a tim
o nadeteschange
OS-C40
100/98
probabilities (second
number).
bard represent
nucleotide
per(E F 6 1 2 3 8 7 )
WP0807B_134
(HQ143872)
100/63
position.
U n cu ltu re d G e m m a tim o nadetes AMGC8 (A M 9 3 5 3 8 2 )
100/91
WP0807B_132 (HQ143870)
100/97
WP0807B_130 (HQ143868)
TP0807B_052 (HQ143953)
0.1
146
Planctomycete and uncultured bacteria
WP0807B_145 (HQ143883)
Uncultured planctomycete g10 (EU979019)
100/100 WP0807B_143 (HQ143881)
100/70
Uncultured planctomycete DEL34 (AJ616275)
Uncultured planctomycete DEL75 (AJ616291)
100/
100/71
WP0807B_144 (HQ143882)
TP0807B_062 (HQ143963)
54/
Uncultured planctomycete COM-33 (AB451756)
100/100
WP0807B_150 (HQ143888)
100/95
WP0807B_141 (HQ143879)
100/100
Uncultured planctomycete delphB8 (FM209076)
100/87
WP0807B_146 (HQ143884)
100/100
Uncultured planctomycete C11 (EF664756)
76/62
100/91
TP0807B_058 (HQ143959)
100/99
TP0807B_056 (HQ143957)
TP0807B_059 (HQ143960)
/96 TP0807B_069 (HQ143970)
/98
TP0807B_070 (HQ143971)
/79
TP0807B_036 (HQ143937)
TP0408B_023 (HQ233638)
Uncultured planctomyceteD1E01 (EU753675)
100/82
100/76
81/53
WP0807B_142 (HQ143880)
WP0807B_074 (HQ143815)
/87
TP0807B_066 (HQ143967)
WP0807B_149 (HQ143887)
/61
/95
/86
TP0807B_057 (HQ143958)
Uncultured planctomycete COM-32 (AB451755)
WP0807B_140 (HQ143878)
/100
Uncultured planctomycete AMBH12 (AM935707)
Uncultured planctomycete MVS-107 (DQ676396)
100/100
WP0807B_148 (HQ143886)
TP0408B_027 (HQ233642)
Uncultured Pirellula sp. CL5.H34 (FM176302)
WP0807B_139 (HQ143877)
Uncultured planctomycete LC1-32 (DQ289903)
100/54
WP0807B_147 (HQ143885)
WP0807B_151 (HQ143889)
100/99
Uncultured planctomycete IODP1319B109.42 (AB433105)
TP0807B_061 (HQ143962)
WP0807B_156 (HQ143894)
/74
Uncultured bacterium DS3-53 (DQ463232)
/92
WP0807B_163 (HQ143901)
/53
/59
Uncultured soil bacterium (FJ621051)
WP0807B_094 (HQ143833)
100/83
/91
Uncultured actinobacterium GASP-MB1W2 (EF664832)
TP0807B_047 (HQ143948)
WP0807B_128 (HQ143866)
WP0807B_154 (HQ143892)
Uncultured anaerobic bacterium C-4 (DQ018787)
TP0807B_071 (HQ143972)
TP0807B_064 (HQ143965)
WP0807B_079
96/82 TP0807B_065 (HQ143966)
100/99 WP0807B_152 (HQ143890)
Uncultured Verrucomicrobia TRK26 (AY874111)
WP0807B_090 (HQ143829)
WP0807B_089 (HQ143828)
TP0807B_050 (HQ143951)
100/100
100/
100/
98/
100/77
100/83
100/84
100/
100/
100/97
100/83
0.1
Figure 4.30. Phylogenetic relationship among Planctomycete and

uncultured bacteria from Willow Point and Three Poles and based upon
Maximum Likelihood analysis of full 16S rRNA gene sequence data.
Sequence codes refer to those given in Table 4.6 a-j, 4.7 a-e, 4.9 & 4.10
a-b. Rooted tree supported by bootstrap values for 1000 replicates (first
number) and Bayesian posterior probabilities (second number). Scale bar
represent nucleotide change per position.
147
4.4 Discussion
Modern microbialites occur in tropical marine environments such
as Exuma Sound, Bahamas (Dravis 1983; Reid et al. 2000) and Shark Bay
West Australia (Hoffman, 1976; Playford and Cockbain, 1976); and
freshwater lakes in Cuatro Cinegas Basin in northern Mexico (Breitbart et
al. 2009) and Pavilion Lake in British Columbia (Lavel et al. 2000). These
two environments are very distinctive by their properties such as pH and
salinity; including the morphological differences in internal and external
structures of microbialites. Extensive research has been carried out in
marine
stromatolites.
However,
little
knowledge
exists
concerning
microbialites structure in freshwater environments. In this chapter,

culturable-independent molecular techniques including 16S rRNA gene
analysis revealed a total bacterial diversity in a freshwater microbialites
system. Interestingly, highly diverse communities in Pavilion Lake were
obtained and closely mirrored the community structures observed in Shark
Bay stromatolites (Goh et al. 2009). This observation supported the quality of
work presented in this study and further suggested certain microbial groups
are essential for microbialites formation regardless of their origins. Bacterial
communities for both marine and freshwater were abundant with
Proteobacteria, especially Alphaproteobacteria (29% Shark Bay; 22% Willow
Point; 24% Three Poles). Cyanobacteria made up 11% of Shark Bay
stromatolites and 14%, 4% of microbialites in Willow Point and Three Poles
respectively. The abundance of Planctomycetes (5% Shark Bay; 8% Willow
148
Point; 5% Three Poles), Deltaproteobacteria (4% Shark Bay; 5% Willow

Point; 7% Three Poles) and Bacteroidetes (8% Shark Bay; 4% Willow Point;
0% Three Poles) were quite consistent in both environmental settings. A few
differences were also observed. 29% of the bacterial community in Shark
Bay was Gammaproteobacteria, whereas Willow Point and Three Poles only
had 5% and 3% respectively; The abundance of Actinobacteria were 11%,
2% and 1% for Shark Bay, Willow Point and Three Poles respectively. High
abundance of, Acidobacteria (11% in Willow Point, 16% in Three Poles), was
only discovered in Pavilion Lake, but not in Shark Bay (Goh et al. 2009).
The similarity in community structure between a marine and freshwater
microbialites system, indicate that there is little selective pressure based
upon salinity. Rather, it may be that a common community assembly is
necessary for biogenesis of carbonate structures such as these.
Coral reefs, microbialites and stromatolites are macro-carbonate
structures and have distinctive formation origins. Coral reefs are formed by
skeletal material by coral animals, while microbialites and stromatolites were
thought to have microbial origins. Despite the difference in deposition
principles and environments; it is interesting to note that the same
Cyanobacteria species were present in both marine corals and freshwater
microbialites. Freshwater provides a very unique habitat for a diverse range
of organisms. In contrast, marine environment displays a totally different type
of salt tolerant organisms. These marine stromatolites environments are
characterized by various physiological challenges. Since these stromatolites
149
are formed at the intertidal zone of open marine, microbial communities are
subjected to salinity, desiccation, chemical and light gradients, as well as
strong erosion at the coastal region (Goh et al. 2009). In particular, salinity is
one major factor influencing marine stromatolites biodiversity. Even though it
is arguable how salinity would affect the bacterial and archaeal biodiversity,
previous
studies
suggested
microbial
diversity
is
salinity
sensitive
(Rodriguez-Valera et al. 1985; Rodriguez-Valera, 1988, 1993; Benlloch et al.

2002; Oren 2002; Jungblut et al. 2005, Vallerie and Jackson 2009). These
factors may result in different metabolic requirements for marine and
freshwater, therefore, it is reasonable to assume freshwater and marine
environments are ecologically distinctive and so predict their microbial
structures are quite different from each other. A comparison of marine and
freshwater setting provides an opportunity to understand microbial diversity
as an adaptive response that reflects both environmental diversity. It was
quite surprising that comparable structures were found in marine
stromatolites in Shark Bay and freshwater microbialites in Pavilion Lake.
This suggests that the abiotic constraints on stromatolite/microbialites
formation are not stringent with regard to marine versus freshwater systems.
Several phyla, including Proteobacteria and Cyanobacteria, are consistently
recovered from varied environments. These recoveries were not affected by
salinity level and these bacteria groups appear essential in the formation of
microbialite communities. High abundance of Acidobacteria in Pavilion Lake
may be unique to freshwater microbialites but not to marine stromatolites,
150
since it has been isolated from freshwater and bacterial mats before (Barns
et al. 1999). Similar microbial structures may also raise the questions about
origin of these microbialites, when the evolution pathways for freshwater and
marine systems are totally different.
Among
many
stromatolite/microbialite
communities,
Cyanobacteria were identified as the dominant phylum. Earlier studies of

Pavilion Lake microbialites revealed a high abundance of Cyanobacteria
detected from microscopy pictures of microbialites surfaces (Lavel et al.
2000).
They
included
Gloeocapsa,
Synechococcus,
Fischerella,
Pseudoanabaena, Oscillatoria and Calothrix. . Previous studies indicated

Cyanobacteria are critical for the accretion and early lithication of
microbialites (Visscher et al. 1998; Visscher et al. 2000; Reid et al. 2000,
Reid et al. 2003). They trap and bind oolitic sediments to form stromatolites.
It is also suggested that remains of dead Cyanobacteria also supply
materials for stromatolite formation. Some authors have suggested that
extracellular polymeric secretions (EPS) of Cyanobacteria are important in
calcium carbonate precipitation (Kawaguchi and Decho 2000, 2001;
Braissant et al. 2007) and are critical to stromatolites accretion (Reid et al.
2000). Even though the biogenicity of microbialites remains unclear whether
the process is microbial mediated, biomineralization of microorganisms or
purely abotic (Walter 1976; Riding 1990; Grotzinger and Rothman 1996;
Summer and Grotzinger 1996, 2000; Riding and Awaramik 2000; Wierzcho
et al. 2005); Cyanobacteria are still considered significant in microbialites
151
ecosystem by their abundance. Clone libraries generated for this study

cultured
eight
genera
Chroococcidiopsis,
of
Cyanobacteria.
Cyanothece,
They
include
Cylindrospermum,
Calothrix,
Gloeotrichia,
Leptolyngbya, Microcoleus and Pseudanabaena. High abundance of

Leptolyngbya (8.8%) isolated from Pavilion Lake microbialites and marine
stromatolites (Foster et al. 2005) suggest it plays a role in microbialites
environment. Leptolyngbya is an Oscillatoriales Cyanobacteria and able to
produce
plenty
Leptolyngbya
of
from
EPS
production.
libraries
show
Moreover,
their
origin
several
from
isolates
hotspring
of
and
cyanonbacterial mats. One particular strain closely affiliated to Leptolyngbya

frigida is a dominant species in benthic mats. This Oscillatoriales
Cyanobacteria genus was suggested to be a key microbial builder for these
laminated carbonates structures in the environments. Moreover, the
inconsistent results obtained by microscopy and 16S rRNA gene analysis
revealed the limitation of both culturable dependent and independent
methods for microbialites. Relatively slow sedimentary rate
(2.5 cm per
thousand years; Lavel et al. 2000; Lim et al. 2009) for these highly calcified
microbialites resulted in highly heterogeneity within structure. Therefore,
traditional DNA extraction method may show limitation for microbialites
diversity studies. The heterogenous recovery of phylotypes in seasonal
clone libraries also suggests that other sources of bias against recovery of
environmental taxa from microbialites exist. However, widely accepted
protocol for environment were applied to ensure data can be compared to
152
similar investigations. In addition, Cyanobacterial clone library using

Cyanobacterial-specific primer are suggested for an in- depth description of
Cyanobacteria in the communities.
Recent studies indicate proteobacteria are ecologically important
in many environments. They are prevalent microbial groups in many
ecosystems,
as
well
as
extreme
environments.
In
traditional
microbialites/stromatolites studies, works were focused on Cyanobacteria.

However recent 16S rDNA-based estimates of microbial diversity pointed out
microbialites systems were not cyano-dominated but dominated by high
abundance
of
non-phototrophic
phylotypes.
These
non-phototrophic
assemblages were dominated by proteobacteria that are capable of

producing EPS as well (Gundlapally and Gracia-Pichel 2006). This result
further
supports
the
significant
of
proteobacteria
in
microbialites
development. Moreover, this trend for recording high numbers of

proteobacteria in niches, thought to be dominated by Cyanobacteria, is
common among different environments, eg. Deserts (Pointing et al. 2007);
Hot springs (Lau et al. 2009).
Pavilion Lake is a relatively small-scale, groundwater-fed
freshwater lake (Lim et al. 2009). It provides a perfect model of a closed
water system to study microbialites, since it is relatively easy to point out the
physical properties (e.g. water chemistry) that relate to microbial diversity in
microbialites, while open marine stromatolites environments do not have
such advantages. It was assumed that the environment is homogeneous.
153
Even though there was a high abundance of Betaproteobacteria (which

suggests a freshwater contaminant) present in Three Poles, In general,
similar microbial structures were present at both Willow Point and Three
Poles, and although community assembly at higher taxonomic ranks was
similar, implying common ecological function (Philippot et al. 2010).
Biodiversity at the genus level was more varied. For example, the
Cyanobacteria, Cyanothece and Microcoleus, were isolated from Three
Poles library but not in Willow Point. Spatial variation in diversity was
discovered between two transects. The two sampling sites are only 1km
apart with no physical barrier in between. Morphological features of
microbialites from two transect were slightly shifted.
For example the
shallow features at Three Poles were found at 10-meter depth when Willow
Point appeared at 21-meter depth. It appears there is little or no invasive
colonization between these structures within the lakes. Bass Becking has
said, Everything is everywhere, but the environment selects. Findings in
this study second this hypothesis. While similar microbial structures and
microbes origins were observed in different microbialites ecosystems.
Several genera of bacterial group were thought to be critical for their
development. However, environmental and localized factors including salinity
vary their diversity within species. Reflecting a slight variation in bacteria
population in spatial scale suggests environmental heterogeneity, founder
effects or maybe past ecological events or disturbances (Dion 2008).
Therefore, studying microbial diversity in microbialites provides implications
154
of how microbial life would possibly evolved on Mars. However, these

variations displayed in this study remain unexplained by currently measured
variables. A multidisciplinary study is suggested to attend this question.
Pavilion Lake microbialites represent a dynamic system where
species composition varies with temporal scale. To date there have been no
studies
comparing
the
temporal
variation
in
microbiology
and
morphologically of microbialites communities. This is particular important to

access the population of specific group that may contribute to unique
morphological features, especially when distinct macroscopic variation were
observed from pavilion lake microbialites from difference seasons. In a
freshwater lake ecosystem, seasonal variation exhibits other physical stress
that is not common to the stromatolites from a tropical marine setting. When
the ocean can act as a big buffer, freeze-thaw action and chemical variation
observed in freshwater Pavilion Lake can be quite substantial for microbial
life. Microbial communities have to able to tolerate these physiological and
chemical stresses. Previous study showed freezing tolerance capacity in
Cyanobacteria is related to their production of EPS (Tamaru et al. 2005).
Therefore, it is interesting to further examine the microbial diversity
difference
between
seasons,
especially
to
the
EPS
producing
microorganism.
Previous
studies
have
identified
archaea
communities
in
microbialites systems. Microscopy studies showed archaea communities

were closely associated with Cyanobacteria below the surface (Goh et al.
155
2009). Recent study indicated microbial activities (in particular archaea)

were proposed to contribute to stromatolites formation (Brady et al. 2010,
Michaelis et al. 2002). Several species were able to fix carbon; at the same
time they are working synthrophically with sulfate reducing bacteria to carry
out anaerobic methane oxidation that can also lead to carbonate
precipitation (Michaelis et al. 2002). Presences of archaea were identified in
freshwater pavilion lake microbialites. While other studies only show the
relative abundance of sample in bulk, this is the first study revealing the
qualitative difference among three domains within stromatolites. This
suggests geochemical process is not uniform throughout microbialites.
Results show the complexity of microbialites system.
The upper (and
possibly younger) parts with more photosynthetic taxa and the basal parts in
the sediment dominated more by archaea mediated process typical of lake
sediments in general.
Microbial structures in modern microbialites present a relic
community of the earliest biosphere on Earth. These analogues provide
insight of into the ancient stromatolites that were dominated early life on
Earth (McNamara and Awramik 1992) and proxies to the paleoenvironmental
conditions. Ancient stromatolites are thought to be microbial in origin and
potentially preserve evidence of the earliest life form and their living
environment
regarding
seawater
chemistry,
climates
and
other
environmental parameters (Walter 1983; Grotzinger and Knoll 1999; van

Kranendonk 2006). Accessing the contemporary freshwater microbialites
156
structures, in particular using molecular approaches, will allow researchers

to reveal that the ancient microbial communities are much more diverse than
originally thought (Burns et al. 2009). They are dominated by bacterial and
archaeal communities with little eukaryotic component, this suggests the
possible life form and their role in carbonate-rich environments found on
early Earth and maybe in early Mars. The dynamic interaction within the
freshwater microbialites system also reveals how life could survive through
such extreme environments and consequently, the evolution of life in Earths
history.
157
4.5 Conclusion
In this chapter, a complex microbial system of unstudied
freshwater microbialites in Pavilion Lake was revealed and identified. To
summarize:
-
Microbial
elucidated;
communities
revealing
of
high
(Alphaproteobacteria),
freshwater
abundance
Cyanobacteria
microbialites
by
were
proteobacteria
(Leptolyngbya)
and
Acidobacteria by rDNA libraries construction, whereas previous

morphological exam only shows the presence of Cyanobacteria
(Gloeocapsa, Synechococcus, Fischerella, Pseudoanabaena,
Oscillatoria and Calothrix) and few Diatoms (Lavel et al. 2000).
-
Very
similar
microbial
community
structures
to
marine
stromatolites were also identified in Pavilion Lake microbialites.

However, the data suggest that the Pavilion Lake microbialites
represent a unique system where species composition varies with
spatial scale. Different microbial species were identified from two
transacts within the same lake.
- Temporal variation was suggested in these freshwater
microbialites at the macroscopic and molecular level (although
this experiment requires further experimental proof). It is proposed
seasonal effects may play a role in the formation of morphological
features in this freshwater microbialites system where seasonal
climate changes are severe.
158
Finally, a dynamic microbial system in freshwater Pavilion Lake was

revealed and data suggestions there are interactions between the biotic and
abiotic environments. Further multidisciplinary research is suggested to
identify such variables and their roles in life evolution.
159
Chapter 5. Microbial diversity of carbonate-rich, microbial mats

in saline, alkaline lakes of the Cariboo Plateau, British
Columbia.
5.1 Introduction
5.1.1. Microbial mats in ecosystems
Microorganisms play a vital role in ecosystems. Modern
microbial mats are present in various environments including desert
crusts (Potts 1994), coastal lagoons (Pinckney et al. 1995), hydrothermal
vents (Moyer et al. 1995), hot springs (Brock 1978; Bauld et al. 1979;
Bauld 1984; Castenholz 1984; Stal et al. 1985; Ward et al. 1986;
Kompantseva and Gorlenko 1988; Skyring et al. 1989; van Gemerden et
al. 1989; Visscher et al. 1992) and aquatic hypersaline settings
(Jrgensen and Cohen 1977; Cohen 1984; Stolz 1984; Gerdes et al.
1985; Pierson et al. 1987; DAmelio et al. 1989; Giani et al. 1989; Mir et
al. 1991; Caumette et al. 1994; Jaknke et al. 2001; Jonkers et al. 2003;
Wieland et al. 2003). Many of these environments are considered as
extreme environments for life. The extreme conditions suppress grazing
animals activity (Cornee et al. 1992; Fenchel 1998), which favor the
formation and population of microbial mats (Buhring et al. 2009).
Microbial mats represent possibly the oldest ecosystems on
Earth and yet are still abundant in the modern biosphere (Tice and Lowe
2004). Microbial mats are vertically multi-layered, sedimentary biofilms
structures mainly formed by bacteria and archaea (Dupraz and Visscher
160
2005). They are mostly consisted of Cyanobacteria, bacteria and algae

(Anderson 1958; Moss and Moss 1969; Walter et al. 1973; Halley 1976;
von der Borch et al. 1977; Pueyo-Mur 1978; Bauld 1981a; Osborne et al.
1982; DeDeckker 1983; Casanova 1986; Hammer 1986; Last and
DeDeckker 1990; Kempe et al. 1991). Microbial mats were very important
in life evolution as they contributed to the modern oxic atmosphere. The
multi-color laminated structures in microbial mat result from vertical
chemical and physical gradients of the environment. Contemporary
microbial mats systems are abundant and recognized as useful analogs
for
the
study
of
biogeochemical
processes
relevant
to
paleoenvironmentsal reconstruction in Precambrian (Bhring et al. 2009).

http://microbes.arc.nasa.gov/images/g3_funtion.swf
09/03/2010
7:08 PM
Six
major functional groups of microbes were identified in microbial
mats
and their biogeochemical cycling between the biomass and the

environments were proposed. A graphical illustration is presented in
Figure 5.1.
Figure 5.1. A conceptual model of biogeochemical cycling in microbial

mats (NASA Ames Research Center).
161
This complicated process involved 6 functional groups of

microorganisms (Visscher et al. 1998; Visscher and Stolz 2005; Van
Gemerden 1993; Dupraz and Visscher 2005). They include: (1)
Cyanobacteria are primary producers who coupling light energy to CO2
fixation and sometimes N2 fixation. They also produce Extracellular
polymeric secretions (EPS) which were suggested to be a critical
structuring agent in the formation of microbialites (Reid et al. 2000; Paerl
et al. 2001); (2) anoxygenic phototrophs utilize HS- as electron donor for
photosynthesis and fix some N2; (3) aerobic heterotrophic bacteria gain
energy from respiration of O2 and organic carbon; (4) fermenters use
organic carbon or sulfur compounds as electron donor and acceptor (Bak
and Cypionka 1987; Visscher et al. 1999); (5) Sulfate reducing bacteria
respire organic carbon with SO42- while producing HS-. They also have an
important geochemical role in the precipitation of carbonate by producing
EPS (Visscher et al. 2000; Dupraz et al. 2004; Baumgartner at al. 2006;
Braissant et al. 2007). (6) sulfide oxidizing bacteria oxidize reduced sulfur
compounds with O2 or nitrate while fixing CO2.
Among microbial mat systems, Those in hypersaline lakes face
severe environmental stress (Oren 1999). Hypersalinity resulting in
extreme abiotic stress could effects water activity and balance, and
induced internal physiological changes in an organism to a level below
some optimal physiological state (Welden and Slauson 1986; Pinckney et
al. 1995). Therefore, abiotic stress would influence processes that control
structure and function (Pinckney et al. 1995). Therefore, it is interesting to
examine the biodiversity of two hypersaline lakes on Cariboo Plateau with
162
different
salinity
and
water
chemistry.
Understanding
microbial
componants assembly under different conditions provides insight of

limiting factors on microorganism and their effects on community
structure and function (Pinckney et al. 1995).
5.1.2 Hypersaline lakes as extreme environment

In this chapter, the biodiversity of microbial mats in two
hypersaline lakes with contrasting aqueous geochemistry was elucidated.
Aquatic hypersaline conditions are thought to be similar to the
evaporative systems in early Earth and on Mars (Clark and van Hart
1891; Rothschild 1990; Mancinelli 2005). It was hypothesized that early
Earth and early Mars were once covered with a massive water body
(Lewis 1972; Baker and Milton 1974; Anders and Owen 1977; McElroy et
al. 1977; Clark and Baird 1979; Pollack and Black 1979; Pieri 1980; Carr
1981; Squyres 1984; Carr 1986; Squyres and Carr 1986; Christianson
1989) The water eventually evaporated and became these individual
water pockets with high concentration of dissolved metal. Nowadays,
hypersaline lakes are globally distributed, they could be found in Cariboo
Plateau in British Columbia, northwestern China, Great Salt Lake in Utah,
the Great Salt Plains of Oklahoma, the Dead Sea, the Mediterranean
Sea, Solar Lake in Sinai, Egypt, Antarctic hypersaline lakes (Oren 2002;
Ventosa 2004; Mancinelli 2005).
Hypersaline lakes are one of the
extreme environments where microbial mats are abundant (Oren 1999).

These microorganisms are called halophiles, because they can thrive in
163
high salt concentration environments (DasSarma and Arora 2001).

Previous studies indicated that microbial taxonomic diversity in the
hypersaline environments is greatly dependent upon salinity. In general,
microbial diversity decreases with increased salinity (Rodriguez-Valera et
al. 1985; Rodriguez-Valera, 1988, 1993; Benlloch et al. 2002; Oren 2002;
Jungblut et al. 2005), however, an opposite situation was suggested from
a more recent study (Jackson and Vallaire 2009). Halophiles are
comprised of a variety of heterotropic and methanogenic archaea
(prodominately
euryarchaeota);
photosynthetic,
lithotrophic
and
heterotrophic bacteria; photosynthetic and heterotrophic eukaryotes

(Jiang et al. 2006; DasSarma and Arora 2001). In aquatic hypersaline
system, microbial mats are generally restricted to green and brown layers
of Cyanobacteria underlain by one and brown layers of purple sulfur
bacteria utilizing sulfide derived from sulfate reduction occurring in the
deeper sediment (Jrgensen and Cohen 1977).
Figure 5.2 Stratified structures in hypersaline microbial mats at Guerrero

Negro, Baja California. Photo credits to John R. Spear and Norman R.
Pace (Copyright 2007, American Society for Microbiology)
164
5.1.3 Hypersaline lakes on Cariboo Plateau
Figure 5.3 Location and geological setting of southern Cariboo Plateau

lakes (Slater 1997).
Cariboo Plateau is a volcanic plateau that lies between the
coastal ranges and the Columbia-Rocky Mountains. It is located in south
of British Columbia with an elevation 1050 to 1250m over sea level. The
semi-arid Cariboo Plateau characterized by disordered drainage and
thousands of lakes which ranged from deep freshwater lakes to shallow
high salinity, high alkalinity lakes (Renaut 1990; Holland 1964). Cariboo
Plateau was first mentioned in 1901 (Hoffmann 1901; Dawson 1902), and
then extensive studies of these hypersaline lakes have been carried out
ever since (Goudge 1926 a, b; Cummings 1940; Renaut and Long 1989).
It is because Cariboo Plateau exhibits a wide collection lakes with
distinctive water chemistry, form and behavior. They range from a thin
biofilm on rock surface, to several meters bacteria mat and stromatolites
(Slater 1997). They not only provide opportunities to investigate the
165
microbial biogeochemical progresses within the lakes at different stage,

they represent potential analogues of the evaporative systems that might
have occurred on early Earth or Mars (Slater 1997).
Benthic, carbonate rich microbial mats were commonly found
in saline alkaline lakes basin on the Cariboo Plateau (Renaut 1993).
These microbial mats are cohesive, stratified microbial communities
develop at the interface between water and solid substrates (Slater
1997). They are mostly associated with Cyanobacteria (filamentous and
coccids), bacteria and algae (Anderson 1958; Moss and Moss 1969;
Walter et al. 1973; Halley 1976; von der Borch et al. 1977; Pueyo-Mur,
1978; Bauld 1981; Osborne et al. 1982; De Deckker 1983; Casanova
1986; Hammer 1986, Last & De Deckker 1990; Kempe et al. 1991). Due
to the relatively small scale size of these mat systems on Cariboo
Plateau, their sources, sinks and fluxes can be easily identified and
therefore to create a mass balance. Cariboo Plateau provides a prefect
model for studying the nutrients recycling in benthic ecosystems (Slater
1997).
5.1.3 Objectives
The objectives of this chapter were to assess microbial
diversity and abundance in two biofilm supporting carbonate-rich
hypersaline lakes with different water chemistry on Cariboo Plateau.
Multi-domain prokaryotic diversity in methane-rich saline lake and sulfaterich saline lake was surveyed. Studying diversity in saline conditions
provide fundamental understanding of survivability and adaptation of
166
halophiles at different levels of tolerance, and therefore provide reference

of the early evolution of life and implication of possibility life on Mars
(Catling 1999; Kunte et al. 2002; Mancinelli 2004; Jiang et al. 2006).
167
5.2 Material and methods

5.2.1 Site Description
Saline lakes in interior British Columbia are clustered in five
major groups. Cariboo Plateau is the largest and most diverse. The
geology of Cariboo Plateau consists of a thick series of Mio-Pliocene
basalt flows covered with a thin layer of till and glaciofluvial sands and
gravels (fig. 5.3). It is bounded by Marble Range at the southwestern and
west, Quesnel-Shuswap Highlands at the east. Very much of the plateau
is surrounded by dense coniferous forest and the whole region is
characterized by extremely cold winters and short hot summers (Renaut
and Long 1989). Most of the saline lakes on Cariboo Plateau are small,
shallow and groundwater-fed (Renaut and Long 1989).
In this chapter, Goodenough Lake and Deer Lake, were
selected to study their prokaryotic communities. Their GPS locations
were shown on Table 5.1.
Table 5.1. Locations and characters of lakes on Cariboo Lakes (Slater
1997).
Lake
Latitude
Longitude
Character
Goodenough
5119.8N
12138.5W
(GEL)
Deer
Low
methane,
high sulfate
5121.2N
12114.5W
(DEER)
High
methane,
low sulfate
5.2.2 Sampling Collection

Microbial mats and sediment samples of Goodenough Lake
and Deer Lake were collected by the Pavilion Lake Research Project
group during the summer season (July to August) of 2008. Samples were
168
collected using forceps and stored in sterile screw-capped Eppendorf

tubes and kept at -4C until they were processed.
5.2.3 Water Chemistry

Water sampling and analysis was carried out by other
members of the Pavilion Lake Research Project, notably Dr. D. Lim
(2009). Water sampling protocols followed those provided by the
environment Canada, Pacific Environmental Science Center (PESC) in
Vancouver, Canada. Water samples were collected, kept cool and dark
for the duration of the field season, and then immediately shipped to
PESC labs for analysis. A total of 37 environmental variables were
measured by PESC, including major ions (Calcium [Ca], magnesium
[Mg], Potassium [K], sodium [Na], sulfur [S], chloride [Cl], fluoride [F],
sulfate [SO4]), metals (aluminum [Al], antimony [Sb], arsenic [As], barium
[Ba], Beryllium [Be], boron [B], Cadmium [Cd], Chromium [Cr], cobalt
[Co], copper [Cu], iron [Fe], lead [Pb], manganese [Mn], Molybdenum
[Mo], nickel [Ni], selenium [Se], silver [Ag], strontium [Sr], tin [Sn], titanium
[Ti], vanadium [V], zinc [Zn]) pH, alkalinity, conductivity, phosphorus,
carbon (dissolved inorganic [DIC], dissolved organic [DOC]), and silicon
[Si]. Additional sulfate and methane concentration data were also
measured.
5.2.4 DNA extraction and Polymerase chain reaction (PCR) amplification

169


A total 4 clone libraries (2 bacterial and 2 archaeal) were
constructed to describe the prokaryotic communities from two sampling
sites. Protocols please refer to Section 2.2.5.


170
5.3 Results
5.3.1 Water Chemistry
Two hypersaline lakes on Cariboo Plateau were examined. To
understand what environmental factors may contribute to the microbial
diversity in the microbial mats systems, the water chemistry of
Goodenough Lake and Deer Lake from June 2006 to October 2008 are
detailed in Appendix 1.
Both Goodenough and Deer Lake are alkaline with pH around 10.
The metals measurements of two lakes are about the same, whereas few
major ions measurements of Goodenough Lake are much higher,
including chloride, sulfate, potassium, sodium, sulfur. Therefore the
conductivity measured in Goodenough Lake was about double of Deer
Lake. DIC and phosphorus contents are also higher in Goodenough
Lake. Methane concentration only taken from August 2006 and August
2007 and both value are about the same.

The Real-time quantitative PCR (qpcr) result reviewed the
absolute and relative abundance of a prokaryotic community in
hypersaline conditions (Table 5.2). All three domains (eukarya, bacteria
and archaea) were detected in both Goodenough Lake and Deer Lake
samples (fig. 5.4 and 5.5). In Goodenough Lake, sample collected from
sediment displayed a different microbial structure than sample from the
microbial mats. The total biomass in mats (8.3x105 copies per gram) was
about twice as much in sediment (4.7x105 copies per gram). The
171
microbial mats in Goodenough Lake were made up from 58.9% of

eukarya, 34.9% of bacteria and 6.2% of archaea. On the other hand, their
sediment sample was made up from 54.8% of bacteria, 26% of archaea
and 19.1% of eukarya.
In Deer Lake, the bacteria mat and sediment samples both
demonstrated very similar community structures. The biomass from
sediment (1.1x106 copies) was 1.5 times more than the one from mats
(2.5x106 copies). They were dominated by bacteria (85.2% and 95.2% for
mats and sediment respectively) and with low percentage of eukarya
(11.8% and 3.3%) and archaea (3.1% and 1.5%).
Table 5.2. Microbial abundance of Goodenough Lake (GEL) and Deer

Lake (DEER) from real-time quantitative PCR analysis.
172
6)78888)
!"#$%&'()*"&+,*+-().-$/0)+$1)/(2)32*45)
&!!"
%#!"
%!!"
(45,67,"
$#!"
8,9-06:,"
;69<,0,"
$!!"
#!"
!"
'()"*+,-."
'()"*/01."
2((3"*+,-." 2((3"*/01."
Figure 5.4. The absolute abundance of archaeal, bacterial and eukaryal

communities in Goodenough Lake (GEL) and Deer Lake (DEER) by realtime quantitative PCR (q-PCR) analysis.
$!!="
B!="
9(%*':;()!"&+,*+-().<5)
A!="
@!="
?!="
(45,67,"
#!="
8,9-06:,"
>!="
;69<,0,"
&!="
%!="
$!="
!="
'()"*+,-."
'()"*/01."
2((3"*+,-."
2((3"*/01."
Figure 5.5. The relative abundance of archaeal, bacterial and eukaryal

communities in Goodenough Lake (GEL) and Deer Lake (DEER) by realtime quantitative PCR (q-PCR) analysis.
173
5.3.3 Bacterial and archaeal diversity in hypersaline lakes

The cloning sizes of bacterial libraries were 106 and 119
clones for Goodenough Lake and Deer Lake respectively, and the
archaeal library size for Goodenough Lake and Deer Lake are 58 and 49
clones respectively (table 5.3).
Rarefaction curve of 4 clone libraries were shown in Figure
5.6, 5.8, 5.10 and 5.12. None of these curves has reached an asymptote.
Two bacterial libraries were more linear, which means the bacterial
communities of both location were very diverse. On the other hand, the
rarefaction curves of two archaeal libraries were not linear and they
already showed the tendency to curve downward under the given
sampling effort. This indicated that the size of clone libraries, although
unequal, were satisfactory to allow the estimation of total OTUs richness.
Both non-parametric richness estimators ACE and Chao1
estimations resulted in similar trends for both lakes (Figure 5.7, 5.9, 5.11
and 5.13). The estimated curves by Chao1 were more stable and had
better concave downward shapes; therefore they were used in estimating
the richness of OTUs.
174
Table 5.3. Coverage summary of clone libraries constructed for microbial

mats in Goodenough and Deer Lakes.
Goodenough
Deer
Bacteria
106
119
61
49
Chao 1 richness
63
52
Coverage, %
53
63
93
93
Goodenough
Deer
Archaea
64
49
13
12
14.2
13.6
91
84
94
91
Chao 1 richness
Coverage, %
175
)!"
!"#$%&'()'*+,-'($-%&.%/'
(!"
'!"
&!"
%!"
$!"
#!"
!"
!"
$!"
&!"
(!"
*!"
#!!"
#$!"
!"#$%&'()'01(2%-'-3#41%/'
Figure 5.6. Rarefaction curves for bacterial clone library from Good
Enough Lakes mat. All curves were averaged over 1000 simulations.
(n=106)
+!"
!"#$%&'()'*+,-'($-%&.%/'
*!"
)!"
(!"
'!"
&!"
,-."
%!"
-/01"#"
$!"
#!"
!"
!"
$!"
&!"
(!"
*!"
#!!"
#$!"
!"#$%&'()'01(2%-'-3#41%/'
Figure 5.7. Plots of estimated OTU richness (using estimators ACE and
Chao1) of bacterial clone library from Good Enough Lakes mat. All
curves were averaged over 1000 simulations. (n=106)
176
(!"
!"#$%&'()'*+,-'($-%&.%/'
'!"
&!"
%!"
$!"
#!"
!"
!"
$!"
&!"
(!"
)!"
#!!"
#$!"
#&!"
!"#$%&'0)'12(3%-'-4#52%/'
Figure 5.8. Rarefaction curves for bacterial clone library from Deer Lakes
mat. All curves were averaged over 1000 simulations. (n=119)
*!"
!"#$%&'()'*+,-'($-%&.%/'
(!"
'!"
&!"
+,-"
%!"
,./0"#"
$!"
#!"
!"
!"
$!"
&!"
(!"
)!"
#!!"
#$!"
#&!"
!"#$%&'()'12(3%-'-4#52%/'
Chao1) for bacterial clone library from Deer Lakes mat. All curves were
averaged over 1000 simulations. (n=119)
177
'$"
!"#$%&'()'*+,-'($-%&.%/'
'#"
'!"
&"
%"
$"
#"
!"
!"
'!"
#!"
(!"
$!"
)!"
%!"
*!"
!"#$%&'()'01(2%-'-3#41%/'
Figure 5.10. Rarefaction curves for archaeal clone library from from Good
Enough Lakes mat. All curves were averaged over 1000 simulations.
(n=58)
'%"
!"#$%&'()'*+,-'($-%&.%/'
'$"
'#"
'!"
&"
+,-"
%"
,./0"'"
$"
#"
!"
!"
'!"
#!"
(!"
$!"
)!"
%!"
*!"
!"#$%&'()'01(2%-'-3#41%/'
Chao1) for archaeal clone library from Good Enough Lakes mat. All
curves were averaged over 1000 simulations. (n=58)
178
'$"
!"#$%&'()'*+,-'($-%&.%/'
'#"
'!"
&"
%"
$"
#"
!"
!"
'!"
#!"
(!"
$!"
)!"
%!"
!"#$%&'()'01(2%-'-3#41%/'
Figure 5.12. Rarefaction curves for archaeal clone library from from Deer
Lakes mat. All curves were averaged over 1000 simulations. (n=49)
'%"
!"#$%&'()'*+,-'($-%&.%/'
'$"
'#"
'!"
&"
*+,""
%"
+-./"'"
$"
#"
!"
!"
'!"
#!"
(!"
$!"
)!"
%!"
!"#$%&'()'01(2%-'-3#41%/'
Chao1) for archaeal clone library from Deer Lakes mat. All curves were
averaged over 1000 simulations. (n= 49)
179
5.3.4 Bacterial communities of Goodenough and Deer Lakes

All 61 unique bacterial phylotypes from Goodenough lake were
sequenced. A diverse variety of bacterial clones were retrieved, and they
include Alpha-, Beta-, Delta- and Gamma-proteobacteria, Acidobacteria,
Actinobacteria, Bacteroidetes, Choroflexi, Cyanobacteria, Fibrobecteres,
Nitrospirae, Planctomycetes and Verrucomicrobia. Detailed descriptions
of the closest blast match of the sequences were listed in Table 5.4 a-d.
Library was dominated by proteobacteria (55%), in particular alpha and
beta sub-phylum. made up to 23.58% of the total bacterial abundance.
Cyanobacteria (10%) was also abundant in the community.
In the Deer Lake bacterial library, 49 phylotypes of bacteria
were recovered. They include Alpha-, Beta-, Delta- and Gammaproteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Choroflexi,
Cyanobacteria,
Firmicutes,
Verrucomicrobia.
It
was
Nitrospirae,
dominated
proteobacteria -- Variovorax paradoxus

Alphaproteobacteria
(10%),
by
an
Planctomycetes
and
EPS-secreting
beta-
(58%); and then followed by
Cyanobacteria
(7%)
and
Gammaproteobacteria (6%). Detailed description of the closet blast

match was shown in Table 5.5 a-c.
A phylogenetic tree of all bacteria clones was generated to
further resolve the relationships among bacteria (fig. 5.14) and the
illustrations for both bacterial libraries were shown in Figure 5.15 and
5.16.
180
Table 5.4a. Identities of 16S rRNA (bacterial) gene sequences obtained from Goodenough Lake (GEL) (NA= possible putative chimeric sequences).
Code
BG-01
Genbank
accession
number
Relative
abundance
(%)
HQ143974
0.94

Putation Phylum
Alphaproteobacteria
Identity
Accession
Number
Rhodobacter gluconicum
AB077986
96
culture
AF292999
99
culture
AF292999
89
culture
Uncultured Green Bay ferromanganous micronodule bacterium

MND8
Uncultured Green Bay ferromanganous micronodule bacterium
MND8
Percent
Source
similarity
BG-02
HQ143975
3.77
Alphaproteobacteria
BG-03
NA
0.94
Alphaproteobacteria
BG-04
HQ143976
0.94
Alphaproteobacteria
Methylocystis sp. m1511
DQ852349
87
culture
BG-05
HQ143977
0.94
Alphaproteobacteria
Rhodobacteraceae bacterium BF A20(17)
DQ628964
98
BG-06
HQ143978
0.94
Alphaproteobacteria
Uncultured Rhodobacteraceae bacterium clone

TDNP_Bbc97_44_3_115
subglacial water or ice

or sediment
FJ516806
97
biofilm
BG-07
HQ143979
0.94
Alphaproteobacteria
Uncultured Hyphomicrobium sp. clone YJQ-3
AY569281
87
pink microbial mat
BG-08
HQ143980
0.94
Alphaproteobacteria
Uncultured Rhodospirillaceae bacterium CM24G1
AM936842
89
soil
BG-09
HQ143981
0.94
Betaproteobacteria
Variovorax paradoxus strain rif200835
FJ527675
99
legume nodule
BG-10
HQ143982
0.94
Betaproteobacteria
FJ527675
96
legume nodule
BG-11
NA
1.89
Betaproteobacteria
Delftia lacustris strain 332
EU888308
99
culture
BG-12
HQ143983
1.89
Betaproteobacteria
Nitrosospira sp. Nsp65
AY123813
90
culture
BG-13
NA
0.94
Betaproteobacteria
Beta proteobacterium HS5/S24542
AY337603
96
soil
BG-14
HQ143984
0.94
Betaproteobacteria
Uncultured beta proteobacterium clone 2630
EF447071
96
cave
BG-15
HQ143985
0.94
Betaproteobacteria
Uncultured beta proteobacterium clone GASP-0KA-539-C08
EU043556
98
soil
BG-16
HQ143986
0.94
Betaproteobacteria
Uncultured beta proteobacterium clone:187
AB252908
97
iron-oxidation biofilm
BG-17
HQ143987
0.94
Deltaproteobacteria
uncultured delta proteobacterium clone AMEB4
AM935293
98
soil
BG-18
HQ143988
0.94
Deltaproteobacteria
Uncultured Syntrophobacterales bacterium clone CMPG5
AM936099
93
soil
181
Table 5.4b (cont.). Identities of 16S rRNA (bacterial) gene sequences obtained from Goodenough Lake (GEL) (NA= possible putative chimeric sequences).
Genbank
accession
number
Relative
abundance
(%)
BG-19
HQ143989
0.94
Deltaproteobacteria
BG-20
HQ143990
0.94
BG-21
HQ143991
BG-22
Code

Identity
Accession
Number
Uncultured Syntrophobacterales bacterium clone AMGC11
AM935385
96
soil
Gammaproteobacteria Uncultured Gamma proteobacterium clone CM41C5
AM936521
94
soil
0.94
Gammaproteobacteria Uncultured gamma proteobacterium clone LiUU-3-334
AY509440
93
freshwater
bacterioplankton
HQ143992
0.94
Gammaproteobacteria Uncultured Gamma proteobacterium clone AMJD7
AM934884
92
soil
BG-23
HQ143993
0.94
Gammaproteobacteria Uncultured Gamma proteobacterium clone CM41C5
AM936521
98
soil
BG-24
HQ143994
0.94
Gammaproteobacteria Uncultured Gamma proteobacterium clone AMBH8
AM935773
99
soil
BG-25
HQ143995
3.77
Acidobacteria
Uncultured Acidobacteriaceae bacterium clone AhedenS12
FJ475356
94
soil
BG-26
HQ143996
0.94
Acidobacteria
Uncultured Acidobacteria bacterium clone AMNF2
AM934751
91
soil
BG-27
HQ143997
0.94
Acidobacteria
Uncultured Acidobacteria bacterium clone RBE1CI-127
EF111087
97
river
BG-28
HQ143998
0.94
Acidobacteria
Uncultured Acidobacteria bacterium clone HAVOmat15
EF032752
96
cyanobacterial mat
BG-29
HQ143999
0.94
Acidobacteria
Uncultured Acidobacteriaceae bacterium clone AMBC2
AM935729
93
soil
BG-30
HQ144000
0.94
Acidobacteria
Uncultured Acidobacteriales bacterium clone Plot29-2C02
EU202757
96
soil
BG-31
NA
0.94
Acidobacteria
Uncultured Acidobacteriaceae bacterium CL5.H26
FM176295
99
rivulet
BG-32
HQ144001
5.66
Actinobacteria
Actinomycetales bacterium XJSS-08
EU598252
99
desert soil
BG-33
HQ144002
1.89
Actinobacteria
Actinomycetales bacterium XJSS-08
EU598252
99
desert soil
BG-34
HQ144003
1.89
Actinobacteria
Uncultured actinobacterium clone Elev_16S_691
EF019440
92
rhizosphere
BG-35
HQ144004
0.94
Bacteroidetes
Sphingobacteriaceae bacterium enrichment culture clone hao06 FJ386545
95
vaccination sludge
BG-36
HQ144005
0.94
Bacteroidetes
Sphingobacteriaceae bacterium enrichment culture clone hao06 FJ386545
85
vaccination sludge
Putation Phylum
182
Percent
Source
similarity
Table 5.4c (cont.). Identities of 16S rRNA (bacterial) gene sequences obtained from Goodenough Lake (GEL) (NA= possible putative chimeric sequences).
Code
Genbank
accession
number
Relative
abundance
(%)
Putation Phylum

Identity
Accession
Number
Percent
Source
similarity
BG-37
NA
0.94
Bacteroidetes
Uncultured Bacteroidetes bacterium clone AS59
EU283378
89
sludge
BG-38
HQ144006
0.94
Bacteroidetes
Uncultured Bacteroidetes bacterium AMKD8
AM935033
95
soil
BG-39
HQ144007
0.94
Chloroflexi
Sphaerobacter thermophilus DSM 20745
AJ420142
83
culture
BG-40
HQ144008
0.94
Chloroflexi
Uncultured Chloroflexi bacterium BuhC-41
FM866303
95
uranium mining wastes
BG-41
HQ144009
0.94
Chloroflexi
Uncultured Chloroflexi bacterium clone 02D2Z61
DQ329894
87
hypersaline microbial
mat
BG-42
HQ144010
1.89
Chloroflexi
Uncultured Chloroflexi bacterium clone C12_WMSP1
DQ450734
79
soil
BG-43
HQ144011
23.58
Cyanobacteria
EF110976
91
cyanobacterial mat
BG-44
HQ144012
3.77
Cyanobacteria
Phormidium pristleyi ANT.LH61.2
AY493582
93
culture
BG-45
HQ144013
2.83
Cyanobacteria
Pseudanabaena sp. PCC 6802
AB039016
95
culture
BG-46
HQ144014
0.94
Cyanobacteria
Coleodesmium sp. ANT.LH52B.5
AY493596
97
culture
BG-47
HQ144015
0.94
Cyanobacteria
Filamentous cyanobacterium LLi71
DQ786167
92
cyanobacterial mat
BG-48
HQ144016
0.94
Cyanobacteria
Leptolyngbya frigida ANT.LH53B.2
AY493576
97
culture
BG-49
HQ144017
0.94
Cyanobacteria
Phormidium sp. SAG 37.90
EF654082
90
culture
BG-50
HQ144018
0.94
Gemmatimonadetes
Uncultured Gemmatimonadetes bacterium clone AKYH1432
AY922110
88
soil
BG-51
HQ144019
0.94
Fibrobacteres
Uncultured Fibrobacteres bacterium clone: 05
AB252948
96
biofilm
BG-52
NA
0.94
Firmicutes
Uncultured Firmicutes bacterium clone GASP-45KB-145-A07
EU044325
92
soil
BG-53
HQ144020
0.94
Firmicutes
Uncultured Firmicutes bacterium clone GASP-WB1S3_B12
EF073490
93
pasture
BG-54
HQ144021
0.94
Firmicutes
Uncultured Firmicutes bacterium clone AUVE_04A01
EF651093
95
cropland
183
Table 5.4d (cont.). Identities of 16S rRNA (bacterial) gene sequences obtained from Goodenough Lake (GEL).
Genbank
accession
number
Relative
abundance
(%)
BG-55
HQ144022
0.94
BG-56
HQ144023
BG-57
Code

Identity
Accession
Number
Nitrospirae
Uncultured Nitrospira sp. clone Amb_16S_847
EF018579
97
rhizosphere
0.94
Nitrospirae
Uncultured Nitrospira sp.
AB113621
93
water
HQ144024
0.94
Planctomycetes
Uncultured Planctomycetaceae bacterium clone AhedenS28
FJ475365
91
soil
BG-58
HQ144025
0.94
Planctomycetes
Uncultured Planctomycetales bacterium clone

TDNP_USbc97_239_1_99
FJ516934
89
upper sediment
BG-59
HQ144026
0.94
Planctomycetes
Uncultured Blastopirellula sp. clone CL5.H54
FM176312
99
rivulet
BG-60
HQ144027
0.94
unidentified clone
Uncultured soil bacterium clone C011
AF507682
92
soil
BG-61
HQ144028
0.94
unidentified clone
Uncultured bacterium clone FFCH17457
EU135302
91
soil
Putation Phylum
184
Percent
Source
similarity
Table 5.5a. Identities of 16S rRNA (bacterial) gene sequences obtained from Deer Lake (DEER) (NA= possible putative chimeric sequences).
Code
Genbank
accession
number
Relative
abundance
(%)
Putation Phylum

Identity
Accession
Number
Percent
Source
similarity
BD-01
HQ144029
0.84
Alphaproteobacteria
Rhodobacter gluconicum
AB077986
96
culture
BD-02
NA
0.84
Alphaproteobacteria
uncultured Green Bay ferromanganous micronodule

bacterium MNG7
AF292997
89
freshwater/ sediment
BD-03
HQ144030
0.84
Alphaproteobacteria
Sphingomonas suberifaciens
D13737
95
culture
BD-04
NA
0.84
Alphaproteobacteria
Rhodobacter sp. TUT3732
AB251408
92
purple microbial mats
BD-05
HQ144031
0.84
Alphaproteobacteria
uncultured alpha proteobacterium
EU050755
77
sediment
BD-06
HQ144032
0.84
Alphaproteobacteria
FM253605
94
biofilm
BD-07
NA
0.84
Alphaproteobacteria
Hyphomicrobium sp. Ellin112
AF408954
89
culture
BD-08
HQ144033
0.84
Alphaproteobacteria
Uncultured Rhodocista sp. clone CL4.E44.
FM175924
94
rivulet
BD-09
HQ144034
0.84
Alphaproteobacteria
uncultured Rhodospirillaceae bacterium
AM935102
99
soil
BD-10
HQ144035
0.84
Alphaproteobacteria
uncultured Rhodospirillaceae bacterium
EF019251
99
trembling aspen
BD-11
HQ144036
0.84
Alphaproteobacteria
AY598790
98
Spongilla lacustris
BD-12
NA
0.84
Alphaproteobacteria
alpha proteobacterium HIBAF003
AB452982
99
freshwater lake
BD-13
HQ144037
1.68
Betaproteobacteria
uncultured Oxalobacteraceae bacterium
EU043565
95
dune fields
BD-14
NA
53.78
Betaproteobacteria
FJ527675
99
legume nodule
BD-15
HQ144038
0.84
Betaproteobacteria
Diaphorobacter sp. J5-51
GU017974
99
cooking water
BD-16
NA
0.84
Betaproteobacteria
Variovorax paradoxus
FJ527675
99
legume nodule
BD-17
HQ144039
0.84
Betaproteobacteria
beta proteobacterium HS5/S24542
AY337603
98
Soil
BD-18
NA
0.84
Deltaproteobacteria
EF662729
89
cropland
185
Table 5.5b(cont). Identities of 16S rRNA (bacterial) gene sequences obtained from Deer Lake (DEER) (NA= possible putative chimeric sequences).
Code
Genbank
accession
number
Relative
abundance
(%)
Putation Phylum

Identity
Accession
Number
Percent
Source
similarity
BD-19
HQ144040
0.84
Deltaproteobacteria
AM936057
91
soil
BD-20
HQ144041
0.84
Gammaproteobacteria
uncultured gamma proteobacterium
DQ640695
95
activated sludge
BD-21
HQ144042
2.52
Gammaproteobacteria
Moraxella sp. D30C2A
AY162144
98
bottled water
BD-22
HQ144043
0.84
Gammaproteobacteria
AY874095
94
tufa
BD-23
HQ144044
0.84
Gammaproteobacteria
uncultured Xanthomonadaceae bacterium
AY360613
95
soil
BD-24
NA
0.84
Gammaproteobacteria
EU810911
99
cave
BD-25
NA
0.84
Acidobacteria
AY281352
95
soil
BD-26
HQ144045
0.84
Actinobacteria
Corynebacterium kroppenstedtii
AY524775
99
blood culture
BD-27
HQ144046
0.84
Bacteroidetes
uncultured Saprospiraceae bacterium
FJ517043
85
water
BD-28
NA
0.84
Bacteroidetes
AM936269
94
soil
BD-29
HQ144047
0.84
Chloroflexi
AY921802
89
soil
BD-30
HQ144048
2.52
Cyanobacteria
Pseudanabaena PCC6802
AB039016
94
culture
BD-31
HQ144049
0.84
Cyanobacteria
Gloeothece membranacea PCC 6501
X78680
91
culture
BD-32
HQ144050
0.84
Cyanobacteria
Coleodesmium sp. ANT.LH52B.5
AY493596
98
culture
BD-33
HQ144051
0.84
Cyanobacteria
Petalonema sp. ANT.GENTNER2.8
AY493624
98
culture
BD-34
HQ144052
0.84
Cyanobacteria
Calothrix sp. KVSF5
EU022730
91
culture
BD-35
HQ144053
0.84
Cyanobacteria
Phormidium persicinum SAG 80.79
EF654085
92
culture
BD-36
HQ144054
0.84
Cyanobacteria
Nostoc sp. 'Pannaria sphinctrina cyanobiont' 1b Ch
EF174207
97
culture
186
Table 5.5c(cont). Identities of 16S rRNA (bacterial) gene sequences obtained from Deer Lake (DEER) (NA= possible putative chimeric sequences).
Code
Genbank
accession
number
Relative
abundance
(%)
Putation Phylum

Identity
Accession
Number
Percent
Source
similarity
BD-37
HQ144055
0.84
Firmicutes
uncultured Streptococcus sp.
AM420055
99
cuclture
BD-38
HQ144056
0.84
Nitrospirae
uncultured Nitrospira sp.
FJ236034
97
drinking water
BD-39
HQ144057
2.52
Planctomycetes
Uncultured planctomycete clone AKYG587
AY921932
91
soil
BD-40
NA
0.84
Planctomycetes
uncultured Planctomycetales bacterium
FJ516887
94
sediment
BD-41
HQ144058
0.84
Planctomycetes
DQ289903
88
Sediment
BD-42
HQ144059
0.84
Planctomycetes
AB247909
90
hydrothermal field
BD-43
HQ144060
0.84
Verrucomicrobia
uncultured Verrucomicrobia bacterium
EF612425
90
soil
BD-44
NA
0.84
unidentified clone
Bacterium TG141
AB308367
92
freshwater sediment
BD-45
HQ144061
0.84
unidentified clone
uncultured anaerobic bacterium
DQ018789
81
lagoon
BD-46
HQ144062
0.84
unidentified clone
AY703464
96
cave
BD-47
HQ144063
0.84
unidentified clone
FM956592
93
roots
BD-48
HQ144064
0.84
unidentified clone
uncultured Crater Lake bacterium CL500-15
AF316773
87
lake
BD-49
HQ144065
0.84
unidentified clone
uncultured marine bacterium
EU010231
84
seawater
187
Figure 5.14. Phylogenetic relationship among bacteria from Goodenough

and Deer Lakes based upon Maximum Likelihood analysis of full 16S
5.5a-d & 5.6a-c. Red text refers to clone from Goodenough lake and
Orange text refers to clone from Deer Lake. Rooted tree supported by
position. (PDF file are available on request)
188
Figure 5.14. (cont).
189
D"%9+('4A+)();- F9G9'H9E0.0C,(&';#,&%):0B,*&'*%+()&);.0!"#$%#&'()'*%+()&,%./0-
=A%9'*%+()&,%.80-
=$"'&'?")@,60-
1%+()&',7)();<
=$"'&'*,>0!+(,9'*%+()&,%:0-
1)(%#&'()'*%+()&,%//0-
!+,7'*%+()&,%80-
3%44%5
#&'()'*%+()&,%60-
2)"(%#&'()'*%+()&,%/0-
Figure 5.15. Relative abundance of bacteria in Goodenough Lake (GEL)

microbial mat.
L)&&K+'4,+&'*,%.0- F9G9'H9- !"#$%#&'()'*%+()&,%B,&4,+K();D"%9+('4A+)();.0.I0:0- C,(&';#,&%)- 60.0-
1%+()&',7)();<
=$"'&'?")@,=$"'&'*,.0/0-
=A%9'*%+()&,%80-
!+(,9'*%+()&,%.0!+,7'*%+()&,%.03%44%#&'()'*%+()&,%60-
2)"(%#&'()'*%+()&,%.0-
1)(%#&'()'*%+()&,%EJ0-
Figure 5.16. Relative abundance of bacteria in Deer Lake (DEER)

microbial mat.
190
5.3.5 Archaeal communities of Goodenough and Deer Lakes

The unique phylotypes recovered from Goodenough and Deer
Lakes are 13 and 12 respectively. Total distinctive compositions of
archaea were present in two locations. Detailed descriptions of the
closest blast match of the sequences were listed in Table 5.6 and Table
5.7.
In Goodenough Lake, 8 isolates of thaumarchaeal were
detected and made up by 91% of the community and only 5 uncultured
Euryarchaeota were found. The dominant clone was an uncultured
thaumarchaeal (GQ302612), it consists of 56% of the total community. In
Deer Lake, the archaeal community was also dominated by two
thaumarchaeal clones by 53.06% and 20.41% (total 73% of the
community). A more diverse collection of Euryarchaeota was isolated (10
clones) and formed 27% of Deer Lake community.
A phylogenetic tree of all archaea clones was generated to
further resolve the relationships among archaea (fig. 5.17) and a bar
chart illustrating the relative abundance for both archaeal libraries is
shown in Figure 5.18.
191
Table 5.6. Identities of 18S rRNA (archaeal) gene sequences obtained from Goodenough Lake (GEL).
Code
Genbank
accession
number
Relative
abundance
(%)
Putation Phylum

Identity
Accession
Number
Percent
Source
similarity
G1
HQ144066
56.25
Thaumarchaeal
Uncultured archaeon clone sw-A433
GQ302612
99
cold spring
G2
HQ144067
12.50
Thaumarchaeal
Uncultured crenarchaeote clone LAR_Cren_54
EU309865
99
rhizosphere
G3
HQ144068
7.81
Thaumarchaeal
Uncultured crenarchaeote clone HAuD-MA13
AB113625
96
water
G4
HQ144069
4.69
Thaumarchaeal
Uncultured crenarchaeote clone Arch_AE_A10
FJ968081
99
sulfur-rich water
G5
HQ144070
3.13
Thaumarchaeal
Uncultured archaeon clone 912
EF188748
100
cave
G6
HQ144071
3.13
Thaumarchaeal
Uncultured crenarchaeote isolate DGGE gel band 1-3 FJ971897
99
soil
G7
HQ144072
1.56
Thaumarchaeal
Uncultured crenarchaeote clone G7
EU910614
99
sediment
G8
HQ144073
1.56
Thaumarchaeal
Uncultured crenarchaeote clone LAR_Cren_54
EU309865
98
rhizosphere
G9
HQ144074
3.13
Euryarchaeota
Uncultured archaeon clone Thp_A_108
EF444613
89
hot springs
G10
HQ144075
1.56
Euryarchaeota
Uncultured archaeon clone Thp_A_134
EF444654
88
hot springs
G11
HQ144076
1.56
Euryarchaeota
Uncultured archaeon clone CaR3b.f11
EU244299
89
river
G12
HQ144077
1.56
Euryarchaeota
Uncultured archaeon clone JMYA24
FJ810531
85
groundwater
G13
HQ144078
1.56
Euryarchaeota
FJ810531
84
groundwater
192
Table 5.7. Identities of 18S rRNA (archaeal) gene sequences obtained from Deer Lake (DEER).
Code
Genbank
accession
number
Relative
abundance
(%)
Putation Phylum

Identity
Accession
Number
Percent
Source
similarity
D1
HQ144079
53.06
Thaumarchaeal
unidentified archaeon LMA226
U87520
99 sediment
D2
HQ144080
20.41
Thaumarchaeal
Uncultured archaeon clone SWA08
AB294264
99 stream
D3
HQ144081
6.12
Euryarchaeota
Uncultured archaeon clone LAa02.34
EU782022
96 culture
D4
HQ144082
4.08
Euryarchaeota
FJ810531
89 groundwater
D5
HQ144083
2.04
Euryarchaeota
Uncultured archaeon clone Dg2003_D_29
AY531715
88 sediment
D6
HQ144084
2.04
Euryarchaeota
Uncultured archaeon clone iBSZ2p.92
DQ146755
89 Arctic Ocean
D7
HQ144085
2.04
Euryarchaeota
EU782007
93 water
D8
HQ144086
2.04
Euryarchaeota
Uncultured archaeon clone mrR2.40
DQ310449
88 Arctic Ocean
D9
HQ144087
2.04
Euryarchaeota
EU750838
85 lake
D10
HQ144088
2.04
Euryarchaeota
Uncultured archaeon clone CaR3s.03
EF014528
97 river
D11
HQ144089
2.04
Euryarchaeota
FJ810531
85 groundwater
D12
HQ144090
2.04
Euryarchaeota
FJ810531
83 groundwater
193
Figure 5.17. Phylogentic relationship among archaea from Goodeough

Lake and Deer Lake based upon Maximum Likelihood analysis of full 16S
5.7 & 5.8. Red text refers to clone from Goodenough lake and Orange
text refer to clone from Deer Lake. Rooted tree supported by bootstrap
values for 1000 replicates (first number) and Bayesian posterior
position. (PDF file are available on request)
194
'!!"
!"#$"%&'(")*+,)
&!"
%!"
5-46147/1+)81"
$!"
9/1-:147/1+1;"
#!"
!"
())*+,)-./"012+"
3++4"012+"
Figure 5.18. Relative abundance of archaeal diversity of Goodenough

Lake and Deer Lake.
195
5.4 Discussion
Prokaryotic diversity of two hypersaline lakes were elucidated
and compared. Communities consisted of archaea, bacteria and eukarya
found in Goodenuogh and Deer Lakes support that halophiles are a
diverse group of organisms (Mancinelli 2005b). Some studies suggested
that microbial diversity decreases with increased salinity (RodriguezValera et al. 1985; Rodriguez-Valera 1988, 1993; Oren 2002).
Conversely, a contradicting result was observed recently (Vallarie and
Jackson 2009). In this chapter, very similar microbial structures were
revealed in two hypersaline lakes with very distinctive water chemistry.
Two very diverse communities that spanned 12 phyla were detected from
two libraries. Both communities were dominated by proteobacteria,
especially high abundance in alpha and beta sub-phylum. Alphaproteobacteria formed 13% of Goodenough Lake and 10% of Deer Lake
while beta-proteobacteria formed 33% of Goodenough Lake and 59% of
Deer Lake. This similar observation of high frequency of proteobacteria
detection from this study is not consistent with recent observations in
other ecosystems, such as marine (Goh et al. 2009) and freshwater
microbialites (Chapter 4), hot springs (Lau et al. 2009) and arid
environments (Pointing et al. 2007).
Results also indicated salinity
enhances the bacterial diversity. High salinity environments would lead to

shift in assembled structure towards a community dominated by
Betaproteobacteria (Vallarie and Jackson 2009). Moreover, bacteria
communities in these hypersaline lakes were not solely comprised of
Cyanobacteria; Cyanobacteria which constituted a large fraction of the
196
surface biomass, may not represent the true composition of the whole
microbial mats systems (Ley et al. 2006). Moreover, proteobacteria
represent a diverse group of microorganisms from heterotroph,
chemoautotrophs to phototrophs, therefore it is quite reasonable that high
abundance of proteobacteria were detected from complex microbial mats
that composed of diverse functional groups. Interestingly, Variovorax is
an EPS-producing bacterium found in soil environment (Jamieson et al.
2009). High abundance of Variovorax of Betaproteobacteria found in both
hypersaline communities (24% in Goodenough Lake and 54% in Deer
Lake) suggests EPS production is maybe critical in hypersaline microbial
mat system. They regulate physiochemical stress in hypersaline extreme
environments as well as play a role in the calcium carbonate precipitation
in microbial mat system (Neu 1994; Costerton et al. 1995; Kawaguchi and
Decho 2000, 2001; Decho 2000; Dupraz and Visscher 2005; Braissant et
al. 2007).
High frequency of Cyanobacteria phylotypes was also detected
from both bacterial libraries (17% in Goodenough Lake; 7% in Deer
Lake), since they are the driving force of biogeochemical cycling in the
mats (Dupraz and Visscher 2005). In some hypersaline environments,
Cyanobacteria are exposed to high concentration of sulfide. Certain
species are able to tolerant and use sulfide as an electron donor in an
anoxygenic type of photosynthesis (Oren 2000). Cyanobacteria in
Goodenough Lake included the genera Coleodesmium, Leptolyngbya,
Phormidium,
Pseudanabaena.
The
dominant
isolated
clone
was
phylogentically affiliated to Pseudanabaena (EF110976) that was
197
previously recovered from similar carbonate structure from Northern

Florida Keys coral reef (Voss et al. 2007). More diverse forms of
Cyanobacteria were recovered from Deer Lake. They included Calothrix,
Coleodesmium, Gloeothece, Nostoc, Petalonema, Phormidium and
Pseudanabaena were isolated. Most of the genera detected from two
libraries are filamentous Cyanobacteria. They were thought to be very
important in lithifying processes in microbial mats through their production
of
EPS.
Particularly,
Pseudanabaena
Calothrix,
were
Leptolyngbya,
suggested
to
be
Nostoc
present
and
in
microbialites/stromatolites systems as well (Lavel et al. 2000; Burns et al.

2004). Another interesting observation is that Chloroflexi were thought to
be major group in hypersaline microbial mats, (Ley et al. 2006) however,
only a small percentage of Chloroflexi was detected in Goodenough and
Deer Lakes. Low abundance of these Chloroflexi may be due to the
occurrence of other phyla that may carry out similar competing functional
roles. For example: proteobacteria (anoxygenic phototrophs) and
filamentous Cyanobacteria.
Previous study showed that Goodenough Lake with its high
concentration of sulfate and low concentration of methane, whereas Deer
Lakes with high concentration of methane and low concentration of
sulfate (Slater 1997). As discussed before, methane concentration was
also an important contributor to microbial diversity other than salinity.
Microbial
communities
that
consume
methane
aerobically
and
anaerobically play an important role in regulating methane gas flux in

both lakes. Methane in Deer Lake was detected 3500-4500 times
198
supersaturated and significant amount were dissolved to the atmosphere

whereas it was only 50 times supersaturated in Goodenough Lake (Slater
1997). Clearly, higher abundance of methane sensitive microbial
communities was expected in Deer Lake than Goodenough Lake.
Detection of few strains that originally discovered from methane-rich
environments was isolated from Deer Lake library but not in Goodenough
Lake library. However, water chemistry presented in this study does not
reveal the expected methane content in Deer Lake. Since methane is a
volatile gas, it is suggested a more sophisticated test of methane could
be applied. Moreover, QPCR revealed the heterogeneity within
Goodenough Lake and earlier studies indicated methane oxidation is
complicated and varies throughout the water column. Further detailed
analysis of individual layers in microbial mat may further describe the
microbial processes and their spatial variation.
Similar
to
bacterial
communities,
archaeal
community
structures in Goodenough and Deer Lakes are very comparable. They

comprised of high abundance of thaumarchaeota (91% in Goodenough
Lake; 74% in Deer Lake) and euryarchaeote (9% in Goodenough Lake;
27% in Deer Lake). It is interesting to note that none of these archeaon
clones are closely related haloarchaeal species (Halobacteriaceas,
Methanospirillaceae and Mahanosacinaceae) (Mancinelli 2005), which
were found abundance in hypersaline microbial system as well as marine
stromatolites (Goh et al. 2009). This suggested halophilic properties are
not only limited to haloarchaeal species. There is a much more diverse
range of halophilic microorganism present in the environment than
199
originally thought. Moreover, archaea were suggested to playing a critical

role in microbial mats. They may coupled with sulfate reducing bacteria
and undergo a complex process, anaerobic oxidation of methane, in
microbial mats system. This is especially common in high methane
environments (Michaelis et al. 2002; Chan 2004). This may suggest the
reason why euryarchaeota formed a larger portion in a high-methane
Deer Lake (methanogens are belongs to euryarchaeota). However, data
represented here do not clearly indicate the linkage between the
recovered archaea and their role, further examinations of microorganism
metabolic gene are suggested to reveal their function in the microbial
mats ecosystem.
The Thaumarchaeota are a new mesophilic archaeal phylum
originally grouped to the crenoarchaeota (Brochier-Armanet et al. 2008).
The dominant G1 thaumarchaeota (56% of total archaeal community) in
Goodenough Lake is closely affiliated to an archaeon clone isolated from
hypersaline lake in China (GQ302612), while the second dominant group
is also thaumarchaeota (G2) (EU309865) (13% of total archaeal
community) that common to both marine and freshwater environments
(Brandt et al. 2007; Herrmann et al. 2008). In contrast, the prevalent
groups in Deer Lake were common in methane sensitive communities.
While D1 (53% of total archaeal community) (U87520) were found
associated with methanogen (MacGregor et al. 1997) and D2 (20% of
total archaeal community) were previously recovered from a deep coal
seam groundwater (Shimizu et al. 2007). These differences in archaeal
communities found in Goodenough Lake and Deer Lake further
200
suggested that archaeal diversity are related to their water chemistry,

particularly in methane concentration.
Only a small fraction of eukarya are able to tolerate
hypersalinity, but they contribute significantly in hypersaline environments
(Mancinelli 2005b). Relatively little was known about halophilic eukarya,
therefore it will be interesting to further look at the eukaryal community in
Goodenough Lake. Low abundance of eukarya recovered from Deer
Lake is probably due to high concentration toxic methane within the lake.
It is interesting to note that there were no microbialites
observed in Deer and Goodenough Lake, when their bacterial
assemblages are similar to Pavilion Lake in Chapter four. The taxa
responsible for biomineralization are unknown, despite claims that certain
cyanobacteria are key. It is not possible to conclude whether biotic or
abiotic (or both) processes are facilitates or prevent microbialites
formation based on current knowledge. Therefore, based on these, we
cannot conclude why lakes do or do not harbor microbialites, but a
possible explanation may include timescale, keystone taxa, and water
chemistry.
From an astrobiology aspect, hypersaline conditions are very
similar to the evaporative systems in early Earth and early Mars
(Mancinelli 2005, Clark and van Hart 1891; Rothschild 1990). If life was
ever on Mars, it was most likely halophilic microorganism. The great
diversity in strategies used by the halophiles to cope with the high salinity
environment like Cariboo Plateau and their distribution throughout in all
three domains suggest that adaptation to life at high salt concentration is
201
easy to evolve, and has probably arisen many times during the evolution
of life (Mancinelli 2005a). Similar to stromatolites, microbial mats were
abundant back in over 3 billion years ago (Tice and Lowe 2004).
Hypersaline microbial mats systems on Cariboo Plateau provide a
valuable
natural
laboratory
for
studying
examining
the
microbiogeochemical interactions with the paleoenvironments on Earth

and Mars (Logan et al. 1999; Summons et al. 1996; Summons et al.
1999). Comparison of diversity level between environments with
contrasting characteristics can yield valuable insight and testable
hypothesis regarding the rules that govern diversity distribution (Ley et al.
2006). Hypersaline microbial mats recorded the information of origin of
life, their evolution from unicellular structure to multicelluar organisms as
well as provide an analogue for searching life that could ever exist on
Mars.
202
5.5 Conclusion
In this chapter, the microbial diversity of microbial mats in high
sulfate low methane and low sulfate high methane lakes were elucidated
and compared. These following points are to summarize their similarities
and differences:
The lake systems of Cariboo Plateau present stresses

to micriobial life, including high salt, pH, methane and
sulfate. Microbial communities comprising archaea,
bacteria and eukarya were present in both microbial
mats on Cariboo Plateau.
Similar prokaryotic communities were revealed from

both hypersaline environments. High abundance of
proteobacteria and Cyanobacteria were isolated from
bacterial assemblages, whereas archaea assemblages
were
dominated
by
thaumarchaea
with
low
percentage of euryarchaeota.
Archaeal and bacterial phylotypes that are closely

affiliated to species found in high methane environment
were isolated from the relatively methane-rich Deer
Lake but not in the sulfate-rich Goodenough Lake.
203
Appendix 1. Chemical proporties of all water samples collected in the Goodenough Lake and Deer Lake. Measurements taken on sampling date were listed in
Red.
204
Appendix 1. (Cont)
205
Appendix 1. (Cont)
206
Chapter 6. Synthesis
6.1 Thesis Summary

Astrobiology is an emerging area that combines knowledge
from geology, chemistry, biology, planetary sciences and many other
areas. Since molecular tools have become a standard in microbial
ecology (Muyzer and Ramsing 1995; Pace 1997a, 1997b; Head et al.
1998;
Gray
and
Head
2001),
molecular
analysis
of
Mars-like
environments on Earth became a focus within astrobiology. Such studies

have identified the microorganisms and their physiological properties
present in Mars-like environments and therefore inform what kind of life
could be predicted in similar niches on Mars. The experiments presented
in this thesis were conducted to determine the microbial communities of
two major Mars-like environments on Earth:
High radiation arid environments. Due to the low

magnetic field observed on Mars, Mars surface is
bombarded with high level of radiation (Squyres 2004).
Therefore, the high UV environments of high altitude
deserts were considered in chapter two and three as
analogs for Mars surface environments.
Carbonate-rich and saline aquatic environments. The

early wet phase of Earth and late wet phase of Mars are
thought
to
have
hypersaline water
been
typified
by
carbonate-rich
(Mancinelli 2005, Clark and van Hart
1891; Rothschild 1990). A large body of water has
207
existed near surface over much of the Mars history

(Squyres 2004). However this water gradually vaporized
and created lakes that were high in dissolved solutes.
Deserts
comprise
35%
of
terrestrial
land
mass
and
characterized by their low moisture and low nutrient conditions (Housman

et al. 2005). Biological soil crusts are ecological important to arid
environments because they stabilize the soil where higher plants are
limited (Belnap and Lange 2003). Many soil crusts of arid environments
across the globe have been studied, however, little is known about the
community of soil crusts in high altitude deserts such as those in Tibet. In
this study, the microbial community of a biological crust from altitude
>5000m in Tibet was reported. Previous studies have emphasized only
bacterial or eukaryal domains but in this study, a multi-domain survey
across all domains was employed. The Tibetan soil crust comprised an
algal-dominated community, largely Stichoococcus bacillaries, plus
Naxibacter sp. and other taxa at lower abundance. Whilst many
phylotypes recovered from this study were most closely affiliated with
those from other deserts, others had close affiliation to phylotypes
recovered from other extreme environments, including marine, glacier,
permafrost and volcanic regions. Tibet high altitudes deserts provide a
model for potential life on Mars and survivability of microorganisms in
extreme environments. The complex community presented here suggests
a diverse range of microorganisms capable of surviving in desiccated
environments with high incident of UV.
208
Radiation is elevated at high altitude environments due to

reduced atmospheric filtering (Blumthaler and Elinger 1996). Therefore, it
is interesting to study the microorganisms from high altitude and clarify if
altitude presents a natural selection for radiation tolerant taxa. In chapter
three, Phylogenetically diverse species of radiation tolerant bacteria were
isolated from Tibet high altitude deserts. They included Deinococci,
Firmicutes, Actinobacteria, Cyanobacteria to Proteobacteria. These
results revealed relatively few radiation tolerate taxa but they spanned
diverse phyla supported findings from the previous studies. This raised a
question over whether the radiation tolerant quality in bacteria is a result
of convergent or divergent evolution. Phylogenetic analysis indicated
these microorganisms were diverse and they were closely affiliated to
species previously isolated from deep-sea, hot spring and permafrost
environments. These assorted origins suggested radiation tolerant
organism with different ancestors might evolve convergently (which may
include lateral gene transfer events). Different microorganisms went
through similar pathway to adapt the extreme arid environments. This is
particular important in astrobiology as they provide implication of life with
the potential to evolve and adapt to the extreme arid and radiated
environments on Mars surface.
In chapter four, a dynamic microbial system of previously
unstudied freshwater microbialites in Pavilion Lake was revealed and
identified using molecular analysis. Results suggested there are
interactions between the biotic and abiotic environments. The microbial
community composition was similar to the marine stromatolites, although
209
they varied significantly in spatial and temporal scales.
They were
comprised of high abundance of proteobacteria (Alphaproteobacteria),

Cyanobacteria (Leptolyngbya) and Acidobacteria by rDNA libraries
construction, whereas previous morphological exam only shows the
presence of Cyanobacteria (Gloeocapsa, Synechococcus, Fischerella,
Pseudoanabaena, Oscillatoria and Calothrix) and few Diatoms (Lavel et
al. 2000). Understanding of these microbialites communities provides
proxies of paleoenvironments conditions when these structures were
formed. Temporal variation observed suggested seasonal effects play a
role in these freshwater microbialites formation. On the other hand spatial
variation observed within same lake may indicate the heterogeneity in
environments. These findings are critical for astrobiology because they
provide implications for the level of biological complexity necessary for
microbialites to farm on Mars.
In chapter five, lakes on Cariboo Plateau display another type
of aquatic environments that are extreme hypersaline. This study
surveyed the microbial composition comprehensively by cultureindependent molecular methods. In this chapter, microbial diversity of
microbial mats in high sulfate low methane and low sulfate high methane
conditions were elucidated and compared. Data indicated the halophilic
communities in both lakes comprised archaea, bacteria and eukarya. The
bacterial assemblage comprise of high abundance of proteobacteria and
Cyanobacteria. In addition, I was also interested in the archaea
community structures; it is because archaea are common in the extreme
environments of high salinity and high methane presented here. These
210
extreme conditions are also very similar to Earths early history. Archaea
can yield energy by utilizing simple substrates from the environments.
These processes can undergo aerobic but mainly anaerobic conditions
(Madigan and Martinko 2006). Archaea revealed here suggested that
Archaea were possibly abundant on early Earth. The results showed the
archaea assemblages were dominated by thaumarchaeal with low
percentage of euryarchaeota. However, species affiliated to high
methane environments were unique to Deer Lake where the methane
availability was higher.
211
6.2 Synthesis of the findings

6.2.1 High radiation arid environments
High altitude desert soil crusts were dominated by chlorophyte

algae of the genus Stichococcus.
The crusts also supported a diverse range of bacteria, dominated

by Naxibacter sp., but also including Alphaproteobacteria,
Betaproteobacteria, Bacteroidetes and gemmatimonadetes.
High altitude soils also supported a diverse range of radiation

tolerant bacteria.
The most radio-tolerant were Deinococci (15kGy), and then

followed by Firmicutes (5kGy).
Phylogenetic analysis revealed the radiation tolerant species are

related to Deinococcus sp., Sphingomonas sp., Leptolyngbya sp.,
Bacilius sp., Micrococcus sp..
New records for radiation-tolerant genera were presented,

including Leptolyngbya and Planomicrobium.
6.2.2. Carbonate-rich lakes
Pavilion Lake in British Columbia and surrounding area are high

carbonate freshwater environment dominated by microbialites.
Microbial communities were dominated by proteobacteria and

Cyanobacteria (Leptolyngbya and other diverse taxa in lower
abundance), and these varied within structures. Basal structures
were more abundant in archaea than the top part of microbialites.
Overall the microbialites shared high levels of community similarity
212
to marine stromatolites, yet nonetheless comprise a novel

community assembly.
The microbial diversity of microbialites varied spatially. Distinctive

communities were observed from two locations on opposing
shores of Pavilion Lake.
The microbial diversity of microbialites also varied temporally.

Microbial communities shifted from Summer (Aug) to Spring (April)
seasons.
Further study of saline lakes surrounding Pavilion Lake with high

sulphate and methane levels revealed additional microbial biofilm
communities supporting taxa adapted to elevated sulphate and
methane levels and involved in methane metabolism, but not
known halophilic taxa.
These
lakes
also
revealed
significant
population
of
Thaumarchaea, a newly proposed phylum within the Archaea

associated with chemolithoautotrophic metabolism.
213
6.2.3 Implications for astrobiology

The radiation levels on Mars surface are magnitudes higher
than Earth (Cockell et al. 2000; Crdoba-Jabonero et al. 2003; Patel et al.
2003). Whilst none of the bacteria could survive on Mars today, however,
diverse range of radiation tolerate bacteria isolated from Earth indicate
potential for evolution of radiation tolerance. This indicates these
microorganisms with different origins may evolve and develop their
radiation toleration through time. Moreover, it is also suggested that
sheltered environments, such as caves or subsurface, on Mars may be
more possibly for life where the radiation level are greatly reduced.
The realization that Mars surface in an arid desert has
stimulated research in arid regions on Earth. It is, however, important to
understand that no prefect analogue exists since Mars is fundamentally
more extreme than any present Earth environment. My study has
investigated the most Mars like of earth extreme environments,
simulating present (arid high altitude desert) and past (microbialites in
carbonate-rich lake) Mars conditions. The soil crusts included in this
study represent an advanced community for Mars, they suggested life
could develop given the right conditions. It is suggested Mars is a
dynamic systems where Mars could vary from a wetter warmer planet to
a recent dry and cold planet. It is possible that there was a period when
all the right ingredients of life were present and life arose from there.
Moreover, microbial mats on Cariboo Plateau show that microbes can
use a diverse range of substrate. Simple molecules like methane and
214
sulphate could be available everywhere as well as Mars. This significantly

raises the probability of life on Mars.
In addition, freshwater Pavilion Lake provides analogue for late
Mars environments. Discovery of unusual assemblage of biomineralized
structures not only suggested similar structures that might have been
present on Mars; Remains of these biomineralized structures are also
suggested as potential biosignatures for searching extraterrestrial life on
Mars.
215
6.3 Limitations of the study and future works

This study has used molecular techniques in determining
microbial community diversity. Several techniques such as QPCR,
TRFLP, clone library and sequencing analysis have been employed in
carrying out experiments in this study. Nonetheless some limitations exist
and these are addressed below:
Lack of phylogenetic analysis in some studies. Much of the

diversity data from this study were obtained by cloning. The
advantages are they can provide quantitative and qualitative
results. Additional phylogenetic analysis could provide positive
identification for recovered clones and therefore provide a more
insightful data to describe the microbial diversity.
Molecular tools in environmental sample.

Discrepancies in the morphotypes and phylotypes in
chapter four suggested the clone libraries may sometimes
underestimate the microbial diversity, which can be due to the bias
in DNA extraction or PCR. Heterogeneity in biological soil crust
and highly calcified microbialites may limit the consistency of
traditional DNA extraction. Moreover, the bias inherent in the
primer used would also limit the microbial diversity. Further
improvement in all molecular techniques and using wide range
molecular tools are needed to improve results.
It is assumed that cloning is more sensitive as diversity
assessment tools than other molecular techniques, such as DGGE
(Lacap 2007). However, cloning sufficiency are greatly depends on
216
the size of clone library. Efforts in cloning size will definitely offer a
clearer picture of microbial community diversity.
New
technologies
in
metagenomics
sequencing,
for
example the application of illumina and pyrosequencing for high

throughput characteristic will allow increased confidence when
estimating community composition.
RNA
genes
of
recovered
phylotypes
requires
further
investigation. Molecular data obtained in this study revealed

information of what taxa are present in the communities. Further
analysis
in
determining
quantitative
expression
profiles
of
metabolic genes involved in photosynthesis and nitrogen fixation

will provide more useful data of physiological properties of these
microbes.
Greater sampling for temporal and spatial studies. Interesting

results indicated microbial communities on Mars-like environments
on Earth vary temporally and spatially. Further sampling and
analysis give a better understand of the community systems.
217
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Title

Molecular microbial ecology of Mars-like environments on earth,

Pointing, SB; Aitchison, JC

Chan, Wai, Olivia.; .

The author retains all proprietary rights, (such as patent rights)

Abstract of thesis entitled

MOLECULAR MICROBIAL ECOLOGY OF

Wai Olivia Chan

Astrobiology is a multidisciplinary topic that addresses the origin,

whilst cold alkaline high-carbonate freshwater lakes were chosen as an analogue

This implies the high-radiation

but not supporting microbialites revealed extensive microbial mats. These

No obvious cues that inhibit or promote

microbialite formation were observed in this study.

MOLECULAR MICROBIAL ECOLOGY

Wai Olivia Chan

A thesis submitted in partial fulfillment of the requirements for the

I declare that this thesis represents my own work, except

Many thanks to Dr Maggie Lau, Dr Donnabella Lacap, Kelly

The beginning of astrobiology

Possible life in space

Mars-like terrestrial environments

Mars-like aquatic environments

Molecular approaches in astrobiology

MOLECULAR DIVERSITY AND

Materials and Methods

DNA extraction and polymerase

chain reaction (PCR) amplification

Real-time quantitative PCR analysis

Bacterial and archaeal diversity in

high altitude deserts

Bacterial community diversity

Eukaryal community diversity

RADIATION TOLERANT BACTERIA

Total organic content and radiation

Culture and Isolation

Analytical Profile Index (API)

Sequence assembly and alignment

Microbialites as a living fossil

Microbialites in Pavilion Lake

Materials and Methods

DNA extraction and polymerase

chain reaction (PCR) amplification

Sites and samples morphology

Terminal restriction fragment length

Summer season vs. budding

Microbial mats in ecosystems

Hypersaline lakes as extreme

DNA extraction and Polymerase

chain reaction (PCR) amplification

Real-time quantitative PCR (q-PCR)

Real-time quantitative PCR (q-PCR)

Goodenough and Deer Lakes

Goodenough and Deer Lakes

Synthesis of the findings

High radiation arid environments

Implications for astrobiology

Limitations of the study and future

Image of the planet Mars

Image Hematite pebbles on Mars surface taken by 9

Image of disappearing ice on Mars subsurface

Mars surface with river bed feature

Schematic drawing of the molecular methods used 21