IDENTIFICATION and COMPARISION of FUNGI from DIFFERENT DEPTHS of ANCIENT GLACIAL ICE Angira Patel a Thesis Submitted to the Grad

Home , Coniochaetales, Dothioraceae, Ice XVI, Penicillium commune, Penicillium crustosum, Penicillium expansum

IDENTIFICATION AND COMPARISION OF FUNGI FROM DIFFERENT DEPTHS OF ANCIENT GLACIAL ICE

Angira Patel

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the Requirements for the degree of

MASTER OF SCIENCE

MAY 2006

Committee:

Dr. Scott Rogers, Advisor

Dr. Stan Smith

Dr. Dawn Hentges

ABSTRACT

Dr. Scott Rogers, Advisor

Glacial ice serves as a unique preservation matrix for contemporary and ancient

microorganisms. The main objective of this study was to evaluate and test the existence of the

fungi encased in ancient glacial ice of Antarctica and Greenland. PCR (polymerase chain

reaction) amplification was used to isolate the DNA followed by DNA sequencing to obtain the

DNA sequences of the ancient microorganisms. Most of the sequences obtained from ancient

microbes were similar to the contemporary fungi. Few fungi cultured were approximately

10,000 years old. Microorganisms isolated from ancient glacial ice have undergone repeated

phases of evolutionary changes, such as irradiation, freezing and thawing, and in the process they

have been archiving various biogenic materials over the period of time. These microorganisms

entrapped in glacial ice provide valuable information about the evolutionary processes, as well as

the rich biodiversity during ancient times. Hence, various species of microorganisms may

appear to be extinct, but factually they might be dormant, entrapped in ice for millions of years

and are capable to reappear amidst suitable conditions. The results of this study can be used in

future to relate the biological, biogeochemical and genetic composition to a unique and well

characterized geologic history of the fungi entrapped in ancient glacial ice.

iii

ACKNOWLEDGMENT

I would like to express my deep gratitude to Dr. Scott Rogers for accepting me as a graduate student pursuing Master of Science in Biological Sciences, and guiding me through the research and thesis work.

I would like to thank Dr. Stan Smith and Dr. Dawn Hentges for serving on my committee and providing me with valuable suggestions and guidelines to continue my research.

My sincere appreciation to Dr.Vincent Theraisnathan for providing constant support and guidelines during my entire research. The development of this manuscript would not have been possible without him.

I would also like to take this opportunity to thank Lorena Harris, a wonderful friend and companion, for helping me out in every way she could and sometimes going out of her way to get me through the difficult times.

My heartfelt appreciation to my husband Jignesh Ladhawala for providing me constant motivation and encouragement which carried me through the hardest times I faced in the past two years.

I also extend my warmest thanks to my parents and my darling younger sister for their support during my stay away from them.

TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

HYPOTHESES AND AIMS ...... 16

MATERIALS AND METHODS...... 18

RESULTS……………...... 25

DISCUSSION...... 30

REFERENCES...... 33

APPENDIX …………………...... 38

A) The sequence alignment of fungal ITS regions. Fungal sequences

from ice core samples are compared with contemporary fungal species from GENBANK

LIST OF TABLES

TABLE Page

1 Fungal isolates and locations from Antarctic and Greenland ice cores...... 19

2 Comparison of fungal sequences from BLAST searches of GENBANK ...... 26

LIST OF FIGURES

FIGURE Page

1 Agarose gel depicting a PCR product compared with DNA ladder in a 1 % agarose

gel………………………..……...... …………………………………………………………. 10

2 Schematic representation of polymerase chain reaction (PCR) amplification………. 13

3 Schematic diagram of eukaryotic ribosomal RNA gene.………………………...... 15

4 Phylogenetic tree derived from rDNA sequence data from ice core

cultures and contemporary taxa...... …………………………………………………………. 29

INTRODUCTION

Glacial ice as a reservoir of microorganism

The close association between glaciers and microbial communities on Earth has been well documented (Ma et al. 1998, 2000; Rogers et al. 1999). Microorganisms in glaciers were reported as early as 1775 by a Russian scientist (Egorowa 1931). Studies have shown the presence of microbial communities on surface snow near the South Pole (Carpenter et al 2000).

Bacterial isolates have been recovered from glaciers in Western China (Christner et al 2003).

Considerable microbial communities are present in the unfrozen sub-glaciers of Southern

Hemisphere in New Zealand (Foght et al 2004). Microorganisms entrapped in glaciers thousands of years ago yield valuable information about the diverse biological history on Earth.

With the course of time, glaciers have entrapped numerous forms of life, including pathogenic microorganisms, forming successive layers of age-specific ancient glacial ice. The extreme temperature and lack of free water in glaciers are the chief factors which ensure the preservation of the trapped biological inclusions from degradation. A worldwide ice age on the Earth for about 10 million years or more, probably completely eradicated any possible source of life

(Williams et al 1998) except microbes. The extreme environments of glaciers in polar regions arrests the biochemical and physiological processes that can deteriorate the preserved microorganisms.

The viability of microbes in the frigid climactic conditions of glaciers has been possible because of “Cryopreservation”. It is because of this that the dormant microbes can become active and viable, thus, advancing our understanding of life processes on Earth.

Cryogenic processes protect the microbial life from mechanical or physical damage. Upon melting, the microbes may resume their metabolic activity, which presents significant 2 implications on the study of past climates and microbial life. Not only the viable, but even the dead microbes from ancient glacial ice are of profound significance due to the preservation of their cellular structures and molecular components.

The highly stable ecosystem within glaciers (due to long periods of constant low temperature) can aid in the studies of the evolution of life on Earth, longevity mechanisms of microbes, and possibly in the studies of microbes in the extraterrestrial environments. This is due to the preservation of living cells, tissues and perhaps even complex life forms for thousands to millions of years.

Glaciers are dynamic in nature, and it is because of this characteristic they also hold the potential to record events of the Earth’s history. The oldest glacial ice known to hold viable microbes is over 750,000 years old, found in glaciers of Western China (Christner et al

2003). However, ice that is well over one million years old exists in Antarctica and now is being tested for the presence of viable microbes, and permafrost many millions of years old has been reported to contain viable microbes. The sensitivity of the glaciers in recording climactic changes is demonstrated in the form of an identifiable pattern in glaciers, in that each layer is distinct to a particular year or vintage. The inhospitable dry environment of the glaciers has harbored a rich population of microorganisms, dense and diverse in species. The glaciers thus, represent an outstanding example of ongoing geological phenomena representing stages of the

Earth's annual changes.

Microbial communities, preserved in glacial ice for centuries, provide evidence for the possibility that similar life forms may thrive in similar environments on Mars or on

Jupiter’s moon Europa (Skidmore et al 2000). The low temperature of permafrost and glacial ice is analogous to the cryogenic environments found on many planets (Paerl and Priscu 1998). The 3

growing scientific evidence, which suggests the survival of microbes under extreme conditions

(Abyzov 1993, Catranis and Starmer 1991, Ma et al 1997), opens up the possibility of life

beyond Earth, as well as interplanetary transport of some microbes. Comparisons of different

environmental periods can provide representations of the development of microbes during their

evolutionary histories. Biogeoscientific studies on the glacial ice present a significant step in

determining the remnant and existing microbial diversity and past climatic histories. Thus,

glacial ice represents an ideal source for both contemporary and ancient microbial life. The

preservation of microbial life forms (including prokaryotes and eukaryotes) provides clues to the

potential for life in Earth’s harshest environments and on other planets. Additionally, this

realization necessitates a novel perspective on different ecosystems on Earth and on other

planets. Microbial life, one of the ancient life-forms on Earth, is diverse and adept in

acclimatizing to hostile environments. Microbes are amazing since the adverse conditions of

glaciers present environmental conditions that are extremely challenging to most organisms.

Microbial communities possess unique characteristics that allow them to survive persistent low

temperatures, various kinds of radiation, and fluctuating freeze-thaw cycles (Vincent et al. 2004).

Currently, it is unknown what gives them the ability to bounce back and become viable after

extended periods of dormancy. In fungi, increases in unsaturated membrane lipids, intracellular

trehalose, polyol concentrations, antifreeze proteins, and enzymes may play significant roles in protecting them from extremely low temperatures (Robinson 2001). Remarkable microbial

communities flourish near hydrothermal vents in deep ocean basins, and now it is known that microbes are found deep in the Earth’s crust and in glaciers and permafrosts.

The remarkable adaptability of some microbes, endows them with protective

mechanisms that make it possible for them to survive in harsh environments. Careful study of the 4

biological adaptation of microbial communities to global environmental changes can yield

information about the changes at their biochemical, physiological and molecular levels. It can

also provide insights into the changes in amino acids, enzymes and proteins at different levels of

evolution.

Earth's history has been marked by constant erosion by wind and water,

components of which sedimented year after year, thus recording the events for the regions of

their origins. These fine particulates have left their marks over thousands and millions of years,

and have become a record of life-forms prevailing at that time. Hence, careful planning and

study of the glacial ice potentially can yield biomarkers of global changes in Earth’s atmosphere.

Most of the trace biological matter entrapped in glacial ice for millions of years has been derived

from distant places, as well as the local environments, and offers a promising resource for future

scientific explorations into microbial diversity. The remnant biotic materials in the glacial ice

consist of pollens, wood fragments, such as vessels, tracheids and fibers, along with fungal and bacterial spores. Powder-fine dust was carried by powerful currents of air deposited minerals and ultra fine particles over the surface of the glaciers. Over the centuries and millennia, the glaciers accumulated nutrient-rich relics with organic matter and species of fungi, bacteria and viruses (Taylor et al 1997). A vast diversity of known and unknown fungi, bacteria, algae, viruses and protists of both local and distant origin has been isolated from Lake Vostok ice cores

(Abyzov 1993).

Microbial adaptability

Viability of microorganisms in the environment has been of interest for many

years (Gest and Mandelstam 1987). However, the knowledge of the mechanisms of microbial

survival is limited. Since glacial ice is an excellent preservation matrix for microbial life, the 5 longevity of microorganisms entrapped in ice can be studied. The intact conservation of microbes in glacial ice is useful for studying the longevity of microbes within specific time frames with regard to chemical, biological, geological, and physiological characters. Species of microorganisms recovered and identified from the glacial ice can be utilized to elucidate some of the processes involved in microbial longevity and survival over long periods of time.

Microorganisms grow and thrive in the extreme environments often by forming spores. Spores are resistant structures that protect the microorganisms from ultraviolet radiations, dessication, and mechanical and chemical damage. Most of the spores have extremely thick cell walls and often are pigmented. These encapsulated structures can remain protected and can revert to viability after millions of years of dormancy (Cano and Borucki 1995,

Gest and Mandelstam 1987). The biochemical adaptation of spores helps the microbes avoid periods unfavorable for growth, thus escaping the harsh conditions and retaining their viability when more favorable conditions return. However, many of the bacteria recovered alive from glaciers are not spore formers. Instead, they have other mechanisms to help them cope with the extremes found in glaciers. Some may to be able to go into a state known as "anabiosis" (Abyzov

1988). Recovery of temperate and tropical fungi in the polar regions has validated that the microorganisms found in the glacial ice travel from distant locales around the globe (Catranis and Starmer 1991; Ma et al. 1998, 2000; Rogers et al. 1999; Starmer et al. 2005). Winds from lower latitudes transport air along with micro-particles that fall onto Antarctic glaciers and become entrapped in the glacial ice (Abyzov 1993). Even in conditions devoid of oxygen and sunlight, under high pressure, extremely low temperature and nutrient level; microbial cells may survive in regions known as acidic or saline liquid veins (Price 2000). A massive network of these veins provide the critical components which sustain the microbial life in harsh 6

environments for hundreds to thousands of years. Additionally, metabolic activity has been

reported in bacteria at well below the freezing point of water.

DNA must be constantly repaired in viable cells, since radiation from space and

from the Earth, as well as oxidative processes, constantly degrade nucleic acids, and other

biomolecules inside the cells. Depending on the availability of free water, ultraviolet irradiation,

acidic conditions, and free oxygen radicals, hydrolysis and oxidation of DNA occurs continually

to a greater or lesser extent (Lindahl 1993). Dehydration greatly reduces the decomposition of

proteins and nucleic acids and thus holds promise in DNA preservation science (Bada et al

1994). In dormant cells, repair of DNA takes place slowly, depending on the availability of

nutrients and environmental conditions, and this explains the survival of encapsulated microbes

over extended periods of time (Morita 2000). Microorganisms buried under ice have either

adapted to the adverse conditions by encapsulation or may have developed other physiological

mechanisms or enzymes and proteins that help them tolerate the extremes of environment. Even

in the presence of dilute nutrients and extremely hostile conditions, microorganisms demonstrate

miraculous metabolic activity in accretion ice at a depth of 3603 m (Karl et al 1999). A greatly

reduced metabolic activity is noticed in microbes at sub-zero temperatures in South Pole

(Carpenter et al 2000). The innate repair mechanisms of bacteria may be able to restore the

macromolecular damage in bacteria immured in ice for hundreds of thousands of years

(Christner 2002).

Human activity and global climactic change

There is an alarming effect of human activity on the Earth’s atmosphere.

Anthropogenic global warming generated because of the rapid increases of greenhouse gases has affected the physical and chemical composition of Earth’s atmosphere. The emission of 7

greenhouse gases, such as carbon dioxide, methane, and chlorofluorocarbons, due to the

industrial revolution might have uncontrollable consequences in the near future. Rising sea levels

due to melting of glacial and polar ice; changes in rain patterns; risks of forest fires, storms,

tornadoes and other weather extremes, such as increases in the daytime temperature, may result

in the deaths of a variety of organisms including humans, and also increases the risk of infectious

diseases due to pathogens released from melting ice. The increase in temperature will cause

melting of ice at the polar regions and thus lead to significant increase of sea level. There is an

enormous risk of release of pathogenic viruses from the melted water of glacial ice. (Rogers et al

2004, Smith et al 2004). Also, the mechanism of “Genome recycling (temporal gene flow)” with increase in frequency due to the release of microorganisms from the melted glacial ice. These

ancient microbes, now free in the environment, can enter extant gene pools and may affect

mutation rates, fitness, survival, pathogenicity, and other characteristics of the organisms

(Rogers et al 2004).

Molecular biology in detection and identification of microorganisms in ancient glacial ice

Methods of molecular biology are successful in extracting ancient DNA (Pace

1997; Rogers and Bendich 1985, 1988, 1994). Recovery and amplification of DNA, millions of

years old, has revolutionized the understanding of evolution (Cano and Borucki 1995).

Molecular techniques can be of fundamental importance in comprehending the role of microbes

to answer the most intriguing questions of evolutionary history. Molecular techniques are

sensitive, reliable and thus, play a significant part in the detection and identification of culturable

and non-culturable diseases. Furthermore, molecular techniques provide sensitive and specific

identifications of ancient microorganisms entrapped in glacial ice. On the contrary, 8

morphological identification techniques often require well defined vegetative, reproductive, or

sporulating structures which may be sometimes absent in microbes. More importantly, the vast

majority of organisms currently cannot be cultured. Estimated range from 0.1% to 17% of organisms from various microbial groups is culturable using present methods. Molecular techniques have shown immense capability in revealing extensive microbial diversity and

identification. Culture dependent and morphological identification are difficult and time consuming processes mainly because of pleomorphic nature of many species of fungi. Also morphological identification focuses on identifying the already known microorganisms while molecular techniques aid in the identification of unknown microorganisms. Even if morphological identification is achieved, phylogenetic relationship is difficult, or impossible, to determine. For example, the complex morphology of cyanobacteria does not result in a phylogenetically reliable cyanobacterial taxonomy, while molecular techniques based on their ribosomal sequences can reliably aid in their identification and phylogenetic affinities (Wilmotte

1994).

Agarose gel electrophoresis

Agarose gel electrophoresis is an analytical tool to separate DNA fragments

according to their size. DNA is a negatively charged molecule, and therefore, migrates through

the gel to the cathode when an electric current sent through the gel. Smaller size DNA molecules

navigate faster than the larger DNA molecules, and thus, the DNA molecules are separated

according to their size. Usually, ethidium bromide is used to stain the gel which fluoresces red-

orange when it intercalates between the bases of DNA (and RNA), and thus, the DNA can be

visualized by exposure of the gel to ultraviolet irradiation (at 230 to 320 nm). The size of the

DNA molecule under investigation is determined by comparing it with a molecular weight DNA 9 ladder that contains DNA molecules of known molecular weights which is also loaded into one of the wells of the gel.

1000 bp 900 bp

800 bp 700 bp PCR product from Rhodotorula laryngis 500 bp visible near 600 bp 400 bp 300 bp 200 bp 100 bp

Figure 1. Agarose gel depicting a PCR product compared with DNA ladder in a 1 % agarose gel. The gel (in 1X TBE with 0.5 ug/ml ethidium bromide) is photographed using ultraviolet illumination (300 nm).

Lane A. Molecular weight markers - 100 bp ladder (Bioline, Massachusetts).

Lane B. Polymerase chain reaction amplicon from a Rhodotorula laryngis species using primers

prITS4 and prITS5 to amplify the rDNA ITS1 and ITS2 regions (including the 5.8S

gene).

Polymerase chain reaction

The polymerase chain reaction (PCR) method was invented and developed by

Kary Mullis in 1985 (Mullis et al 1986), a Nobel Prize recipient for chemistry in 1993. The polymerase chain reaction (PCR) is an amplification technique used to create numerous copies of short, specific regions of DNA beginning with a template molecule. Thus, millions or billions of

DNA molecules can be derived from a single molecule of double stranded DNA. With the help of heat stable DNA polymerases, deoxynucleotide triphosphates (dNTPs), and two oligonucleotide primers, DNA undergoes heat denaturation separating two strands of the template DNA. This is followed by cooling so that the primers can anneal to their complementary segments on the template DNA, and new complimentary strands of DNA are synthesized by the incorporation of new nucleotides by the action of DNA polymerase. Hence by repeated cycles of controlled heating and cooling, DNA is amplified, each cycle doubling the number of DNA molecules. Thus, a single molecule of DNA gets amplified into more than a million copies at the end of Polymerase Chain Reaction. Originally, a standard DNA polymerase was used, and had to be added after each denaturations sttep, since the heating also denatured the enzyme. Soon after, a heat stable DNA polymerase, Taq DNA polymerase (from the thermophilic Thermus aquaticus) was used so that repeated additions of polymerase were not required. This enzyme, and similar heat stable enzymes, continue to be used in this methods.

Figure 2.0 is a schematic diagram of a typical polymerase chain reaction.

Figure 2. Schematic representation of polymerase chain reaction (PCR) amplification.

The double stranded DNA is separated by heat denaturation (at 94 C). The primers (short, synthetic DNA strands) are then annealed to each strand by reducing the temperature to approximately 5 C below the Tm of the primers, and then extended by DNA polymerase (usually at 72 C). The two newly formed relatively long DNA strands act as the template DNA for the next cycle.The next strands formed are shorter, and are of defined length. At each cycle the number of short, defined length, DNA amplicons essentially doubles. The entire amplification process requires 30 – 40 similar cycles.

5’ 3’ 3’ 5’ + primer 1 Heat denaturation 94°C + primer 2

5’ 3’ primer 2 primer 1 separated DNA strands 1st cycle 3’ 5’ + dNTPs + buffer with Mg 2+ Primer annealing, extension 72°C + DNA polymerase 5’ 3’ Primer 2 New long strands Primer 1

3’ 5’

Heat denaturation 94°C

5’ 3’

3’ 5’

5’ 3’

3’ 5’

Primer annealing, extension 72°C nd 2 5’ 3’ cycle

primer 2 primer 1

New short strands

primer 2 primer 1 3’ 5’

Ribosomal DNA and phylogenetic analysis

Eukaryotic ribosomal RNA genes (ribosomal DNA or rDNA) occur as repeat

units. Each repeat unit has a transcribed region for18S (nuclear small subunit, SSU), 5.8S and

26S (nuclear large subunit, LSU) that are separated from each other by an intergenic spacer

(IGS) region that includes an external transcribed spacer (ETS), and two internal transcribed

spacers (ITS 1and ITS 2), that are present on either side of 5.8S ribosomal RNA gene. Some

regions of ribosomal RNAs play vital roles in translation of mRNAs, and hence have highly

conserved DNA regions that can be utilized for DNA sequence analysis, while some other

segments undergo rapid mutations. Since ribosomal DNA (rDNA) occurs in all cellular

organisms on Earth, it is extremely useful for studying the phylogenetic relationships and

molecular systematics. The conserved gene regions can be used for broad phylogenetic studies, for example, comparing prokaryotes with eukaryotes, while the variable regions of the spacers can be used to study phylogenies at the genus, species, and varietal levels.

Repeat Unit

IGS Transcription Unit IGS

prITS 5

ITS 1 ITS 1 ITS 2 ETS 18 S rRNA 28 S rRNA

B 5.8 S prITS 4

Figure 3. Schematic diagram of eukaryotic ribosomal RNA gene.

A Diagram of eukaryotic rDNA depicting an entire repeat unit, including the IGS.

B Diagram of the rDNA transcription unit. One repeat unit consists of an external transcribed

spacer (that includes the promoter), a small subunit (e.g., 18S) gene, a 5.8S gene surrounded

by two internal transcribed spacers (ITS1 and ITS2), a large subunit (e.g., 26S) gene, and an

intergenic spacer (IGS). Primers used to amplify ITS 1 and ITS 2 are designated by prITS 5

and prITS 4, respectively.

HYPOTHESES AND AIMS OF THE STUDY

Antarctica, the largest ice sheet on Earth, and Greenland are considered to have

one of the most arid ecosystems (Priscu 1999). Various species of bacteria have been isolated

from Antarctica ice sheets (Karl et al. 1999, Priscu 1999), and viable bacteria and fungi have

been reported from both Antarctica and Greenland ice sheets (Castello et al. 1999; Catranis and

Starmer 1991; Ma et al. 1997, 2000; Rogers et al. 1999). The recovery of viable fungi, bacteria, and viruses have been made from deep ice cores of ancient glacial ice in Antarctica and

Greenland (Abyzov 1993; Ma et al. 1997, 2000; Rogers et al. 1999).

This study was initiated to identify fungi in ancient glacial ice at different depths from Greenland and Antarctica. The study was carried out with the following hypotheses:

1. Glacial ice is a long-term preservation matrix for ancient microorganisms entrapped in

glaciers that may remain viable and/or detectable over thousands and hundreds of years

2. Fungi are present in ancient glacial ice from Antarctica and Greenland ice cores.

3. Novel species of fungi will be detected in glacial ice from Antarctica and Greenland ice cores.

4. Some of the fungi are viable in ice and are detectable using conventional culture methods.

5. Some of the fungi are detectable only by amplification of their DNA using polymerase chain

reaction (PCR) amplification.

The specific objectives of the study were as below:

1. Isolate viable fungi from Antarctica and Greenland ice cores using conventional culture

methods.

2. Amplify DNA from viable and nonviable fungi using polymerase chain reaction (PCR)

amplification.

3. Obtain gene sequences from the amplified fungal ribosomal DNA. 17

4. Obtain a reliable phylogenetic reconstruction using rDNA ITS sequesnces from the ancient ice

fungi and contemporary taxa.

MATERIALS AND METHODS

Ice cores from Greenland and Antarctica were obtained from the National Ice

Core Laboratory (NICL, Denver, Colorado). Ice cores (Table 1) of various depths , ranging in

age from <500 ybp to approximately 100,000 ybp, were used. Clorox (10%) and ethanol (70%),

respectively, were used before and after the experiment to decontaminate all work surfaces.

Ultraviolet light exposure (941 700 J/m2) for 10-15 minutes was carried out every time before

and after use of Clorox and ethanol, respectively. The Vostok ice cores were treated for 10s with

cold (4 °C) Clorox (5.25 % sodium hypochlorite, stabilized with sodium hydroxide) and then

rinsed twice with large volumes of cold (4°C) purified water (18.2 MΩ, <1 ppm total organic

carbon, TOC). The meltwater was collected in in a sequence of five different containers, each

with 30 ml capacity, noted as shell 1 (outermost) through shell 5 (innermost). Plates containing

the MEA media (12.75 g/L maltose, 2.75 g/L dextrin, 2.35 g/L glycerol, 0.78 g/L peptone, 15

g/L agar; final pH 4.7 were inoculated with 200 ul of meltwater. Inoculated plates were

incubated at 8°C for 1 week, at 15°C for 2 weeks and then incubated at 22°C for a year. The

culture plates were examined once every week for growth.

Subculturing

Fungi were subcultured on the slant test tubes made from MEA (12.75 g/L

maltose, 2.75 g/L dextrin, 2.35 g/L glycerol, 0.78 g/L peptone, 15 g/L agar; final pH 4.7). The agar (33.6 g of MEA/1L) was sterilized by autoclaving at 15 psi and 120°C for 25 min. It was

poured into the sterilized petri plates, and these were then left untouched for at least 2 hours and

when solidified, were stored at 4°C. For subculturing, the fungus samples were transferred onto

the agar by using sterilized plastic loops. Then, the slant tubes were incubate at 22 °C for 3 days. 19

TABLE 1. Fungal isolates and locations from Antarctic and Greenland ice cores.

ISOLATE SOURCE ICE CORE DEPTH (m) AGE (ybp)

GI 16 Greenland GISP 2D 134.000-134.200 < 500

GI 228 Greenland GISP 2D 130.000-130.200 < 500

GI 232 Greenland GISP 2D 130.000-130.200 < 500

GI 237 Greenland GISP 2D 130.000-130.200 < 500

GI 336 Greenland Dye 3 71 158.569-158.869 < 500

GI 341 Greenland Dye 3 71 158.569-158.869 < 500

GI 343 Greenland Dye 3 71 158.569-158.869 < 500

GI 371 Greenland Dye 3 71 200.710-200.920 1000

GI 497 Greenland Dye 3 71 369.013-369.313 2000

GI 545 Greenland Dye 3 79-81 798.680-798.980 2200

GI 594 Greenland Dye 3 79-81 1007.235-1007.535 3000

GI 731 Greenland Dye 3 79-81 1405.515-1405.735 5500

GI 829 Antarctica Vostok 5G-91 section 21 3567.995-3568.315 < 10,000

(accretion ice) GI 831 Antarctica Byrd 68 section 24 2132.110-2132.350 ~ 200,000

GI 840 Antarctica Vostok 5G-91 section 22 3581.525-3581.795 ~ 10,000 (accretion ice) GI 848 Antarctica Vostok 5G-91 3567.995-3568.315 ~ 10,000

GI 850 Antarctica Byrd 68 section 24 388.560-389.040 <500

GI 854 Antarctica Byrd 68 section 24 388.560-389.040 <500 20

During this process, the disinfection of working areas was carried out by using Clorox (10%) and

thereafter by ethanol (70%). On the third day most of the cultures showed growth of their colonies, and then they were used for subsequent PCR amplification.

POLYMERASE CHAIN REACTION

Fungal cells grown on the agar were taken and diluted into a 50 ul sterilized

distilled water in an Eppendorf tubes. This produced the required dilution of the DNA sample for

the PCR reaction. The diluted fungal samples were then vortexed vigorously to break open the

fungal cells to expose the DNA for amplification. Primer pair prITS 4 and prITS 5 were used to amplify the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS 1 and ITS 2) and

5.8S regions (White et al 1990).

DNA was amplified using a GeneAmp PCR Reagent Kit (Applied Biosystems,

Foster City, California). A preamixed reaction cocktail ("mastermix") contained 280 ul dNTP mix (10 mM each of ATP, CTP, GTP, and TTP), 175 ul 10 X buffer (15 mM MgCl2, 100 mM

Tris HCl, 100 mM KCl; pH 8.3?) 15 ul Taq DNA polymerase (5 units/ul), and 280 ul water. The

dNTP mix was made by mixing 125 ul ATP (10 mM), 125 ul CTP (10 mM), 125 ul GTP (10

mM), 125 ul TTP (10 mM), and 500 ul water. Each reaction consisted of 10 ul master mix, 1.25

ul primer 1 (prITS 4, 10 uM, 20 bp, TCCTCCGCTTATTGATATGC), 1.25 ul primer 2 (prITS 5,

10 uM, 22 bp, GGAAGTAAAAGTCGTAACAAGG), 1ul DNA (5 ng/ul) and 11.5 ul water. The

thermal cycling program (Gene Amp PCR system 9700, Applied Biosystems, Foster City,

California) was 95°C for 2 min, then 35 cycles of 1 min at 95°C, 30 seconds at 54°C and 4 min

at 60°C. This was followed by 7 min incubation at 72°C. 21

Upon the completion of the PCR cycle, the samples were stored at -20°C and then

were separated by electrophoresis on 1% agarose gels (1 X TBE, 2 mM TBE = Tris-Borate-

EDTA with 0.5 ug/ml ethidium bromide).

AGAROSE GEL ELECTROPHORESIS Agarose gels (1%) were made using 0.25 g of agarose powder dissolved in 25 ml

1X TBE buffer (Tris 89 mM, Boric acid 89 mM, disodium EDTA 2mM; pH 8.0) and was boiled

in a microwave for 1 minute. It was allowed to cool for a 2 minutes at room temperature.

Meanwhile, the gel tray with comb was adjusted such that the distance between the comb and the

tray was approximately 1 mm. Ethidium bromide (1.25 ul, 10 mg/ml stock solution, final

solution in the gel 0.5 ug/ml) was added to the mixture and then poured into the tray. The gel was left undisturbed for about 45 minutes until the gel had solidified and then kept at 4 ° C in the

refrigerator until needed.

The gel apparatus (Minnie submarine, Hoefer Scientific Instruments, San

Francisco, California) contains 250 ml TBE buffer with 0.5 ug/ml ethidium bromide. For

loading of the gel, 5 ul of the “100 base pair ladder” DNA (Bioline, Massachusetts) used as a

control DNA was loaded in the well immediately adjacent to the first well, and in the subsequent

wells, a mixture of 3 ul bromophenol blue dye (0.05% bromophenol blue, 50 mM EDTA, 20%

ficoll) and 4 ul of each PCR product were loaded into the wells of the gel. The starting current

was kept at 100 V for 30 sec followed by stopping the current for 15 sec. This was repeated

twice. Finally, the gel was subjected to electrohoresis at 25 V for 30 min and at 100 V for about

1 hr. When two combs one at each end of the gel were used to load more samples on a single gel,

the gel was subjected to electrophoresis at 25 V for 15 min and at 100 V for about 30 min or

until the bromophenol blue dye had migrated 2/3rd of the length of the gel. 22

The gel was photographed using an ultraviolet light source and a Polaroid camera

(Polaroid Type 55 4x5 film, Fotodyne Incorporated, Hartland, Wisconsin). When sharp bright bands were observed, the sample was used for sequencing.

Purification of the PCR product using ethanol precipitation:

The PCR product was transferred into a 1.5 ml microfuge tube containing Qiagen

MinElute spin column (QIAGEN, Valencia, California). Next, 100 ul of PB buffer (DNA

purification kit, Qiagen, Valencia, California) was added. Then, the microfuge tube was

centrifuged at 13,000 rpm in a microfuge for 1 min. The supernatant was discarded. Next, 750 ul

of PE buffer (750 ul) (DNA purification kit, Qiagen, Valencia, California) was added to the tube

and centrifuged at 13,000 rpm for 1 min, and the supernatant was discarded. The microfuge tube

was centrifuged again at 13,000 rpm for 1 min. The QIAGEN quick column was placed in

another 1.5 ml microfuge tube, and 30 ul of EB buffer (DNA purification kit, Qiagen, Valencia,

California) was added on the center of the column. After 2 min, it was centrifuged again at

13,000 rpm for 1 min. The eluent was saved for further use. Electrophoresis on 1% agarose gels

(conditions as above) was carried out to estimate the concentration of the sample DNA.

Cycle sequencing on the PCR product:

Using the gel picture of the PCR amplifications as a guide, the amplification products were diluted to 5-10 ng/ul and were used for cycle sequencing reactions. The reaction

consisted of 8.0 ul terminator ready reaction mix (dye terminators, deoxynucleoside

triphosphates, amplitaq DNA polymerase FS, magnesium chloride and buffer 10 mM Tris- HCl

pH 9.0), (Applied Biosystems, Foster City, California), 1-2 ul PCR product, 0.5-1 ul primer (10

mM stock solution), and 9.0-10.5 ul water (to reach a final volume of 20.0 ul). The cycling 23

program, performed on a Gene Amp PCR system 9700 (Applied Biosystems, Foster City,

California), was 94°C (1 min), and 25-30 cycles of 94°C (10 sec), 54°C (30 sec), 60°C (4 min).

Cycle sequence PCR ethanol precipitation

Following amplification, 2 ul of 3 M sodium acetate and 12.5 ul of 100% ethanol were added to the cycle sequenced PCR tube. It was then chilled for 20 min at -20 °C and then

centrifuged at 13,000 rpm for 15 min. The supernatant was discarded. Next, 50 ul of 70% ethanol was added, followed by centrifugation for 10 min at 13,000 rpm. The supernatant was discarded, and the pellets were dried in a Speed Vac for 20 min. Then, 12 ul of template suppression reagent (80% Formamide, 5 mM EDTA, 10 mg/ml blue dextran) (Applied

Biosystems, Foster City, California), was added, and after 1 min, the mixture was centrifuged for

10 sec. The contents were transferred into 0.2 ml microfuge tubes and heat denatured for 4 min at 95°C by using a thermal cycler if sequenced immediately after or stored at -20 °C. was performed on an ABI (Foster City, California) 310 Automated DNA Analyzer using 0.2 ml

MicroAmp reaction tubes, 0.2 ml (Applied Biosystems, Foster City, California).

PHYLOGENETIC ANALYSIS

A gapped BLAST search (Altschul et al 1997) of GenBank (National Center for

Biotechnology Information Internet site, http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/) was then performed with each of the resulting sequences. The most closely related taxon to each sequence and BLAST similarity calculations was determined. Sequences were aligned using ClustalW 1.7

(National Laboratory for Computational Science and Engineering at the University of California,

San Diego, California) and adjusted manually. Phylogenic reconstructions in the form of 24

Neighbor-Joining unrooted trees were conducted using Mega 3.1 software (Integrated Software for Molecular Evolutionary Genetics Analysis and Sequence Alignment, Version 3.1. Kumar et al 2004). Gaps were not considered in the analyses. Neighbour Joining unrooted tree was constructed using nucleotide p-distance, and branch lengths are arbitrary.

RESULTS

Isolation of fungi from culture:

Ice cores sections from GISP 2 and Dye 3 from Greenland, and Vostok and Byrd

from Antarctica were used for the study. The cores were from 130 to 3581 m in depth, ranging

in age from <500 ybp to approximately 200,000 ybp (Table 1). Viable fungi from cultures were

used for amplification and subsequent sequencing of the ribosomal DNA ITS region. Twenty

different fungal cultures were successfully amplified by polymerase chain reaction (PCR) using fungal primers prITS4 and prITS5, followed by DNA sequencing (White et al 1990).

The analysis of the representative fungal rDNA ITS sequences represented a

wide diversity of fungal taxa. BLAST searches of GenBank indicated that fifteen of the fungal

sequences resembled to ascomycetes and three resembled basidiomycetes. Species of

ascomycetes that included the hyphomycetes or conidial fungi were Penicillium crustosum

AY373907, Penicillium griseoroseum AY425983, Penicillium dipodomydis PDJOO4896, Penicillium

commune AY360403, Penicillium expansum AY425984, Penicillium farinosum AF527057,

Penicillium aurantiogriseum AY280955, Penicillium chrysogenum AY373902, Cladosporium

oxysporum AY391832. Other ascomycetes were Cladophialophora minourae AF393716, Pichia

guilliermondii AF455495, Togninia minima AY786143, Coniochaeta ligniaria AY198390,

Aureobasidium pullulans AY141180, and Lecythophora hoffmannii AY781227. Basidiomycetous

fungal species included Sporobolomyces sp. AF444603, Rhodotorula slooffiae ABO25994, and

Rhodotorula laryngis AF444617. Table 2 presents the contemporary fungal taxa from GenBank,

which were closest to the fungi isolated from the ice cores.

TABLE 2. Comparison of fungal sequences from BLAST search of GENBANK

GI SOURCE ICE AGE Closest Taxon

No. CORE (ybp) (Accession No.)

GI 16 Greenland GISP 2D < 500 Penicillium crustosum AY373907

GI 228 Greenland GISP 2D < 500 Penicillium griseoroseum AY425983

GI 232 Greenland GISP 2D < 500 Sporobolomyces sp. AF444603

GI 237 Greenland GISP 2D < 500 Aureobasidium pullulans AY141180

GI 336 Greenland Dye 3 71 < 500 Lecythophora hoffmannii AY781227

GI 341 Greenland Dye 3 71 < 500 Penicillium aurantiogriseum AY280955

GI 343 Greenland Dye 3 71 < 500 Coniochaeta ligniaria AY198390

GI 371 Greenland Dye 3 71 1000 Cladosporium oxysporum AY391832

GI 497 Greenland Dye 3 71 2000 Pichia guilliermondii AF455495

GI 545 Greenland Dye 3 79-81 2200 Penicillium dipodomydis PDJOO4896

GI 594 Greenland Dye 3 79-81 3000 Penicillium expansum AY425984

GI 731 Greenland Dye 3 79-81 5500 Cladophialophora minourae AF393716

GI 829 Antarctica Vostok 5G-91 < 10,000 Penicillium commune AY360403

GI 831 Antarctica Byrd 68 section ~ 200,000 Penicillium chrysogenum AY373902

GI 840 Antarctica Vostok 5G-91 ~ 10,000 Rhodotorula slooffiae ABO25994

GI 848 Antarctica Byrd 68 section ~ 10,000 Rhodotorula laryngis AF444617 24 GI 850 Antarctica Byrd 68 section <500 Togninia minima AY786143

GI 854 Antarctica Byrd 68 section <500 Penicillium farinosum AF527057

The DNA sequences from the samples were compared to the selected contemporary

sequences in GenBank. A majority of the cluster belonged to the Penicillium species followed

by Rhotorula. Two fungal isolate GI 831 showed close similarity to Penicillium chrysogenum.

Penicillium species dominated the fungal isolates from Greenland where three Antarctica

cultures, GI 840 and GI 848 were identified as Rhodotorula species.

Phylogenetic analysis of the fungal sequences:

The ribosomal DNA sequences of fungal cultures were compared to the existing

contemporary sequences from GENBANK. These were grouped into different branches

representing different lineages by generating a phylogram (Figure 4.)

Subclades identified amongst nonflagellated Ascomycetes were Trichocomaceae,

Mycosphaerellaceae, Herpotrichiellaceae, Sordariomycetideae, Calosphariaceae,

Coniochaetaceae, and Dothioraceae while Basidiomycetes included subclade

Microbotryomycetideae.

Members of subclade Microbotryomycetideae included two Rhodotorula sp. and one Sporobolomyces sp. Trichocomaceae sublclade included eight Penicillium sp. Two fungal cultures GI 371 and GI 237 identified as Cladosporium oxysporum and Aureobasidium pullans, respectively, were grouped in Dothiodeales clade. Other sequences closely resembling to

Lecythophora hoffmanni and Coniochaeta ligniria were included in Coniochaetales clade.

Isolates GI 731, and GI 850 resembling to contemporary sequences Cladophialophora minourae

and Togninia minima, respectively, were included in Chaetothyriales and Calophaeriaceae

clades.

Figure 4. Phylogenetic tree derived from rDNA ITS sequence data from ice core cultures and contemporary taxa. GI represents ice core specimens (glacial isolates). Phylogenetic reconstruction was conducted using Mega 3.1 software (Integrated Software for Molecular

Evolutionary Genetics Analysis and Sequence Alignment, Version 3.1. Kumar et al 2004).

Phylogenetic tree construction is Nucleotide-p-distance and gaps were not included in the analyses. Sequence database in GenBank was used for comparision.

GI 840 Rhodotorula slooffiae GI 848 Rhodotorula laryngis GI 497 Pichia guilliermondii Aureobasidium pullulans GI 343 Coniochaeta ligniaria GI 371 Cladosporium oxysporum GI 731 Cladophialophora minourae GI 850 Togninia minima GI 232 GI 545 Penicillium dipodomyis GI 831 Penicillium chrysogenum GI 854 Penicillium farinosum GI 228 Penicillium griseoroseum GI 594 Penicillium expansum GI 341 Penicillium aurantiogriseum GI 829 Penicillium commune GI 16 Penicillium crustosum GI 237 Sporobolomyces sp GI 336 Lecythophora hoffmannii

0.05 30

DISCUSSION

Studying ancient glacial ice at the molecular level demonstrates a high level of

corroboration in the field of biological research. The diversity of fungi in ancient glacial ice can

be used to distinguish the phylogenetic positions of ancient fungi with distinct characteristics

compared to contemporary species. The existence of viable microbes entrapped in ancient

glacial ice under unique conditions has been demonstrated by several researchers (Ma et al.

1998, 2000; Rogers et al. 1999). Thus, the recovery and identification of viable fungi from

ancient ice has wide ranging implications because of its prospects to increase the understanding

of ancient microbial communities and their changes through time.

Glacial ice has accumulated huge quantities of wind-blown organic material

including viruses, as well as bacterial and fungal spores from distant sources and is dominated by

species found in surrounding regions (Gordon et al 2000). It is the general perception of ecology

that as conditions become more extreme, survival becomes less likely (Casamayor et al 2002).

In contrast, the extreme environments of Antarctica and Greenland hold a large diversity of

microorganisms. For example, Penicillium species often referred as “opportunistic fungi” have been recovered from the Antarctic and Arctic, as well as from temperate and tropical soils

(Mahaney et al 2000). Most of these species thrive on a variety of organic carbon and nitrogen sources. Hundreds of viable and non-viable fungi, bacteria and viruses have been recovered from

Greenland and Antarctic ice cores (Ma et al 2000, Rogers et al 1999).

Glacial ice provides a clue to the response of microorganisms to the extremes of climate, and their survival in conditions of nutrient deprivation. In the presence of nutrient

deficient mediums, the microorganisms deal with the repair of their cell damage and sustain

growth and metabolism (Christner et al 2000). Most of the species of Rhodotorula, 31

Cladosporium, and Lecythophora are capable of surviving harsh environments through spore

formation. Species like Penicillium are found almost everywhere on Earth, and the data

indicating several Penicillium spp. are not surprising.

Isolates GI 336, GI 371, and GI 850 exhibited relatively low BLAST similarities to all GenBank sequences, but came closest to Togninia minima, Cladosporium oxysporium, and

Lecythophora hoffmanni, respectively. All other isolates yielded higher similarities to

contemporary fungal ITS sequences. Fungi have been found prevalent on continental Antarctica,

especially the hyphomycetes, such as Cladosporium and Penicillium.

Classification of the sequences is reliant on a number of parameters including the quality of PCR products, quality of the sequence and the reference source. In many instances, poor quality sequences are included in the reference database, and hence, the final phylogenetic

trees might be more or less inaccurate. Hence, for the present study, sequences with

comparatively higher similarity with those from GenBank were included for determining the

phylogenetic relationship. Also, the 600 bp long DNA fragments amplified by prITS4 and

prITS5 are sufficient to yield robust phylogenetic information.

The phylogenetic tree (Figure 4) revealed higher number of Ascomycotes than

Basidiomycetes. Basidiomycete fungi are more prevalent in temperate regions of the Earth, while

the cold climates are represented to a greater extent by the Ascomycotes clade of fungi.

Phylogenetic data can dictate the correct place of different microbes and also find the missing

links in the Earth’s evolutionary history.

Microbes, one of the few life-forms to retain viability after being dormant for

such long periods of time (some animals and plants can remain viable in ice for hundreds to 32 thousands of years), thus present a unique window into the biogeochemical cycles, and their adaptation to the fluctuations in climates seems astounding.

With the present scientific knowledge, the effect of climate and other environmental changes over several centuries on the Earth’s dynamics can be related.

However, there are several compelling questions related to the risks of pathogen release from the melting of the glacial ice, to the greenhouse effects, and to the longevity of these microbes in ancient ice that can be solved in corroboration with other studies in this area. The ancient microbes also may be possible sources and may be directly relevant to the science of antibiotic resistance. Thus, these studies on ancient ice have wide ranging implications on various strata of life and provide information which can significantly increase and challenge the understanding of

Earth’s biological frontier.

. .

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APPENDIX A

The sequence alignment of fungal ITS regions. Fungal sequences from ice core samples are compared with contemporary fungal species from GenBank (at NCBI).

GI = Fungal sequences from ice core samples.

All other sequences are used for comparison and are obtained from GenBank.

GI_840 ??????????????????????????????????????????????GCC?GC?AC?ACCCGGGCGCCGGGGT Rhodotorula_slooffiae ??????????????????????????????????????????????????????????????????CTGCGG GI_848 ???????????????????????????????????????????????????????????????????????C Rhodotorula_laryngis ???????????????????????????????????????????????????????????????????????- GI_831 ???????????????????????????????CCTCATTAGCGGAC—GAGAAAGGGTCATTCTCGCTCCCGCG Penicillium_chrysogenum ??????????????????????????????-TCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGAGGG GI_228 ????????????????????CACTCATTATGCGGGAGGATGGGAAGTAAAAGTCGTTACAGGGCGATATGAA Penicillium_griseoroseum ?????????????????????ACTAGTGATTGGTGAACCTGCGG---AAGGATCATTACCGAGTGA---GGG GI_16 ?????????????????????????????????CAGGTGAACATACGGAAGACATCATTCCCGGGTGTGGGA Penicillium_crustosum ?????????????????????????????TCCGTAGGTGAACCTGCGGAAGG-ATCATTACCGAGTGAGGGC GI_854 ??????????????????????????????????CTTCATTAGGCGGACGAGGAAGGACAGACCCGTGAGGG Penicillium_farinosum ??????????????????????????????????????????????????AAGGATCATTACCGAGTGAGGG GI_545 ?????????????????????????CTCCGGGGCTCGG?G?TAGCTCG?GC??AATCCGACTGAGGGGCCGA Penicillium_dipodomyis ????????????????????????????????????????????????????????CCGAGTGAGGGCCCTC GI_341 ???????????????????GTATCAATAGCGGCGGGGAGGGAG----GGAAAGTTCATTACCGGGCCAGGGC Penicillium_aurantiogriseum ?????????????AAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGAGGGC GI_594 ???????????????????CGTTCTAAGAAGCCGACCTGGA?GGGAAAGTAAAAGTCGCGATCAAGGCCCTA Penicillium_expansum ??????????????ACTAGTGATTTCCGTAGGTGAACCTGC---GGAAGGATCATTACCGAGTGAGGGCCCT GI_829 ????????????????????????????????????????????GGGCGGAGGATCCACGCCCACTGAGGGA Penicillium_commune ??????TAAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGAGGGC GI_371 ?????????????????????????????????????????????????????????CA?CAT?TACAAAGG Cladosporium_oxysporum ????????????????????????????????????????????????????????????????????AGGG GI_731 ??????????????????????????????????????????TCAAGACTTAAGTAGGCAAGCCGGAAGTAT Cladophialophora_minourae ???TTGGTCAAACCCGGTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGG-AG GI_336 ???????????????????CTGCTGGTTGTGC?C?G?CGG??A?GC?GCACA??????GAA???TTTTTCCC Lecythophora_hoffmannii ???????????????????????????????????????????????????????????????????????? GI_850 CGTAAAAGGCCTTCATCAATAAGC-GGAGGATGGGTTCCAAAAGTCGTAAGAAGGCATATCAATAAGCGGAG Togninia_minima CGTAACAAGGTCTCCGTTGGTGAACCAGCGGAGGGATCA------TTATCGAGTTTCGTA-CTCCAAACCCT GI_232 ??????????????????????????????????????????????????TCATCGTTAAAAGTGAGTTAGA Sporobolomyces_sp ?????????????????????????????????????????????????TCCGTAGGTGAACCTGCGGAAGG GI_497 ??????????????????G??????CAGGGGGGCGCGGGG?TACGCGGCGAGGAGATTCGTATTCTTTTGCG Pichia_guilliermondii ?????????????????????????????????????????TAACCTGCGAAGACATCAGTATTCTTTTGCC GI_343 ??------TACGGG-CTCCG--GGGGGATCGCTCGAGGCTTAAGTCTAAGAAGCGGGAAAACGA----GGAG Coniochaeta_ligniaria AGTCGTAACAAGGTCTCCGTTGGTGAACCAGCGGAGGGATCATTACAAGAAGCCGAAAGGCTACTTCAAACC GI_237 ?????????????????????NTCNGNNNCNGNNTTNGNGNNNTNNTCCNCNNGGGANAGAATTCTTTTT-C Aureobasidium_pullulans ????????????????????????????????????????????????????????????ACAAGAGCCGAA

GI_840 GAAGATAACTCATGCTAAACAGGCGGGATGAATGTTATGCGGAGGGATCGGTTCCCTTTCATCTGTTAA?TA Rhodotorula_slooffiae AAGGATCATTAATGAATTTTAGG------ACGTTCTTTTTAGAAGTCCGA-CCCTTTCATTTTCT---TA GI_848 TCCATTAGCGG-CCTGAGGAAGGATCATTAATTAATTTTAGGAGAAAAATTTTTAGAGGTCCGATCCTTTCA Rhodotorula_laryngis TCCGTAGGTGAACCTGCGGAAGGATCATTAATGAATTTTAGGACTCTC-TTTTTAGAGGTCCGACCCTTTCA GI_831 GCGAAGGGGTCAAACTCCCTTCCCCTGTTTATTGGAA-TTGTTGCTTCGGCGGGCCCGCCTTAACTGGCCGC Penicillium_chrysogenum CCCTCTGGGTCCAACCTCCCACCCGTGTTTATTTTACCTTGTTGCTTCGGCGGGCCCGCCTTAACTGGCCGC GI_228 TAAGCGGAAGGCAAGAGTCCAGCTGAATTTATTATAACGTGTTGCTTCGGCGGGCCCGACTTAACTGGCCGC Penicillium_griseoroseum CCCTCTGGGTCCAACCTCCCACCCGTGTTTATTTTACCTTGTTGCTTCGGCGGGCCCGCCTTAACTGGCCGC GI_16 A---TGGGC---GTTCCCGGAAGGTGATTATTTTACCTTGTTGCTTCGGCGGGCCCGCCTTAACTGGCCGCC Penicillium_crustosum CCTCTGGGTCCAACCTCCCACCCGTGTTTATTTTACCTTGTTGCTTCGGCGGGCCCGCCTTAACTGGCCGCC GI_854 CCCAAAGGGTCCAACCTCCCATCC-TGTTTATTTTAACTTGTTGCTTCGGCGGGCCCGCCTTAACTGGCCGC Penicillium_farinosum CCCTCTGGGTCCAACCTCCCACCCGTGTTTATTTTACCTTGTTGCTTCGGCGGGCCCGCCTTAACTGGCCGC GI_545 AAGGGTCCAACCTCCTTCCCGTGTTTAGGTAAAATTGGTTGCTTCGGCGGGCCCGCCTTAACTGGCCGCCGG Penicillium_dipodomyis T-GGGTCCAACCTCCCACCCGTGTTTATTTTACCTTG-TTGCTTCGGCGGGCCCGCCTTAACTGGCCGCCGG GI_341 A---AGAGGCCGAAGGATTACCCGGGTTTAGGAAACCT-GTTGCTTCGGCGGGCCCGCCTTAACTGGCCGCC Penicillium_aurantiogriseum CCTCTGGGTCCAACCTCCCACCCGTGTTTATTTTACCTTGTTGCTTCGGCGGGCCCGCCTTAACTGGCCGCC GI_594 TTAGTACGCGGCAGGAATG?TCGTAAAAGTCGTAACACGGCTGATCCCTATGCGCAGGATGGAAGTAAAAGT Penicillium_expansum CTGGGTCCAACCTCCCA---CCCGTGTTTATTTTACCTTGTTGCTTCGGCGGGCCCGCCTT—AACTGGCCGC GI_829 ------GGGTTTACCCCCCAAAAGAGGT—AAAAAACCTTGTTGCTTCGGCGGGCCCGCCTTAACCGGCCGCC Penicillium_commune CCTCTGGGTCCAACCTCCCACCCGTGTTTATTTTACCTTGTTGCTTCGGCGGGCCCGCCTTAACTGGCCGCC GI_371 GTCATTTGAGTAGACCGCGGATAATA?GCTCCGGATGTTCGTAAAACTCTTGTCACGGCGTATCTTTATTCG Cladosporium_oxysporum ATCATTACAAGTGACCCCGGTCTAAC-CACCGGGATGTTCATAACCCTTTGTTGTCCGACTCTGTTGCCTCC GI_731 AGATTCGTACAAGGCGTATCTGTATTCG--CCGGATGTTCGTAAAACTCTGTT-CACGGCGTATCTTTATCC Cladophialophora_minourae GGATCATTACAAGAACGCCCGGGCTTCGGCCTGGTTATTCATAACCCTTTGTTGTCCGACTCTGTTGCCTCC GI_336 CTCGGGAAAAAAAACAAGTTGCTTCGGCGGCGCGGGGCCCCCTCTCACCGGGGCGCCGCAGCCCTCCGCCCC Lecythophora_hoffmannii -TCGCGAACACGTCCAAGTTGCTTCGGCGGCGCGGGGCCCCTTC---CCGGGGCGCCGCTGCCCTCCGCCCC GI_850 GAGGGAAGTAAAAGTCGTAACTTTTTCCGATCAATAAGCGGAGGATGCACGCACGTCTTTTGGGGGGTTTTT Togninia_minima TTGTGAACATA-----CCTGTTTTCGTTGCTTCGGCAGGTCGGGGGCCAAC-CCCGCCCGCCGCCGGACTCC GI_232 GACAATACCGAATATTAGAAAGTT----TTTCCCCGAAACACGAGAAAAAACTTTCTAACCCTGTGCACTTG Sporobolomyces_sp ATCATTAGTGAATATTAGGACGTTCAATTTAACTTGAAGTCCGAACTCTCACTTTCTAACCCTGTGCATTTG GI_497 AGGGCAATACGGGCGTCAAAGGAATT?CCAGAATCTTAACAATAGTGCTCTTTTTTTCCACAGAACTCTTGC Pichia_guilliermondii AGCGCTTAACTGCGCGGCGAAAAAC------CTTACACACAGTGTCTTTTTGAT—ACAGAACTCTTGC GI_343 GAGGGCTAACCCGTCCAAGGTGAAACGGCGGCGCGGGGGCCCCCCTCACCGGGGCGCCGCAGCCCTGCCCCC Coniochaeta_ligniaria ATCGCGAACTCGTCCAAGTTGCTTCGGCGGCGCGGCA--CCCCTTAACGGGGGCGCCGCAGCCCTGCCTCTC GI_237 CCNCGGGAACTCGTCCAAGTTGCTTCGGGGGGGCGGGGCCCCCTCTCACCGGGGCGCCGCAGCCCTCCGCCC Aureobasidium_pullulans AGCTACTTAAAACCATCGCGAACTTGTCCAAGTTGTTTCGGCGGCGCGGGACCCCTTGGGGGACCGCAGCGC

GI_840 CACCGTGTCACACACTTCTTGTGTTTGACACACACTTTTAACACCTTAGCATAAGAATGTAATAGTCTCTTA Rhodotorula_slooffiae CACCGTG-CACACACTTCTT---TTTTACACACACTTTTAACACCTTAGTATAAGAATGTAATAGTCTCTTA GI_848 TTTCCATACACTGTGCACACACTTCTTTTCACACATTTTAACACTATAGTATAAGAATGTAACAGTCTCTTT Rhodotorula_laryngis TTTCCATACACTGTGCACACACTTCTTTTCACACATTTTAACACTATAGTATAAGAATGTAACAGTCTCTTT GI_831 CGGGGGTCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGTAGTCTGA Penicillium_chrysogenum CGGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGTAGTCTGA GI_228 CGGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGAAGTCTGA Penicillium_griseoroseum CGGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGAAGTCTGA GI_16 GGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGAAGTCTGAG Penicillium_crustosum GGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGAAGTCTGAG GI_854 CGGGGGGNTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGAAGTCTGA Penicillium_farinosum CGGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGAAGTCTGA GI_545 GGGTTTCCCGCCCCCGGGGCGGAAGCCCGCCGAAAGACACCCTCGGACTCTGTCCGAAGATTGTAGTCTGAG Penicillium_dipodomyis GGGGCTCACGCCCCCGGGCCCGC-GCCCGCCGAA-GACACCCTCGAACTCTGTCTGAAGATTGTAGTCTGAG GI_341 GGGTTTGCTCACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTGGGGCTCTGTCTGAAGATTGAAGTCTGA Penicillium_aurantiogriseum GGGGGGGCTCACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGAAGTCTGA GI_594 CGGATCACTTCCCCTCCCGATCCGGAGGACGGGGGAAACACCCTCGACCTCTGTCCGCACATTGCGGTCTGT Penicillium_expansum CGGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGAAGTCTGA GI_829 GGGGGGCACACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGACTGAAGATTGAAGTCTGAG Penicillium_commune GGGGGGCTCACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGAAGTCTGAG GI_371 CGGGCGGCCCGGACAAATCTCCGCACCTCGGATTGGAAACTTCAGACTCTGGGAAAACTTTGC?AGCCTCCC Cladosporium_oxysporum GGGGCGACCCTGCCTTCGGGCGGGGGCTCCGGGTGGACACTTCAAACTCTTGCGTAACTTTGC—AGTCTGAG GI_731 GCGGGGAGCCTGCCTAAATCTGCGCACCCCGGGTCTATACTAGCAGCTCTTGCGTAACTTTGCAGTCTCACC Cladophialophora_minourae GGGGCGACCCTGCCTTCGGGCGGGGGCTCCGGGTGGACACTTCAAACTCTTGCGTAACTTTGCAGTCTGAGT GI_336 CGGGTTCCGCCCGGAGGTGTGGGGCGCCCGCCGGAGGTACGAAACTCTCATGTATTATAGTGGCATCTCTGA Lecythophora_hoffmannii CGGGTCTC--CGGGAGGTGCGGGGCGCCCGCCGGAGGTACGAAACTCTTATGTATCACAGTGGCATCTCTGA GI_850 GCCTCCTTGGGGGGACCGCCCACCATTTGCGCCCCCCTGAGCCTTTTGCAGTCGAACACG-GCGTCGCTGAG Togninia_minima CCCTCGCGGGGCTGCGCCGGCGGGCCTGCCGGAGGGCACAGAC—TCTGTATTCAAAAACGTACCTCTCTGAG GI_232 GTTGGTCATAGAACCTCTCGCAAGAGAGAGAACTCCTATACACTTACAAACACAAAGGGGGGGGGTGGG?AT Sporobolomyces_sp TTTTGGCTAGTAGGATCTTGTATCTGA----ACGCCTCTTCATTTACAAACACAAAGTCTATGAATGT---T GI_497 TTTGGTTTGGCCTTTACCAAGGTTGGGCCAAAGGTTTAACAAAACACAATTTATGTATTTTTACAGTTAGTC Pichia_guilliermondii TTTGGTTTGGCCTAGAGATAGGTTGGGCCAGAGGTTTAACAAAACACAATTTAATTATTTTTACAGTTAGTC GI_343 CCGGGTTC?GAAAGGAGGTGTGGGGCGCCCGCCGGAGGTACGAAACTCTCATGTATTATAGGGGCATCTCTG Coniochaeta_ligniaria CGG------AGGTTCGGGGCGCCCGCCGGCAGGTACGAAACTCT---GTATTATAGTGGCATCTCTG GI_237 CCGGGTTCCGCCCGGAGGTGTGGGGCGCCCGCCGGAGGTACGAAACTCTCATGTATTATAGTGGCATCTCTG Aureobasidium_pullulans CGCTTCCCCCCCCGGGGGTTGCGGGGCGCCCGCCGGAGGTCACAAACTCTCATGTATTATAGTGGATCTCTG

GI_840 ATTGAGCATAAATAAAAACAAAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGA Rhodotorula_slooffiae ATTGAGCATAAATAAAAACAAAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGA GI_848 ATTGAGCATAAATAAAAATAAAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGA Rhodotorula_laryngis ATTGAGCATAAATAAAAATAAAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGA GI_831 GTGAAAATATAAATTACTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA Penicillium_chrysogenum GTGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA GI_228 GTGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA Penicillium_griseoroseum GTGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA GI_16 TGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAAGAACGCAGCGA Penicillium_crustosum TGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAA-GAACGCAGCGA GI_854 GTGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA Penicillium_farinosum GTGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA GI_545 GGAAAATATTAACCCCCTAAAACTTTCCCACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA Penicillium_dipodomyis TGAAAATATAAATTATTTAAAACTTTCA-ACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA GI_341 GTGAAAATTTAAATCATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA Penicillium_aurantiogriseum GTGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA GI_594 GTGAAAATATAAATTATTTAAAACTTTCACCAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA Penicillium_expansum GTGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA GI_829 TGAAAATATACCACTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA Penicillium_commune TGAAAATATAA-ATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA GI_371 CACACTTAATTAATAAATTAAAACTTTTAACACCGGATCTCTTGGTTCTGGCATCGATGAAGAACGCACCGA Cladosporium_oxysporum TAAACTTAATTAATAAATTAAAACTTTTAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGA GI_731 ACACTTAATTAATAAATTAGAGCTTTTAAACAACGGAACTCTTGGTTCTGGCATCGATGAAGAACGCAGCGA Cladophialophora_minourae AAACTTAATTAATAAATTAAAACTTTTAA-CAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGA GI_336 GTACAAAACAAATAAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAAGAACGCAGCGA Lecythophora_hoffmannii GTGAAAAACAAATAAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAA-GAACGCAGCGA GI_850 T------ACAAATTTGTTACAACTTTCA-CAACGGATCTCTTGGTTCTGGCATCGATGATGATTGCAGCGA Togninia_minima TTATCTTTACAAATAAGTAAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGA GI_ 232 TAAATTTTATAACAAAACGAAAACTTTCAACAACGGATCTCTTGGCTCTCGCATCGATGAAGACCGCAGCGA Sporobolomyces_sp TAAATTTTATAACAAAAC-AAAACTTTCAACAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGA GI_497 AAATTTTGAATTAATCTTCAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGA Pichia_guilliermondii AAATTTTGAATTAATCTTCAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGA GI_343 AGTATAAAACAAATAAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGA Coniochaeta_ligniaria AGTATAAAACAAATAAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGA GI_237 AGTACAAAACAAATAAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGA Aureobasidium_pullulans AGTAAAAAACAAATAAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGA

GI_840 ATTGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCACTCTTTGG Rhodotorula_slooffiae ATTGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCACTCTTTGG GI_848 ATTGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCACTCTTTGG Rhodotorula_laryngis ATTGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCACTCTTTGG GI_831 AATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGT Penicillium_chrysogenum AATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGT GI_228 AATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGT Penicillium_griseoroseum AATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGT GI_16 AATGCGATACGTAATGTGAATTGCAAATTCAGTTTTATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGG Penicillium_crustosum AATGCGATACGTAATGTGAATTGCAAATTCAGTGA-ATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGG GI_854 AATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGTT Penicillium_farinosum AATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGT GI_ 545 AATGCGATACGTAATGGGAATTGCAAATTCAGTGANTTATCGAGTCTTTGAACGCACATTGCGCCCCCTGGT Penicillium_dipodomyis AATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGT GI_341 AATGCGATACGTAATGTGAATTGCAAATTCAGTGTTTCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGT Penicillium_aurantiogriseum AATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGT GI_594 AATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGT Penicillium_expansum AATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGT GI_829 AATGCGCATACGACAACGCGAATTGCTTTTTTTTTTGAATCACCGAGTCTTTGCAACGCCACATGGCGCCCC Penicillium_commune AATGCG-ATACG-TAATGTGAATTGCTTTTTCAGT-GAATCATCGAGTCTTTG-AACGCACATTGCGCCCCC GI_371 AATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGCAATCTTTTTTTTCACATTGCGCCCCCTG Cladosporium_oxysporum AATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCG-AATCTTTGAACGCACATTGCGCCCCCTG GI_731 AATGCGATAAGTAATGTGAATTGCAAAATTCAGTGAATCATCGAATCTTTTTTTCGCACATTGCGCCCCCTG Cladophialophora_minourae AATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAA-CGCACATTGCGCCCCCTG GI_ 336 AATGCGATAAGTTTTTTTTAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGGCA Lecythophora_hoffmannii AATGCGATAAGTAATGTG-AATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGGCA GI_850 ACCCCCATAAGGAGGGTGTTTTTTTTT-TTCAGTGGATCATCGGAATCTTTTTTCGCACATTGCGCCGGGGG Togninia_minima AATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCG-AATCTTTGAACGCACATTGCGCCCGCTA GI_232 AATGCGACAAGAGAATGTGGAATGCAGAATTCAGGTGAATCATCGGAATCTTTTTTTTTCATCTGCGCTCCA Sporobolomyces_sp AATGTGACAAGT-AATGTGAATTGCAGAATTCAG-TGAATCATCG-AATCTTTGAACGCATCTTGCACTCCT GI_497 AATGCGATAAGTAATATGAATTGCAGATTTTCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGG Pichia_guilliermondii AATGCGATAAGTAATATGAATTGCAGATTTTCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGG GI_343 AATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGGAAG Coniochaeta_ligniaria AATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGGTAG GI_237 AATGCGATAAGTAATTTGTTTTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGGTAG Aureobasidium_pullulans AATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCTAG

GI_840 TATTCCGAAGAGTATGTCTGTTTGAGTGTCATGAAACTCTCAACCCCCCTATTATTGTAATGAAATGGGCGT Rhodotorula_slooffiae TATTCCGAAGAGTATGTCTGTTTGAGTGTCATGAAACTCTCAACCCCCCTATT-TTGTAATGAGATGGGCGT GI_848 TATTCCGAAGAGTATGTCTGTTTGAGTGTCATGAAACTCTCAACCCCCCTATTTTGTAATGAGATGGGCGTG Rhodotorula_laryngis TATTCCGAAGAGTATGTCTGTTTGAGTGTCATGAAACTCTCAACCCCCCTATTTTGTAATGAGATGGGCGTG GI_831 ATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCACGGCTTGTGTGTTGGGCCCCGTCCTC Penicillium_chrysogenum ATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCACGGCTTGTGTGTTGGGCCCCGTCCTC GI_228 ATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCCGTCCCC Penicillium_griseoroseum ATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCCGTCCCC GI_16 TATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCCGTCCC Penicillium_crustosum TATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCCGTCCC GI_854 ATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCCGTCCCC Penicillium_farinosum ATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCCGTCCCC GI_545 ATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCGAGGACGGCTTGTGGGTTGGGCCCCGTCCTC Penicillium_dipodomyis ATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCACGGCTTGTGTGTTGGGCCCCGTCCTC GI_341 ATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTGGGGCCCGGCTTGTGTGTTGGGCCCCGTCCTC Penicillium_aurantiogriseum ATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCCGTCCTC GI_594 ATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCCGTCCCC Penicillium_expansum ATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCCGTCCCC GI_829 CGGAAAATCCCGGGGGGACCCTGCCGGACCCGAGCCGGGGGGGGGAGGGGCCCCCCCCCCCCCGGGCTGGGG Penicillium_commune TGGTA-TTCCGGGGGGCA--TGCCTG-TCCGAGC------GTCATTGCTGCCCTCAAGCCCGGCTTGTGT GI_371 GTATTCCGGGGGGCATGCCTGTTCCAAGCGTCATTTCCCCNCTCAAAGCCTCGCTTGGGGAGTGGGCACCCC Cladosporium_oxysporum GTATTCCGGGGGGCATGCCTGTTC-GAGCGTCATTTCACCACTCAA-GCCTCGCTTGGT-ATTGGGCAACGC GI_731 GTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCACTCAAGCCTCGCTCCCCATTGGGCAACGCGGT Cladophialophora_minourae GTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCACTCAAGCCTCGCTTGGTATTGGGCAACGCGGT GI_336 GTACTCTGCCGGGCATGCCTGCCCCCCCCCTCATTTCAACCCTCAAGCCCTGCTTGGAGTTGGGGTCCCACG Lecythophora_hoffmannii GTACTCTGCCGGGCATGCCTGTTCGAGCG-TCATTTCAACCCTCAAGCCCTGCTTGGTGTTGGGGCCCTACG GI_850 GGTATTCCCGAGGGCATGCCTGGTCGAGCGGTCTTTTTTTACCCCTCAGGGCCTGGT-GTTGGGGGTTGGGG Togninia_minima G-TATTCTGGCGGGCATGCCTGTCCGAGCG---TCATTTCAACCCTCAGGCCCTGGTTGCCTGGTGTTGGGG GI_232 GGGTAATCCGGGGGGTATGATGCCCAGTTTGAGTCGTCCTGAATTACTTAAAAGGGGGGGGGGGGGG?CCCC Sporobolomyces_sp TGGTATTCCGAGGAGTATG----TCTGTTTGAGT-GTCATGAATTCTTCAA------CCC GI_497 TATTCCAGAGGGCATGCCTGTTTGAGCGTCATTTCTCTCTCAAACCCCCGGGTTTGGTATTGAGTGATACTC Pichia_guilliermondii TATTCCAGAGGGCATGCCTGTTTGAGCGTCATTTCTCTCTCAAACCCCCGGGTTTGGTATTGAGTGATACTC GI_343 AACTCTGCCGGGCATGCCTGTTCGAGCGGCGGTTCAACCCTCAAGACCTGCTTGGTGGTGGGGCCCCACGGC Coniochaeta_ligniaria TACTCTACCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCCTGCTTGGTGTTGGGGCCCTACGGC GI_237 TTACTCTGCCGGGCATGCCTGTTCGAGCGCCATTTCAACCCTCAAGCCCTGCTTGGTGTTGGGGTCCCACGG Aureobasidium_pullulans TACTCTAGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCCTGCTTGGTGTTGGGGCCCTACGGC

GI_840 GGGCTTGGAGGAGGGTTGCCTGTTGGCGAAATTGCCGGCTCAACTGAAATACACGAGCAACCCTACTGAAAT Rhodotorula_slooffiae GGGCTTGGATTATGGTTGTCTGTTGGCGTAATTGCCGGCTCAACTGAAATACACGAGCAACCCTACTGAAAT GI_848 GGCTTGGATTATGACTGCTGTCGGCGTAATTGCCGGCTCAGTTGAAATACACGAGCAACCCATTTGAAATAA Rhodotorula_laryngis GGCTTGGATTATGACTGCTGTCGGCGTAATTGCCGGCTCAGTTGAAATACACGAGCAACCCATTTGAAATAA GI_831 CGATCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTCA Penicillium_chrysogenum CGATCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTCA GI_228 CGATCTCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTC Penicillium_griseoroseum CGATCTCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTC GI_16 CCGATCTCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGT Penicillium_crustosum CCGATCTCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGT GI_854 CGATCTCCGGGGGGGACGGGCCCGGAAAGGCAGCGGGTGGGGCACCGCGTCCGGGTCCTCGAACGGTTATGG Penicillium_farinosum CGATCTCCGGGGG--ACGGGCCCG-AAAGGCAGCGGC---GGCACCGCGTCCGG-TCCTCGAGCG—TATGGG GI_545 CGATCCCGGGGGACGGGCCCGAAAGGGAGCGGCGGCACCGCGTCCGGTGCCTCGAGCGGATGGGGCTTTGGC Penicillium_dipodomyis CGATCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGT-CCTCGAGCGTATGGGGCTTTGTC GI_341 CGATTTCCGGGGGACGGGCCCGAAAGGCAGCGGGGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTC Penicillium_aurantiogriseum CGATCTCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTC GI_594 CGATCTCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTC Penicillium_expansum CGATCTCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTC GI_829 GGATGGGCCCCGTCCTACCGAACCCCCGGGGGGAACGGGGCCCCGAAAAGGAAGGGGACGCCACCGCCGCCC Penicillium_commune GTTGGGCCCCGTCCT-CCGATCTCC---GGGGGACGGGCCC---GAAAGGCAGCGG-CGGCACCGCGTCCGG GI_ 371 CCCCCC?ACCGCGAGCCTCAAATCGACCGGCTGGT?TCTTCTGTCCCCCTAACCGTTGATGGAAACCTATTC Cladosporium_oxysporum GGTCC—-GCCGCGTGCCTCAAATCGACCGGCTGG-GTCTTCTGTCCCC-TAAGCGTTG--TGGAAACTATTC GI_731 CCGCCGCGTGCCTCAAATCGTCCGGCTGGGTCTTCTGTCCCCTAAGCGTTGTGGAAACTATTCGCTAAAGGG Cladophialophora_minourae CCGCCGCGTGCCTCAAATCGTCCGGCTGGGTCTTCTGTCCCCTAAGCGTTGTGGAAACTATTCGCTAAAGGG GI_336 GCTGCCGATGGGCCCTGAAAGGAAGTGGCGGGCTCGCTACAACCTCCGAGCGGAAGTAATTCATTAATCTCG Lecythophora_hoffmannii GCTGCCG-TAGGCCCTGAAAGGAAGTGGCGGGCTCGCTGCAAC-TCCGAGCGTA-GTAACTCATTA-TCTCG GI_850 CGCTTTGGTCT-TCACGGGGCTCGGGCTCGGAGAATCAGGGTGGGAGTCGGGCGGAGGGGGGGTTTGGAGGT Togninia_minima CGCCGCGTCCCCTCAGCGGGCGCGGGCCCCGAAAGTCAGTGGCGGGCTCGCCAGGACTCCGAGCGCAGTAGT GI_232 CCCCCCTTAATAGTAAGTTGAGGGGCGCCTGGAGCGCAAGTGAGGGTAACCTAGG?GGG?GAGGGGTCGAAC Sporobolomyces_sp TCCCTTTTCTTAGTGAATCGAGAGGTGTTTGGATTCTGAGTGTTGCT—CCTAAAT------CGAGCTCATTC GI_497 TTAGTCGGACTAGGCGTTTGCTTGAAAAGGATTGGCATGGGTAGTACTGGATAGTGCTGTCGACCTCTCAAT Pichia_guilliermondii TTAGTCGGACTAGGCGTTTGCTTGAAAAGTATTGGCATGGGTAGTACTGGATAGTGCTGTCGACCTCTCAAT GI_343 TGCCGGGGGCCCTGAAAGGAAGTGGCGGGCTCGCTACAACTCCGAGCGGAGTAATTCATTATCTCGCTAGGG Coniochaeta_ligniaria TGCCGTAGGCCCTGAAAGGAAGTGGCGGGCTCGCTACAACTCCGAGCGTAGTAATTCATTATCTCGCTAGGG GI_237 CTGCCGTGGGCCCTGAAAGGAAGTGGCGGGCTCGCTACAACTCCGAGCGTAGTAATTCATTATCTCGCTAGG Aureobasidium_pullulans TGCCGTAGGCCCTGAAAGGAAGTGGCGGGCTCGCTACAACTCCGAGCGTAGTAATTCATTATCTCGCTTAGG

GI_840 AAACGGTTTGACTTGGCGAAATAATTATTTCGCTAAGGACGTTTTCTTCAATTATAAGAGGGGCTTCTAATT Rhodotorula_slooffiae AAACGGTTTGACTTGGCGTAATAATTATTTCGCTAAGGACGTTTTCTTCAATTATAAGAGGTGCTTCTAATT GI_848 ACGGCTTGACTCGGCGTAATAATTATTTCGCTGAGGACGTTTTCTTCAAAAGTTAGGAGGTGCTTCTAATGC Rhodotorula_laryngis ACGGCTTGACTCGGCGTAATAATTATTTCGCTGAGGACGTTTTCTTCAAAAGTTAG-AGGTGCTTCTAATGC GI_831 CCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGATCAGGTA Penicillium_chrysogenum CCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGATCAGGTA GI_228 ACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGATCAGGT Penicillium_griseoroseum ACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGATCAGGT GI_16 CACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGAATCAG Penicillium_crustosum CACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGA-TCAG GI_854 GGGCCTTTGTCACCCGGCTCTTGGTAGGCCCCGGGGCGGCGCCTTGCCGGATCAAACCCAAATTTGTTATCC Penicillium_farinosum G--CTTTGTCACCCG---CTCGGTAGGCCC---GGCCGGCGCTTGCCG-ATCAA-CCCAAATTT-TTATCCA GI_545 ACCCGCTCTGTAGGCCCGGCCGGGGCTTGCCGATCAACCCAAATTTTTATCCAGGGGTGACCTCGGATCAGG Penicillium_dipodomyis ACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGT-TGACCTCGGATCAGG GI_341 ACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGGATCAACCCAAATTTTTATCCAGGGTTGACCTCGGGATCA Penicillium_aurantiogriseum ACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCG-ATCAACCCAAATTTTTATCCAGG-TTGACCTCGG-ATCA GI_594 ACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGATCAGGT Penicillium_expansum ACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGATCAGGT GI_829 CGGG?CCCC?GAGCG?AA?GGGGG?CCT??G???CACCCCGC?TC??G??AAG?CCC?CGGGA?AGG?CGC? Penicillium_commune TCCTCGAGCGTATGGGG—CTTTGTCACCCGCTCTCCC??????????????????????????????????? GI_371 GCCTAAAAGGGTGTTTCGCACAGGCTACCCCCGT?AAAACAACCCCCATTTTCTAAAGGTTGACCCCCCGGA Cladosporium_oxysporum GCTAAAGGGTGTTCGGGA—-GGCTACGCCGT--AAAACAACCCCATTTCT---AAGGTTGACCTC--GGATC GI_731 TGTTCGGGAGGCTACGCCGTAAAACAACCCCATTTCTAAGGTTGACCTCGGAATCAGGTAGGGATACCCGCT Cladophialophora_minourae TGTTCGGGAGGCTACGCCGTAAAACAACCCCATTTCTAAGGTTGACCTCGGA-TCAGGTAGGGATACCCGCT GI_336 CTAGGGAGGTTGCGGCCGCGCTCCCCGCCGTTAAAGACCACATCTTTTAAACAAAGGT?GGACCTCGG?A?? Lecythophora_hoffmannii CTAGGGACGTTGCGGC-GCGCTCCT-GCCGTTAAAGAACCCATCTTT—AACCAAGGT??????????????? GI_850 GTGGGCACGGGAGCGGAGGGCCCGGCCGATGGGAATGGCGGGGCGGGCAGGGAGGCCGGGACTTTTAGGATC Togninia_minima TTACACCTCGCTGCGGAGGACCTGGC-----GGGTTACCCAGCTCGTAAAACACACCCAAACTTCTAAGGTT GI_232 CGGGTAATGCAATCAGCAATCCCCATAACGAACATACG??AATGGDACTGGGCAGGAAATCAGGAGATATCT Sporobolomyces_sp GGAGTGTTGCT--CCTAAAT------CGAGCTCATTCG--TAATGCA-TTAGCA-TCCATAT-TCGAATTT- GI_497 GTATTAGGTTTATCCAACTCGTTGAATGGTGTGGCGGGATATTTCTGGTATTGTTGGCCCGGCCTTACAACA Pichia_guilliermondii GTATTAGGTTTATCCAACTCGTTGAATGGTGTGGCGGGATATTTCTGGTATTGTTGGCCCGGCCTTACAACA GI_343 AGGGTGCGGCGGGCTCCTGGCGGTAAAGACCACATCTTTAACCAAGGGTGAACTCGGAACAGGGAGGAATAC Coniochaeta_ligniaria AGGTTGCGGCGTGCTCCTGCCGTTAAAGACC-CATCTTTAACCAAGGTTGACCTCGGATCAGGTAGGAATAC GI_237 GAGGTTGCGGCGTGCTCCTGGCCGTTTAAAAAACNACATCTTTAACCAAGTTTGAACCTCGAATCAGGTAAG Aureobasidium_pullulans GTGTTGTGCGGTGCTCAGCCCGTTAAAGACCATCTTTAACTCAAGGTTGAC?????????????????????

GI_840 CGCTTCTAATAGCATTTAAGCTTTAAACCTCAAATCAGTCAGGACTACCCGCTGAACTTAAGCTTCACAAAN Rhodotorula_slooffiae CGCTTCTAATAGCATTTAAGCTTTAGACCTCAAATCAGTCAGGACTACCCGCTGAACTTAAGCATATCAATA GI_848 GCTATTACAGCACTTTAAATTTTAGACCTCAAATCAGTCAGGACTACCCGCTGAACTTAAGCATATCATAAA Rhodotorula_laryngis GCTATTACAGCACTTTAAATTTTAGACCTCAAATCAGTCAGGACTACCCGCTGAACTTAAGCATATCAATAA GI_831 GGGGATACCCGCTGAACTTAAGCATATCATAAAGCGGGAGGAAGGGTGGTGGGGGGGGGGGGCGGGGGG?GG Penicillium_chrysogenum GGG-ATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCCCC??????? GI_228 AGGGATACCCGCTGAACTTAAGCATATAAAATGAGGGGAGGGAATC?????????????????????????? Penicillium_griseoroseum AGGGATACCCGCTGAACTTAAGCATATCAA-TAAGCGGAGGAAATC?????????????????????????? GI_16 GTAGGGGATACCCGCTGAAACTTAACCATATCATTAAGCGGAGGAA????????????????????????A? Penicillium_crustosum GTAGGG-ATACCCGCTGAA-CTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCCCC??? GI_854 GAGGGTTGGACCTCGGGATCAGGGGAGGGGATTACCCGGCTGGAACTTAAG?CAT?ATCGATTAGGCGGGA? Penicillium_farinosum GG---TTGACCTCGG-ATCAGGTAGGGA---TACCCG-CTGAACTTAAGCATATCAATAAGCGGAGGAAAAG GI_545 GTGGGGATACCCGCTGAACTTAAGCATATCAATAAGGGGGGGGG?G???G?G?G?GG?G?G?G?G??GG?GG Penicillium_dipodomyis -TAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA????????????????????????????? GI_341 GGTGGGGGATACCCGCTGGAACTTGAGCATATCATTGGGGGCGG?GGGA?GGTG?GGGGGGG?GGG?GGGGG Penicillium_aurantiogriseum GGTAGGG-ATACCCGCTGAA???????????????????????????????????????????????????? GI_594 AGGGAATACCCGCTGAACTTAACCATATCATTAAGCGGGAGGAATC?????????????????????????? Penicillium_expansum AGGGA-TACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAATC?????????????????????????? GI_829 CGAGGCCC??AACCAACCCC?AACA?A?CT?AACCC??GGCTGGA??CC?CGG?GACC?GGGCAA?GGGA?A Penicillium_commune ???????????????????????????????????????????????????????????????????????? GI_371 TCCAGCTCACGCATACCCCCCTCCACCTTAAA?C?TATCCAAAT?ACCC??A?AAA???C?C??CCC??CC? Cladosporium_oxysporum TCAGGT--AGGGATACCCGCTG—AACTTAA?????????????????????????????????????????? GI_731 GAACTTAAGCATATCAATAAA?C??A??AAA????????????????????????????????????????? Cladophialophora_minourae GAACTTAAGCATATCAATAAC??????????????????????????????????????????????????? GI_336 CA?GGCAGGGAATACCCGCCTGGAACTTTAAGC?ATAC?CA?ATAA?CCGGAGGGAAACTCGCCT?CAC?G? Lecythophora_hoffmannii ???????????????????????????????????????????????????????????????????????? GI_850 GAT?GGG?CCTCGGGGGCAGGTGGGGGATACCCGGGGTGAGCTTGAGGGTATTAGGGTAGGGTT?GGGGAGA Togninia_minima GA------CCTCGGAT-CAGGTAGGA-ATACCCGC--TGAACTTAAGCATATCA--ATAGGG----GGAAGA GI_232 ?GAGGGAGGGATTGCCGAGGCAGAACGGAA?AAAAGGACGGAAGCCGAAACAGGAAGAGAGAGCAATCTGAA Sporobolomyces_sp CGGATTGACTTGGCGTAATAGACT----ATTCGCTGAGGAATCTAACTTCGGTTAGAGCCGGATTTGAAC-- GI_497 ACCAAACAAGTTTGACCTCAAATCAGGTAGGAATACCCGCTGAACTTAAGCATATCATTAAGG??GGAGG?A Pichia_guilliermondii ACCAAACAAGCTTGACCTCAAATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGAGG???? GI_343 CCGCTGAACTTAAGCATACA???AA???GGGGGAAA???????????????????????????????G???? Coniochaeta_ligniaria CCGCTGAACTTAAGCATATCAATAAGCGGAGGA??????????????????????????????????????? GI_237 GAATACCCGCTGAACTTTAAGCATAATCAATNAAGCNGAAGGAACTCNACTTCATCNGTNCCTNTTATNNTC Aureobasidium_pullulans ????????????????????????????????????????????????????????????????????????

GI_840 ???GGGGGGGAAA??????????????????????????????????????????????????????????? Rhodotorula_slooffiae AGCGGAGGAAAAGAAACTAACAAGGATTCCCCTAGTAACGGCGAGTGAAGTGGGAAAAGCTCAACTTTGAAA GI_848 GGGGGAGGGAAG?G?GGG?G???GGGGGGGGG?G?GGG?GGG?GGG?GGG?GGG?GGG?GGG?GGGGGGGGG Rhodotorula_laryngis GCGGAGGA???????????????????????????????????????????????????????????????? GI_831 GGGGGGGGGGGAGGGGGGGGGGGGGTT?GGGGGG?GGGGG?GGGGGGGAG?GGGGGGGGGGG?GGG?T?GGG Penicillium_chrysogenum ???????????????????????????????????????????????????????????????????????? GI_228 ???????????????????????????????????????????????????????????????G?G?????? Penicillium_griseoroseum ???????????????????????????????????????????????????????????????????????? GI_16 ????????????????????????????????????????????????A??????????????????????T Penicillium_crustosum ???????????????????????????????????????????????????????????????????????? GI_854 GGAA-----AGGGGGTCCGGAGGGGCATGGGGTGGGGGGGGAAGGTGGGAAAGTGGAGAGGGGGGTTGTTCG Penicillium_farinosum AAACCAACAGGGATTGCCCCAGTAACGGCGAGTGAAGCGGCAAGAGCTCAAATTTGAAAGCTGGCTCCTTCG GI_545 GGG????G??G????G?GGGGGGG??G?G??GGG?G?G?GG?G?G???G?G????GG??GGGG??GGGGGGG Penicillium_dipodomyis ???????????????????????????????????????????????????????????????????????? GI_341 GGGGGGGGGGGGGGGGGTGGGGGGGTGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGTGGGGGGGGGGGGGGG Penicillium_aurantiogriseum ???????????????????????????????????????????????????????????????????????? GI_594 ????????????????????????????????????????????????C??????????????????????? Penicillium_expansum ???????????????????????????????????????????????????????????????????????? GI_829 A??CC?GGGCGGA??CCT?A??GCAAAA?CCAG?AA??GGGGAGGCAA?G?A?GCCACCC?C?AAGG?AAA? Penicillium_commune ???????????????????????????????????????????????????????????????????????? GI_371 CC??C??CAAC?C?C??AC?T??C?C?CAACCAAT?AC?CC?????CCC????ACCC?C????T?C?C??C? Cladosporium_oxysporum ???????????????????????????????????????????????????????????????????????? GI_731 ???????????????????????????????????????????????????????????????????????? Cladophialophora_minourae ???????????????????????????????????????????????????????????????????????? GI_336 ?CA?A?ACAC?CA?AACCCC?A?A??T?ATACTCACGACTG?ATT?A?TC?TCACT?G?ACTC??C?CT??C Lecythophora_hoffmannii ???????????????????????????????????????????????????????????????????????? GI_850 ATGGGCTGGGGGGTGGGGGTGGGTGGGACGGGG?T?GGGGGGGGTGTGGGGGTGGGGGGGG?GGGGGGGGGG Togninia_minima ATCCGCTTATTGATATGCCTTGTTACGAC??????????????????????????????????????????? GI_232 TACGAGAACTGGTCATATCGTGTAGGAGAACCCAAGTTT????AGCACCCCAGGAAAGCGTAAGAACCCGGT Sporobolomyces_sp TA-GGAAGCTTATAATCTAGCTTAGTCTACTTTAAGTTT----AGACCTCAAATCAGATAGGATTACCCGCT GI_497 A?????????????????????????????????G?????????????????????????????G???G??? Pichia_guilliermondii ???????????????????????????????????????????????????????????????????????? GI_343 ???????????????????????????????????????????????????????????????????????? Coniochaeta_ligniaria ???????????????????????????????????????????????????????????????????????? GI_237 TTNCTNAAACTATNAANATCATNAANNTCTNNNATNACTNCTACTNCANNCAATATNTNTTTCCNTATNATT Aureobasidium_pullulans ????????????????????????????????????????????????????????????????????????