- Email: [email protected]
Revival and characterization of fungi from ancient polar ice
..
Volume 13, Part 2, May 1999
Revival and characterization of fungi from ancient polar ice l
LIJUN MA, CATHARINE M. CATRANIS, WILLIAM T. STARMER , AND SCOTT O. ROGERS 2 Environmental and Forest Biology, State University of New York, College of Environmental Science and Forestry, Syracuse, NY 13210 USA. Tel./Fax: 315-470-6935/315-470-6934 'Department of Biology, Syracuse University, Syracuse, NY 13244 'Author for correspondence
Glacial ice provides a unique global source of micro-organisms that allows study of both contemporary and ancient fungal diversity. The aim of this research was to revive and/or characterize fungi and fungal DNA entrapped in ancient ice cores from Greenland. Two main methods were developed to detect these micro-organisms in glacial ice: (1) Culturing to obtain viable isolates which were characterized by microscopy, PCR (polymerase chain reaction) amplification and DNA sequencing, using fungal ribosomal DNA internal transcribed spacer primers; and (2) PCR amplification and DNA sequencing directly from glacial ice. Hundreds of viable fungi (as well as bacteria) were isolated, and DNA sequences were obtained from Greenland ice cores up to 140,000 years old. Some sequences derived directly from ice melt resembled those of contemporary species, while others exhibited little similarity to wellcharacterized present-day fungi. Species diversity varied among the ice cores. Some fungi remain viable for over 100,000 years in ice. This leads to the possibility that a species can appear to become extinct, but may be capable of reappearing at a later point in time. Keywords: ancient DNA, Arctic, Antarctic, ice cores, fungi, fungus Ice in the environment, whether from glaciers, polar regions, or elsewhere, is a natural airsampling medium that can entrap microorganisms. These micro-organisms may have originated in the vicinity of the deposited ice, or they may have travelled from other parts of the earth or beyond. Bacteria and fungi have been reported to remain viable for at least thousands of years (Abyzov, 1993; Catranis & Starmer, 1991; Ma et al., 1997; Shi et al., 1997; Vishniac, 1996). A surprising number and variety of microorganisms can be recovered from the interior of deep-core samples of Arctic and Antarctic ice, perhaps dating back three million years (Taylor et al., 1997). We have detected filamentous fungi (Catranis & Starmer, 1991; Ma et al., 1997; Fig 1) and other organisms (yeasts, bacteria, and algae; Table 1) in samples of this ancient ice. Ice cores were examined and selected from the National Ice Core Laboratory and transported to a sterile hood kept in a -20 "C room in our laboratory(Fig 2). Ice cores having any signs of
fractures were eliminated. Sterilized black paper and aluminium foil were used to cover the middle portion of the cores, followed by exposure to a UV source. The centre portion of the ice cores then was removed using a sterile drill and saw. All extractions were done aseptically in a laminar flow hood located in a -20°C cold room. The inner and outer cores were then melted (aseptically at 22 °C) and the ice melts (filtered and unfiltered) were examined microscopically and were used to inoculate eight types of agar media plates, incubated at three different temperatures, to isolate fungi. DNA was extracted (using a variation of the Rogers & Bendich, 1985 method), followed by PCR amplification and DNA sequencing (according to the methods in Liu et al., 1997). The ice melts also were used in PCR amplifications (without prior DNA extraction) followed by DNA sequencing and comparisons to the sequences from the cultures and to extant micro-organisms. Glacial ice apparently is an excellent matrix for long-term preservation of micro-organisms, having trapped wind-transported microorganisms through geological time. These
Volume 13, Part 2, May 1999 organisms (representing taxa that Dermocybe crocea Cortinarius limo nius are probably endemic to the polar 100 Peride rmium harknessii Cronartium ribicola regions, as well as exotics from Pisolithu s tinctorius temperate and tropical regions) Acremonium alternatum Phaeococcomyces exophialae may have originated from ocean Hortaea werneckii mist, wind -borne pollen and soil CU 180 (Ulocladium sp.) [400 ybp) particles, infected plant surfaces, 100 CU 356 (Cladosporium sp.) [800 yhp] 1M 1571-1 [9,000 ybp) and many other sources. They 1M 939-1 [2,500 ybp) may have been transported and 1M 49-2 [100 ybp) 1M 135-1 [200 ybp) deposited by the action of waves, 1M 60-1 [100 ybp) wind, rain, snow, animals, or by 100 Capronia acutiseta other means. Thus, glacial ice Exophia la salmo nis Exophia la jeanselmei provides a unique global source of ' - - -- 1M 1945-1 [140,000) S9 micro-organisms that presents a 1M 724-1 [2,000 ybp) view of both contemporary and Neuro spora crassa Epichloe clarkii ancient microbial diversity. Eurotium rubrum There is an inherent interest in CU 141 (Penicillium sp. 2) [200 ybp ] S9 CU 351 (Penicillium sp, 5) [800 ybp) knowing how long microCU 224 (Penicillium sp, 2) [600 ybp) organisms can remain viable S3 CU 536 (Pen icillium sp , 4) [1,500 ybp) (Gest & Mandelstam, 1987). Penicillium crustosum 91 Eupenicil lium javanicum Studies on deep mud cores Talrom yces j7avus provide evidence for survival of Eremascus albu s Botrytis cinerea thermoactinomycete endospores 100 Monilin ia fructig ena in excess of 1,000 years old (Cross & Attwell, 1974). Viable microbes Fig 1 Phylogenetic tree based on sequence data of ribosomal DNA internal transcribed have been isolated from 320 year- spacer regions from ice specimens and cultures, as well as from contemporary taxa. Ice old herbarium specimens samples are designated as 1M (sequenced directly from ice melts) or CD (sequenced from cultured isolates). For CD isolates, preliminary identifications are indicated in (Sneath, 1962); thermophilic parentheses. All other sequences are from our sequence database (Capronia, Exophiala, bacteria have been isolated Hortaea, and Phaeococcomyces species) or GenBank (all others). Bootstrap support valfrom ocean basin sediment ues (>50%) are indicated on branches (500 replications). The tree was generated using a cores estimated to be 5,800 heuristic search on PADP (Swofford, 1993). Approximate ages in years before present (ybp) are indicated for the fungal sequences from ice (ages determined from Reeh et al., years old (Bartholomew & Paik, 1985) 1966); and bacteria have been recovered from Roman archaeological sites expectations assume that the immediate 1,900 years old (Seaward et al., 1976). environment protects the spores from Investigators isolated viable bacteria from ultraviolet (UV) irradiation, oxidation, and ice cores at Vostok Station in Antarctica, some chemical damage common with hydrated of which were deposited more than 10,000 nucleic acids (Lindahi, 1993). years ago (Abyzov, 1993), and from Siberian Glacial ice provides a frigid and stable permafrost dated to 3 million years before environment for micro-organisms and nucleic present (Taylor et al., 1997). Theoretical acids. Spores are protected from UV irradiation, expectations on the longevity of bacterial spores and because they become desiccated, DNA damage is minimized. We have recovered place the limit for recovering viable bacterial spores at about 200,000 years (Gest & hundreds of fungal isolates from Greenland ice Mandelstam, 1987; Sneath, 1962). Empirically cores that are up to 140,000 years old (Catranis & based DNA mutation rates that result in Starmer, 1991). The ribosomal DNA regions of detrimental effects on spore viability have been some of these fungi have been sequenced and compared to contemporary taxa (Fig 1). In used to estimate the half-life of Bacillus subtilis at 7,000 years. Assuming an exponential death addition, sequences have been obtained directly rate, a large population of viable spores would from the same ice melt samples using PCR (polymerase chain reaction) methods. The fungi be detectable after several hundred thousand, and possibly for several million years. These characterized to date represent a broad range of
•
Volume 13, Part 2, May 1999
Table 1. List of selected micro-organisms identified from Greenland ice core sections (dry drilled). Bacteria are underlined, filamentous fungi are italicized, yeasts are in bold lettering, and algae are in plain letters. Core No.
Sampling Depth Im)'
GISP2-B-63
62.300
Outer Core
Inner Core --_._- -~--~~~_
.._--------_ .. _._- --_
._ ._--
Cladosporium sphaerospermum Geotrichum sp. Phoma sp. 1 Exophiala sp. 1 Cryptococcus uniguttulatus Rhodotorula rubra R. rubra Sarcina sp. 1 Sarcina sp. 2 Micrococcus sp. 1 Penicillium sp. 1 R. rubra Candida sp. Cryptococcus sp. Sarcina sp. 1
GISP2-D-131
130.185
GISP2-D-135
134.203
Sarcina sp. 1
GISP2-D-226
225.205
Penicillium sp. 1 Aspergillus sp. 2 R. rubra Chrysophyta sp. 1
GISP2-D-326
325.160
Dye3-1971-49
158.879
Exophiala sp. 1 Penicillium variabile Aspergillus sp. 2 Sarcina sp. 1 Chrysophyta sp. 3
0_-
-
--------------.---
--
--_."
------._ .._-.__..-.------_..--.
Acremonium sp. Aureobasidium pullulans Basipetospora sp. 1 Cladosporium herbarum Phoma sp. 1 dematiaceous sp. 1 dematiaceous sp. 2 R. rubra mycelial yeast Penicillium sp. 1 Alternaria sp. C. herbarum Exophiala sp. 1 Cryptococcus albidus Chloromonas polyptera Sarcina sp. 1 Phoma sp. 1 R. rubra Chrysophyta sp. 1 Acremonium sp. 1 Basipetospora sp. 1 C. herbarum Penicillium sp. 1 Phoma sp. 2 R. rubra Chrysophyta sp. 2
Phoma sp. 1
'Depths correspond to the following approximate ages: 62.300m = 100 years before present (ybp); 130.185m = 300 ybp; 134.203m = 300 ybp; 158.879m = 350 ybp; 225.205m = 550 ybp; 325.160m = 700 ybp (determined using Reeh et al. 1985)
taxa, including ascomycetes, basidomycetes, and hyphomycetes. Some may have originated in the polar regions, while others may represent temperate or tropical taxa, whose spores or cells have been blown in from various locations around the world. The sequence data obtained from cultured isolates, and from direct sequencing of the ice melts, has already provided valuable information about fungal diversity, evolution, population structure through time and gene flow. The research is continuing with more ice cores from Greenland and Antarctica. Species detected in a continuous chronological sequence may be of particular value in future phylogenetic studies,
especially when older ice cores are sampled. In addition, this information can provide clues about global climatic changes, geological changes (volcanism,bolide impacts, etc.), human influences, and disease epidemics. Viable micro-organisms entrapped in ice are destined to be released during polar warming and glacial melts, or after the calving of icebergs into the ocean, perhaps releasing organisms that may not have been active in the biosphere for thousands of years. This could have immediate effects on populations of extant organisms of the same species, as well as influences on other organisms that may be hosts (including humans) or competitors of the recently released micro-organisms.
Volume 13, Part 2, May 1999 Acknowledgments. We thank C. J. K. Wang for providing assistance in the cultural and taxonomic aspects of this study. We also thank H. Fan for technical assistance and J. D. Castello for reviewing the manuscript.
Ice Core From Stor a ge Facility
I
11111/1/ I \ \ \'
~ III
~~~~~ Section Selected
Surface T rea ted with UV Irradiation (middle covered with aluminum foil) /
Inner Cor e Separated Fr om Outer Shell Aseptica lly @ _20°C
--lJ
References Abyzov, S. S. G. (1993) Micro-organisms in the Antarctic Filtered Su bsa mple ice, in Antarctic Microbiology (ed. E. 1. Friedman), pp. .. (ta ken through same processes as Unfi lter ed 265-295. New York: John Wiley & Sons, Inc. with unfiltered sam ples below) Suh sample Bartholomew, J. W. & Paik, G. (1966) Isolation and Inn er Co re Melted identificationofobligatethermophilic sporeformingbacilli @ 22°C (Stored @ .20°C) from ocean basin cores.Journal of Bacteriology92:635-638. Catranis, C. & Starmer, W. T. (1991) Micro-organisms ]BACT ERIA AND FUN GUS ASSAYS entrapped in glacial ice. Antarctic Journal of the VIRUS ASSAYS / \ United States 26: 234-236. Cultures Cross, T. & Atwell, R. W. (1974) Recovery of viable thermo actinomycete endospores from deep mud cores, in Spore Research (eds. A. N. Barker, G. W. 8 Media (with replicates) Gould & J. Wold), pp. 11-20.London: Academic Press. Gest,H. & Mandelstam, J. (1987)Longevity ofmicro-organisms Direct PCR Amplifica tion, Sequ encing in natural environments. Microbiological Sciences4:69-71. Various From Melted Ice Lindahl, T. (1993) Instability and decay of the primary Incuhation T imes and structure of DNA. Nature 362: 709-715. Temp eratures Liu, Y., Ammirati, J. F. & Rogers, S. O. (1997) Phylogenetic Relationships of Dermocybe and Cortinarius species based on nuclear ribosomal DNA Extra ction, Examinations PCR Am plilication, internal transcribed spacers. Canadian Journal of '" Sequencing " Botany. 75: 519-532. Characterize Sequ ence Com pa r isons Ma,1. J., Fan, H., Catranis, C.,Rogers,S. 0., & Starmer, W. T. and/or Identify .. (DNA Database Searches and (1997) Isolation and characterization of fungi entrapped Ph ylogenetic Ana lyses) in glacial ice. Inoculum 48(3):MSA Abstracts, p.23. Reeh, N., Johnsen, S. J. & Dahl-Jensen, D. (1985) Dating Fig 2 Protocol for ice core isolation, culturing, and DNA sequencing. the Dye 3 deep ice core by flow model calculations, in Sneath, P. H. A. (1962) Longevity of micro-organisms. Greenland Ice Core: Geophysics, geochemistry, and the Nature 195: 643-646. Environment, Geophysical Monograph 33 (eds. C. C. Swofford, D. (1993) PAUP: phylogenetic analysis using Langway, Jr., H. Oeschger & W. Dansgaard), pp. 57parsimony, Version 3.1.1, University of Illinois, 65. Washington, D. C.: American Geophysical Union. Champaign, 11. Rogers, S. O. & Bendich, A. J. (1985) Extraction of DNA Taylor, K. C., Mayewski, P. A., Alley, R. B., Brook, E. J., from milligram amounts of fresh, herbarium and Gow, A. J., Grootes, P. M., Meese, D. A., Saltzman, E. mummified plant tissues.Plant MolecularBiology 5:69-76. S., Severinghaus, J. P., Twickler, M. S., White, J. W. Seaward, A. C., Cross, T. & Unsworth, B. A. (1976)Viable C., Whitlow, S., & Zielinski, G. A. (1997) The bacterial spores recovered from an archaeological Holocene-Younger dryas transition recorded at excavation. Nature 261: 407-408. Summit, Greenland. Science 278: 825-827. Shi, T., Reeves, R. H., Gilichinsky, D. A. & Friedman, E. Vishniac, H. S. (1996) Biodiversity of yeasts and filamentous 1. (1997) Characterization of viable bacteria from microfungi in terrestrial Antarctic ecosystems. Siberian permafrost by 16S rDNA sequencing. Biodiversity and Conservation 5:1365-1378. Microbial Ecology 33: 169-179.
~
~ /
+
Micros~ 1~
The BMS Homepage The following are recent additions and amendments to the BMS Homepage (underlined entries are links): • • • • •
Foray Box (References for Identifying Fungi), has been updated to January 1999. Library: Books, has been reformatted and updated to 1994by integrating a supplement. Below Ground Research at York describes their work on roots and mycorrhizas. The Warwickshire Fungus Survey has been added to Fungus Groups. Photographs of fungi: The Quoditch Moor Nature Research has a nice selection. RTM
•
Volume 13, Part 2, May 1999
Revival and characterization of fungi from ancient polar ice l
LIJUN MA, CATHARINE M. CATRANIS, WILLIAM T. STARMER , AND SCOTT O. ROGERS 2 Environmental and Forest Biology, State University of New York, College of Environmental Science and Forestry, Syracuse, NY 13210 USA. Tel./Fax: 315-470-6935/315-470-6934 'Department of Biology, Syracuse University, Syracuse, NY 13244 'Author for correspondence
Glacial ice provides a unique global source of micro-organisms that allows study of both contemporary and ancient fungal diversity. The aim of this research was to revive and/or characterize fungi and fungal DNA entrapped in ancient ice cores from Greenland. Two main methods were developed to detect these micro-organisms in glacial ice: (1) Culturing to obtain viable isolates which were characterized by microscopy, PCR (polymerase chain reaction) amplification and DNA sequencing, using fungal ribosomal DNA internal transcribed spacer primers; and (2) PCR amplification and DNA sequencing directly from glacial ice. Hundreds of viable fungi (as well as bacteria) were isolated, and DNA sequences were obtained from Greenland ice cores up to 140,000 years old. Some sequences derived directly from ice melt resembled those of contemporary species, while others exhibited little similarity to wellcharacterized present-day fungi. Species diversity varied among the ice cores. Some fungi remain viable for over 100,000 years in ice. This leads to the possibility that a species can appear to become extinct, but may be capable of reappearing at a later point in time. Keywords: ancient DNA, Arctic, Antarctic, ice cores, fungi, fungus Ice in the environment, whether from glaciers, polar regions, or elsewhere, is a natural airsampling medium that can entrap microorganisms. These micro-organisms may have originated in the vicinity of the deposited ice, or they may have travelled from other parts of the earth or beyond. Bacteria and fungi have been reported to remain viable for at least thousands of years (Abyzov, 1993; Catranis & Starmer, 1991; Ma et al., 1997; Shi et al., 1997; Vishniac, 1996). A surprising number and variety of microorganisms can be recovered from the interior of deep-core samples of Arctic and Antarctic ice, perhaps dating back three million years (Taylor et al., 1997). We have detected filamentous fungi (Catranis & Starmer, 1991; Ma et al., 1997; Fig 1) and other organisms (yeasts, bacteria, and algae; Table 1) in samples of this ancient ice. Ice cores were examined and selected from the National Ice Core Laboratory and transported to a sterile hood kept in a -20 "C room in our laboratory(Fig 2). Ice cores having any signs of
fractures were eliminated. Sterilized black paper and aluminium foil were used to cover the middle portion of the cores, followed by exposure to a UV source. The centre portion of the ice cores then was removed using a sterile drill and saw. All extractions were done aseptically in a laminar flow hood located in a -20°C cold room. The inner and outer cores were then melted (aseptically at 22 °C) and the ice melts (filtered and unfiltered) were examined microscopically and were used to inoculate eight types of agar media plates, incubated at three different temperatures, to isolate fungi. DNA was extracted (using a variation of the Rogers & Bendich, 1985 method), followed by PCR amplification and DNA sequencing (according to the methods in Liu et al., 1997). The ice melts also were used in PCR amplifications (without prior DNA extraction) followed by DNA sequencing and comparisons to the sequences from the cultures and to extant micro-organisms. Glacial ice apparently is an excellent matrix for long-term preservation of micro-organisms, having trapped wind-transported microorganisms through geological time. These
Volume 13, Part 2, May 1999 organisms (representing taxa that Dermocybe crocea Cortinarius limo nius are probably endemic to the polar 100 Peride rmium harknessii Cronartium ribicola regions, as well as exotics from Pisolithu s tinctorius temperate and tropical regions) Acremonium alternatum Phaeococcomyces exophialae may have originated from ocean Hortaea werneckii mist, wind -borne pollen and soil CU 180 (Ulocladium sp.) [400 ybp) particles, infected plant surfaces, 100 CU 356 (Cladosporium sp.) [800 yhp] 1M 1571-1 [9,000 ybp) and many other sources. They 1M 939-1 [2,500 ybp) may have been transported and 1M 49-2 [100 ybp) 1M 135-1 [200 ybp) deposited by the action of waves, 1M 60-1 [100 ybp) wind, rain, snow, animals, or by 100 Capronia acutiseta other means. Thus, glacial ice Exophia la salmo nis Exophia la jeanselmei provides a unique global source of ' - - -- 1M 1945-1 [140,000) S9 micro-organisms that presents a 1M 724-1 [2,000 ybp) view of both contemporary and Neuro spora crassa Epichloe clarkii ancient microbial diversity. Eurotium rubrum There is an inherent interest in CU 141 (Penicillium sp. 2) [200 ybp ] S9 CU 351 (Penicillium sp, 5) [800 ybp) knowing how long microCU 224 (Penicillium sp, 2) [600 ybp) organisms can remain viable S3 CU 536 (Pen icillium sp , 4) [1,500 ybp) (Gest & Mandelstam, 1987). Penicillium crustosum 91 Eupenicil lium javanicum Studies on deep mud cores Talrom yces j7avus provide evidence for survival of Eremascus albu s Botrytis cinerea thermoactinomycete endospores 100 Monilin ia fructig ena in excess of 1,000 years old (Cross & Attwell, 1974). Viable microbes Fig 1 Phylogenetic tree based on sequence data of ribosomal DNA internal transcribed have been isolated from 320 year- spacer regions from ice specimens and cultures, as well as from contemporary taxa. Ice old herbarium specimens samples are designated as 1M (sequenced directly from ice melts) or CD (sequenced from cultured isolates). For CD isolates, preliminary identifications are indicated in (Sneath, 1962); thermophilic parentheses. All other sequences are from our sequence database (Capronia, Exophiala, bacteria have been isolated Hortaea, and Phaeococcomyces species) or GenBank (all others). Bootstrap support valfrom ocean basin sediment ues (>50%) are indicated on branches (500 replications). The tree was generated using a cores estimated to be 5,800 heuristic search on PADP (Swofford, 1993). Approximate ages in years before present (ybp) are indicated for the fungal sequences from ice (ages determined from Reeh et al., years old (Bartholomew & Paik, 1985) 1966); and bacteria have been recovered from Roman archaeological sites expectations assume that the immediate 1,900 years old (Seaward et al., 1976). environment protects the spores from Investigators isolated viable bacteria from ultraviolet (UV) irradiation, oxidation, and ice cores at Vostok Station in Antarctica, some chemical damage common with hydrated of which were deposited more than 10,000 nucleic acids (Lindahi, 1993). years ago (Abyzov, 1993), and from Siberian Glacial ice provides a frigid and stable permafrost dated to 3 million years before environment for micro-organisms and nucleic present (Taylor et al., 1997). Theoretical acids. Spores are protected from UV irradiation, expectations on the longevity of bacterial spores and because they become desiccated, DNA damage is minimized. We have recovered place the limit for recovering viable bacterial spores at about 200,000 years (Gest & hundreds of fungal isolates from Greenland ice Mandelstam, 1987; Sneath, 1962). Empirically cores that are up to 140,000 years old (Catranis & based DNA mutation rates that result in Starmer, 1991). The ribosomal DNA regions of detrimental effects on spore viability have been some of these fungi have been sequenced and compared to contemporary taxa (Fig 1). In used to estimate the half-life of Bacillus subtilis at 7,000 years. Assuming an exponential death addition, sequences have been obtained directly rate, a large population of viable spores would from the same ice melt samples using PCR (polymerase chain reaction) methods. The fungi be detectable after several hundred thousand, and possibly for several million years. These characterized to date represent a broad range of
•
Volume 13, Part 2, May 1999
Table 1. List of selected micro-organisms identified from Greenland ice core sections (dry drilled). Bacteria are underlined, filamentous fungi are italicized, yeasts are in bold lettering, and algae are in plain letters. Core No.
Sampling Depth Im)'
GISP2-B-63
62.300
Outer Core
Inner Core --_._- -~--~~~_
.._--------_ .. _._- --_
._ ._--
Cladosporium sphaerospermum Geotrichum sp. Phoma sp. 1 Exophiala sp. 1 Cryptococcus uniguttulatus Rhodotorula rubra R. rubra Sarcina sp. 1 Sarcina sp. 2 Micrococcus sp. 1 Penicillium sp. 1 R. rubra Candida sp. Cryptococcus sp. Sarcina sp. 1
GISP2-D-131
130.185
GISP2-D-135
134.203
Sarcina sp. 1
GISP2-D-226
225.205
Penicillium sp. 1 Aspergillus sp. 2 R. rubra Chrysophyta sp. 1
GISP2-D-326
325.160
Dye3-1971-49
158.879
Exophiala sp. 1 Penicillium variabile Aspergillus sp. 2 Sarcina sp. 1 Chrysophyta sp. 3
0_-
-
--------------.---
--
--_."
------._ .._-.__..-.------_..--.
Acremonium sp. Aureobasidium pullulans Basipetospora sp. 1 Cladosporium herbarum Phoma sp. 1 dematiaceous sp. 1 dematiaceous sp. 2 R. rubra mycelial yeast Penicillium sp. 1 Alternaria sp. C. herbarum Exophiala sp. 1 Cryptococcus albidus Chloromonas polyptera Sarcina sp. 1 Phoma sp. 1 R. rubra Chrysophyta sp. 1 Acremonium sp. 1 Basipetospora sp. 1 C. herbarum Penicillium sp. 1 Phoma sp. 2 R. rubra Chrysophyta sp. 2
Phoma sp. 1
'Depths correspond to the following approximate ages: 62.300m = 100 years before present (ybp); 130.185m = 300 ybp; 134.203m = 300 ybp; 158.879m = 350 ybp; 225.205m = 550 ybp; 325.160m = 700 ybp (determined using Reeh et al. 1985)
taxa, including ascomycetes, basidomycetes, and hyphomycetes. Some may have originated in the polar regions, while others may represent temperate or tropical taxa, whose spores or cells have been blown in from various locations around the world. The sequence data obtained from cultured isolates, and from direct sequencing of the ice melts, has already provided valuable information about fungal diversity, evolution, population structure through time and gene flow. The research is continuing with more ice cores from Greenland and Antarctica. Species detected in a continuous chronological sequence may be of particular value in future phylogenetic studies,
especially when older ice cores are sampled. In addition, this information can provide clues about global climatic changes, geological changes (volcanism,bolide impacts, etc.), human influences, and disease epidemics. Viable micro-organisms entrapped in ice are destined to be released during polar warming and glacial melts, or after the calving of icebergs into the ocean, perhaps releasing organisms that may not have been active in the biosphere for thousands of years. This could have immediate effects on populations of extant organisms of the same species, as well as influences on other organisms that may be hosts (including humans) or competitors of the recently released micro-organisms.
Volume 13, Part 2, May 1999 Acknowledgments. We thank C. J. K. Wang for providing assistance in the cultural and taxonomic aspects of this study. We also thank H. Fan for technical assistance and J. D. Castello for reviewing the manuscript.
Ice Core From Stor a ge Facility
I
11111/1/ I \ \ \'
~ III
~~~~~ Section Selected
Surface T rea ted with UV Irradiation (middle covered with aluminum foil) /
Inner Cor e Separated Fr om Outer Shell Aseptica lly @ _20°C
--lJ
References Abyzov, S. S. G. (1993) Micro-organisms in the Antarctic Filtered Su bsa mple ice, in Antarctic Microbiology (ed. E. 1. Friedman), pp. .. (ta ken through same processes as Unfi lter ed 265-295. New York: John Wiley & Sons, Inc. with unfiltered sam ples below) Suh sample Bartholomew, J. W. & Paik, G. (1966) Isolation and Inn er Co re Melted identificationofobligatethermophilic sporeformingbacilli @ 22°C (Stored @ .20°C) from ocean basin cores.Journal of Bacteriology92:635-638. Catranis, C. & Starmer, W. T. (1991) Micro-organisms ]BACT ERIA AND FUN GUS ASSAYS entrapped in glacial ice. Antarctic Journal of the VIRUS ASSAYS / \ United States 26: 234-236. Cultures Cross, T. & Atwell, R. W. (1974) Recovery of viable thermo actinomycete endospores from deep mud cores, in Spore Research (eds. A. N. Barker, G. W. 8 Media (with replicates) Gould & J. Wold), pp. 11-20.London: Academic Press. Gest,H. & Mandelstam, J. (1987)Longevity ofmicro-organisms Direct PCR Amplifica tion, Sequ encing in natural environments. Microbiological Sciences4:69-71. Various From Melted Ice Lindahl, T. (1993) Instability and decay of the primary Incuhation T imes and structure of DNA. Nature 362: 709-715. Temp eratures Liu, Y., Ammirati, J. F. & Rogers, S. O. (1997) Phylogenetic Relationships of Dermocybe and Cortinarius species based on nuclear ribosomal DNA Extra ction, Examinations PCR Am plilication, internal transcribed spacers. Canadian Journal of '" Sequencing " Botany. 75: 519-532. Characterize Sequ ence Com pa r isons Ma,1. J., Fan, H., Catranis, C.,Rogers,S. 0., & Starmer, W. T. and/or Identify .. (DNA Database Searches and (1997) Isolation and characterization of fungi entrapped Ph ylogenetic Ana lyses) in glacial ice. Inoculum 48(3):MSA Abstracts, p.23. Reeh, N., Johnsen, S. J. & Dahl-Jensen, D. (1985) Dating Fig 2 Protocol for ice core isolation, culturing, and DNA sequencing. the Dye 3 deep ice core by flow model calculations, in Sneath, P. H. A. (1962) Longevity of micro-organisms. Greenland Ice Core: Geophysics, geochemistry, and the Nature 195: 643-646. Environment, Geophysical Monograph 33 (eds. C. C. Swofford, D. (1993) PAUP: phylogenetic analysis using Langway, Jr., H. Oeschger & W. Dansgaard), pp. 57parsimony, Version 3.1.1, University of Illinois, 65. Washington, D. C.: American Geophysical Union. Champaign, 11. Rogers, S. O. & Bendich, A. J. (1985) Extraction of DNA Taylor, K. C., Mayewski, P. A., Alley, R. B., Brook, E. J., from milligram amounts of fresh, herbarium and Gow, A. J., Grootes, P. M., Meese, D. A., Saltzman, E. mummified plant tissues.Plant MolecularBiology 5:69-76. S., Severinghaus, J. P., Twickler, M. S., White, J. W. Seaward, A. C., Cross, T. & Unsworth, B. A. (1976)Viable C., Whitlow, S., & Zielinski, G. A. (1997) The bacterial spores recovered from an archaeological Holocene-Younger dryas transition recorded at excavation. Nature 261: 407-408. Summit, Greenland. Science 278: 825-827. Shi, T., Reeves, R. H., Gilichinsky, D. A. & Friedman, E. Vishniac, H. S. (1996) Biodiversity of yeasts and filamentous 1. (1997) Characterization of viable bacteria from microfungi in terrestrial Antarctic ecosystems. Siberian permafrost by 16S rDNA sequencing. Biodiversity and Conservation 5:1365-1378. Microbial Ecology 33: 169-179.
~
~ /
+
Micros~ 1~
The BMS Homepage The following are recent additions and amendments to the BMS Homepage (underlined entries are links): • • • • •
Foray Box (References for Identifying Fungi), has been updated to January 1999. Library: Books, has been reformatted and updated to 1994by integrating a supplement. Below Ground Research at York describes their work on roots and mycorrhizas. The Warwickshire Fungus Survey has been added to Fungus Groups. Photographs of fungi: The Quoditch Moor Nature Research has a nice selection. RTM
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