Options and constraints in rapid diversity analysis of ... · Fungal Diversity 2 (March 1999) ecosystems in response to perturbation is large as many species with subtly different

Fungal Diversity 2 (March 1999)

Options and constraints in rapid diversity analysis of fungi innatural ecosystems

PaulF.Cannon

CAB! Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UK; email: [email protected]

Cannon, P.F. (1999). Options and constraints in rapid diversity analysis of fungi in naturalecosystems. Fungal Diversity 2: 1-15.

Fungal diversity is particularly difficult to assess and monitor. The number of species is largeand most are not effectively characterized. Many are small and require specialized samplingtechniques. Fruit-bodies (which are essential for identification) are ephemeral and theirproduction can rarely be reliably predicted. There is nevertheless a pressing need to makeassessments of fungal diversity and richness due to their prominence within ecosystems. Theaim must be to develop assessments which are both repeatable and comparable, so samplingprotocols must be far more tightly defined than is usually the case for fungal recording. Onelong-term solution to fungal diversity assessment may be development of formulas based onplant diversity and abiotic factors such as temperature and rainfall, which could be used inconjunction with indicator species to assess the likely extent of diversity. Such methods wouldnot directly count fungal species, but could be used to judge the likely effect of removal ofparticular plants or destruction of habitats. Options using currently available technology arelimited, but there is considerable potential for the recording of species associated with tightlydefined microhabitats such as individual living or fallen leaves. There is rather littleinformation on the small-scale patchiness of fungal distributions, which means that efficientsampling protocols cannot be designed, and we need much more information on the effects of

environmental factors such as water availability on fungal species profiles. There is scope forthe development of DNA-based diversity assessments for fungi similar to those which arealready available for bacteria. These involve extraction of whole-kingdom ribosomal DNAfrom samples using universal primers, and separation of individual sequences using denaturingor temperature gradient gel electrophoresis. Individual species identification is not possiblewithout sequencing or the separate development of probes, but very rapid diversity snapshotscan be achieved. Development is required before such techniques could be used on a widescale, especially in efficient DNA extraction and in the specificity of fungal primers. There isalso a need to extend the coverage of fungal rDNA sequences within phylogenies, and to relatevariation within particular parts of the genome more effectively to the taxonomic hierarchy.

Key words: conservation, fungi, molecular ecology, rapid diversity analysis,sampling strategies.

Introduction

Fungi play crucial roles in ecosystem function (Christensen, 1989). Fungiand bacteria constitute the dominant organism groups involved in CIN cycling,primarily in the degradation of organic matter. Critical stages in lignindecomposition are almost exclusively carried out by fungi, and the breakdownof complex biomolecules such as cellulose and tannins in soils is due mostly tofungal enzymatic activity. Fungi mediate plant health and promote growth viamycorrhizal and parasitic associations. They provide a food source for a widerange of invertebrates including Collembola, mites and nematodes (Shaw,1992), and gain nutrition in turn from living or dead animals as well as plants.Hyphallength has frequently been estimated at over 1 km g.l in grassland soil(Kjoller and Struwe, 1982), and considerable more in forested areas. Fungiaccount for up to 90 % of total living biomass in forest soils (Frankland, 1982),and soil fungal respiration has been found to be two to four times greater thanthat of bacteria (Faegri, Lid- Torsvik and Goks0yr, 1977; Anderson andDomsch, 1978). The long-term stability of ecosystems is therefore dependenton the continued contribution of fungal activities.

Despite their importance, we currently do not have objective methods for thedescription, characterization and function of fungal communities (Cannon,1997), and protocols for their analysis are neither timely nor comparable. Thenumber of species of fungi is very large, and many informed sources estimatefigures in excess of 1 million (Hawksworth, 1991; Hammond, 1995). Probablyless than 100000 of these have received even basic description, althoughperhaps 300000 names of fungi have been published (Rossman, 1994). Muchof the information about described species is inaccessible, with criticalshortages of up-to-date monographs and compilations. Lack of agreement overfungal species concepts (Brasier, 1997) and inconsistencies in their application(especially for necrotrophic fungi) are further uncertainties which place barriersin the path of objective diversity analysis. Quantification is also a major issue,as there is no agreement on the definition of the individual for most fungi(Cannon and Hawksworth, 1995). Even reasonably comprehensive surveys offungal diversity at the species level constitute major research projects, anddonor agencies have recently been unwilling to assign the very large sums ofmoney needed (Rossman et al., 1998).

There is currently very little information available on the relationshipsbetween organismal diversity and ecosystem function, especially forhyperdiverse taxa such as the fungi. There has been considerable attentiondevoted in recent years to theories of ecological redundancy (Walker, 1992;Lawton and Brown, 1993). These postulate that the buffering potential of

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ecosystems in response to perturbation is large as many species with subtlydifferent environmental requirements can perform the same essential ecologicalfunctions. In contrast, other recent studies consider that niche specialismrequires high species diversity in complex ecosystems such as soil (Finlay,

Maberly and Cooper, 1997). This means that the loss even of individual speciesmay have deleterious consequences for overall ecosystem function, especiallythrough knock-on effects caused by disruption of interorganismal interactions.Preliminary mesocosm experiments have suggested that soil fungal speciesprofiles change significantly in response to relatively small perturbations(lones et al., 1998), and differing management regimes and environmentalpressures may have a very great effect on the number and identity of the fungipresent (Persiani et al., 1998; Sampo et al., 1997). This research underlines theneed for rapid and cost-effective methods for characterizing and comparing thesystematic profiles of the fungi in environmental samples. A major benefit willbe to allow considered and timely decision-making on the fate of threatenednatural habitats, where unsustainable exploitation such as logging, urbandevelopment, or pollution is feared. Information on habitat value must beprovided very much faster than is currently the case, if the effect ofenvironmental change on fungi and other diverse organism groups is to beproperly addressed.

Comprehensive surveys or rapid estimates of fungal diversity?Fungi have been studied within natural ecosystems for hundreds of years,

but often in an incomplete and ill-considered manner. Organized fungal"forays" have often been treated more as social events than serious samplingexercises, despite the very considerable field skills of their participants. Even inthe few instances where locations have been studied over the long term,strategies have depended on the spare time of small numbers of scientists withvery restricted financial resources. Two sites in southern UK, Slapton Ley inDevon and Esher Common in Surrey, are amongst the most comprehensivelysurveyed in the world, with around 2500 species currently known from eachafter more than 25 years work (D.L. Hawksworth and P.M. Kirk, pers. comm.).However, there is no sign of the species/effort curve flattening in either case,and the overlap between the lists is only about 30 %, suggesting that thesurveys are not yet nearing completion. A workshop in Costa Rica in 1996 toplan an all-species fungal inventory of the Guanacaste Conservation Area(Rossman et al., 1998) concluded that seven years and US$ 31 million wouldbe required in order to carry out a comprehensive survey. Even with that

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magnitude of human and financial resources, none of the participants were ableto predict that all species would be detected.

With current technology, a reasonably detailed survey of the fungal specieseven in a simple system such as a single soil sample takes a matter of months.For example, Christensen (1989) recorded between 185 and 286 species fromsagebrush grassland, which involved culture and examination of between 1000

and 1400 propagules for each sample, and even at this stage there was noindication that the species number/isolate asymptote had levelled. In anotherstudy (Domsch, 1975) 250 species were counted from no less than 17000

separate isolations. At this level of work, studies involving comparison ormanipulation are rarely feasible, and the small numbers of replicates possiblemean that results cannot be effectively tested in statistical terms. There is alsoan almost complete lack of information on the small-scale patchiness of fungaldistributions within soil, which means that it is currently impossible to designeffective sampling strategies. Evidence from spatial analysis of leaf endophytes

(Lodge, Fisher and Sutton, 1996) suggests that fungal communities arestructurally complex even in simple, small-scale substrata.

All this information forces the conclusion that even moderately completefungal surveys are impractical, and that rapid estimates must be considered as ameans of providing evidence of the extent of fungal diversity. The challenge istherefore to develop protocols which are efficient, reliable, repeatable andcomparable. In order to achieve these aims, sampling strategies must be tightlydefined, and designed to provide thorough analysis of a restricted sample base,rather than a superficial though wide-ranging examination. For protocolsinvolving direct observation, the latter option will result in over-sampling oflarge and obvious taxa which are not necessarily representative of the fungalcommunity as a whole. Many current culture-based protocols are stronglybiased towards ruderal species which form large numbers of propagules andgrow rapidly in artificial conditions (Bills and Polishook, 1994; Bills, 1995).Basidiomycetes in particular are poorly sampled using standard techniques, butare known from (very labour-intensive) visual studies of mycelia to comprise amajor component of soil fungal biomass (Frankland, 1982).

There has been considerable interest in recent years in rapid diversityestimates which use trained technicians rather than taxonomic specialists(Beattie and Oliver, 1994). Although in such cases identifications to speciescan rarely be provided, species numbers and some basic impressions ofsystematic diversity are obtainable. Oliver and Beattie (1993) achieveddiversity assessments of spiders, ants, polychaetes and mosses usingbiodiversity technicians with only three hours training which were comparable

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to those made by specialist taxonomists. Fungi present greater challenges dueto their less well-defined morphological features, but in all probabilityacceptable results could be obtained from technical staff after a short trainingperiod. Such approaches are tempting, as reference collections and up-to-datelibraries are inaccessible to many scientists (especially in developingcountries), and the number of specialist taxonomists is now far too small toprovide such services. The major drawback to these strategies is thatcommunities cannot effectively be compared, as the number of speciescommon to pairs of sites cannot be calculated. This has major implications forconservation policy, as in many cases the trigger for protection is the presenceof rare species rather than the overall number recorded. For this reason,diversity analysis without direct reference to species identification is of limitedvalue, unless it is very rapid indeed.

The most effective approach to fungal diversity assessment is therefore tomake careful studies of very tightly defined samples. Our patchy knowledge offungal distribution patterns and the environmental factors which determinethem makes meaningful comparison of samples or sample sites almostimpossible unless the degrees of freedom are minimized. Comparing specieslists from standard collecting trips is of very limited value without anexhaustive survey of the differences in climatic conditions and current and

preceding local weather patterns, soil type, aspect, plant cover and compositionetc. The most easily defined and compared samples are small items such asindividual leaves or soil samples. Even here, account must be taken of factorssuch as the degree of decomposition of the plant part surveyed, or the depthbelow the surface of soil samples.

There are four main categories of protocols for fungal diversity assessmentand analysis. These are direct observational methods, cultural procedures,'direct molecular analysis and association methods. The aim for all is to restrictthe amount of sampling and analysis time, while maintaining the comparabilityof results.

Direct observationDirect observation has been the traditional method of choice for most

analyses of fungal communities. Direct field observations are problematic inmost circumstances, as the large majority of fungi are small, inconspicuous,and ephemeral in their appearance. Their detection is critically dependent onthe skill of the observer, and even sampling by specialists may have verydifferent results depending on minor, often unconcious, differences inprocedure and ability. Exceptions might include many groups of lichenized

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fungi which have perennial and often conspicuous thalli, for which surveys ofparticular rock or tree types can be relatively simple to standardize.

The large basidiomycetes are currently difficult to sample indirectly. Due totheir often short-lived and strongly seasonal fruit-bodies there is no easy way toensure consistency of sampling except over small geographical scales, and littlecan be done other than to make comprehensive notes of environmental andother criteria, and completing comparative sampling programmes within asshort a timescale as possible. Sampling is confined to fruiting specimens,which may give a distorted picture of overall basidiomycete diversity. There islittle recent information on the effectiveness of different sampling programmes,

but Mueller, Leacock and Murphy (1998) have briefly described methods for

rapid analysis using these means, which resulted in acceptable assessmentscompared with those obtained from long-term studies. The same team (Schmit,Murphy and Mueller, 1998) examined the effort required to obtain a goodestimate of total species richness, and found that sampling a single plot for

several years resulted in higher species numbers than studying two plots in thesame year. This demonstrates the difficulties in obtaining acceptable measuresof basidiomycete diversity using fruit bodies as samples.

A more effective protocol for direct observation is to collect samples andobserve them periodically in damp chambers. In this way, the samples (such asindividual leaves or pieces of wood from the same plant species) can be easilydefined, the effect of climatic differences on sample episodes can beminimized, and account can be taken of seasonal and successional effects. Forobvious practical reasons, such procedures are restricted to microfungi.Disadvantages over cultural methods include competition effects between

species within the damp chamber, which may result in less aggressive taxa notdeveloping or remaining sterile. However, other species will produce spores inthese circumstances and thus be recognizable, but fail to grow or sporulate inculture. The overall diversity obtainable from direct observational techniquesversus culture is significantly lower at least in some circumstances (Bills, pers.comm.), but the results are not necessarily less comparable. The most extensivesurvey of this type published is that of Rambelli et al. (1983), who carried out alarge-scale study of the effect of disturbance on forest ecosystems in the IvoryCoast.

Cultural proceduresCulture of fungi from samples such as soil, litter and plant parts can result in

a comprehensive and comparable estimate of diversity, although the protocolsare very labour-intensive (Bills and Polishook, 1994; Bills and Christensen, in

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press). Typically, samples are fragmented with a blender, and particles of

defined sizes plated onto agar in various concentrations. Germinating sporesand mycelium are then transferred to nutrient agar to allow colonydevelopment in isolation. Petri dishes containing too much inoculum will resultin overgrowth of fastidious species by ruderal taxa, and modern methods uselow-nutrient agar incubated at low temperatures and with antifungal additivessuch as cYclosporin to minimize competition between developing colonies. Thedilution plate protocol for soil samples also ensures an appropriateconcentration of inoculum. Other methods include plating out subsamples ofleaves (especially for endophyte work) made with cork-borers or by slicingthem with a sharp knife. Species numbers obtained by these methods may belarge; 220 species were isolated from wheat field soil in Germany (Gams andDomsch, 1969), 228 species from desert soils in Arizona and Utah (States,1978), and 250 species from soil and litter on the Galapagos Islands (Mahoney,1972). Bills and Polishook (1994) discovered between 78 and 134 species innon-exhaustive surveys of small litter samples from Costa Rica, and their ownevidence that sampling processes were incomplete was reinforced by Lodgeand Cantrell (1995), who re-analyzed their data and found only a 15-28 %overlap of species between sites.

Direct molecular diversity analysisThe most innovative procedures for biodiversity analysis of the environment

are based on analysis of ribosomal DNA (rDNA) which is extracted directlyfrom samples without prior culture. The techniques have rapidly becomeestablished for bacteria, but have not yet been widely used for study ofeukaryotic groups. While such approaches do not result in formal inventories,they provide a means of generating data to describe the variety of taxa present,and allow the design of reagents which can be used in the description,quantification and analysis of organismal diversity and community dynamics insoil. Such approaches have only become feasible for fungi as data sets increasefollowing the design of specific oligonucleotide primers directed to conservedregions across the known taxa. Applications are numerous, including effectiveand timely comparisons of management practices, charting the effects ofexternal perturbations, and assessment of overall ecosystem health. Thepreferred processes involve the extraction of total DNA from environmentalsamples, PCR-mediated amplification of fungal sequences using universalprimers, and separation of the sequences using DGGE (density-gradient gelelectrophoresis) or TGGE (temperature-gradient gel electrophoresis). Thisprocess uses differences in denaturation between AfT and G/C rich sequences

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to separate mixed samples. The gel bands can then be sequenced or probed toobtain identifications.

Though many problems remain to be properly resolved, there have been asmall number of attempts to apply this technology to fungal analysis.Kowalchuk, Gerards and Woldendorp (1997) applied these protocols to thestudy of fungal diversity within Ammophila roots. They were able to extractribosomal DNA from a number of fungal species, some of which could be

identified by sequence comparison with cultured taxa. Others did not closelymatch sequences in published rDNA databases. Their work is most interesting,but requires development if such techniques are to be used effectively fordiversity estimation rather than detection of particular species. The communitystructure within single root pieces is much simpler than, for example, soilsamples, and DNA extraction procedures for root material are relatively

problem-free compared with those for more heterogenous environments. Onlyone to four fungal species were detected from each sample. The only otherstudy to date is that of Pine, Dawson and Pace (1998), who presentedpreliminary results of the fungi in anoxic contaminated soil and sedimentsamples. Here also the number of species detected was low, although fungaldiversity within anoxic environments would be expected to be restricted. Theyisolated 14 different environmental sequences which clustered within diverse

fungal lineages. It is not clear whether the samples were of actively growingfungi, or derived from spores which were present but dormant.

A central issue in the analysis of diversity is whether the very largeestimates of bacterial diversity obtained by DNA-based techniques comparedwith traditional cultural methods (e.g. Torsvik, Goks0yr and Daae, 1990a;

Torsvik et al., 1990b; Hopkins, MacNaughton and O'Donnell, 1993; Bomemanet al., 1996) are reflected in other organism groups. Current predictions are thatthis extreme imbalance in diversity estimation will not be encountered to the

same degree for fungi, although many species are difficult or impossible toculture using current technologies. The main evidence which leads to thishypothesis is that fungal structures are far larger than bacteria and show moremorphological variation, and are thus more likely to be detected usingtraditional methods.

A major restraint to the development of DNA-based diversity assessmentsof fungal populations is the large number of gaps in published databases. rDNAsequences of many pathogenic taxa (both human and plant) are known, butsaprobic species are very under-represented. Any major programme to developDNA-based diversity protocols for fungi must therefore be accompanied by

sequence generation based on traditional cultural methods, and/or direct

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extraction from fruit-bodies. These sequences will also allow the improved

design of primers for detection of key species in environmental samples.Primers with enhanced specificity for fungal rDNA sequences have been

developed for analysis of environmental samples, particularly for mycorrhizal,lichen-forming and clinical fungi (Gardes and Bruns, 1993; Gargas and Taylor,1992; Hughes, Rogers and Haynes, 1998). The two most commonly studiedsections of the ribosomal genome are the small subunit (18S) and ITS regions.18S is generally considered to resolve taxa at higher levels of the taxonomichierarchy than ITS rDNA, and has more commonly been used for phylogeneticstudies, while ITS is typically used for species distinction and diagnosis (Whiteet al., 1990; Bruns, White and Taylor, 1991). The choice of region for differentpurposes appears not always to have been made rigorously, and may relate todiffering species concepts within the major fungal lineages. Berbee et al.(1998) have discovered differences in the sequence length needed todistinguish groups within the major lineages of the Ascomycota, and Carboneand Kohn (1998) have found similar variation during their work on primerdesign. Some of the most commonly used primers actually amplify DNA fromboth regions. Useful phylogenetic information can be obtained from ITSsequences (Mugnier, 1998), sequence diversity is likely to be greater inabsolute terms, and the region is less subject to introns. Fungal 18S rDNAfrequently contains insertion sites (17 are known) which have been reported tocontain both conserved and optional introns (Gargas, DePriest and Taylor,1995) which may distort species profiles.

Published universal fungal primers (White et al., 1990; Mugnier, 1994)appear to have been tested against a limited range of organisms, and most aredescribed only as allowing selective amplification of fungal DNA. While theiruse by subsequent researchers has been widespread and some publishedsequences are clearly useful for amplification of a wide range of fungal rDNAs,there seems to have been little attempt to explore the range of other organismsfor which amplification will also occur. This is not an issue for fungalphylogeny studies where sequences from specific strains are amplified, but it isclearly important for ecosystem community analyses where mixtures of DNAfrom a wide variety of organisms are encountered. Primer specificity iscurrently also a greater concern for fungal studies compared with those ofbacteria, as in comparison prokaryote rDNA sequences are distinctive andcomprehensively researched (the proportion of known species for which 16SrDNA has been sequenced is orders of magnitude greater than for the fungalequivalent, 18S rDNA), although the increasing prominence in researchprogrammes of major lineages such as the Archaea will no doubt result in

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modifications to primer design. It is unlikely that universal fungal rDNAprimers can be developed which do not amplify DNA from any otherorganisms, but so long as we are aware of their limitations, they can be used atleast for rapid assessment of organismal diversity with a strong bias towardsfungi. Such measures would in themselves be novel and valuable.

Molecular diversity analyses using current technology will not solve allproblems. PCR is an inherently competitive process which favours theamplification of common over rare sequences (Reysenbach et al., 1992;Farrelly, Rainey and Stackebrandt, 1995), and stringent conditions must beemployed (especially for DNA concentration) to maximise the diversity ofsequences amplified. The overall genome size also has a significant impact onthe efficiency of amplification, and work on bacteria suggests that theamplification process may select against certain groups of bacteria in complexsamples (Ward et al., 1994). Imperfections of amplification may also producephantom bands as a result of chimeric sequence production (Wang and Wang,1996). The DGGE/TGGE process needs careful optimisation in order to

maximize gel band separation, and many of the machines on the market are notwell suited to analysis of complex DNA mixtures due to the small size of thegels they accommodate. The distance travelled by the DNA fragments beforethey denature is not related to their systematic affinities, and identification ofindividual band sequences is currently problematic due to deficiencies in thepublished molecular databases. Despite all these limitations, the methods showgreat promise, and are the only technologies with realistic potential for reallyrapid diversity analysis.

Association methodsCorrelations of incidence, abundance and diversity of fungi with other

organisms or features of natural environments may prove to be effectivevehicles for decision-making, when conservation or monitoring of fungi isneeded. In practice, if fungal diversity can be linked to other measurementssuch as plant diversity, vegetational type or architecture, climatic factors etc, itshould be possible to predict rough species numbers and systematiccomposition for particular sites which may be under threat. This approachcould be combined with monitoring of indicator species, or taxa which are

particularly valued such as edible mushrooms or fungi known to be rare. Thedrawbacks of such measures are that initial calibration of the system will be

very time-consuming, and the degree of accuracy of predicted species numberswill also be expensive to measure.

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__ • __ •• n _. __ n - •••• - -.--- ----- -- - - - - -


Much of the evidence so far available of correlations between diversity of

major organismal groups unfortunately suggests that such systems may not be

reliable (Hammond, 1994a; Prendergast et al., 1993). Gaston (1992) and

Hammond (1994b) demonstrated that moisture levels are more relevant than

plant diversity when estimating species numbers of insects. This suggests thatif such systems are to be useful, complex equations may have to be developedwhich include meteorological and geological measurements as well as speciesnumbers of other groups. This ties in with the suggestions of May (1991), whosuggested that fungal diversity in the tropics might be more closely allied to thephysical variety of tree habitats and the pattern of plant species distribution,than to the number of plant species present. Similar suggestions were made byHuston (1994), who predicted that the diversity of saprobic organisms shouldbe highest where plant production was greatest, irrespective of its diversity.These approaches may be most appropriate for monitoring and prediction ofpopulation levels for particularly valued species, as has been explored byPeterson and McCune (1998) for Caliciales in Pacific Northwest old-growthforests.

Conclusions

Mycologists must come to terms with the fact that for the forseeable future,the available human and financial resources will be inadequate forcomprehensive survey of fungi in natural ecosystems. Nevertheless, if fungi areto take their place within conservation strategies, the tools for assessing theirpresence and diversity must be provided. There is a clear need to makeavailable, at a minimum, indications of species numbers and systematic profilefor sites of interest. There is also a need to develop methods for fungaldiversity estimation and species monitoring which do not require sustainedinputs from experienced specialists. The number of these is now completelyinadequate for survey work even in developed countries.

The various methods for diversity assessment described above and inCannon (1997) vary considerably in their cost, reliability and technicaldemands. Considerable advances may be possible in traditional diversityestimation methods involving analysis of tightly defined samples such asindividual plant leaves or twigs, either using culture or damp-chamberprotocols. The potential for association techniques may be very considerable,but the start-up costs could only be met by a coordinated research programmeinvolving many different disciplines. Molecular methods need much furtherdevelopment, but these are the only known options for truly rapid diversityassessment and monitoring.

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AcknowledgementsThis is an expanded version of a paper presented at the Asia-Pacific Mycological Congress

on Biodiversity and Biotechnology, held at Hua Hin, Thailand on 6-9 July 1998. I would liketo acknowledge the contributions of Drs. Paul Bridge and Gerry Saddler (CAB! Bioscience),and Dr. Mark Bailey (Institute of Virology and Environmental Microbiology, Oxford) for their

contributions to the molecular aspects of this paper.

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Options and constraints in rapid diversity analysis of ... · Fungal Diversity 2 (March 1999) ecosystems in response to perturbation is large as many species with subtly different