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 ii 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. iv 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 v 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 1 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