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Fungal Diversity 2 (March 1999)
Options and constraints in rapid diversity analysis of fungi in
natural 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 natural
ecosystems. Fungal Diversity 2: 1-15.
Fungal diversity is particularly difficult to assess and monitor. The number of species is large
and most are not effectively characterized. Many are small and require specialized sampling
techniques. Fruit-bodies (which are essential for identification) are ephemeral and their
production can rarely be reliably predicted. There is nevertheless a pressing need to make
assessments of fungal diversity and richness due to their prominence within ecosystems. The
aim must be to develop assessments which are both repeatable and comparable, so sampling
protocols must be far more tightly defined than is usually the case for fungal recording. One
long-term solution to fungal diversity assessment may be development of formulas based on
plant diversity and abiotic factors such as temperature and rainfall, which could be used in
conjunction with indicator species to assess the likely extent of diversity. Such methods would
not directly count fungal species, but could be used to judge the likely effect of removal of
particular plants or destruction of habitats. Options using currently available technology are
limited, but there is considerable potential for the recording of species associated with tightly
defined microhabitats such as individual living or fallen leaves. There is rather little
information on the small-scale patchiness of fungal distributions, which means that efficient
sampling 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 for
the development of DNA-based diversity assessments for fungi similar to those which are
already available for bacteria. These involve extraction of whole-kingdom ribosomal DNA
from samples using universal primers, and separation of individual sequences using denaturing
or temperature gradient gel electrophoresis. Individual species identification is not possible
without sequencing or the separate development of probes, but very rapid diversity snapshots
can be achieved. Development is required before such techniques could be used on a wide
scale, especially in efficient DNA extraction and in the specificity of fungal primers. There is
also a need to extend the coverage of fungal rDNA sequences within phylogenies, and to relate
variation 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). Fungi
and bacteria constitute the dominant organism groups involved in CIN cycling,
primarily in the degradation of organic matter. Critical stages in lignin
decomposition are almost exclusively carried out by fungi, and the breakdown
of complex biomolecules such as cellulose and tannins in soils is due mostly to
fungal enzymatic activity. Fungi mediate plant health and promote growth via
mycorrhizal and parasitic associations. They provide a food source for a wide
range 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. Fungi
account 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 than
that of bacteria (Faegri, Lid- Torsvik and Goks0yr, 1977; Anderson and
Domsch, 1978). The long-term stability of ecosystems is therefore dependent
on the continued contribution of fungal activities.
Despite their importance, we currently do not have objective methods for the
description, characterization and function of fungal communities (Cannon,
1997), and protocols for their analysis are neither timely nor comparable. The
number of species of fungi is very large, and many informed sources estimate
figures in excess of 1 million (Hawksworth, 1991; Hammond, 1995). Probably
less than 100000 of these have received even basic description, although
perhaps 300000 names of fungi have been published (Rossman, 1994). Much
of the information about described species is inaccessible, with critical
shortages of up-to-date monographs and compilations. Lack of agreement over
fungal species concepts (Brasier, 1997) and inconsistencies in their application
(especially for necrotrophic fungi) are further uncertainties which place barriers
in 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 of
fungal diversity at the species level constitute major research projects, and
donor agencies have recently been unwilling to assign the very large sums of
money needed (Rossman et al., 1998).
There is currently very little information available on the relationships
between organismal diversity and ecosystem function, especially for
hyperdiverse taxa such as the fungi. There has been considerable attention
devoted 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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Fungal Diversity 2 (March 1999)
ecosystems in response to perturbation is large as many species with subtly
different environmental requirements can perform the same essential ecological
functions. In contrast, other recent studies consider that niche specialism
requires high species diversity in complex ecosystems such as soil (Finlay,
Maberly and Cooper, 1997). This means that the loss even of individual species
may have deleterious consequences for overall ecosystem function, especially
through knock-on effects caused by disruption of interorganismal interactions.
Preliminary mesocosm experiments have suggested that soil fungal species
profiles change significantly in response to relatively small perturbations
(lones et al., 1998), and differing management regimes and environmental
pressures may have a very great effect on the number and identity of the fungi
present (Persiani et al., 1998; Sampo et al., 1997). This research underlines the
need for rapid and cost-effective methods for characterizing and comparing the
systematic profiles of the fungi in environmental samples. A major benefit will
be to allow considered and timely decision-making on the fate of threatened
natural habitats, where unsustainable exploitation such as logging, urban
development, or pollution is feared. Information on habitat value must be
provided very much faster than is currently the case, if the effect of
environmental change on fungi and other diverse organism groups is to be
properly 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 sampling
exercises, despite the very considerable field skills of their participants. Even in
the few instances where locations have been studied over the long term,
strategies have depended on the spare time of small numbers of scientists with
very restricted financial resources. Two sites in southern UK, Slapton Ley in
Devon and Esher Common in Surrey, are amongst the most comprehensively
surveyed in the world, with around 2500 species currently known from each
after 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 the
surveys are not yet nearing completion. A workshop in Costa Rica in 1996 to
plan an all-species fungal inventory of the Guanacaste Conservation Area
(Rossman et al., 1998) concluded that seven years and US$ 31 million would
be 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 able
to predict that all species would be detected.
With current technology, a reasonably detailed survey of the fungal species
even 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 from
sagebrush grassland, which involved culture and examination of between 1000
and 1400 propagules for each sample, and even at this stage there was no
indication that the species number/isolate asymptote had levelled. In another
study (Domsch, 1975) 250 species were counted from no less than 17000
separate isolations. At this level of work, studies involving comparison or
manipulation are rarely feasible, and the small numbers of replicates possible
mean that results cannot be effectively tested in statistical terms. There is also
an almost complete lack of information on the small-scale patchiness of fungal
distributions within soil, which means that it is currently impossible to design
effective sampling strategies. Evidence from spatial analysis of leaf endophytes
(Lodge, Fisher and Sutton, 1996) suggests that fungal communities are
structurally complex even in simple, small-scale substrata.
All this information forces the conclusion that even moderately complete
fungal surveys are impractical, and that rapid estimates must be considered as a
means of providing evidence of the extent of fungal diversity. The challenge is
therefore to develop protocols which are efficient, reliable, repeatable and
comparable. In order to achieve these aims, sampling strategies must be tightly
defined, and designed to provide thorough analysis of a restricted sample base,
rather than a superficial though wide-ranging examination. For protocols
involving direct observation, the latter option will result in over-sampling of
large and obvious taxa which are not necessarily representative of the fungal
community as a whole. Many current culture-based protocols are strongly
biased towards ruderal species which form large numbers of propagules and
grow rapidly in artificial conditions (Bills and Polishook, 1994; Bills, 1995).
Basidiomycetes in particular are poorly sampled using standard techniques, but
are known from (very labour-intensive) visual studies of mycelia to comprise a
major component of soil fungal biomass (Frankland, 1982).
There has been considerable interest in recent years in rapid diversity
estimates which use trained technicians rather than taxonomic specialists
(Beattie and Oliver, 1994). Although in such cases identifications to species
can rarely be provided, species numbers and some basic impressions of
systematic diversity are obtainable. Oliver and Beattie (1993) achieved
diversity assessments of spiders, ants, polychaetes and mosses using
biodiversity technicians with only three hours training which were comparable
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Fungal Diversity 2 (March 1999)
to those made by specialist taxonomists. Fungi present greater challenges due
to their less well-defined morphological features, but in all probability
acceptable results could be obtained from technical staff after a short training
period. Such approaches are tempting, as reference collections and up-to-date
libraries are inaccessible to many scientists (especially in developing
countries), and the number of specialist taxonomists is now far too small to
provide such services. The major drawback to these strategies is that
communities cannot effectively be compared, as the number of species
common to pairs of sites cannot be calculated. This has major implications for
conservation policy, as in many cases the trigger for protection is the presence
of rare species rather than the overall number recorded. For this reason,
diversity analysis without direct reference to species identification is of limited
value, unless it is very rapid indeed.
The most effective approach to fungal diversity assessment is therefore to
make careful studies of very tightly defined samples. Our patchy knowledge of
fungal distribution patterns and the environmental factors which determine
them makes meaningful comparison of samples or sample sites almost
impossible unless the degrees of freedom are minimized. Comparing species
lists from standard collecting trips is of very limited value without an
exhaustive survey of the differences in climatic conditions and current and
preceding local weather patterns, soil type, aspect, plant cover and composition
etc. The most easily defined and compared samples are small items such as
individual leaves or soil samples. Even here, account must be taken of factors
such as the degree of decomposition of the plant part surveyed, or the depth
below the surface of soil samples.
There are four main categories of protocols for fungal diversity assessment
and analysis. These are direct observational methods, cultural procedures,
'direct molecular analysis and association methods. The aim for all is to restrict
the amount of sampling and analysis time, while maintaining the comparability
of results.
Direct observation
Direct observation
has been the traditional method of choice for most
analyses of fungal communities. Direct field observations are problematic in
most circumstances, as the large majority of fungi are small, inconspicuous,
and ephemeral in their appearance. Their detection is critically dependent on
the skill of the observer, and even sampling by specialists may have very
different results depending on minor, often unconcious, differences in
procedure and ability. Exceptions might include many groups of lichenized
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fungi which have perennial and often conspicuous thalli, for which surveys of
particular rock or tree types can be relatively simple to standardize.
The large basidiomycetes are currently difficult to sample indirectly. Due to
their often short-lived and strongly seasonal fruit-bodies there is no easy way to
ensure consistency of sampling except over small geographical scales, and little
can be done other than to make comprehensive notes of environmental and
other criteria, and completing comparative sampling programmes within as
short a timescale as possible. Sampling is confined to fruiting specimens,
which may give a distorted picture of overall basidiomycete diversity. There is
little 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 assessments
compared with those obtained from long-term studies. The same team (Schmit,
Murphy and Mueller, 1998) examined the effort required to obtain a good
estimate of total species richness, and found that sampling a single plot for
several years resulted in higher species numbers than studying two plots in the
same year. This demonstrates the difficulties in obtaining acceptable measures
of basidiomycete diversity using fruit bodies as samples.
A more effective protocol for direct observation is to collect samples and
observe them periodically in damp chambers. In this way, the samples (such as
individual leaves or pieces of wood from the same plant species) can be easily
defined, the effect of climatic differences on sample episodes can be
minimized, and account can be taken of seasonal and successional effects. For
obvious 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 not
developing or remaining sterile. However, other species will produce spores in
these circumstances and thus be recognizable, but fail to grow or sporulate in
culture. The overall diversity obtainable from direct observational techniques
versus culture is significantly lower at least in some circumstances (Bills, pers.
comm.), but the results are not necessarily less comparable. The most extensive
survey of this type published is that of Rambelli et al. (1983), who carried out a
large-scale study of the effect of disturbance on forest ecosystems in the Ivory
Coast.
Cultural procedures
Culture of fungi from samples such as soil, litter and plant parts can result in
a comprehensive and comparable estimate of diversity, although the protocols
are 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 spores
and mycelium are then transferred to nutrient agar to allow colony
development in isolation. Petri dishes containing too much inoculum will result
in overgrowth of fastidious species by ruderal taxa, and modern methods use
low-nutrient agar incubated at low temperatures and with antifungal additives
such as cYclosporin to minimize competition between developing colonies. The
dilution plate protocol for soil samples also ensures an appropriate
concentration of inoculum. Other methods include plating out subsamples of
leaves (especially for endophyte work) made with cork-borers or by slicing
them with a sharp knife. Species numbers obtained by these methods may be
large; 220 species were isolated from wheat field soil in Germany (Gams and
Domsch, 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 in
non-exhaustive surveys of small litter samples from Costa Rica, and their own
evidence that sampling processes were incomplete was reinforced by Lodge
and Cantrell (1995), who re-analyzed their data and found only a 15-28 %
overlap of species between sites.
Direct molecular diversity analysis
The most innovative procedures for biodiversity analysis of the environment
are based on analysis of ribosomal DNA (rDNA) which is extracted directly
from samples without prior culture. The techniques have rapidly become
established for bacteria, but have not yet been widely used for study of
eukaryotic 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 in
soil. Such approaches have only become feasible for fungi as data sets increase
following the design of specific oligonucleotide primers directed to conserved
regions across the known taxa. Applications are numerous, including effective
and timely comparisons of management practices, charting the effects of
external perturbations, and assessment of overall ecosystem health. The
preferred processes involve the extraction of total DNA from environmental
samples, PCR-mediated amplification of fungal sequences using universal
primers, and separation of the sequences using DGGE (density-gradient gel
electrophoresis) or TGGE (temperature-gradient gel electrophoresis). This
process 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 to
obtain identifications.
Though many problems remain to be properly resolved, there have been a
small number of attempts to apply this technology to fungal analysis.
Kowalchuk, Gerards and Woldendorp (1997) applied these protocols to the
study of fungal diversity within Ammophila roots. They were able to extract
ribosomal DNA from a number of fungal species, some of which could be
identified by sequence comparison with cultured taxa. Others did not closely
match sequences in published rDNA databases. Their work is most interesting,
but requires development if such techniques are to be used effectively for
diversity estimation rather than detection of particular species. The community
structure within single root pieces is much simpler than, for example, soil
samples, and DNA extraction procedures for root material are relatively
problem-free compared with those for more heterogenous environments. Only
one to four fungal species were detected from each sample. The only other
study to date is that of Pine, Dawson and Pace (1998), who presented
preliminary results of the fungi in anoxic contaminated soil and sediment
samples. Here also the number of species detected was low, although fungal
diversity within anoxic environments would be expected to be restricted. They
isolated 14 different environmental sequences which clustered within diverse
fungal lineages. It is not clear whether the samples were of actively growing
fungi, or derived from spores which were present but dormant.
A central issue in the analysis of diversity is whether the very large
estimates of bacterial diversity obtained by DNA-based techniques compared
with traditional cultural methods (e.g. Torsvik, Goks0yr and Daae, 1990a;
Torsvik et al., 1990b; Hopkins, MacNaughton and O'Donnell, 1993; Bomeman
et al., 1996) are reflected in other organism groups. Current predictions are that
this extreme imbalance in diversity estimation will not be encountered to the
same degree for fungi, although many species are difficult or impossible to
culture using current technologies. The main evidence which leads to this
hypothesis is that fungal structures are far larger than bacteria and show more
morphological variation, and are thus more likely to be detected using
traditional methods.
A major restraint to the development of DNA-based diversity assessments
of fungal populations is the large number of gaps in published databases. rDNA
sequences of many pathogenic taxa (both human and plant) are known, but
saprobic species are very under-represented. Any major programme to develop
DNA-based diversity protocols for fungi must therefore be accompanied by
sequence generation based on traditional cultural methods, and/or direct
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Fungal Diversity 2 (March 1999)
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 studied
sections of the ribosomal genome are the small subunit (18S) and ITS regions.
18S is generally considered to resolve taxa at higher levels of the taxonomic
hierarchy than ITS rDNA, and has more commonly been used for phylogenetic
studies, while ITS is typically used for species distinction and diagnosis (White
et al., 1990; Bruns, White and Taylor, 1991). The choice of region for different
purposes appears not always to have been made rigorously, and may relate to
differing species concepts within the major fungal lineages. Berbee et al.
(1998) have discovered differences in the sequence length needed to
distinguish groups within the major lineages of the Ascomycota, and Carbone
and Kohn (1998) have found similar variation during their work on primer
design. Some of the most commonly used primers actually amplify DNA from
both regions. Useful phylogenetic information can be obtained from ITS
sequences (Mugnier, 1998), sequence diversity is likely to be greater in
absolute terms, and the region is less subject to introns. Fungal 18S rDNA
frequently contains insertion sites (17 are known) which have been reported to
contain 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 are
described only as allowing selective amplification of fungal DNA. While their
use by subsequent researchers has been widespread and some published
sequences 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 organisms
for which amplification will also occur. This is not an issue for fungal
phylogeny studies where sequences from specific strains are amplified, but it is
clearly important for ecosystem community analyses where mixtures of DNA
from a wide variety of organisms are encountered. Primer specificity is
currently also a greater concern for fungal studies compared with those of
bacteria, as in comparison prokaryote rDNA sequences are distinctive and
comprehensively researched (the proportion of known species for which 16S
rDNA has been sequenced is orders of magnitude greater than for the fungal
equivalent, 18S rDNA), although the increasing prominence in research
programmes of major lineages such as the Archaea will no doubt result in
9
modifications to primer design. It is unlikely that universal fungal rDNA
primers can be developed which do not amplify DNA from any other
organisms, but so long as we are aware of their limitations, they can be used at
least for rapid assessment of organismal diversity with a strong bias towards
fungi. Such measures would in themselves be novel and valuable.
Molecular diversity analyses using current technology will not solve all
problems. PCR is an inherently competitive process which favours the
amplification of common over rare sequences (Reysenbach et al., 1992;
Farrelly, Rainey and Stackebrandt, 1995), and stringent conditions must be
employed (especially for DNA concentration) to maximise the diversity of
sequences amplified. The overall genome size also has a significant impact on
the efficiency of amplification, and work on bacteria suggests that the
amplification process may select against certain groups of bacteria in complex
samples (Ward et al., 1994). Imperfections of amplification may also produce
phantom 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 not
well suited to analysis of complex DNA mixtures due to the small size of the
gels they accommodate. The distance travelled by the DNA fragments before
they denature is not related to their systematic affinities, and identification of
individual band sequences is currently problematic due to deficiencies in the
published molecular databases. Despite all these limitations, the methods show
great promise, and are the only technologies with realistic potential for really
rapid diversity analysis.
Association methods
Correlations of incidence, abundance and diversity of fungi with other
organisms or features of natural environments may prove to be effective
vehicles for decision-making, when conservation or monitoring of fungi is
needed. In practice, if fungal diversity can be linked to other measurements
such as plant diversity, vegetational type or architecture, climatic factors etc, it
should be possible to predict rough species numbers and systematic
composition for particular sites which may be under threat. This approach
could be combined with monitoring of indicator species, or taxa which are
particularly valued such as edible mushrooms or fungi known to be rare. The
drawbacks of such measures are that initial calibration of the system will be
very time-consuming, and the degree of accuracy of predicted species numbers
will also be expensive to measure.
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Fungal Diversity 2 (March 1999)
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 that
if such systems are to be useful, complex equations may have to be developed
which include meteorological and geological measurements as well as species
numbers of other groups. This ties in with the suggestions of May (1991), who
suggested that fungal diversity in the tropics might be more closely allied to the
physical variety of tree habitats and the pattern of plant species distribution,
than to the number of plant species present. Similar suggestions were made by
Huston (1994), who predicted that the diversity of saprobic organisms should
be highest where plant production was greatest, irrespective of its diversity.
These approaches may be most appropriate for monitoring and prediction of
population levels for particularly valued species, as has been explored by
Peterson and McCune (1998) for Caliciales in Pacific Northwest old-growth
forests.
Conclusions
Mycologists must come to terms with the fact that for the forseeable future,
the available human and financial resources will be inadequate for
comprehensive survey of fungi in natural ecosystems. Nevertheless, if fungi are
to take their place within conservation strategies, the tools for assessing their
presence and diversity must be provided. There is a clear need to make
available, at a minimum, indications of species numbers and systematic profile
for sites of interest. There is also a need to develop methods for fungal
diversity estimation and species monitoring which do not require sustained
inputs from experienced specialists. The number of these is now completely
inadequate for survey work even in developed countries.
The various methods for diversity assessment described above and in
Cannon (1997) vary considerably in their cost, reliability and technical
demands. Considerable advances may be possible in traditional diversity
estimation methods involving analysis of tightly defined samples such as
individual plant leaves or twigs, either using culture or damp-chamber
protocols. The potential for association techniques may be very considerable,
but the start-up costs could only be met by a coordinated research programme
involving many different disciplines. Molecular methods need much further
development, but these are the only known options for truly rapid diversity
assessment and monitoring.
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Acknowledgements
This 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 like
to 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.
References
Anderson, lP.E. and Domsch, K.H. (1978). A physiological method for the quantitative
measurement of microbial biomass in soils. Soil Biology and Biochemistry 10: 215-221.
Beattie, AJ. and Oliver, I. (1994). Taxonomic minimalism. Trends in Ecology and Evolution
9: 488-490.
Berbee, M.L., Carmean, D., Winka, K. and Eriksson, O. (1998). Phylogenetic resolution and
the radiations of the Ascomycota [abstract]. Inoculum 49: 8.
Bills, G.F. (1995). Analyses of microfungal diversity from a user's perspective. Canadian
Journal of Botany 73 (supplement I): 833-841.
Bills, G.F. andChristensen, M. (In press). Evaluating diversity of soil fungi. In: Measuring and
Monitoring Biological Diversity. Standard Methods for Fungi (eds. G. Mueller, G.F. Bills,
HA
Burdsall and
AY
Rossman). Smithsonian University Press, Washington and London.
Bills, G.F. and Polishook, J.D. (1994). Abundance and diversity of microfungi in leaf litter of a
lowland rain forest in Costa Rica. Mycologia 86: 187-198.
Borneman, l, Skroch, P.W., Jansen, lA., O'Sullivan, K.L., Palus, lA., Rumjanek, N.G.,
Nienhuis, land
Triplett, E.W. (1996). Microbial diversity of an agricultural soil in
Wisconsin. Applied and Environmental Microbiology 62: 1935-1943.
Brasier, C.M. (1997). Fungal species in practice: identifying species units in fungi. In: Species:
The Units of Biodiversity (eds. M.F. Claridge, H.A. Dawah and M.R. Wilson). Chapman
and Hall, London etc.: 135-170.
Bruns, T.D., White, TJ. and Taylor, J.W. (1991). Fungal molecular systematics. Annual
Review of Ecology and Systematics 22: 525-564.
Cannon, P.F. (1997). Strategies for rapid assessment of fungal diversity. Biodiversity and
Conservation 6: 669-680.
Cannon, P.F. and Hawksworth, D.L. (1995). The diversity of fungi associated with vascular
plants: the known, the unknown and the need to bridge the knowledge gap. Advances in
Plant Pathology 11: 277-302.
Carbone, 1. and Kohn, L.M. (1998). A strategy for designing primer sets for speciation studies
[abstract]. Inoculum 49: 12.
Christensen, M. (1989). A view offungal ecology. Mycologia 81: 1-19.
Domsch, K.H. (1975). Distribution of soil fungi. In: Proceedings of the First International
Congress lAMS (ed. T. Hasegawa). 2: 340-353.
Faegri, A., Lid-Torsvik, V. and Goks0yr, l (1977). Bacterial and fungal activities in soil:
separation of bacteria and fungi by a rapid fractionated centrifugation technique. Soil
Biology and Biochemistry 9: 105-112.
Farrelly, V., Rainey, F.A. and Stackebrandt, E. (1995). Effect of genome size and rrn gene
copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species.
Applied and Environmental Microbiology 61: 2798-2801.
12
Fungal Diversity 2 (March 1999)
Finlay, B.1., Maberly, S.C. and Cooper,
function. Oikos 80: 209-213.
l1. (1997). Microbial
diversity
and ecosystem
Frankland, le. (1982). Biomass and nutrient recycling by decomposer basidiomycetes. In:
Decomposer Basidiomycetes (eds. lC. Frankland, IN. Hedger and M.1. Swift). 241-261.
Gams, W. and Domsch, K.H. (1969). The spatial and seasonal distribution of microscopic
fungi in arable soils. Transactions of the British Mycological Society 52: 301-308.
Gardes, M. and Bruns, T.D. (1993). ITS primers with enhanced specificity for basidiomycetes
- application to the identification of mycorrhizae and smuts. Molecular Ecology 2: 113118.
Gargas, A., DePriest, P.T. and Taylor, l.W. (1995). Positions of multiple insertions in SSU
rDNA of lichen-forming fungi. Molecular Biology and Evolution 12: 208-218.
Gargas, A. and Taylor, lW. (1992). Polymerase chain reaction (PCR) primers for amplifying
and sequencing nuclear 18S rDNA from lichenized fungi. Mycologia 84: 589-592.
Gaston, K.1. (1992). Regional numbers of insect and plant species. Functional Ecology 6: 243247.
Hammond, P.M. (1994a). Practical approaches to the estimation of the extent ofbiodiversity in
speciose groups. Philosophical Transactions of the Royal Society of London, Biological
Sciences series B, 345 (no. 1311): 119-136.
Hammond, P .M. (1994b). Described and estimated species numbers: an objective assessment
of current knowledge. In: Microbial Diversity and Ecosystem Function (eds. D. Allsopp,
R.R. Colwell and D.L. Hawksworth). CAB International, Wallingford, UK: 29-71.
Hammond, P.M. (1995). Magnitude and distribution of biodiversity. In: Global Biodiversity
Assessment (ed. V.H. Heywood). Cambridge University Press, Cambridge, UK: 107-191.
Hawksworth, D.L. (1991). The fungal dimension ofbiodiversity: magnitude, significance, and
conservation. Mycological Research 95: 641-655.
Hopkins, D.W., MacNaughton, S.l and O'Donnell, A.G. (1993). A dispersion and differential
centrifugation technique for representatively sampling microorganisms. Soil Biology and
Biochemistry 23: 217-225.
Hughes, T., Rogers, T.R. and Haynes, K. (1998). PCR diagnostics in medical mycology. In:
Applications of peR in Mycology (eds. P.D. Bridge, D.K. Arora, C.A. Reddy and R.P.
Elander). CAB International, Wallingford, UK.
Huston, M.A. (1994). Biological Diversity. The Coexistence of Species on Changing
Landscapes. Cambridge University Press, Cambridge, UK.
Jones, T.R, Thompson, L.1., Lawton, lH., Bezemer, T.M., Bardgett, R.D., Blackburn, T.M.,
Bruce, K.D., Cannon, P.F., Hall, G.S., Howson, G., lones, e.G., Kampichler, C., Kandeler,
E. and Ritchie, D.A. (1998). Impacts of rising CO2 on soil biota and processes in terrestrial
ecosystems. Science 280: 441-443.
Kjoller, A. and Struwe, S. (1982). Microfungi in ecosystems: fungal occurrence and acitivity in
litter and soil. Oikos 39: 391-422.
Kowalchuk, G.A., Gerards, S. and Woldendorp, lW. (1997). Detection and characterization of
fungal infections of Ammophila arenaria (marram grass) roots by denaturing gradient gel
electrophoresis
of specifically amplified 18S rDNA. Applied and Environmental
Microbiology 63: 3858-3865.
Lawton, lH. and Brown, V.K. (1993). Redundancy in ecosystems. In: Biodiversity and
Ecosystem Function (eds. E.D. Schulze and RA. Mooney). 255-270.
Lodge, DJ. and Cantrell, S. (1995). Fungal communities in wet tropical forests: variation in
time and space. Canadian Journal of Botany 73 (supplement I): S 1391-S 1398.
13
Lodge, 0.1., Fisher, P.1. and Sutton, RC. (1996). Endophytic fungi of Manilkara bidentata
leaves in Puerto Rico. Mycologia 88: 733-738.
Mahoney, D.P. (1972). Soil and Litter Fungi of the Galapagos Islands. Ph.D. thesis. University
of Wisconsin.
May, R.M. (1991). A fondness for fungi. Nature 352: 475-476.
Mueller, G.M., Leacock, P.R. and Murphy, J.F. (1998). Rapid methods for assessing the
diversity and distribution of macro fungi [abstract]. Inoculum 49: 38.
Mugnier, 1. (1994). PCR Catalogue. Fungi. Rhone-Poulenc Agro, Lyon.
Mugnier, 1. (1998). Molecular evolution and phylogenetic implications of ITS sequences in
fungi and plants. In: Molecular Variability of Fungal Pathogens (eds. P.D.Bridge,
Y.
Couteaudier and 1.M. Clarkson). CAB International, Wallingford, UK: 253-277.
Oliver, 1. and Beattie, AJ. (1993). A possible method for the rapid assessment of biodiversity.
Conservation Biology 7: 562-568.
Persiani, A.M., Maggi, 0., Casado, MA and Pineda, F.D. (1998). Diversity and variability in
soil fungi from a disturbed tropical rain forest. Mycologia 90: 206-214.
Peterson, B.B. and McCune, B. (1998). Predicting community composition
with a
phenomenological model based on direct gradient analysis [abstract]. Inoculum 49: 61.
Pine, E.M., Dawson, S.C. and Pace, N.R. (1998). Fungal diversity of two anoxic environments
assessed by sequencing of 18S rRNA from total environmental DNA [abstract]. Inoculum
49: 42.
Prendergast, 1.R., Quinn, KM., Lawton, 1.H., Eversham, RC. and Gibbons, D.W. (1993). Rare
species, the coincidence of diversity hots pots and conservation strategies. Nature 365: 335337.
Rambelli, A., Persiani, A.M., Maggi, 0., Lunghini, D., Onofri, S., Riess, S., Dowgiallo, G. and
Puppi, G. (1983). Comparative Studies on Microfungi in Tropical Ecosystems. UNESCO,
Rome.
Reysenbach, A.L., Giver, LJ., Wickham, G.S. and Pace, N.R. (1992). Differential
amplification of rRNA genes by polymerase chain reaction. Applied and Environmental
Microbiology 58: 3417-3418.
Rossman, A.Y. (1994). A strategy for an all-taxa inventory of fungal biodiversity. In:
Biodiversity and Terrestrial Ecosystems (eds. C.l. Peng and C.H. Chou). Academia Sinica
Monograph Series no 14. Academia Sinica, Taipei, Taiwan: 169-194.
Rossman, AY, Tulloss, R.E., O'Dell, T. and Thorn, R.G. (1998). Protocols for an All Taxa
Biodiversity Inventory of Fungi in a Costa Rican Conservation Area. Parkway, Boone,
North Carolina.
Sampo, S., Bergero, R., Buffa, G. and Luppi-Mosca, A.M. (1997). Soil fungal communities in
a young and an old Alnus viridis coenosis. Mycologia 89: 837-845.
Schmit, 1.P., Murphy, 1.F. and Mueller, G.M. (1998). Macrofungal diversity in a temperate oak
forest [abstract]. Inoculum 49: 47.
Shaw, PJ.A. (1992). Fungi, fungivores and fungal food webs. In: The Fungal Community. Its
Organization and Role in the Ecosystem (eds. G.c. Carroll and D.T. Wicklow). 295-310.
States, J.S. (1978). Soil fungi of cool-desert plant communities in northern Arizona and
southern Utah. Journal of the Arizona-Nevada Academy of Science 13: 13-17.
Torsvik, V., Goksoyr, 1. and Daae, F.L. (1990a). High diversity in DNA of soil bacteria.
Applied and Environmental Microbiology 56: 782-787.
14
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Fungal Diversity 2 (March 1999)
Torsvik, V., Salte, K., Sorheim, R. and Goks0yr, 1. (1990b). Comparison of phenotypic
diversity and DNA heterogeneity in a population of soil bacteria. Applied and
Environmental
Walker,
Microbiology 56: 776·781.
RH. (1992). Biodiversity and ecological redundancy. Conservation Biology 6: 18·23.
Wang, G.c.Y. and Wang, Y. (1996). The frequency of chimeric molecules as a consequence of
PCR co-amplification
142: 1107·1114.
of 168 rRNA genes from different bacterial species. Microbiology
Ward, N., Rainey, F.A., Goebel, B. and Stackebrandt, E. (1994). Identifying and culturing the
"unculturables": a challenge for microbiologists. In: Microbial Diversity and Ecosystem
Function (eds. D. AIIsopp, R.R. Colwell, R.R. and D.L. Hawksworth). CAB International,
Wallingford, UK: 89-110.
White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990). Amplification and direct sequencing of
fungal ribosomal RNA genes for phylogenetics. In: peR Protocols: a Guide to Methods
and Applications (eds. MA Innis, D.H. Gelfand, U. Sninsky and T.J. White). 315-322.
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