[5] Collection and storage of fungal and algal samples

[5] Collection and storage of fungal and algal samples

[5] OBTAINING FUNGAL AND ALGAL SAMPLES 65 ies, care should be taken not to allow the tissue to warm after initial storage. Similarly, if protein an...

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ies, care should be taken not to allow the tissue to warm after initial storage. Similarly, if protein analysis is the goal of the study, extreme care should be taken to maintain the specimens at temperatures that will prevent denaturation of proteins. Although high molecular weight DNA is the preferred template for PCR, degraded DNAs will often amplify if the target fragment is small enough and the palmers specific enough. DNA slated for PCR analysis can often be used from alcohol-preserved or museum specimens, which sometimes produce fairly degraded DNA. Summary The validity of any comparative study is dependent on the reliability of the identification of the samples in the study. Not all researchers are experts in the field of identification of samples, nor do all researchers have quick and ready access to expert systemetists who can accomplish the task of identification. The importance of verification of sample identity for comparative studies is vital. We describe several methods by which researchers can obtain and identify samples from the wild, and we suggest methods by which voucher samples can be obtained for future reference to these collected samples. We outline alternatives to collection of samples from the wild, such as purchase from stock centers and biological supply companies. Museum collections can also be extremely helpful in obtaining complete organismal samples for comparative studies.

[5] C o l l e c t i o n a n d S t o r a g e o f F u n g a l a n d A l g a l S a m p l e s

By MEREDITh BLACKWELLand RUSSELLL. CHAPMAN Introduction Samples to be used for taxonomic and systematic study of aloe, fungi, and lichens always have been important, but the current combination of explicit phylogenetic analysis and molecular approaches in studies of the systematics and evolutionary biology of these taxa requires some special considerations and approaches. Obtaining and storing fungal and algal samples is a major component in an exciting new assault on interesting, and previously untractable, questions in the biology of fungi and algae. The problems of applying evolutionary analysis to organisms such as fungi, lichen symbionts, and algae have been due in part to (1) the availability of relatively few morphological characters, some of which may be METHODS IN ENZYMOLOGY, VOL 224

~ t © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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missing in certain closely related taxa; (2) an inability to distinguish morphological features as discrete characters or multiple states of the same character; and (3) the difficulty in establishing character polarity by outgroup rooting or other means. The emphasis of taxonomists usually has been on distinguishing among or recognizing distinct taxa of these difficult organisms; thus many groups, especially at higher taxonomic levels, are based on autapomorphic characters. Until recently, few attempts 1-4 were made to apply phylogenetic analysis methods to fungi and algae. The increasing application of explicit phylogenetic methods has occurred concurrently with the use of molecular data. The use of molecular approaches such as nucleic acid sequencing has required greater reliance on computer-aided data analysis to deal with the vast number of new characters. The combination of new sources of characters with new methods of data analysis having the capability of establishing character polarity in the absence of an extensive fossil record has kindled an exciting new era in the taxonomy and systematics of fungi and algae. The advent of simpler molecular techniques, particularly those requiring small amounts of sample, allows a new group of biologists to exploit the methods in conjunction with morphological characters. The advances hold hope for eventual answers to several hundred years of evolutionary questions by providing for the testing of hypotheses based primarily on morphological characters with independent data sets. The revolution brought about by additional molecular characters is just beginning. The importance of obtaining and storing fungal and algal samples for study remains fundamentally important in the new era. This chapter provides a cursory review of the practical aspects of obtaining fungal and algal samples for molecular studies. It is directed toward the molecular biologist interested in initiating studies on the organisms, but it also should generally be useful to mycologists and phycologists, particularly beginning students. Culture Collections The obvious place to obtain fungi and algae for molecular analysis is from culture collections where they are maintained in pure culture, usually with species determinations. Because the services of culture collections, 1 Q. Wheeler and M. Blackwell, in "Fungus-Insect Relations, Perspectives in Ecology and Evolution" (Q. Wheeler and M. Blackwell, eds.), p. 5. Columbia Univ. Press, New York, 1984. 2 R. Currah, Am. J. Bot. 71, 161 (Abstr.) (1984). 3 D. R. Reynolds, Mycotaxon 27, 377 (1986). 4 E. C. Thefiot, J. Phvcol. 25, 407 (1989).

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inducting maintenance, are expensive, costs must be defrayed by user fees in many cases. Researchers must remember to budget for these necessary expenses. In addition to well-known major collections with broad general holdings, there are many smaller or specialized culture collections found throughout the world. For example, if isolates from a particular geographical locality are required, a smaller collection in the region of interest might be best for providing the culture; an insect fungal pathogen can be obtained from the specialist collection of entomopathogenic fungi maintained by the U.S. Department of Agriculture (USDA) (ARSEF) at the U.S. Plant, Soil, and Nutrition Laboratory (Ithaca, NY). Cultures of many isolates of wood-rotting basidiomycetes are available from the USDA Forest Service Center for Forest Mycology (Madison, WI); besides having large numbers of multiple isolates of species, the laboratory maintains voucher specimens for every culture on hand. Among examples of specialized algal collections are the Chlamydomonas Genetics Center maintained at Duke University (Durham, NC), which houses a large collection of Chlamydomonas reinhardtii mutants and numerous strains of other Chlamydomonas species; all strains are axenic, and a printout of file information on each strain is provided. A collection of prokaryotic nitrogen-fixing blue-green algae is maintained by the Soil Microbiology Division of the International Rice Research Institute (Manila, Philippines), and a collection of algae with a high potential for use in biomass energy production is available at the Solar Energy Research Institute (Golden, CO). More detailed information on the specialized collections mentioned above and on hundreds of collections worldwide is available from the World Federation for Culture Collections (WFCC) (see address below), which maintains a data center, the World Data Center on Microorganisms (WDC), also a component of the Microbial Resources Centers (MIRCEN) network. In its major role of disseminating information on culture collections, the WDC has two important publications that are essential guides to cooperating fungal and algal collections throughout the world. The World Catalogue of Algaes was published with the cooperation of 39 algal collections in 16 countries. The World Directory of Collections of Cultures of Microorganisms6 in its third edition, includes fungi from 345 culture collections worldwide. In addition, users may search the listings of all the 5 S. Miyachi, O. Nakayama, Y. Yokohama, Y. Hara, M. Ohmori, K. Komagata, H. Sugawara, and Y. Ugawa, "World Catalogue of Algae," 2nd Ed. Japan Scientific Societies Press, Tokyo, 1989. J. E. Staines, V. F. McGowan, and V. A. D. Skerman, "World Directory of Collections of Cultures of Microorganisms," 3rd Ed. World Data Centre, Univ. of Queensland, Brisbane, Australia, 1986.

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collections for individual species on the WDC electronic database that is accessible 24 hr a day. A guide to the database (Guide to World Data Center on Microorganisms--A List of Culture Collections in the World) is available from the WFCC, WDC, RIKEN, Wako, Saitama, Japan. Users must first register with the WDC to obtain a "user-ID" and "password" for access. Registration may be done easily by electronic mail, telex, or postal service using the following addresses: DIALCOM: 42:CDT0007 JUNET: [email protected] Telex: 2962818 RIKEN J Postal service: WDC/RIKEN 2-1 Hirosawa, Wako Saitama 351-01, Japan Cultures of some fungi are stored lyophilized and can be shipped immediately on order; however, others must be grown out on agar or in liquid culture so that shipping may be delayed several weeks. Additional delays can be encountered by the necessity of obtaining permits for shipping of certain organisms, particularly plant pathogenic fungi. This process involves both state and federal agriculture official approval in the United States that may take several months7 ,8 Some fungi or their secondary products are the cause of virulent or chronic disease in animals and require special p r o c e d u r e s . 7 No one should attempt to work with these organisms without proper facilities and a thorough appreciation of the risks involved. Culture collection staff can advise on the correct procedures that may be necessary. Literature on worldwide importation and postal requirements is cited in the International Mycological Directory? Algal samples are usually provided as liquid- or agar-grown cultures and are rarely subject to any requirements for special permits; however, the viability of the cultures (especially those with specific temperature requirements) is often a greater concern. Although many phototrophic algae can survive several days without light, a prolonged delay in shipping time will kill some algae, as will exposure to extreme temperatures. Most culture collection curators take elaborate precautions to ensure that cultures are correctly identified. Some even return transferred cultures S. C. Jong and W. B. Atkins, in "Fungi Pathogenic for Humans and Animals" (D. H. Howard, ed.), p. 153. Dekker, New York, 1985. s D. L. Hawksworth and K. Allner, in "Living Resources for Biotechnology, Filamentous Fungi" (D. L. Hawksworth and B. E. Kirsop, eds.), p. 54. University Press, New York, 1988. 9 G. S. Hall and D. L. Hawksworth, "International Mycological Directory," 2rid Ed. International Mycological Association, CAB International Mycological Institute, Oxon, United Kingdom, 1990.

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to the depositing scientist for authentication before cataloging. The vast majority of fungal and algal cultures will be identified correctly, but mistakes can be made by the depositor, collection employees, or even the researcher. Similarly, contamination with other cultures is a real possibility. To ensure against incorrect identifications we routinely grow out cultures to confirm identity if possible, and we always keep the original or verified subcultures stored. For fungi, it is sometimes useful also to keep dried culture vouchers. If there is any doubt about the purity and identity of batch cultures used for nucleic acid extraction, they should be grown on agar and examined microscopically for comparison with the stored culture. When a number of similar isolates are being handled, extreme care must be taken to avoid contamination that cannot be detected easily. Even with great care against contamination during all phases of work, problems may occur, and a final check is the inclusion of two closely related forms that occur as nearest relatives in the analysis. Ploidy or nuclear number should be a consideration when choosing cultures. Highly variable DNA regions in single-copy or repetitive genes (e.g., ribosomal RNA genes) may vary at single base positions in the homologous chromosomes of diploid or dikaryotic isolates.~° Haploid cultures of many fungi with gametic meiosis can be acquired easily from single spores, whereas isolates from basidiomata, ascomata, or mass spore cultures of heterothallic basidiomycetes and ascomycetes provide strains with nuclear variation. Diploid material is difficult to avoid in some organisms such as oomycetes, but it is important to recognize the possibility of variation. DNA can be extracted from single spores H and may eliminate some ploidy problems as well as provide the means for a variety of intraspecific level studies. For most algae in culture collections, the ploidy is known or can be inferred from what is known about the life cycles. Life cycle information can be obtained from a number of sources such as mycology and phycology textbooks.~2.~3 Larger culture collections usually offer multiple strains of species. Here again, informed choices should be made. Whenever possible one should use so-called type cultures, that is, those isolated from the type collection of a species at the time of its description. If generic level questions are l0 M. Gardes, T. J. White, J. A. Fortin, T. D. Bruns, and J. W. Taylor, Can. J. Bot. 69, 180 (1991). l~ S. B. Lee and J. W. Taylor, in "PCR Protocols: A Guide to Methods and Applications" (M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, eds.), p. 282. Academic Press, San Diego, California, 1990. ~2C. J. Alexopoulos and C. W. Mires, "Introductory Mycology." Wiley, New York, 1979. ~3H. C. Bold and M. J. Wynne, "Introduction to the Algae." Prentice-Hall, Englewood Cliffs, New Jersey, 1985.

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involved in the project, cultures of the type species of the genus always should be used. Strains may differ in metabolism and other genetic characters, and these differences may be important in choosing a culture. The American Type Culture Collection (Rockville, MD) has a helpful publication 14that fists processes and products associated with many isolates in the ATCC collection, and some culture collection catalogs contain information including literature references to strains. 15 Herbaria There is renewed interest in herbaria because they provide a source of potentially useful DNA from identified organisms, including type specimens. 16-18 Herbarium specimens of fungi as old as 50 years 17 and almost 100 years 19have provided adequate template for polymerase chain reaction (PCR) amplification of ribosomal genes. In fact, Swarm and colleagues 19 were able to obtain DNA sequences from a sample of only about 1000 spores of a rust fungus specimen and a single powdery mildew ascoma. As Bruns and co-workers 17have shown, cultures established at the time of specimen collection are sometimes available and provide for comparisons to demonstrate the integrity and usefulness of DNA from herbarium specimens. Although DNA may be partially degraded, primer-directed amplification, especially of multiple copy genes, may overcome the problem. Generally, specimens fast-dried at moderate temperatures or edges of larger specimens provide the best DNA template. 17 Herbarium curators should consider including provisions for maintenance of DNA samples as part of their guidelines (see below). Herbarium specimens are especially important for rare or nonculturable species; they will be important to type studies. Finally, in addition to providing DNA template from tissue, herbarium specimens of fungi and algae may contain viable propagules for establishing cultures.2°~! ~4M. J. Edwards, "Microbes and Cells at Work: An Index to ATCC Strains with Special Applications." American Type Culture Collection, 12301 Parklawn Drive, RockviUe, Maryland, 1988. t5 M. I. Krickevsky, B. O. Fabficius, and H. Sugawara, in "Living Resources for Biotechnology, Filamentous Fungi" (D. L. Hawksworth and B. E. Kirsop, eds.), p. 31. Cambridge Univ. Press, New York, 1988. 16F. Rollo, A. Amici, R. Sabvi, and A. Garbuglia, Nature (London) 335, 774 (1988). ~7T. D. Bruns, R. Fogel, and J. W. Taylor, Mycologia82, 175 (1990). ~s E. M. Golenberg, D. E. Giannasi, M. T. Clegg, C. J. Smiley, M. Durbin, D. Henderson, and G. Zurawski, Nature (London) 344, 656 (1990). ~9E. C. Swann, G. S. Saenz, and J. W. Taylor, MSA Newsl. 42, 36 (Abstr.) (1991). 2oA. S. Sussman, in "The Fungi" (G. C. Ainsworth and A. S. Sussman, eds.), Vol. 3, p. 447. Academic Press, New York, 1968. 2~ D. J. Young, R. L. Gilbertson, and S. M. Alcorn, Mycologia 74, 504 (1982).

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Field Collections Field collectionsof fungi,lichens,and algae arc useful for establishing cultures or extracting D N A directly.The Mycology Guidebook 22 is an important resource for information on isolationof fungi from nature,and Volume 1 of the Handbook of Phycological Methods 23 provides comparable information for the isolation of algae. Many species of fungi and algae have never been cultured, so fresh collections may be essential, especially with species for which inadequate herbarium material is available. Although the amount of material needed for DNA extraction has been reduced by the use of the PCR, culturing even small amounts or using herbarium specimens of some fungi and algae may require more time and resources than would a collection from nature. If extraction cannot be performed in a reasonable time, field-collected specimens should be refrigerated or even frozen. Liquid nitrogen is useful for indefinite storage of moderate-sized specimens. An important caution to be heeded in using fresh material directly is to ensure that all contaminating organisms are removed (washed, picked, etc.) from the desired specimen. This may not always be possible, and use of taxon-specific primers in the PCR may overcome the problem. For example, the presence of bacterial contaminants on an eukaryote would be of little consequence ff eukaryote-specific primers were being used; in the case of lichens, fungus- or alga-specific primers might be used.24 Voucher specimens must be prepared, so save enough of the specimens, being certain they contain diagnostic features for positive identification. Vouchers should be deposited in established herbaria if they warrant it. Most herbaria probably would not have space for or desire large numbers of specimens of a single species from one locality or specimens lacking diagnostic characters. We feel it is desirable to keep vouchers for a number of years in case questions should arise about published work. Voucher specimens should be kept dry and insect-free in a special herbarium cabinet, preferably in a herbarium and perhaps at the home institution or in the care of the researcher if they are not appropriate for the herbarium. Another potential problem in using field-collected specimens is obtaining species identifications. This problem is addressed more fully below. However, it may be necessary to contact a specialist in the group of interest. If a home institution mycologist or phycologist cannot be of help, he or she can usually suggest a specialist in the group of interest. Publishing 22 R. B. Stevens, "Mycology Guidebook." Univ. of Wa.~hington Press, Seattle, 1974. 23 j. R. Stein, "Handbook of Phycological Methods: Culture Methods and Growth Measurements." Cambridge Univ. Press, New York, 1973. 24 A. Gargas and J. W. Taylor, MSA NewsL 42, 14 (Abstr.)(1991).

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taxonomists also can be located by looking through taxon indices of primary or reference journals such as Biological Abstracts. If all else fails, officers of mycological and phycological societies (usually listed in societypublished journals) can make suggestions of appropriate taxonomists. Society newsletters often will publish requests for specific specimens. Fungal and algal "forays" are held periodically by both professional and amateur groups. It is especially important to check identifications of specimens that are acquired more informally, and, again, collectors are cautioned to keep vouchers in case there are questions later. For fungi and some algae fresh field-collected specimens often are a source of insects and mites. Not only can these arthropods destroy specimens, but they can also destroy a culture collection by contamination as they move from one tightly sealed culture to the next. Mites also are suspected vectors in horizontal gene transfer between Drosophila spp., 25 and perhaps this form of "contamination" may be detected in fungi and algae one day. Storage of Cultures Length of culture viability is dependent on species (or strain in some cases), age of culture at storage, propagules present, culture medium (affecting nutritional state), and method of storage. For these reasons storage of cultures is a research area in itself and cannot be covered adequately here. In-depth discussions of the techniques as well as primary references can be found in various reviews.7~-28 Some culture collections provide the service of specimen storage preparation, and the price may well be worth it if long-term storage is the goal. Over a period of 2 - 4 weeks most fungi survive well at room temperature. Parafilm wrapping retards desiccation of the medium and increases length of viability; contamination is also lessened. Fungal stock cultures usually remain viable for at least 6 months on agar slants stored in a refrigerator (5°). Cultures in screw-capped bottles (15 to 20 ml capacity) remain viable for longer periods in our experience. Although there are some auxotrophic algae that may be quite similar to fungi in short-term storage requirements, most are phototrophic or photoauxotrophic and 2s M. A. Houck, J. B. Clark, K. R. Petcrson, and M. G. Kidwell, Science 253, l 125 (1991). 2~S. C. Jong, in "Biotic Diversity and Gcrmplasm Preservation, Global Imperatives" (L. Knutson and A. K. Stoner, eds.), p. 241. Kluwer Academic Publ., Dordrecht, The Netherlands, 1989.

27D. Smith, in "Living Resourcesfor Biotechnology,FilamentousFungi" (D. L. Hawksworth and B. E. Kirsop, eds.),p. 75. CambridgeUniv. Press,New York, 1988. z80. Holm-Hanson,in "Handbookof PhycologicalMethods:CultureMethodsand Growth Measurements"(J. R. Stein,¢d.), p. 195.CambridgeUniv. Press,New York, 1973.

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require adequate lighting. Although exact requirements will vary greatly among different types of algae, temperatures slightly below room temperature ( - 2 1 *) and dim lighting are best for storage of cultures in liquid medium or on agar. Some algae will require special conditions (e.g., bubbring with air or CO2) even for short-term storage periods of 2 - 4 weeks. One c o m m o n practice that leads to disaster is placing algal cultures on window sills where they will be exposed to direct sunlight for part of the day. Such exposure will readily kill many algae. Detailed information on standard practices are presented in Handbook of Phycology. 23 Many fungal and algal cultures are viable after lyophilization. 29-32 This type of storage is time consuming over the short term, but over a long period it may save time, space, and expense. Lyophilization of cultures has the additional advantage of preserving the genetic integrity of strains that might change during years of active g r o w t h . 7,26,27 Fungi that produce spores also have been stored, usually for shorter periods of time, by drying in soil a3-35 or silica gel. 36,37 Cultures on agar may be covered with sterile mineral oil to lower oxygen levels (but not deplete them completely) or kept in sterile distilled water at 4*. 7,26,27 Smith 27 considers freezing and storage with a cryoprotectant in liquid nitrogen 38 to be the best general method of preserving filamentous fungi. Algae can be stored as frozen samples with cryoprotectants such as glycerol, dimethyl sulfoxide, or dried milk solids. For short-term storage a frozen sample can be kept in a standard freezer at - 2 0 " , in a mixture of glycol and dry ice (about - 7 8 ° ) , or in liquid nitrogen at - 1 9 6 °. For long-term storage a temperature of - 4 0 * or colder is considered more likely to maintain viability than a normal freezer for most algae. 28 The complexity of the processes involved in freezing fungi and algae and maintaining cell viability requires that optimal conditions be determined for each culture. 7,~,27 Even when empirical results demonstrate such optimal conditions, a program of routine, periodic monitoring of viability is probably essential for very long-term (decade or longer) storage. 29 R. H. Haskins and J. Anastasiou, Mycologia 45, 523 (1953). 3o R. H. Haskins, Can. J. Microbiol. 3, 477 (1957). 31 W. C. Haynes, L. J. Wiekerham, and C. W. Hesseltine, Appl. Microbiol. 3, 361 (1955). 32 C. W. Hesseltine, B. J. Bradle, and C. R. Benjamin, Mycologia 52, 762 (1960). 33 R. G. Atkinson, Can. J. Bot. 32, 673 (1954). D. I. Fennell, Bot. Rev. 26, 79 (1960). 3s C. Booth, "The Genus Fusarium." Commonwealth Mycological Institute, Kew, Australia, 1971. D. D. Perkins, Can. J. Microbiol. 8, 591 (1962). 37 D. D. Perkins, Neurospora Newsl. 24, 16 (1977). 38 S. C. Jong, in "The Biology and Cultivation of Edible Mushrooms" (S. T. Chang and W. A. Hays, eds.), p. 119. Academic Press, London, 1978.

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Inclusion of Taxonomists in Molecular Studies

Alpha-level taxonomists are an essential but dwindling resource. It is these individuals who use their data (generally morphological characters of organisms) to propose level one hypotheses (erection of species, genera, and higher taxa) essential to later molecular studies. In addition to species identification, taxonomists can provide important information on taxon selection and particularly interesting questions about unresolved relationships and good model systems for studying broad biological questions, such as speciation processes in wind-dispersed species, broad intercontinental distributions, coevolution of symbionts, or chloroplast evolution. Molecular techniques already are bringing new evidence to provide independent data in 100-year-old morphologically based hypotheses. For example, an extremely close relationship between basidiomycetes with highly divergent basidiomes (boletes and hypogeous forms) has been supported by molecular data} ? On the other hand, a long-standing hypothesized relationship between red algae and ascomycetes that periodically resurfaces 39 is not supported by molecular evidence.4°,4! In 1858, Pringsheim42 suggested a controversial relationship between some oomycetes and the Vaucheriaceae (which contain plastids with chlorophylls a and c); additional morphological evidence from flagellar apparatus structure supports this view.43,*~The topic is the exciting subject of current molecular studies by at least two groups of workers to provide additional independent data that appear to support the idea of Pringsheim.45,~ Long-standing nomenclatural problems in mycology are even being addressed. Some species of ascomycetes and basidiomycetes have several morphs. Asexual stages without known sexual stages long have been placed as form species in the "Deuteromycota." Reynolds and Taylor47,4s have questioned the need for this practice now that molecular characters are

39 V. Demoulin, Biosystems 18, 347 (1985). 4o S. Kwok, T. J. White, and J. W. Taylor, Exp. Mycol. 10, 196 (1986). 41D. Bhattacharya, H. J. Elwood, L. J. Goff, and M. L. Soon, J. Phycol. 26, 181 (1990). 42 N. Pringsheim, Pringsh. Jahrb. Wiss. Bot. 1, 284 (1859). 43 D. J. S. Barr, in "Zoosporic Plant Pathogens" (S. T. Buczacki, ed.), p. 43. Academic Press, New York, 1983. D. J. S. Barr, Mycologia 84, 1 (1992). 45 G. R. Klassen, G. Hausner, and A. Belkhiri, MSA Newsl. 42, 22 (Abstract) (1991). S. B. Lee and M. S. Fuller, MSA Newsl. 42, 23 (Abstract) (1991). 47 D. R. Reynolds and J. W. Taylor, Taxon 40, 311 (1991). 48 D. R. Reynolds and J. W. Taylor, in "Improving the Stability of Names: Needs and Options" (D. L. Hawksworth, ed.), p. 171. Koeltz, Konigstein, Germany, 1991.

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available; they have initiated discussion of the use of DNA as the type element in some cases and its implications for classification under the International Code of Botanical Nomenclature. They envision the possibility that some species even may be discovered and known only by their DNA. Ward and colleagues49 certainly have found this to be true of bacteria. Although an undergraduate student can be trained to perform most of the techniques used in modern phylogenetic studies, a depth of knowledge of fungal and algal taxonomy, including the ability to identify the organisms to the species level, takes many years. Given the tens of thousands of species of algae and perhaps more than 1.5 million species of fungi,5° a large cadre of taxonomic experts is needed in both disciplines, but the modest numbers of these scientists are declining rather than increasing. It is ironic that this shortage should occur at a time when interest in biodiversity is high and molecular approaches combined with explicit phylogenetic analysis of the data are providing the means for exciting advances in systematics and evolutionary biology. The suggestion that Pneumocystis carinii is a fungus 51,52 has important implications for disease treatment; there is an interest in fungal phylogenetic relationships to decrease time and expense in fungicide trials. The exciting recognition that coastal nanoplankton organisms are far more important in total primary productivity than previously thought increases the practical need to be able to identify these minute organisms, for which morphology is of limited usefulness.53 These applications underscore the fundamental importance of taxonomy. Unfortunately, the number of taxonomists and students of major groups of fungi, lichens, and algae continues to decrease. The condition is critical and warrants mention here. Because taxonomists are essential, they should be totally involved in molecular evolution studies as principal investigators. They should not simply be consulted toward the end of a study when identifications or discussions for papers are needed, but rather should be included from the very beginning in the project design. In fact, the pairing of taxonomists and molecular biologists should be encouraged both for efficiency and for well-conceived science. 49 D. M. Ward, R. Weller, and M. M. Bateson, Nature (London) 345, 63 (1990). so D. L. I-Iawksworth, MycoL Res. 95, 641 ( 1991). 5~ j. Edman, J. A. Kovacs, H. Masur, D. V. Santi, H. J. Elwood, and M. L. Soon, Nature (London) 334, 519 (1988). 52 j. Edman, J. A. Kovacs, H. Masur, D. V. Santi, H. J. Elwood, and M. L. Soon, J. Protozool. 36, 185 (1989). 53 L. S. Murphy and E. M. Haugen, Limnol. Oceanogr. 30, 47 (1985).

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Herbarium Guidelines

We have already mentioned that herbarium curators should begin to formulate guidelines concerning extraction of DNA from specimens. Hans should be made for the best preservation of incoming specimens so that they will provide minimally degraded samples. Pilot studies can be made to obtain information needed to make informed decisions of this kind. Herbaria also should make provisions for storage of excess DNA samples once they are obtained from specimens, either at their institution or at a cooperative institution. Certain zoological museums already store frozen tissues for isozyme and other molecular studies. Ideally DNA extractions from new type specimens could be performed routinely soon after they are described. We need to begin planning for curation of these new resources that provide more characters than we have ever known. Conclusions Given the small amounts of nucleic acid needed for many studies, one can propose that major coordinated regional collections of samples eventually could serve the needs of the scientific community, just as major herbaria and culture collections do. Perhaps nucleic acid sample collections will be established as additions to major herbaria and/or culture collections. The potential usefulness of, and need for, major, coordinated regional collections is clear, but the feasibility is not. The concept warrants attention because the historical record has proved that individual local collections are often maintained and sustained by the commitment and effort of one or more individual researchers, rather than by a long-term commitment of the institution of the collection. Thus, orphaned collections of specimens of all types often are lost (literally lost, discarded, or allowed to deteriorate), and long-term protection and continuity are needed both for traditional fungal and algal material (e.g., herbarium specimens, cultures) and nucleic acid samples. The latest, most modern advances on the molecular front have underscored dramatically the importance of very basic, mostly traditional, aspects of obtaining and storing fungal and algal specimens. Newer aspects, such as cryostorage and nucleic acid inventories, are now part of the game plan. Our samples are literally bits of the biodiversity of the world; as such, they are useful sources of many kinds of information. Because we do not know all there is to know, we cannot appreciate how critically important some specimens will be in future research. Some of the specimens will prove to be scientific treasurers, and since we do not know which specimens will be the most valuable, we must treat all of them a priori as invaluable.

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Acknowledgments We thank Drs. S. C. Jongand D. L. Hawksworth for providing helpful literature. Dr. John W. Taylor was a helpful reviewer of the manuscript. Financial assistance from the National Science Foundation (BSR-8918157 to M.B. BSR-8918564 to M. A. Buchheim and R.L.C., and BSR-9107389 to R.L.C.) is gratefuBy acknowledged.