Fungal genetic resource centres and the genomic challenge

Fungal genetic resource centres and the genomic challenge

Mycol. Res. 108 (12): 1351–1362 (December 2004). f The British Mycological Society 1351 DOI: 10.1017/S0953756204001650 Printed in the United Kingdom...

198KB Sizes 0 Downloads 2 Views

Mycol. Res. 108 (12): 1351–1362 (December 2004). f The British Mycological Society


DOI: 10.1017/S0953756204001650 Printed in the United Kingdom.


Fungal genetic resource centres and the genomic challenge

Matthew J. RYAN* and David SMITH Genetic Resources Collection, CABI Bioscience UK Centre, Bakeham Lane, Egham, Surrey TW20 9TY, UK. E-mail : [email protected] Received 16 August 2004; accepted 28 September 2004.

Fungal research and education has for many years been supported by public service genetic resource centres, whose roles have been to maintain, preserve and supply living cultures to the research community. In the genomic era, genetic resource centres are perhaps more important than ever before. The cultures held, many of which are described and validated by expert biosystematists, are valuable resources for the future. There is a need to supply genomic and proteomic research programmes with fully characterised organisms, as usage of organisms from unreliable sources can prove disastrous, not least in economical terms. However, mycologists often require more than just the organisms, for example, their associated information is vital for bioinformatic applications and some researchers may only require genomic DNA from the organism rather than the organism per se. Genetic resource centres are continually adapting to meet the needs of their users and the wider mycological research community, this associated with OECD international initiatives should ensure they exist to support research for many years to come. This review considers the impact of such initiatives, the current roles of fungal genetic resource centres, the mechanisms used to preserve organisms in a stable manner and the range of resources that are offered for genomic research.

BACKGROUND Current status The 21st century dawned with the expectation that it would be the century of biotechnology and would particularly see the harnessing of the hidden potential of microorganisms. Yet we have only touched the tip of the iceberg with our knowledge and understanding of the largest group of microorganisms, the fungi, of which less than 100 000 of the estimated 1.5 million species have been described (Hawksworth 2001). Using traditional means of isolation and description by physical and morphological characters it will take some 1400 years to describe them as only around 1000 new names are published annually. A focussed approach using molecular technologies is critical. Of the described fungi, only a minority have been grown in culture, hence only 10 % of the described species are held by the world’s collections of fungus cultures. The World Data Centre for Microorganisms (2004 ; WDCM, lists 262 genetic resource centres throughout the world, which hold fungi. * Corresponding author.

These collections hold 375 052 fungal strains, which are catalogued using 23 807 names. However, the former figure does not take into account duplicate strains held in more than one collection and the latter figure does not take into account the overlap in anamorph and teleomorph names, synonyms, and variations in spelling. The strain data on many of these fungi are available via the electronic catalogues provided directly by the individual collections or via national or regional databases such as the WDCM. Therefore, collections of fungus cultures can, at best, only contain a small representative selection of fungal species. Some of the larger centres (Table 1) may be supported by expert taxonomists and other specialized technical staff, allowing the collections to operate to an extremely high standard, ensuring that their holdings are correctly characterised and authenticated. Unfortunately, this luxury may not be available in smaller genetic resource centres, and even in the larger ones coverage of all major fungal groups is seldom possible. The task of isolating, growing and storing fungi is immense. In addition, genetic resource centres must now deal with the vast diversity of new genetic entities generated by life scientists as they seek to reveal the genomes of

Fungal genetic resources for genomics


Table 1. A list of some public service collections of fungal culture with significant (>10 000 strains) holdings. Collection (acronym)


Total fungia

American Type Culture Collection (ATCC) Belgium Co-ordinated Collection (BCCM) CABI Bioscience (IMI) Canadian Collection of Fungal Cultures (CCFC) Centraalbureau voor Schimmelcultures (CBS) Institute for Fermentation Osaka (IFO) Fungal Genetic Stock Centre (FGSC) IBT Culture Collection of Fungi (IBT) MAFF Genebank USDA ARS Culture Collection (NRRL)

USA Belgium UK Canada Netherlands Japan USA Denmark Japan USA

46 000 28 500 28 000 10 000 55 000 10 849 16 000 22 500 10 181 59 500


Including yeasts, straminipilous fungi, etc. Data mainly obtained from World Data Center for Microorganisms (WDCM 2004).

many organisms and to engineer new strains with novel properties. Mycology has often been left behind when compared to advances in bacteriology, but there are now many studies that apply molecular techniques to fungi resulting in extensive fungal sequence data being available. Sequencing each fungus means hundreds of new entities need to be preserved (OECD 2001). In addition to the public service genetic resource centres, there are many private collections, for example those started and maintained by individuals in academia, and those held by biotechnology, industrial, and pharmaceutical corporations. There are numerous specialist centres concentrating on specific groups of organisms, such as entomopathogens and mycorrhizal fungi, and those focussing on individual genera such as Fusarium. Many of the latter have developed preservation protocols specifically for the organisms held, which can be difficult to reproduce in less specialized centres. Some examples of specialist collections of fungus cultures include the International Culture Collection of Vesicular Arbuscular Mycorrhizal fungi (IVAM) in West Virginia, the Culture Collection of Basidiomycetes (CCBAS) in Prague, the CSIRO Insect Pathogen Culture Collection (DE-CSIRO) in Canberra, and the Fusarium Research Centre in Pennsylvania.

The OECD Initiative The Organisation for Economic Co-operation and Development (OECD) Biological Resource Centre (BRC) Task Force consider that genomic studies are generating extraordinary amounts of information and taxing the capabilities of informatics for analysing and using data. Mycologists will spend the next few decades analysing and exploiting the information provided by these genome-sequencing efforts. These sequence data, and their by-products such as genome libraries, have to be preserved and made easily accessible. The quest to obtain information on each of the thousands of genes, gene products and other characteristics of each organism highlights the daunting task of storing, maintaining

and disseminating this information faced by BRC data banks. In 1998, the OECD Working Party on Biotechnology endorsed a proposal by Japan to examine support for Biological Resource Centres (BRCs) as a key element of the scientific and technological infrastructure of the life sciences and biotechnology. A task force on BRCs identified the needs and challenges facing BRCs and they published the report : Biological Resource Centres : Underpinning the Future of Life Sciences and Biotechnology ( The report argues the case for biological resource centres, strengthened and modified to meet the requirements of the 21st century, and recommends the creation of a Global Biological Resource Centre Network (GBRCN). Mycological genetic resource collections are an essential component of the infrastructure underpinning biotechnology. They consist of service providers and repositories of the living cells, genomes of organisms, and information relating to heredity and the functions of biological systems. The collections must be entities compliant with appropriate national laws, regulations and policies, and have been constituted to fulfil many crucial roles, which include, as listed in the report : . Preservation and supply of biological resources for scientific, industrial, agricultural, environmental and medical research and development applications. . Performance of research and development on these biological resources. . Conservation of biodiversity. . Repositories of biological resources for protection on intellectual property. . Resources for public information and policy formulation. The collections must employ best practice in carrying out these duties, but must also ensure that they ensure maintenance of reference strains and ex-type strains needed to fix the application of scientific names worldwide and to serve as repositories for voucher materials of strains used in published research papers of all kinds. The setting of common standards for operation is not

M. J. Ryan and D. Smith new ; it has been the goal of the World Federation for Culture Collections (WFCC) since its inception over three decades ago. Indeed, the WFCC has discussed and published the key requirements for the establishment and operation of culture collections (Hawksworth et al. 1990). Through subsequent committee work, these guidlines have been further developed to meet increasing demands ( html). Such standards provide a useful target for new collections to achieve. However, they do not cover all protocols or procedures, nor do they set minimum requirements. Standards are necessary to maintain quality in collections and to ensure they offer the service to science and industry that is required today and provide stable reference material for the future. The OECD BRC Task Force has provided the rules and criteria in the OECD Standard for the operation of Biological Resource Centres. This is being formulated at two levels, the general criteria that can be applied to all BRCs, and organism domain specific criteria that are applied to BRCs based on the kinds of biological materials they hold. The domains cover microorganisms (including viruses), animal, plant and human genetic material, cell lines, and tissues as outlined in the OECD definition (OECD 2001). The BRCs applying for accreditation to be part of the GBRCN must comply with relevant national agreements, policies, frameworks and recommendations and the regulations of the countries of supply or receipt when moving biological materials across national boundaries. No one collection, or country, will be able to meet these challenges alone. The OECD Report on BRC’s stresses that to cope with the massive expansion of biological resources, including living biological materials and data on genomics, BRC’s need to: (1) Contribute to the co-ordination of efforts to conserve biodiversity and to provide access to natural and engineered biological resources. (2) Assist in the development of a co-ordinated international system for decision making to guide appropriate acquisition, maintenance and distribution of biological resources so as to avoid unnecessary duplication of effort while preserving critical levels of biodiversity. (3) Modernise to incorporate the latest developments in web-based electronic communication, bioinformational science, and informatics technologies. (4) Coordinate and unify catalogues and databases to meet the requirements of science in the developing post-genomics era. (5) Develop new systems and technologies for the long-term maintenance and distribution of large numbers of diverse biological resources. (6) Co-ordinate curation, as well as development and networking of informatics tools for data analysis, comparison and visualisation. (7) Ensure that the scientific community has access to affordable products and services.

1353 IMPORTANCE OF GENETIC RESOURCE C E N T R E S : I M P A C T S F O R M Y C OL O G Y Roles Most public service collections are unique reserves of well-preserved, authenticated fungi. Traditionally, genetic resource centres have supported taxonomy by providing a broad range of cultures, including ex-type cultures. More recently any permanently preserved, metabolically inactive culture can be designated as a type of the name (Art. 8.4) : Type specimens of names of taxa must be preserved permanently and may not be living plants or cultures. However, cultures of fungi and algae, if preserved in a metabolically inactive state (e.g. by lyophilization or deep-freezing), are acceptable as nomenclatural types (Greuter et al. 2000). Although some genetic resource centres will remain facilities to deposit and store organisms, the majority are responding to the needs of the scientific community and are diversifying to increase the range of services available and to improve their standards of operation, as envisaged in the OECD initiative. Some offer identification services for external clients, such as spoilage and pathogenic fungi, using traditional mycological culture methods, molecular methods, or a combination of both. General mycological services can include mould susceptibility testing, biochemical and molecular characterisation, toxicity testing, patent and legal deposits, biological control, and the capacity to preserve and store organisms for external clients, especially vouchers for published research (Hawksworth 2004). Legislative considerations The GBRCN is being established to address legislative needs through co-ordinated and harmonised approaches and sharing tasks in a cost effective and appropriate manner. European collections are already collaborating and making progress in these areas, initially through the Common Access to Biological Resources and Information (CABRI) program, an electronic catalogue project setting common guidelines for operation of member collections ( Currently this work continues through the EU project (QLRT-2000-00221) European Biological Resource Centres Network (EBRCN). There is an ever-increasing demand for reliably named cultures as more and more industries are adopting certification or accreditation as a means of demonstrating quality and competence. There is also an increasing requirement to satisfy the sponsors of research who seek high quality science and solutions. The ability to demonstrate the competence to carry out and manage high quality research is being recognised by Research Councils and government departments in the UK (Anon. 2002, 2003). Third-party evaluation through accreditation or certification may be the only way to demonstrate this. It is imperative that organisms

Fungal genetic resources for genomics utilised in biotechnology are maintained in a way that will ensure that they retain their full capacity. Genetic resource centres must ensure a quality product providing standard reference material that will give reproducible results. To achieve this, collections must apply quality control and assurance measures to maintain these standards and this often requires them to harness any new technologies available.

MAINTENANCE OF GENOMIC INTEGRITY Importance of long-term preservation Examples of fungal variability, whether phenotypic or genotypic, are well-documented (Kistler & Miao 1992, Clutterbuck 2004). The maintenance of phenotypic and genomic integrity is paramount but this is difficult for some fungi. Dimorphism is common in yeasts (Ernst & Schmidt 2000) but less so in filamentous fungi. However, dimorphisms may be associated with changes at the chromosomal level (Gow 1995). Similarly transposons, have been found in many fungi (Daboussi & Capy 2003) and have been associated with instability, for example in Fusarium oxysporum (Daviere, Langin & Daboussi 2001). An example of the variable nature of fungi in culture is illustrated in Fig. 1. A nonspecialist could describe ‘unstable ’ cultures quite differently and an inexperienced technician may sub culture from an area of mycelium not representative of the parent fungus. Variation in culture morphology may also be exhibited following preservation by nonoptimal preservation methodology (Fig. 2). Despite the unstable nature of some fungi, the aim of preservation is to ensure long-term survival of an organism without change in its physiological and genomic integrity (Ryan & Smith 2003). Some mycologists still regard viability as a suitable criterion for assessing whether an organism is suitable for any given application. However, this approach is misguided and simple assessments of viability are grossly inadequate. Many authors have recorded differences in postpreservation stability following storage in genetic resource centres. Kuhls, Liekfeldt & Borner (1995) noticed that presumably identical strains of Trichoderma obtained from different centres had deviating PCR fingerprints. Kelly et al. (1994) found that an isolate of Fusarium oxysporum f.sp. ciceris maintained for 12 yr did not conform to the typical PCR fingerprints of other strains of the same species, and concluded that the strain had deteriorated after this period of maintenance. Horgen et al. (1996) found that chromosomal abnormalities and polymorphisms in RFLP fingerprints were associated with strain degeneration in Agaricus bisporus. More recently, Ryan et al. (2002) reported that strain degeneration subsequent to sector formation was associated with changes in physiology in a strain of Metarhizium anisopliae. Changes in physiological integrity following preservation were also

1354 noted in Penicillium expansum where production of the secondary metabolites patulin and citrinin was disrupted (Santos et al. 2002). More worryingly, PCR fingerprinting of replicates of an isolate of M. anisopliae subjected to non-optimised preservation techniques revealed evidence of polymorphisms (Ryan et al. 2001). Therefore, at the very least, collections should be adopting techniques to determine if their preservation methods are indeed preserving the strains without change. Such examples are important ; an inexperienced scientist could inadvertently work with a degenerate strain, which would ultimately produce spurious results. Such events would be less likely to occur with organisms that have been correctly characterized, maintained and stored. The need to ensure the stability of strains used for biotechnological applications (Clutterbuck 2004, Smith & Ryan 2004) is of particular importance. If the integrity of a production strain is compromised, a ‘stable’ stock of the original and production strain must be available. Good practice requires that potentially important isolates are preserved as the earliest opportunity. A little cash outlay for optimised preservation early in the development process of a product could save tens of thousands of dollars in the event of deterioration of a production strain. Research aimed at developing cryopreservation protocols should be associated with mechanisms to assess the maintenance of physiological or genomic integrity. These should include, at the very least, analysis of cultural characteristics or growth rate, but could also include analyses of physiology, extrolites (secondary metabolites), enzymes, and the genome. Any method used must be reproducible and include built-in replication. No genetic resource centre can guarantee total conservation of genomic characteristics, but it may be able to optimise procedures to ensure that the prospect of strain deterioration during preservation and storage is substantially reduced. Preservation techniques The aim of preservation is to maintain purity, viability, sporulation capacity and genomic integrity, avoid selection of variants from within a population, and lessen the prospects of strain deterioration (Smith & Ryan 2004). There are a plethora of methods described in the literature for the preservation of fungi, but no single preservation method can be guaranteed to be successful for all (Table 2). Ryan, Smith & Jeffries (2000) devised a decision-based key, which incorporated scientific, logistical and financial criteria to aid mycologists in the selection of preservation regime. Basic methods such as continual subculture/serial transfer (Smith & Onions 1994), storage under oil (Fennell 1960, Smith, Ryan & Day 2001), in water (Boesewinkel 1976, Burdsall 1994), in sand or soil (Booth 1971, Smith et al. 2001), or on silica gel (Perkins 1962, Smith et al. 2001), may be suitable in the absence

M. J. Ryan and D. Smith


Fig. 1. An example of culture variability in Penicillium aureocephalum (IMI 387181). Both cultures were inoculated from the same source, but exhibit quite different cultural morphology.

Fig. 2. Culture variation in an isolate of Fusarium oxysporum (IMI 370367). Both cultures are of the same strain, but have been maintained by different preservation methods. One (left) was preserved for 2 yr as a mycelial plug in water, and the other (right) was stored for 2 yr at x20 xC.

of alternatives. Although such methods are initially cost-effective and straight-forward to undertake, the length of time of storage before reprocessing is required can be as little as 2–3 weeks for some fungi, but longterm stability cannot always be guaranteed. The majority of large public service collections ensure that each of their strains is preserved by at least two methods. For most, the preservation methods of choice are freeze-drying (Tan 1997, Smith & Ryan 2004) and cryopreservation in the vapour phase of liquid nitrogen

(Polge, Smith & Parkes 1949, Hwang 1960, Smith & Ryan 2004). These methods were first introduced in the 1960s and their use is now universal where infrastructure permits. Freeze-drying (lyophilization) is routinely undertaken with sporulating fungi, particularly ascomycetes and allied conidial fungi. However, it is not so suitable for non-sporulating cultures. Although it is only spores that are routinely freeze-dried, research has been carried out to establish whether lyophilized hyphae can

Fungal genetic resources for genomics Receipt of culture

1356 Screen for mites or other contaminants

Taxonomic characterisation

Molecular and physiological characterisation

Digitise culture and bioinformatic data

Selected preservation methods*

Preserve using optimised methodologies



Viability test

Culture for preservation

Post preservation analysis of molecular and physiological integrity


Distribution * The choice of preservation method is dependent on the taxonomy, structure and water content of the organism, which can vary intraspecifically. Most culture collections generally preserve their organisms by at least two methods

Fig. 3. Progress of a culture through a fungal genetic resource collection. Table 2. Advantages, disadvantages and storage lengths of various fungal preservation regimes. Preservation Regime



Storage timea

Repeated sub-culture (serial transfer, continuous culture)

Cost effective in the short-term Method straight-forward

Short time before re-sub culture is required Risk of contamination Strain drift

From 2 weeks to 1 yearb

Storage in water

Cost effective in the short-term Method straight-forward

Culture deterioration Risk of contamination Strain drift

Up to 2 years

Storage under oil

Cost effective in the short-term Method straight-forward

Culture deterioration Risk of contamination Strain drift

Up to 10 years

Storage in sand or soil

Cost effective in the short-term Method straight-forward

Culture degeneration if moisture content too high Strain drift

Up to 10 years

Storage on silica gel

Cost effective Method straight-forward

Toxic effects of silica gel Only suitable for some sporing fungi

Up to 25 years

Lyophilisation (freeze drying)

Long storage time Easy storage and distribution of ampoules

Method complex Time consuming Equipment expensive Not suitable for non-sporing fungi

Up to of 50 years

Cryopreservation (mechanical deep-freeze)

Method straightforward Reduced prospects of strain deterioration

Equipment expensive Guaranteed supply of electricity required. Some cryoprotectants may be toxic to some fungi

Up to 40 years

Cryopreservation (liquid nitrogen)

Suitable for most fungi Methodology can be optimised for individual genera Reduced prospects of strain deterioration

Equipment expensive Controlled rate coolers required Guaranteed supply of liquid nitrogen required Cryoprotectants may be toxic to some fungi

Potentially indefinitely

a For optimal storage for most organisms, as recommended by Smith et al. (2001). For most regimes there are examples where organisms have survived for many years longer than noted above. b Storage of cultures at 4 xC (for example, in screw capped tubes) may prolong the length of time before subculture is required.

be revitalised successfully after preservation. In most cases this has met with mixed success (Tan, Stalpers & van Ingen 1991, Tan, van Ingen & Stalpers 1991) although Claviceps spp. (Pertot et al. 1977) are reported to have survived.

The standard method involves freezing the specimens and drying them from the frozen state by the sublimation of ice under reduced pressure (Smith 1983). Drying must avoid the liquid phase and be executed at temperatures below x15 xC until the water content of

M. J. Ryan and D. Smith the culture is reduced to less than 5 % (Smith 1983). Lyoinjury (Tan 1997), damage caused by the freezedrying process, can occur during the cooling and/or drying stages. The phase changes encountered during the drying process can cause the liquid crystalline structure of the cell membranes to degenerate to the gel phase, which disrupts the fluid-mosaic structure of the membrane (Tan 1997). Lyoprotectants such as serum, skimmed milk, inositol, trehalose and peptone can be added to the suspending medium to reduce the prospects of damage. Once preserved, cultures are easy to handle and occupy relatively little storage space with a proven shelf life of 20–40 yr or longer (Smith & Onions 1994). Ampoules can also be dispatched to clients without having to be revitalised beforehand, cutting the cost of postal charges and avoiding potential damage to the organism during transport. Cryopreservation The ability of living organisms to survive freezing and thawing was first realised in 1663 when Henry Power successfully froze and revived nematodes (Morris 1981). Polge et al. (1949) became the first modern day scientists to report the freezing of living organisms when they successfully froze and thawed avian spermatozoa. Cryopreservation of fungi was first noted by Hwang (1960). Like other preservation methods, cryopreservation has disadvantages. Controlled rate coolers, cryo-refrigerators and safety equipment can be expensive. Although for low throughput volumes, cryopreservation at a controlled rate of x1x minx1 can be achieved without the need for a controlled rate cooler using the commercial ‘Mr Frosty ’ (Nalgene, Rochester, NY) bench top cooler. The availability and high variable cost of liquid nitrogen can also present a problem to some laboratories, and the process may become uneconomic, especially because a reliable and continuous supply of nitrogen is required. Reports of cryopreservation of specific genera or groups of fungi are often published. Recent examples include the ectomycorrhizal fungus Cantharellus cibarius (Danell & Flygh 2002), the ‘ mushroom ’ Volvariella volvacea (Chen 1999), tropical wood-inhabiting basidiomycetes (Croan, Burdsall & Rentmeester 1999) and white-rot basidiomycetes (Stoychev et al. 1998). The standard method for cryopreservation involves controlled cooling at x1 x minx1, typically in the presence of a suitable cryoprotectant such as glycerol, trehalose or DMSO (Smith et al. 2001). The application of controlled rate cooling with cryoprotectants lessens the risks of cryo-injury (Smith & Thomas 1998). Smith (1983) suggests that cryo-injury in fungi is a result of the interaction of several stresses, such as the rupturing of tissue by ice damage or concentration/solution effects. The later includes pH changes caused by precipitation of buffers, dissolved gases, electrolyte concentration, intracellular crystallisation resulting

1357 from loss of the water of hydration from macromolecules, and cell shrinkage (Merryman, Williams & Douglas 1977). Membrane damage may be a result of solution effects, but could also be caused by ice damage. Indeed, Roquebert & Bury (1993), who studied the effects of cooling on Lentinula edodes, suggest that ice crystals may exhort a physical destructive pressure on membranes causing death. For fungi where cryo-injury is a problem, and in others that exhibit poor viability following preservation, specific methods may need to be tailored to ensure optimal cryopreservation. Examples of these are some members of the Basidiomycota and Straminipila such as Phytophthora and Saprolegnia spp. Such organisms are often termed preservation recalcitrant and finding a suitable method for the long-term preservation of such fungi can be difficult. This has given rise to the field of preservation technology, but few collections are actively involved in preservation protocol research and development.

Quality assessment The preservation regimes utilized by genetic resource centres have been developed and optimised over many years. To ensure cultures maintain their integrity, they should be routinely examined by appropriately skilled mycological systematists. Unfortunately, most collections do not have access to sufficient numbers of systematists, so other methods must be used to assess the success of preservation regimes. These should be more than assessments of growth rate and cultural morphology, and could include the analysis of extrolites or enzyme production, or ideally assessment at the molecular level. Studies on post-preservation integrity are particularly important for strains used in biotechnology. The effects of cryopreservation regimes on pathogenicity has been assessed for entomopthoralean fungi (Lopez Laster, Hajek & Humber 2001) and Fusarium moniliforme (Horita, Kita & Tsuchiya 1994), while fruit body production of cryopreserved test cultures of Flammulina velutipes was also reported (Ohmasa et al. 1996).

Preservation technology Research has been focussed on improvements in methodology and assessment of stability of specimens in culture. Some organisms are preservation recalcitrant and difficult to store long-term. Recalcitrant fungi include generally unculturable obligate fungi such as microcylic rusts, straminiples such as Halophythophthora, Saprolegnia, and Aphanomyces spp., some Basidiomycota, and the Glomeromycota. Research aims to develop improved cryopreservation techniques for such organisms, which are frequently of economic significance. The techniques employed include vitrification cryopreservation (Smith & Ryan

Fungal genetic resources for genomics 2004), immobilisation (Ryan 2001), and cryopreservation with the host substrate (Ryan & Ellison 2003). An alternative approach to cryopreservation is the cryopreservation of air-dried conidia, notably of the grapevine powdery mildew Uncinula necator (Stummer, Zanker & Scott 1999). Vitrification (Tan & Stalpers 1996) is a technique involving the application of very highly concentrated cryoprotectant solutions and has been applied to organisms of many cell types, especially plant cells (Benson 1994). The vitrification solution that surrounds the cells forms an amorphous glass upon cooling ; this prevents the onset of ‘concentration effects ’ or ice damage. Samples are plunge-cooled in liquid nitrogen which nullifies the need for controlled rate cooling. On resuscitation from the frozen state, care must be taken to ensure that samples do not ‘ crack ’ which could cause physical damage to the mycelium. More importantly, samples must be immediately washed to remove the toxic vitrification solution. The technique has been applied to a range of fungi with some success (Ryan & Smith 2003). However, vitrification solutions are extremely toxic to most living organisms, so routine use may not be advantageous. Immobilisation (encapsulation cryopreservation) is a technique where cells are encapsulated in calcium alginate beads prior to preservation (Benson et al. 1996). Examples of the use of this technology are now well documented for fungi ; for example Serpula lacrymans (Ryan 2001) and monoxenically produced spores of Glomeromycota (Declerck & Coppenolle 2000). The application of encapsulation has two main functions ; first, it allows cells to be easily manipulated by providing a suitable suspending matrix. Secondly, the water content of the cells can be reduced by osmotic treatment or drying which decreases the prospects of ice damage or concentration effects during the cooling stage of the cryopreservation procedure. Vitrification and immobilisation cryopreservation techniques have immense potential for preserving recalcitrant fungi that would otherwise not be stored in the long-term. However, these methods are extremely time consuming to undertake and are still relatively under-researched and therefore not broadly tested. The approach to cryopreservation where organisms are preserved with its host or alternative growth substrate have been applied in various guises for many years. For example, plant seeds have been used to support members of the Straminipila when cryopreserved. For obligate pathogens or mutualists, cryopreservation has been attempted where the fungus is preserved with its host substrate. This approach has been used for many recalcitrant organisms, such as the microcyclic rust Puccinia spegazzini where the teliospores were preserved on the stem or petiole tissue (Ryan & Ellison 2003). Wood, Pritchard & Miller (2000) used a combinational approach of cryopreservation with a substrate and encapsulation cryopreservation. Seeds of the common spotted orchid

1358 (Dactylorhiza fuchsii) and green-winged orchid (Anacamptis morio) were encapsulated in alginate beads with hyphae of the basidomycete Ceratobasidium cornigerum with no adverse effects post-cryopreservation. The combination of cryopreservation with an appropriate substrate/encapsulation cryopreservation approach may also be suitable for the cryopreservation of other mutualistic associations such as endophytes and lichens. These alternative approaches to cryopreservation has immense potential for the huge numbers of unculturable fungi that are otherwise not maintained in a living state by genetic resource centres. Until now, such fungi have only been stored as air-dried herbarium specimens.

SERVING THE GENOMIC REVOLUTION Any mycologists undertaking genomic research will require authenticated reference strains. These must also have retained the characteristics when first deposited and not have been at risk of strain drift during maintenance. Failure to deposit or use ‘voucher ’ cultures could have unfortunate consequences. If an organism used in genomic research is not fully representative of its species, has been incorrectly maintained, or wrongly identified, the comparability and interpretations will be incorrect ; this could have serious scientific repercussions. Therefore, any mycologist wishing to ensure high quality results, should obtain authentificated, properly identified strains from a reliable source. The practice of exchanging cultures between laboratories is a common occurrence, but potentially calamitous from a scientific one as such cultures may have been contaminated and subjected to drift during repeated subculturing. Reliable sequences Molecular phylogenetics has had a major impact on biosystematics. However, as aluded to above, the use of correctly named material is essential. Doubt has been expressed at the reliability of sequences available in publicly available sequence databases (Crous 2002, Deckert, Hsiang & Peterson 2002, Bridge et al. 2003). The veracity of published sequence data is dependent on the journal. Mycological Research and other leading-edge journals, require the deposition of vouchers (Agerer et al. 2000, Hawksworth 2004) before a paper is published. It also represents good cultural practice (Crous 2002). However, not all journals require vouchers of organisms used in papers they published to be preserved. If the material is not available, it may be impossible to check the validity of any sequence deposited in a nucleotide or protein sequence database; this ultimately means that scientists using sequences from databases must rely on the integrity of those who lodge the sequences. Until recently, nucleotide sequence databases, such as GenBank and EMBL, have only allowed changes to be made by those who submit

M. J. Ryan and D. Smith the primary sequence information; at least third parties can now provide annotations of existing entries, in a third part annotation (TPA) data set (Kulikova et al. 2004). However, for optimal reliance on data, there should be a minimum requirement that any sequence deposition made should be associated with either an available living culture or other material, to allow critical evaluation and allow others to verify sequence data. Many strains in public collevtions have been used to generate sequence data. For example, the Centraalbureau voor Schimmelcultures (CBS) has generated, from its holdings of filamentous fungi, an estimated 8–10 000 sequences (Joost A. Stalpers pers. com.). Further there are 14 589 direct references to CBS fungal strains in GenBank (Stalpers, pers. com.). However, despite the progress being made by researchers and genetic resource centres, the vast majority of strains remain unsequenced. Progress is being made in the computational analysis of sequences to provide a greater understanding of phylogeny. For example, Tehler, Little & Farris (2003) analysed 1551 ribosomal sequences of chitinous fungi. However, it is vital that a situation is achieved where examination of the unknown can be compared with a more representative sample of the known. Bioinformatics Genetic resource centres have a responsibility to ensure that the information on a strain is correctly recorded and updated. The validity and extent of information on a strain is just as important as the stability and viability of the organism on which it is based. Types of information associated with strains can be wide-ranging and complex. It is a common misconception that bioinformatic analysis incorporates only sequence data. The term incorporates all types of data, such as isolate history, growth requirements, physiological information, proteomic, and metabolomic data. Some database formats also allow for anatomical and cultural images to be stored. There are many commercial software packages available which enable genetic resource centres to compile and store information. Software packages have different strengths and weaknesses ; some simply provide mechanisms for storing information while others allow for identifications and comparisons. However, there is no standardised system in place, which can complicate transferring information from one collection to another. Bioinformatics has become a discipline in its own right, and the future will see those concerned with the maintenance, storage and exploitation of fungi, employing specialist bioinformaticians. The ‘free ’ availability of information to aid bioinformatics is paramount. The Global Biodiversity Information Facility (GBIF) is a framework for facilitating the digitization of biodiversity data (Edwards, Lane & Nielsen 2000), and is attempting to coordinate

1359 universal access to data regarding the world’s biodiversity, while components of the OECD BRC initiative will seek to collate information across the web. Web Biolomics, an association between Bioaware SA and CBS, provides an example of a cutting edge approach to how collections are harnessing and handling a bioinformatic approach ( databases/index.htm). Hundreds of characters are already detailed for 850 species and 5500 yeast strains. The database includes a bibliography of 10 000 references, a taxonomic database featuring 23 500 scientific names, and allows users to align their own sequences with a database containing up to 450 000 fungal sequences. The filamentous fungal and bacterial database are due to be made available shortly (Crous, Samson & Summerbell 2004). The AFTOL project (Assembling the Fungal Tree of Life) project, is a major initiative lead by mycologists at Duke University (Durham, NC). The aim of the project is to make available on the internet, broad datasets of molecular (focusing on seven molecular regions) and non-molecular (e.g., morphological) characters, in a suite of continually updated databases. A summary of this project is available at, and the first major paper from the project has just appeared (Lutzoni et al. 2004).

Resources for genomics DNA supply In addition to the organisms and the information associated with them, some genetic resource centres provide additional resources ; for example, genomic DNA extracted from collection holdings. Obtaining DNA from a collection can have many advantages ; it saves time because the researcher does not have to grow up and extract the material themselves, and ensures a high degree of reproducibility based on extraction from an authenticated source. Exchange of DNA between researchers is common practice, but with no guarantees of authentificaton or compliance with legislation, this should not be encouraged. Currently, DNA supply is being achieved through either a DNA banking approach or a responsive DNA extraction service from collection holdings. Establishment of a DNA bank requires that DNA extraction and storage procedures are optimised and that sufficient stocks of DNA are built up. Genetic resource centres generally charge for the extraction, purification and provision of DNA, but some organisations offer DNA without charge. The Nationaal Herbarium Nederland ( operates a DNA bank covering a broad range of organism types, but makes DNA available from the fungal genera Boletus, Chalciporus, Leccinum and Tylopilus. A DNA extraction service, has less risk involved as DNA is extracted from collection holdings on a request basis. This approach does not require excessive

Fungal genetic resources for genomics investment to build up stocks but does have disadvantages in that customers may have to wait a number of days or weeks while the organism is grown up and the DNA extracted. Genetic collections A search of the WDCM revealed that most of the collections that hold significant numbers of mutants are bacterial collections. Indeed, collections of fungal mutants have often been restricted to model organisms used in genetic studies. Many public service genetic resource centres have expanded their collections to incorporate mutant and modified strains, mating type strains, cDNA libraries, transposons, and organisms with full genome sequences. However, several specialist collections hold organisms specifically for applications in genetics, genomics or genetic engineering. For example the School of Biological Sciences at Flinders University, Australia, holds 4000 Neurospora crassa mutants, and the Laboratory of Molecular Genetics and Breeding of Edible Mushrooms University of Bordeaux, holds 3500 strains. The Fungal Genetics Stock Center (University of Kansas Medical Centre) holds over 5000 mutant strains, mainly Neurospora and Aspergillus species (McCluskey 2003). The total holdings of this collection are 16 000 fungi, 300 plasmids, 20 cDNA libraries, and 10 genomic libraries. The genomic approach to genetic resource centres, where genomic technologies are applied to genetic resources, will become increasingly the norm. T H E FU T U R E As the technologies for characterisation and screening and for optimal preservation evolve, new challenges are placed on genetic resource centres, both scientific and ethical. The UK National Institute of Agricultural Botany (NIAB, has described four methods by which DNA can be made to hold information in a binary or other number base format as a DNA barcode (Matthews 2003). The encoding of non-genetic information has the overall major benefit of providing a means of ready identification and authentication of goods and organisms. Their patent also describes ways in which the encoded information can be compressed to save space and how error correction methods can be introduced. DNA bar coding can also be used by pharmaceutical companies to protect their intellectual property, and by agricultural producers to mark the authenticity of produce and products. Similarly barcodes could be implanted in organisms ensuring that scientists use authenticated materials. Genetic resource centres will continue to serve the mycological community through the supply of authenticated organisms and their roles in ex-situ conservation, while complying with legislation and regulations. Many are responding positively to the demands placed on them by the genomics challenge,

1360 but this will inevitably mean establishing partnerships, as no single collection will have all the required expertise and facilities. Unfortunately, some public collections are increasingly being forced to become more commercial in their operations, the result of which is that their basic science remits become compromised. Collections need to find innovative income lines to counter reducing government inputs to support their activities, and they must get the balance right between supporting the life sciences and commercialisation. There is a need for more international action. The OECD BRC initiative tackles broad issues related to collections such as quality management and control, but does not extend to the financial support of collections, or to collections outside its member countries.

ACKNOWLEDGEMENTS We thank David L. Hawksworth for suggestions on the content of this article, Paul M. Kirk for advising on fungal names, and Joost A. Stalpers for providing information on CBS genomic activities.

REFERENCES Agerer, R., Ammirati, J., Baroni, T. J., Blanz, P., Courtecuisse, R., Desjardin, D. E., Gams, W., Hallenberg, N., Halling, R., Hawksworth, D. L., Horak, E., Korf, R. P., Mueller, G. M., Oberwinkler, F., Rambold, G., Summerbell, R. C., Triebel, D. & Watling, R. (2000) Always deposit vouchers. Mycological Research 104: 642–644. Anon. (2002) Universities face up to accreditation demands. Research Fortnight 179 : 1. Anon. (2003) Delivering the Evidence: DEFRA’s science and innovation strategy (2003–06). DEFRA Publications, London. Benson, E. E. (1994) Cryopreservation In Practical Plant Cell Culture: a practical approach (R. A. Dixon, ed.) : 147–166. IRL Press, Oxford. Benson, E. E., Wilkinson, M., Todd, A., Ekuere, U. & Lyon, J. (1996) Developmental competence and ploidy stability in plants regenerated from cryopreserved potato shoot tips. CryoLetters 17: 119–128. Boesewinkel, H. J. (1976) Storage of fungal cultures in water. Transactions of the British Mycological Society 66: 183–185. Booth, C. (1971) The Genus Fusarium. Commonwealth Mycological Institute, Kew. Bridge, P. D., Roberts, P. J., Spooner, B. M. & Panchal, G. (2003) On the unreliability of published DNA sequences. New Phytologist. 160: 43–48. Burdsall, H. H. & Dorworth, E. B. (1994) Preserving cultures of wood decaying Basdiomycotina using sterile distilled water in cryovials. Mycologia 86: 275–280. Chen, C. J. (1999) Preservation of Volvariella volvacea in culture through cryogenic storage. Fungal Science 14 : 99–104. Clutterbuck, A. J. (2004) Stability and Instability of fungal genomes. In Handbook of Fungal Biotechnology (D. K. Arora, ed.): 69–80. 2nd edn. Marcel Dekker, New York. Croan, S. C., Burdsall, H. H. jr & Rentmeester, R. M. (1999) Preservation of tropical wood-inhabiting basidiomycetes. Mycologia 91 : 908–916. Crous, P. (2002) Adhering to good cultural practice. Mycological Research 106: 1378–1379. Crous, P. W., Samson, R. A. & Summerbell, R. C. (2004) CBS Progress Report 2002–2003. Centraalbureau voor Schimmelcultures, Utrecht.

M. J. Ryan and D. Smith Daboussi, M. J. & Capy, P. (2003) Transposable elements in filamentous fungi. Annual Review of Microbiology 257 : 275–299. Danell, E. & Flygh, G. (2002) Cryopreservation of the ectomycorrhizal mushroom Cantharellus cibarius. Mycological Research 106: 1340–1342. Daviere, J. M., Langin, T. & Daboussi, M. J. (2001) Potential role of transposable elements in the rapid reorganisation of the Fusarium oxysporum genome. Fungal Genetics and Biology 35: 177–192. Deckert, R. J., Hsiang, T. & Peterson, R. L. (2002) Genetic relationships of endophytic Lophodermium nitens isolates from needles of Pinus strobus. Mycological Research 106 : 305–313. Declerck, S. & Coppenolle, M. G. A. V. (2000) Cryopreservation of entrapped monoxenically produced spores of an arbuscular mycorrhizal fungus. New Phytologist 148: 169–176. Edwards, J. L., Lane, M. A. & Nielsen, E. S. (2000) Interoperability of biodiversity databases : biodiversity information on every desktop. Science 289: 2312–2314. Ernst, J. F. & Schmidt, A. (2000) Dimorphism in Human Pathogenic and Apathogenic Yeasts. Karger, Basel. Fennell, D. I. (1960) Conservation of fungus cultures. Botanical Reviews 26: 79–141. Greuter, W., McNeill, J., Barrie, F. R., Burdet, H. M. V., Demoulin, V., Filgueiras, T. S., Nicolson, D. H., Silva, P. C., Skog, J. E., Trehane, P., Turland, N. J. & Hawksworth, D. L. (2000) International Code of Botanical Nomenclature (Saint Louis Code). [Regnum Vegetabile No. 138.] Koeltz Scientific Books, Ko¨nigstein. Hawksworth, D. L. (2001) The magnitude of fungal diversity: the 1.5 million species estimate revisited. Mycological Research 105: 1422–1432. Hawksworth, D. L. (2004) Biological papers without cited voucher material are so much waste paper. European Science Editing 30: 81–83. Hawksworth, D. L., Sastramihardja, K. I., Kokke, R. & Stevenson, R. (1990) Guidelines for the Establishment and Operation of Collections of Cultures of Microorganisms. World Federation of Culture Collections, Campinas, Brazil. Horita, M., Kita, K. & Tsuchiya, K. (1994) Stable preservation of pathogenicity of Fusarium moniliforme strains by freeze-drying method. Proceedings of the Kanto Tosan Plant Protection Society 41: 13–14. Horgen, P., Carvalho, D., Sonnenberg, A., Li, A. & van Griensven, L. J. L. D. (1996) Chromosomal abnormalities associated with strain degeneration in the cultivated mushroom, Agaricus bisporus. Fungal Genetics and Biology 20: 229–241. Hwang, S. W. (1960) Long term preservation of fungus cultures with liquid nitrogen refrigeration. Applied Microbiology 14: 784–788. Gow, N. A. R. (1995) Yeast-hyphal dimorphism. In The Growing Fungus (N. A. R. Gow & G. M. Gadd, eds): 403–422. Chapman and Hall, London. Kelly, A., Alcala Jimenez, A. R., Bainbridge, B. W., Heale, J. B., Perez Artes, E. & Jimenez Diaz, R. M. (1994) Use of genetic fingerprinting and random amplified polymorphic DNA to characterize pathotypes of Fusarium oxysporum f.sp. ciceris infecting chickpea. Phytopathology 84: 1293–1298. Kistler, H. C. & Miao, V. P. W. (1992) New modes of genetic change in filamentous fungi. Annual Review of Phytopathology 30: 131–152. Kuhls, K., Liekfeldt, E. & Borner, T. (1995) PCR fingerprinting used for comparison of ex type strains of Trichoderma species deposited in different culture collections. Microbiological Research 150: 363–371. Kulikova, T., Aldebert, P., Althorpe, N., Baker, W., Bates, K., Browne, P., van den Broek, A., Cochrane, G., Duggan, K., Eberhardt, R., Faruque, N., Garcia-Pastor, M., Harte, N., Kanz, C., Leinonen, R., Lin, Q., Lombard, V., Lopez, R., Mancuso, R., McHale, M., Nardone, F., Silventoinen, V., Stoehr, P., Stoesser, G., Tuli, M. A., Tzouvara, K., Vaughan, R., Wu, D., Zhu, W. & Apweiler, R. (2004) The EMBL Nucleotide Sequence Database. Nucleic Acids Research 32: (Database issue): D27–D30.

1361 Lopez Lastra, C. C., Hajek, A. E. & Humber, R. A. (2001) Effects of two cryopreservation techniques on viability and pathogenicity of entomophthoralean fungi. Canadian Journal of Botany 79: 861–864. Lutzoni, F., Kauff, F., Cox, C. J., McLaughlin, D., Celio, G., Dentinger, B., Padamsee, M., Hibbett, D., James, T. Y., Baloch, E., Grube, M., Reeb, V., Hofstetter, V., Schoch, C., Arnold, A. E., Miadlikowska, J., Spatafora, J., Johnson, D., Hambleton, S., Crockett, M., Shoemaker, R., Sung, G.-H., Lucking, R., Lumbsch, T., O’Donnell, K., Binder, M., Diederich, P., Ertz, D., Gueidan, C., Hall, B., Hansen, K., Harris, R. C., Hosaka, K., Lim, Y.-W., Liu, Y., Matheny, B., Nishida, N., Pfister, D., Rogers, J., Rossman, A., Schmitt, I., Sipman, H., Stone, J., Sugiyama, J., Yahr, R. & Vilgalys, R. (2004) Where are we in assembling the fungal tree of life, classifying the fungi, and understanding the evolution of their subcellular traits? American Journal of Botany: in press. Matthews, D. (2003) Technical Explanation of the NIAB Barcoding Patent. McCluskey, K. (2003) Fungal germplasm and databases. In Fungal Genomics (D. K. Arora & G. C. Khachatourians, eds): 295–309. Elsevier, Amsterdam. Merryman, H. T., Williams, R. J. & Douglas, M. S. J. (1977) Freezing injury from solution effects and its prevention by natural or artificial cryoprotection. Cryobiology 14: 287–302. Morris, G. J. (1981) Liposomes as a model system for investigating freezing injury. In Effects of Low Temperatures on Biological Membranes (G. J. Morris & A. Clarke, eds): 241–262. Academic Press, London. OECD (2001) Biological Resource Centres Underpinning the future of life sciences and biotechnology. OECD Publications, Paris. Ohmasa, M., Tsunoda, M., Babasaki, K., Hiraide, M. & Harigae, H. (1996) Fruit-body production of test cultures of Flammulina velutipes preserved for seven years by freezing at three different temperatures. Mycoscience 37: 449–454. Perkins, D. D. (1962) Preservation of Neurospora stock cultures in anhydrous silica gel. Canadian Journal of Microbiology 8 : 591–594. Pertot, E., Puc, A. & Kresmer, M. (1977) Lyophilization of nonsporulating strains of the fungus Claviceps. European Journal of Applied Microbiology 4: 289–294. Polge, C., Smith, A. U. & Parkes, S. (1949) Revival of spermatozoa after dehydration at low temperatures. Nature 164: 666. Roquebert, M. F. & Bury, E. (1993) Effects of freezing and thawing on cell membrane of Lentinus edodes, the Shiitake mushroom. World Journal of Microbiology and Biotechnology 9: 641–647. Ryan, M. J. (2001) The use of immobilisation for the preservation of Serpula lacrymans. Mycologist 15: 66–69. Ryan, M. J. & Ellison, C. (2003) Development of a cryopreservation protocol for the microcyclic rust-fungus Puccinia spegazzinii. CryoLetters 24 : 43–48. Ryan, M. J., Bridge, P. D., Smith, D. & Jeffries, P. (2002) Phenotypic degeneration occurs during sector formation in Metarhizium anisopliae. Journal of Applied Microbiology 93: 163–168. Ryan, M. J., Jeffries, P., Bridge, P. D. & Smith, D. (2001) Developing cryopreservation protocols to secure fungal gene function. CryoLetters 22 : 115–124. Ryan, M. J. & Smith, D. (2003) Preserving a microorganism’s capacity to produce active biomolecules. In Biological Resource Centres and the Use of Microbes (N. Lima & D. Smith, eds): 197–216. Livraria Minho, Braga, Portugal. Ryan, M. J., Smith, D. & Jeffries, P. (2000) A decision-based key to determine the most appropriate protocol for the preservation of fungi. World Journal of Microbiology and Biotechnology 16: 183–186. Santos, I. M., Abrunhosa, L., Venancio, A. & Lima, N. (2002) The effect of culture preservation techniques on patulin and citrinin production by Penicillium expansum Link. Letters in Applied Microbiology 35 : 272–275. Smith, D. (1983) Cryoprotectants and the cryopreservation of fungi. Transactions of the British Mycological Society 80: 360–363.

Fungal genetic resources for genomics Smith, D. & Onions, A. H. S. (1994) The Preservation and Maintenance of Living Fungi. 2nd edn. [IMI Technical Handbooks No. 2.] CAB International, Wallingford. Smith, D. & Ryan, M. J. (2004) Current status of fungal collections and their role in biotechnology. In Handbook of Fungal Biotechnology (D. K. Arora, ed.): 527–538. 2nd edn. Marcel Dekker, New York. Smith, D., Ryan, M. J. & Day, J. G. (2001) The UKNCC Biological Resource : properties, maintenance and management. United Kingdom National Culture Collection Secretariat, Egham. Smith, D. & Thomas, V. E. (1998) Cryogenic light microscopy and the development of cooling protocols for the cryopreservation of filamentous fungi. World Journal of Microbiology and Biotechnology 14 : 49–57. Stoychev, I., Homolka, L., Nerud, F. & Lisa, L. (1998) Activities of ligninolytic enzymes in some white-rot basidiomycete strains after recovering from cryopreservation in liquid nitrogen.necator. Antonie van Leeuwenhoek 73: 211–214. Stummer, B. E., Zanker, T. & Scott, E. S. (1999) Cryopreservation of air-dried conidia of Uncinula. Australasian Plant Pathology 28: 82–84. Tan, C. S. (1997) Preservation of fungi. Cryptogamic Mycology 18: 157–163.

1362 Tan, C. S. & Stalpers, J. A. (1996) Vitrification of fungi. In Biodiversity, International Biodiversity Seminar ECCO XIV meeting (A. Cimerman & N. Gunde-Cimerman eds): 189–193. European Culture Collection Organization, Ljubljana. Tan, C. S., Stalpers, J. A. & van Ingen, C. W. (1991) Freeze drying of fungal hyphae. Mycologia 83 : 654–657. Tan, C. S., Van Ingen, C. W. & Stalpers, J. A. (1991) Freeze-drying of fungal hyphae and stability of the product. In Genetics and Breeding of Agarics (A. Van Griensven ed.): Pudoc, Wageningen, The Netherlands. Tehler, A., Little, D. P. & Farris, J. S. (2003) The full length phylogenetic tree from 1551 ribosomal sequences of chitinous fungi. Mycological Research 107: 901–916. WDCM. (2004) World Data Centre for Microorganisms. http:// Wood, C. B., Pritchard, H. W. & Miller, A. P. (2000) Simultaneous preservation of orchid seed and its fungal symbiont using encapsulation-dehydration is dependent on moisture content and storage temperature. CryoLetters 21: 125–136.

Corresponding Editor: D. L. Hawksworth