Fungal Diversity Revisited: 2.2 to 3.8 Million Species

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Fungal Diversity Revisited:
2.2 to 3.8 Million Species 4
David L. Hawksworth
1
and Robert Lu
¨cking
2
BACKGROUND
In 1825, Elias Magnus Fries (1794–1878) predicted that
the fungi would prove to be the largest group in the
vegetable kingdom, analogous to the insects in the ani-
mal kingdom. Notwithstanding that fungi are not actu-
ally part of the plant kingdom, how right he has proved
to be as the bicentenary of his prediction approaches.
By the 1960s a few mycologists were speculating that
there might be as many fungal as plant species, but
almost no attempts to calculate estimates from the
available data were made. As concern over the conser-
vation of biodiversity in general grew in the subsequent
decades, culminating in the signing of the Convention
on Biological Diversity in 1992, more precise figures
on species numbers of all kinds of organisms were
required. A series of estimates of the number of fungi
settled on figures ranging from 500,000 to almost 10
million species, with 1.5 to perhaps 5 million receiving
most support among mycologists. A recent study even
predicts up to a trillion species of microorganisms glob-
ally (1); how many of these are supposed to be fungi is
not specified, but if this estimate holds true and only
1% of these were fungi, the global estimate of fungal
diversity would be a thousand times higher than the
current highest estimate of 10 million species.
Different extrapolation techniques have been used to
arrive at global fungal species richness estimates, in-
cluding publication rates of new taxa (2), plant:fungus
ratios (3, 4) similar to plant:insect ratios first used in
Erwin’s famous study (5), quantitative macroecological
grid-based approaches (6–8), methods based on envi-
ronmental sequence data including plant:fungus ratios
(9, 10), and ecological scaling laws (1).
This article provides updated background informa-
tion on the number of described species and explores
new evidence obtained from rates of species discovery,
extrapolations from fungus:plant ratios, and molecular
sequence data from environmental samples. We con-
clude by indicating where undescribed species are likely
to be found and recommend a working number suit-
able for general use in estimates of global and regional
species richness.
We focus here on the numbers of species in the king-
dom Fungi, that is, inclusive of the Cryptomycota and
Microsporidia, and also including lichen-forming fungi.
We do not, however, consider other organisms that my-
cologists study as fungi, such as the fungal analogues
Mycetozoa (as an unranked supergroup or in the king-
dom Protozoa, phylum Amoebozoa) and Oomycota
and Hyphochytriomycota (in the kingdom Heterokonta
=Straminipila). However, we caution that some data
sets and papers discussed in the following sections
are based on fungi in the broad sense and thus include
information from the analogue phyla. While readers
should bear this in mind, the numbers of known taxa in
these groups are relatively modest, so their occasional
unavoidable inclusion will not materially affect the in-
terpretations presented here.
EVIDENCE FROM PUBLICATION
RATES OF NEW TAXA
Numbers of Described Species
The number of new species described in each decade,
as recorded in the Index Fungorum database (http://
www.indexfungorum.org), shows three distinct phases
(Fig. 1): an ascending phase in which progress was fast
in the 1750s to 1860s, a steep phase as microscopy
came into general use and there was intensive collecting
1
Department of Life Sciences, The Natural History Museum, London SW7 5BD, United Kingdom, and Comparative Plant and Fungal Biology,
Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, United Kingdom;
2
Botanischer Garten und Botanisches Museum, Freie Universita
¨t
Berlin, 14195 Berlin, Germany.
The Fungal Kingdom
Edited by J. Heitman, B. J. Howlett, P. W. Crous, E. H. Stukenbrock, T. Y. James, and N. A. R. Gow
©2018 American Society for Microbiology, Washington, DC
doi:10.1128/microbiolspec.FUNK-0052-2016
79

in hitherto barely explored parts of the world in the
1870s to 1880s, and then a relatively constant phase
from the 1890s to the present day. The partially higher
figures in the first decades of the “constant phase” re-
flect continuing exploration and a number of prolific
individual mycologists. The rate of description over the
past 40 years has, however, remained fairly constant at
around 1,300 per year (Fig. 2), with peaks generally re-
lating to particular major monographic works.
Of special note is the somewhat steep increase in the
rate since 2010 to around 1,800 per year. This resur-
gence in species description is largely attributed to the
increasing use of molecular techniques for species de-
limitation and is of particular note because this increase
followed, and even accelerated after, the ending of the
separate naming of morphs of the same species in 2011
(when the provision for a Latin diagnosis or description
was also eliminated). Prior to that date, the totals for
Figure 1 Numbers of newly introduced species names of fungi for each decade from 1750
to 2010. Based on data from the Index Fungorum database provided by P. M. Kirk.
Figure 2 Numbers of newly introduced species names of fungi for each year from 1975 to
2015. Note that the data for 2015 were incomplete when this work went to press. Based on
data from the Index Fungorum database provided by P. M. Kirk.
80 FUNGAL BRANCHES ON THE EUKARYOTIC TREE OF LIFE

annually described “species” included separate names
given to different morphs of the same species.
The number of newly named species does not, how-
ever, equal the number that have been truly described
for the first time. It is much easier to name a fungus as
new to science rather than to ascertain that it has previ-
ously been named. This is not simply a matter of com-
paring material to that of previously recognized species
in the same genus but requires meticulous detective
work, because not uncommonly, species were described
in genera they do not belong to. Further, species
concepts change, for example, in genera where mor-
phologically identical fungi were regarded as different
species if they occurred on different plant species. The
number of accepted fungi can only be obtained by
counting those recognized as “good” in each genus.
This has been done since 1943 through the 10 editions
of Ainsworth & Bisby’s Dictionary of the Fungi and
now annually through Species Fungorum inputs to the
Catalogue of Life (http://www.catalogueoflife.org/an-
nual-checklist/). An analysis of these data shows that
the total number of existing species names runs at
around 2 to 3 times that of currently accepted species
(Fig. 3), a figure consistent with the ratio of 2.6 derived
from an analysis of names in monographic works (11).
The number of described good fungal species currently
stands at around 120,000.
Unlike the situation in many other groups of orga-
nisms (12), the number of good fungal species continues
to rise steeply decade by decade, with no indication of
leveling off. The steepness of the curve would be even
greater if there were more mycologists actively involved
in species discovery, using either morphological or mo-
lecular approaches.
The number of species being recognized is neces-
sarily impacted by the species concepts used. While in
the premolecular era, species circumscriptions were
based almost entirely on morphological and sometimes
biochemical and cultural features, the incorporation
of molecular information has led to a refining of spe-
cies concepts. There is, however, a plethora of species
concepts in biology (13, 14) and no single objective cri-
terion that can be applied universally. Because the pur-
pose of names is to provide a means of communication,
a pragmatic species concept is essential, which may
be defined as the following: “a species is what it is use-
ful to give a species name to.” (15). There have been
numerous attempts to apply mechanistic systems for
species recognition in phylogenetic trees, but the con-
solidated species concept has much to commend it and
ultimately provides a broad consensus on how to rec-
ognize fungal species. The consolidated species concept
adopts a polyphasic approach combining morphologi-
cal, ecological, and phylogenetic species concepts (16)
and is being increasingly taken up by mycologists. While
in some situations this may lead to more species being
recognized than otherwise would have been the case,
in others it results in formerly separately distinguished
Figure 3 Growth in the total catalogued number of species names of fungi by decade from
1750 compared with the global number of accepted species. Based on figures adopted in
the 10 editions of Ainsworth & Bisby’s Dictionary of the Fungi for 1943–2008 and data
in the Index Fungorum and Species Fungorum (Catalogue of Life) databases provided by
P. M. Kirk.
4. FUNGAL DIVERSITY REVISITED 81

species being united. Therefore, over- and underestima-
tions in particular cases are usually balanced out. A
rarely considered notion is the time scale: a proportion
of “species” is in active speciation, with incomplete line-
age sorting, yielding it difficult to resolve good species
(17); in such cases, determining the number of species
is philosophical, since that number is in active flux. A
large number of new fungal species continue to be de-
scribed with no molecular data, either due to the age
of the underlying material (e.g., unveiled in older col-
lections) or by taxonomists who do not have access to
molecular techniques; assessing the proportion of good
species in such taxa is therefore difficult.
Patterns in Taxon Discovery
Some predictable patterns in the recognition of pub-
lished taxa in the rank of genus and above have been
recognized and applied to a broad set of organisms,
including fungi (2). By combining the full classifica-
tions of around 1.2 million species of all groups of
organisms, these authors found, as might have been
expected, that accumulation curves of higher taxa over
time showed that these were more completely described
and in many cases approached an asymptote. The use
of the higher taxon approach was then validated by
comparing the actual number of known species with
that predicted from the accumulation curves. The au-
thors then fitted asymptotic regression models to the
actual curves for different groups to provide estimates
of undiscovered species numbers. In the case of fungi,
this led to a total number of expected fungal species of
611,000 (standard error ± 297,000). This conclusion
was, however, misleading, because the analysis was
based on the assumption that there were only 43,271
catalogued fungal species, rather than the nearly
100,000 accepted at that time (18). Intriguingly, a re-
calculation using their method with the latter species
number generates a figure of around 1,400,000—not
far out of line with estimates derived from extrapola-
tions based on plant:fungus ratios (see below).
EVIDENCE FROM SPECIES
RECOGNITION STUDIES
Unknown fungi are now considered to come mostly
from two sources: (i) newly discovered species by
means of traditional inventory methods in little-studied
areas and habitats and (ii) newly discovered lineages
through environmental sequencing. A further, neglected
but significant source of unrecognized fungal diversity
is the restudy of known taxa, applying species recogni-
tion techniques to molecular data, using either the fun-
gal barcoding locus ITS (19) or multilocus approaches.
Exemplar taxa are the fly agaric, Amanita muscaria,
and the golden chanterelle, Cantharellus cibarius, which
contain numerous, often regionally distributed species
(20, 21). The record is currently set by the basidio-
lichen fungus Cora glabrata, which has been shown
to contain at least 189 species (6, 22). We surveyed
45 such studies published between 1998 and 2016,
including studies of non-lichenized and lichenized
forms, and computed the ratio of species recognized in
the target group before and after the study (Table 1).
Most studies started out with a single or few species,
whereas two looked at complexes of up to 18 species—
Protoparmelia (23) and Usnea (24)—and one revised a
complex of 71 species accepted prior to the study, in the
genus Coprinellus (25). The posterior:prior ratios in the
number of species recognized ranged from 0.67 (from
18 to 12 species in a group of Usnea [24]) to 189 (from
1 to 189 species in the basidiolichen Cora glabrata)(6,
22). Other high ratios were found for Dictyonema with
59 (26), Cladosporium cladosporioides with 22 (27),
Sticta weigelii with 15 (28), Microbotryum violaceum
with 11 (29), and Fusarium graminearum with 7 (30).
Environmental sequence and genomic data revealed
high ratios for the genera Archaeorhizomyces, with 145
(31), and Cyphobasidium with 26 (32), as well as for
the still barely explored Cryptomycota, with 138 (33,
34), in which the true figure could be much higher. The
median ratio over all evaluated studies was 3.0, and the
arithmetic mean was 14.5 (Table 1).
To obtain a more reliable picture, we adjusted these
ratios by geographic area covered. If a species complex
had a global distribution prior to the study, and if based
on material from a single geographic area, it was found
to represent several species with presumably restricted
distribution, the detected ratio was adjusted by the fac-
tor 2 (expanding to both hemispheres in cases in which
one hemisphere was covered), the factor 3 (for tropical
fungi for which a single tropical region was covered),
and up to the factor 6 (for cosmopolitan fungi for
which a single region within a single continent was
studied). These factors were derived from selected glob-
al studies, for example, in the genera Cladosporium
(27), Cora (6, 22), Fusarium (30), and Sticta (28). In
some cases, we added unpublished data to the adjusted
values; thus, for Archaeorhizomyces we currently rec-
ognize at least 500 species (B. E. Smith and R. Lu
¨cking,
unpublished data), and for Cyphobasidium, we recog-
nize about 100 species from deposited GenBank se-
quences (32). The thus adjusted ratios ranged from 0.7
(Usnea) to 435 (Archaeorhizomyces), with a median
value of 10.5 and an arithmetic mean of 39.3.
82 FUNGAL BRANCHES ON THE EUKARYOTIC TREE OF LIFE

To predict the global species richness of Fungi using
these raw and adjusted ratios, we employed two further
adjustments. First, we weighted the ratios according
to the number of species recognized in a genus in the
2008 Dictionary of the Fungi (18). For instance, a ge-
nus in which a single species had been recognized re-
ceived a low weight (1.0), since even a high number
of newly recognized species will only contribute mini-
mally to global fungal species richness. This adjustment
avoided a high impact of extraordinary ratios such as
in the previously monotypic genus Cora. In contrast,
for groups in which a large number of species had al-
ready been recognized, the ratios based on a study of
one or a few species were upweighted, e.g., in the genus
Puccinia (35), with 4,000 accepted species. In addition,
to account for ecological bias in the evaluated studies,
we applied a group weight according to the major life-
style attributed to each genus; to that end, we divided
the approximately 100,000 known fungi in 2008 as fol-
lows: 40,000 saprotrophic (weight 4.0), 20,000 phyto-
pathogenic (weight 2.0), 20,000 lichenized (weight
2.0), 8,000 ectomycorrhizal (weight 0.8), 5,000 endo-
phytic (weight 0.5), 3,000 entomopathogenic (weight
0.2), 2,000 aquatic (weight 0.2), 500 endomycorrhizal
(weight 0.05), 500 marine (weight 0.05), 400 soil
(weight 0.04), and 100 human pathogenic (weight 0.01).
The categories aquatic, marine, and soil are used here
for cases where the habitat is known but not the biology.
These proportions denote estimates based on known
fungi in 2008, not estimated proportions of extrapolated
global species richness, in which, presumably, categories
such as plant pathogens and soil fungi will have much
higher values. Using these weights, the overall arithmetic
mean of the posterior:prior ratio for all evaluated studies
amounted to 11.3. This suggests that even without in-
cluding important sources of new species discoveries,
the already accepted Fungi may contain up to 10 times
as many species as currently recognized, resulting in
more than 1 million estimated species. Remarkably, this
is about 4 times higher than the currently estimated syn-
onymy ratio of 2.6, suggesting that a large proportion
of these unrecognized species have no names available.
EVIDENCE FROM EXTRAPOLATIONS
BASED ON PLANT:FUNGUS RATIOS
The widely cited and accepted plant:fungus ratio-
derived global estimate of 1.5 million species of fungi
was based on information from several independent
sources (3): (i) the numbers of fungi reported from all
habitats in the United Kingdom and sites within the
United Kingdom compared with the number of plant
species present, (ii) the numbers of host-specific fungi
on particular plant species, (iii) percentages of new
species being discovered, and (iv) extrapolations to
the global level based on conservative estimates for
the number of plant species, making allowances to dis-
count separately named morphs of the same species.
The 1.5 million figure was considered conservative be-
cause, while numbers of fungi from all sources, includ-
ing organisms other than plants and soil, were included
in the species totals for particular places, no allowances
were made for undiscovered fungi in and on the mil-
lions of insects predicted to exist.
Some figures used in these early extrapolations have
subsequently been revised upward. Ten years later, it
was pointed out that the number of plant species and
the fungus:plant ratio were too conservative and the
vast number of insect fungi had not been considered.
As a consequence, some revised estimates ranged be-
tween 2.7 and 9.9 million fungal species (4).
The total inventory of fungal species found in field
surveys in a particular site continues to increase year by
year, even with regular visits spanning several decades,
while the number of plant species remains more or less
unchanged. Esher Common (Surrey, United Kingdom)
is by far the most investigated site by field mycologists
in the world, and to date about 3,400 species have been
found (B. M. Spooner, personal communication). Be-
cause the number of vascular plant species remains at
420, this now gives a fungus:plant ratio of 8:1.
Some preliminary results from next-generation se-
quencing of soil samples from a wood within the sec-
ond most field-surveyed site in the world, the Slapton
Ley National Nature Reserve in southwest England,
are illuminating in relation to the fungus:plant ratios
(G. W. Griffith, B. S. Dentinger, and D. L. Hawksworth,
unpublished). The wood studied has 1,136 species of
fungi and 88 vascular plant species recorded during
field surveys, a ratio of 12.9:1. However, just 686 se-
quence-based species (accounting only for taxa repre-
sented by five or more sequences) were recovered from
soil samples (and a further 153 taxa were represented
by one to four sequences). What is intriguing is that
of the 164 named genera represented in the next-
generation sequencing data set, only 32 were among
the 549 genera collected in the fieldwork, which in-
cluded fungi on plant parts and plant litter, not just
sporophores emerging from the soil. The actual ratio in
a site may therefore be substantially higher than indicat-
ed by traditional inventory techniques.
The interpretation of some data sets needs to be
considered in the light of such long-term investiga-
tions. Ratios comparing fungal sequences recovered
4. FUNGAL DIVERSITY REVISITED 83

Table 1 Selected species recognition studies in different groups of fungi
Taxon Genus
a
Lifestyle Coverage
b
Range
c
Ratio
d
Factor
e
Adjusted
f
Weight
g
Reference
Acantholichen pannarioides 1 Lichenized Neotropical Neotropical 6 1 6 8 100
Amanita muscaria 500 Ectomycorrhizal N. Hemisphere Global 6 2 12 6,000 20
Archaeorhizomycetes 0 Soil fungus N. Hemisphere Global 145 3 500 500 31
Aspergillus flavus 266 Saprotrophic Australia Global 2 6 12 3,192 101
Aspergillus fumigatus 266 Human pathogenic Global Global 2 1 2 532 102
Auxarthron zuffianum 15 Saprotrophic Global Global 2 1 2 30 103
Beauveria 9 Entomopathogenic Global Global 4 1 4 36 104
Blastomyces dermatitidis 1 Human pathogenic N. America Global 2 1 2 2 105
Botrytis cinerea 54 Phytopathogenic Europe Global 2 6 12 648 106
Calonectria pauciramosa 34 Phytopathogenic Global Global 3 2 6 204 107
Cenococcum geophilum 1 Ectomycorrhizal N. America Global 5 6 30 30 108
Cladosporium cladosporioides 150 Saprotrophic Global Global 22 1 22 3,300 27
Coccidioides immitis 3 Human pathogenic N. America N. America 2 1 2 6 103
Coniophora puteana 20 Saprotrophic Global Global 3 1 3 60 109
Coprinellus 100 Saprotrophic Global Global 1,2 1 1.2 120 25
Cora 1 Lichenized Subglobal Global 189 1 189 189 6, 22
Corella 0 Lichenized Central/S. America Central/S. America 11 1 11 11 26
Cryptomycota 0 Aquatic N. America Global 39 6 234 234 33
Cyphellostereum 1 Lichenized Central/S. America Global 15 3 45 45 26
Cyphobasidium 0 Lichenicolous N. America Global 26 4 100 100 32
Dictyonema 6 Lichenized Subglobal Global 59 3 177 1062 26
Fusarium graminearum 111 Phytopathogenic Global Global 7 1 7 777 30
Grosmannia clavigera 1 Phytopathogenic N. America N. America 2 1 2 2 110
Hymenoscyphus albidus 155 Phytopathogenic Europe Europe 2 1 2 310 111
Lasiodiplodia theobromae 9 Phytopathogenic Pantropical Pantropical 3 1 3 27 112
84 FUNGAL BRANCHES ON THE EUKARYOTIC TREE OF LIFE

Taxon Genus
a
Lifestyle Coverage
b
Range
c
Ratio
d
Factor
e
Adjusted
f
Weight
g
Reference
Letharia 2 Lichenized N. Hemisphere N. Hemisphere 3 1 3 6 113
Lobariella 5 Lichenized Neotropical Neotropical 3.8 1 3.8 19 114
Metarhizium anisopliae 9 Entomopathogenic N. America Global 2 6 12 108 115
Microbotryum violaceum 87 Phytopathogenic Europe/N. America Global 11 4 44 3,828 29
Neofusicoccum parvum/ribis 13 Phytopathogenic Regional/host Global 2 60 150 1,950 116
Paracoccidioides brasiliensis 1 Human pathogenic S. America S. America 4.0 1 4 4 117
Parmelia 95 Lichenized Europe/N. America N. Hemisphere 3 1.5 4.5 428 118
Parmelia saxatilis 1 Lichenized N. America Global 4 3 12 12 119
Parmotrema reticulatum 348 Lichenized Subglobal Subglobal 4 1 4 1,392 120
Phialocephala fortinii 1 Endophytic Europe Global 7 6 42 42 121
Protoparmelia 20 Lichenized Global Global 1.6 1 1.6 32 23
Puccinia monoica 4,000 Phytopathogenic N. America Global 1.8 6 10.5 42,000 35
Rhizoplaca melanophthalma 11 Lichenized N. America Global 2.5 6 15 165 122
Stachybotrys chartarum 44 Saprotrophic N. America Global 2 1 2 88 123
Sticta fuliginosa 114 Lichenized Global Global 15 1 45 5,130 28
Sticta weigelii 114 Lichenized Global Global 23 1 23 2,622 28
Strobilomyces 20 Ectomycorrhizal Asia Global 3.5 6 21 420 124, 125
Tricholoma scalpturatum 200 Ectomycorrhizal Europe Global 2 6 12 2,400 126
Uncinocarpus reesii 3 Saprotrophic Global Global 2 1 2 6 103
Usnea 338 Lichenized N. Hemisphere N. Hemisphere 0.7 1 1 338 24
a
Number of species recognized in the corresponding genus in reference 18.
b
Origin of material used in the study.
c
Global distribution of the group.
d
Ratio of the number of species recognized after versus before the study.
e
Geographic adjustment.
f
Adjusted ratio based on raw ratio and geographic adjustment.
g
Weight based on adjusted ratio and original number of species recognized in the corresponding genus in reference 18 (in some instances [Acantholichen,Sticta,Usnea] further adjusted based on unpub-
lished data).
4. FUNGAL DIVERSITY REVISITED 85

from environmental samples with plants growing on
the sample sites have given some surprising ratios. For
example, data from a forest in North Carolina gave
fungus:plant ratios of 19:1 (491 fungi and 26 plants)
and 13:1 (616 fungi and 48 plants), leading to a sugges-
tion that there may be 3.5 to 5.1 million fungal species
worldwide (9). A more modest ratio of 7.5:1 (1,500
fungi and <200 plants) was obtained from boreal forest
soils in Alaska (36). In contrast, Tedersoo et al.’s (37)
impressive study of 365 globally distributed soil sam-
ples using metabarcoding molecular methods (see be-
low) led the authors to conclude that fungal species
richness had actually been overestimated by 1.5 to 2.5
times from that based on plant:fungus species ratios.
Any such extrapolation from soil samples alone to the
total fungal biota at a site is, however, unsound as
evidenced by the above data from the Slapton reserve.
Much of the actual fungal species diversity in a site
will inevitably be missed in soil sampling, because most
is above ground, on and in plants and animals of all
kinds, in water, in lichens, on rocks, etc.
Collections made in brief smash-and-grab collecting
trips or even extending over several years will only start
to reveal what fungi may actually be present at a site.
Studies such as that of Piepenbring and collaborators
(38), in which 567 species of fungi and 311 species of
plants were found along a 500-m pathway in Panama
over 2 years, giving a ratio of just 1.8:1, have to be
interpreted in this context. The ratio resulting from
that study would be expected to rise significantly if the
period over which it was conducted had been 2 decades
rather than 2 years, and with increased attention to mi-
croscopic fungi.
When considering fungi of all biologies and in all
ecological niches in a site, the ratio can be expected to
vary depending on the geographical location, as recog-
nized some 25 years ago (3). It is not surprising, there-
fore, that the studies of fungus:plant ratios in a site,
obtained by field survey and molecular approaches,
have generated ranges from 1.8 to 19.1:1. The mean of
the figures cited in this section yields a ratio of 9.8:1,
suggesting that the ratio of around 6:1 arrived at in
1991 may be conservative. With the currently accepted
number of approximately 380,000 vascular plant spe-
cies (39), this adjusted ratio would predict a number of
nearly 3.8 million fungal species.
EVIDENCE FROM ENVIRONMENTAL
SEQUENCING TECHNIQUES
The advent of obtaining taxon-specific, molecular se-
quence data from environmental samples, most com-
monly soils but also water and plant tissues, has
generated a new source of data for estimating global
species numbers. It is commonly found that numerous
sequences thus obtained do not have any matches with
those from named fungi in GenBank (see below), and
this has led to speculations that previous estimates have
been too low.
Generalizing data from environmental samples is
problematic for several reasons, including that the
broader geographic context of detected operational tax-
onomic units (OTUs) is usually unknown. For exam-
ple, using 454-sequencing technology, two 0.25-g soil
samples from an Alaskan forest collected just 1 m apart
had only 14% of fungal species in common (36). In a
pyrosequencing study in the Andes, 1,839 species-like
taxa were found, of which 25% were most similar to
other unidentified environmental samples, with signifi-
cant differences between forests at different altitudes
(40). The most comprehensive study of soil samples to
date is that of Tedersoo et al. (37), who analyzed 2-g
soil samples from pooled soil in 5-cm deep cores taken
from 365 global sites. They obtained 80,486 fungal
OTUs, used a 98% sequence similarity for species rec-
ognition, and did not consider almost half in further
analyses because they were singletons and potentially
artifacts; in that study, plant:fungus richness declined
toward the poles. However, in concluding that extra-
polations from plant:fungus ratios were unjustified
(see above), the authors did not take into account fungi
other than those in soil, such as those in and on aerial
parts of plants, in decaying vegetation, lichen-fungi,
and entomogenous fungi.
Especially instructive is a study of 928 swabs of dust
samples across the continental United States by direct
PCR and using high-throughput sequencing (HTS) to
analyze them (41); not only were 38,473 fungal taxa
detected, but there were clear geographic patterns with
a predictive value of placing a sample within 230 km.
Of the 38,473 taxa, however, sequences of about 40%
could not be matched with any named species (R. R.
Dunn, personal communication).
Just how many unidentified sequences generated
from environmental samples represent undescribed spe-
cies is uncertain. Compared with the 120,000 formally
recognized species (see above), as of 25 November
2016, there were only 34,878 named fungal species in
GenBank, but there were a further 94,059 species-level
OTUs with no names (C. L. Schoch, personal com-
munication); these figures have increased by 68% and
a staggering 117%, respectively, in the past 5 years.
However, only a minute fraction of environmental se-
quences are deposited individually in GenBank; the vast
86 FUNGAL BRANCHES ON THE EUKARYOTIC TREE OF LIFE

majority is placed in the Sequence Read Archive (SRA)
(42). Currently, the SRA, on the query “(fungal OR
Fungi) AND (internal transcribed spacer [or, ITS] OR
ITS1 OR ITS2),” returns 179 studies, 1,822 biosam-
ples (i.e., environmental samples), and 14,334 experi-
ments or HTS runs, containing 928 million fungal
ITS reads, with an average length of 353 bases (SRA:
https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=
search_obj; accessed 12 March 2017). In contrast, using
the same query, GenBank (43) returns 993,987 sequences
obtained predominantly through Sanger sequencing
(GenBank: https://www.ncbi.nlm.nih.gov/genbank; ac-
cessed 12 March 2017). Thus, at present there are almost
1,000 times more HTS reads than Sanger sequences.
Only 3 years ago, this ratio was 18:1, which means it
hasincreasedbyafactorofmorethan50inthisshort
period.
Obviously, a proportion of the unidentified se-
quences can be expected to represent known but as yet
unsequenced species, especially considering that about
85,000 of the currently accepted fungal species have no
sequence data available. In the case of Mortierella,a
reference set of type and other strains was sequenced and
then compared with unnamed sequences in GenBank
(44); the authors modeled the effects of increasing type
strain sequencing and found a linear relationship with
the number of strains that could be identified, and they
predicted a number close to that of the already described
species.
One of the most significant problems with using en-
vironmental sequence data to extrapolate global fungal
species richness is the approach to define OTUs. Due
to the volume of data generated and the usual broad
taxonomic range detected, alignment-based methods
that would allow us to critically define species as phylo-
genetic lineages are impossible to use as routine tech-
niques. Thus, environmental sequence data are analyzed
using clustering techniques, and OTUs as equivalents to
species are defined on thresholds, ranging between 95
and 99% depending on the study. This approach has
several shortcomings. First, species do not exhibit fixed
sequence thresholds; in contrast, sequence divergence
and hence the barcoding gap depend on the evolution-
ary age of the species complex. Second, commonly used
thresholds of 95 to 97% are unrealistic. Assuming that
the ITS barcoding locus has a length of, e.g., 500 to
600 bases, 5% divergence would correspond to 25
to 30 bases. In most studied species, infraspecific ITS
variation is much lower, with an average of about 1%.
Theoretically, a more realistic threshold of 99% would
greatly increase the number of OTUs detected in a data
set. However, this effect is more than outweighed by the
sensitivity of clustering methods to variation in the
data, including the common CAFIE (carry-forward-
incomplete-extension) sequencing error common to all
base flow technologies (45) (see below).
This error and other problems with clustering tech-
niques can lead to an overestimation of the number of
OTUs at several orders of magnitude, which may result
in serious bias when using such estimates for global
extrapolations. We tested this effect with SRA data of
the soil fungus Archaeorhizomyces (Smith and Lu
¨cking,
unpublished). Through a blast script, we obtained
106,563 ITS reads from the SRA that were reasonably
close to the type species, Archaeorhizomyces finlayi.
Clustering analysis using USEARCH (46) resulted in
the following numbers of OTUs depending on the set
threshold: 2,658 at 95%, 5,793 at 97%, 10,630 at
98%, and 28,435 at 99%. Thus, the difference between
the 95% and the 99% threshold is an order of mag-
nitude. Alignment-based phylogenetic analysis suggests
the presence of approximately 500 species-level line-
ages in this data set. Thus, depending on the threshold
level used for species-level separations, clustering could
overestimate the actual species richness in this case by
a factor of between 5 and 50.
Lu
¨cking and colleagues (45) showed that severe
problems with estimating OTUs through clustering
emerge from even minor sequencing errors. The CAFIE
error is common to all next-generation sequencing
technologies, because it represents a statistical error
of the distribution of bases among wells on a plate
through a given base flow. Through careful adjustment,
this error can be held to a minimum of less than one
erroneously phased insertion per read, i.e., about 0.3%
assuming an ITS1 or ITS2 read length of approximately
300 bases. This proportion is well within the expected
infraspecific variation even at a high level of 99% simi-
larity and thus theoretically should disappear as back-
ground signal. However, the random distribution of
this error across the entire sequence, which cannot be
recognized by quality filtering methods, especially not
in environmental samples where no expected sequence
patterns exist for comparison, causes clustering meth-
ods to interpret these differences as taxonomic, leading
to serious overestimations by distribution sequences of
the same taxon, but with different minor errors, into
separate clusters. This is because clustering methods
work with local alignments of small, highly similar se-
quences, whereas phylogenetic methods require align-
ment of sequences of a data set to template sequences
obtained from Sanger sequencing. As a consequence,
in a broad alignment including Sanger reference se-
quences, erroneous and phased indels will be located
4. FUNGAL DIVERSITY REVISITED 87

in mostly gapped columns, which have practically no in-
fluence on the resulting topology, whereas in clustering
methods, they will frequently be aligned with non-
homologous base calls and falsely interpreted as substi-
tutions (45).
In the above (45), a proportion of less than 1% erro-
neously phased indels caused by CAFIE errors in the
ITS of a single species caused overestimation of the
number of taxa by a factor of 35 at a 95% threshold
level, 137 at 97%, and 980 at 99%. Thus, instead
of disappearing as background signal, the sequencing
error had a tremendous effect on OTU evaluation
when in that case it was known that all sequence data
came from a single target species. Even after removing
all erroneous indels, clustering continued to overesti-
mate species richness by a factor of 4 at a 95% thresh-
old level, 8 at 97%, and 116 at 99%, due to minor
remaining infragenomic variation and other sequencing
errors. In contrast, alignment-based phylogenetic analy-
sis of a broad set of nearly 2,000 reads recovered a
single species, even with all sequence errors included.
Thus, while environmental sequencing continues to
accumulate a vast amount of important data on un-
recognized fungi, extrapolations from these data must
be taken with great care when based on clustering
methods. An extreme example is a recent study which
predicts up to a trillion species of microorganisms glob-
ally based on OTUs derived from environmental se-
quence data (1), using ecological scaling laws which,
put in simple terms, predict how the global, limited
number of Nindividuals would divide into Sspecies,
taking into account resource competition based on body
size and other factors. This number, while certainly in-
triguing, appears to be exaggerated, and it would be
interesting to see what alternative prediction would be
obtained when defining the underlying taxonomic enti-
ties in this analysis through alignment-based phyloge-
netic methods.
In spite of these shortcomings, it is clear that envi-
ronmental sequencing is now the major source of dis-
covering novel fungal taxa, at least in the same order of
magnitude as taxa recovered in species recognition stud-
ies or perhaps much higher. It follows that to properly
catalog these fungi, a naming system for voucherless se-
quences must be developed, and proposals on how to
do that are currently under consideration (47–51).
In addition to discovering new species, environmen-
tal sequencing may reveal unknown higher fungal taxa
up to the class level. The best-known example is the re-
cently discovered class Archaeorhizomycetes (31, 52).
In Tedersoo and colleagues’ study (37), ∼6% of all fun-
gal OTUs below the phylum level could not be assigned
to any known class. Further clustering of unidentified
fungal sequences at 70% sequence similarity revealed
14 distinct taxonomic groups comprising >7 OTUs,
suggesting that there are several deeply divergent class-
level fungal lineages that have not yet been described or
previously sequenced. A follow-up study by the same
group (53) also revealed numerous new lineages at least
at the order level.
WHERE ARE THE UNDESCRIBED FUNGI?
If there are at least 1.5 million or more fungi on Earth,
and we know of just 120,000, where can the missing
1.38 million species be? As outlined, three major sources
for unrecognized fungal diversity seem to emerge: (i)
geographic areas and ecological habitats that are largely
understudied, particularly in tropical regions and bio-
diversity hot spots, (ii) ecologically cryptic fungi that
occur in the environment but usually do not manifest
themselves via discernible structures other than micro-
scopic hyphae and mycelia, and (iii) so-called cryptic
species hidden under well-established names.
Biodiversity Hot Spots
It is generally recognized that missing species of all
groups of organisms will be found in biodiversity hot
spots (54). A novel quantitative method to identify hot
spots of unrecognized species richness is the grid ap-
proach (6–8), which relies on linear interpolation and
hence gives more reliable estimates than nonlinear ex-
trapolation. The area in question is divided into grids,
and observed richness per grid is linearly correlated
with predictive macroenvironmental parameters and
sampling effort, using at least one well-sampled grid
for calibration. This method can be applied to both tra-
ditional inventories and data based on environmental
sequencing, as long as species are reliably delimited
and macroenvironmental predictors can be reasonably
derived from the data. Thus far, the method has been
employed for lichen-forming fungi in the families
Graphidaceae and Trypetheliaceae and the genus Cora,
resulting in estimates ranging between 1.5 and 4 times
the number of known species.
South America is generally recognized as having ma-
jor hot spots, and various studies point to high levels of
novelty there. By combining data from basidiomes and
sequence data from ectomycorrhizal roots in a Dicymbe
corymbosa forest in Guyana, Henkel and collaborators
(55) estimated that over 250 species were associated
with this one tree species in this single site. Lo
´pez-
Quintero and colleagues (56) found that of 632 macro-
mycete species in Colombian Amazonian forests, 52%
88 FUNGAL BRANCHES ON THE EUKARYOTIC TREE OF LIFE

could not be identified to the species level, a signifi-
cant proportion likely representing undescribed species.
Truong and coworkers (57) collected 1,430 basidiomes
during just four collecting expeditions in southern South
American Nothofagaceae-dominated forests; they gen-
erated 439 OTUs out of 957 specimens, of which 308
(ca. 70%) did not match any in the UNITE dynamic
database at 97 to 99% similarity and thus did not
correspond to any “species hypothesis” currently in the
database.
Little-Explored Habitats
There are an enormous number of potential sites for
fungi in any locality, and in the case of the tropics at
least 31 ecological niches meriting study can be recog-
nized, many requiring different techniques and specialists
to explore (58). While no site on Earth can yet be consid-
ered to have a complete inventory of the fungi present,
some habitats stand out as particularly underexplored.
The situation can be exemplified by the fungi which
obligately grow on lichens, the lichenicolous fungi.
With a few notable exceptions, this niche was largely
overlooked by lichenologists and other mycologists
until the mid-1970s. In 1976, there were just 457 spe-
cies known worldwide (59), but by 2016 that number
exceeded 1,800 (http://www.lichenicolous.net/); that
represents a 394% increase over 40 years, or 98.5%
per decade. This growth is reflected at the more local
level, where in Great Britain and Ireland the number
known rose from just 218 species in 1983 to 403 in
2003, an increase of 85% over 20 years (60); the num-
ber has now passed 500 and continues to rise (D. L.
Hawksworth, unpublished). That figure compares with
around 1,900 lichenized species in the same region, im-
plying by extrapolation to the global scale that there
may be as many as 25% as many lichenicolous fungi as
lichenized species, i.e., around 5,000 species. This hy-
pothesis is supported by studies of these fungi in areas
where they have not previously been investigated; for
example, of 189 species reported from Isla Navarino
in Chile on the basis primarily of collections made in
just 1 year, 6 genera and 60 species (32%) were new
to science (61). And these figures do not include the
so-called endolichenic fungi that can be cultured from
lichen thalli or are only known from sequence data
(e.g., 62, 63). In addition, emerging studies suggest the
presence of highly specific, yet morphologically cryptic,
basidiomycete yeasts and other basidiomycetes in the
cortex of lichen thalli, which add yet another dimen-
sion to unknown fungal diversity (32, 51).
The experience with lichenicolous fungi appears or
can be expected to be paralleled in other little-explored
habitats, including bryophytes (64–66), algae (67, 68),
endophytic fungi inside vascular plants (69–71), tropi-
cal foliicolous and fungicolous fungi (72), mammal guts
(73), insect guts (74–77) and exoskeletons (78, 79),
on and in rocks (80, 81), and in deep sea and ocean
sediments (82), to name just a few.
Cryptic Species
Biologically distinct species which are morphologically
indistinguishable, so-called cryptic species, were recog-
nized 2 decades ago as one place where some of the
predicted “missing fungi” might be discovered, since
physiological and other biological divergence evidently
often precedes morphological divergence in the evolu-
tion of fungal species. Based on studies of selected com-
plexes then available, it was suggested that the number
of known fungi might rise by a factor of 5 or more for
this single reason (83). Molecular studies reveal that
cryptic speciation is widespread through diverse groups
of fungi, including plant pathogens, clinically impor-
tant fungi, lichen-forming fungi, and mushrooms. Rec-
ognition of such cryptic speciation is indeed playing
an increasingly important role in species discovery,
as outlined above, and the ratio derived from current
studies, 11.3:1, is about twice as high as estimated by
Hawksworth and Rossman (83).
Existing Reference Collections
It has been estimated that over half of the projected
70,000 undiscovered plant species have already been
collected and await description (84). Similarly, most
reference collections of fungi, whether of dried speci-
mens (fungaria) or living cultures (culture or micro-
bial resource collections), include material that is not
named to the species level, and in some cases not even
to the genus level. Further, some collections and isolates
referred to named fungi not uncommonly prove, on
critical examination, to be different species. It has
been suggested that around 20,000 fungal species have
been collected but are as yet undescribed (83), but that
is likely to be an underestimate because many mycol-
ogists have large backlogs of material awaiting formal
description.
One of the challenges in the linking of newly
obtained sequence data with named material in col-
lections, particularly those that are type material, is fix-
ing the application of the species name. Currently, the
34,878 species with sequences in GenBank (see above)
represent just 29% of the 120,000 known species.
Obtaining sequences for the missing 71% of named ac-
cepted species is essential to determine the novelty of
recovered sequences from the environment, specimens,
4. FUNGAL DIVERSITY REVISITED 89

or isolates. While some researchers have had consider-
able success in recovering DNA from dried specimens
(85), even from a dried Hygrophorus basidiome col-
lected as far back as 1794 (86), this is not always pos-
sible (87). Aged dried material generally exhibits high
levels of DNA degradation, making access through
conventional extraction and PCR methods difficult (88,
89). In view of the serendipity of obtaining satisfactory
results, collection curators are often unwilling to allow
parts of often scant, irreplaceable type specimens to be
destroyed for DNA extraction. Several attempts are
now being made at using high-throughput sequencing
technologies to obtain sequence data from highly frag-
mented DNA (88). One of the main problems in this re-
spect is contamination, since even for highly variable
loci, it will be a challenge to piece small fragments to-
gether without the risk of generating artificial chimeras.
Therefore, efforts should be made to identify highly
diagnostic regions in the ITS or other loci that allow
the identification of a taxon even when only short frag-
ments are at hand (89), as exemplified by comparing
the ITS1 and ITS2 subregions (90).
There is a particular problem regarding the type
species of generic names. An impressive 5,317 generic
names of fungi are represented in GenBank (C. L.
Schoch, personal communication), but in many cases
the sequences are not from the name-fixing type spe-
cies. A concerted attempt has been initiated to sequence
the type species of genera currently not represented
in DNA databases. This involves recollection and/or
isolation from the geographical area of the original
material and from the same host or substrate when
no DNA has been recovered from the type material
(91). Sequenced material of species can then in some
cases be designated as an interpretive type—an epitype
(92). Such an effort involves global collaboration of
sequencing laboratories with colleagues in countries
where high unknown diversity is located.
TOWARD A WORKING NUMBER OF
GLOBAL FUNGAL SPECIES RICHNESS
There is general acceptance among mycologists that the
global number of fungal species is a seven-digit figure, in
the range 1 to 5 million (10), with “at least” 1.5 million
now predominating, though a possible order of mag-
nitude higher has been hinted at (93) and a minimum
figure of 611,000 to 712,000 was arrived at (2, 94),
leaving aside the figure of potentially billions of fungi
extrapolated by Locey and Lennon (1). A recalcula-
tion of the number computed via Mora and colleagues’
approach (2) gives a figure of about 1.4 million (see
above), close to the 1.5 million estimate which has come
to be widely accepted by other biologists (54, 95–97),
notwithstanding a few estimates that fail to appreciate
the difficulty of inventorying fungi (98) or that equate
numbers detected in soil alone as indicative of the total
number at a site (37).
Unfortunately, the different techniques used to esti-
mate global species richness are in part additive and in
part overlapping or redundant, so that a combination
of estimates from these studies is difficult. Thus, the re-
vised estimate of nearly 3.8 million fungal species based
on an updated fungus:plant ratio of 9.8:1 and 380,000
vascular plant species should include fungal species
of all sorts with all types of ecologies and detected by
different methodologies, including environmental se-
quencing and species delimitation methods. This num-
ber should then be congruent with approaches such
as publication rates or scaling methods, but these ap-
proaches result in widely disparate estimates. Alter-
natively, an additive approach would be based on the
three major sources of unrecognized fungal species
richness, which can be reasonably sorted in descending
order into (i) environmental sequencing, (ii) cryptic spe-
ciation, and (iii) other novel discoveries through tradi-
tional field work. For cryptic speciation, the above
survey of established literature results in a weighted
ratio of about an order of magnitude, i.e., more than
1 million additional species if based on 120,000 previ-
ously known species. If novel species from environ-
mental sequencing are at least as high in number, this
would add at least another million.
We conservatively assume that other novel discoveries
(excluding species delimitation methods and environ-
mental sequencing) will occur at a proportion of at best
10% of the latter two approaches. This assumption is
based on the rate of 1,300 newly described species per
year until about 2010, before the onset of rigorous spe-
cies delimitation methods and environmental sequencing
(see above). At this rate, it would take nearly 100 years
to double the number of known species. Thus, while en-
vironmental sequencing and species delimitation meth-
ods would contribute about 2 million new taxa, other
novel discoveries would add at best another 120,000
taxa within a reasonable time frame, for a total of a lit-
tle over 2.2 million. This would give a range of between
2.2 (additive approach) and 3.8 million (global ratio ap-
proach), very much in line with the previously updated
estimates of between 2.3 and 3 million (4, 99) and pre-
cisely narrowing down the range of 1 to 5.1 million given
by Blackwell (10) by 1.2 to 1.3 million on either side.
We therefore propose to replace previous estimates
of global fungal species richness with this updated range
90 FUNGAL BRANCHES ON THE EUKARYOTIC TREE OF LIFE

of 2.2 to 3.8 million. This estimate is likely to be further
improved on when reliable statistical approaches to
analyze the huge amount of environmental sequence
data become available. An interesting approach would
be to combine the following techniques: (i) a geographi-
cally and ecologically broad sample of environmental
sequence data, (ii) alignment-based species recogni-
tion methods to properly estimate OTU diversity, (iii)
species-based niche modeling to establish macro- and
microecological patterns, and (iv) a grid-based inter-
polation of global species richness, taking into account
sampling effort. We predict that such an analysis will
result in estimates that might lie well above the revised
conservative estimate of 2.2 million species and likely
even beyond 3.8 million species. But even if continuing
to adopt the previous, now appearing highly conser-
vative number of 1.5 million species, the discovery and
formal description of the missing fungi remain a daunt-
ing task.
Acknowledgments. We thank Robert R. Dunn, Gareth W.
Griffiths, Conrad L. Schoch, and Brian M. Spooner for pro-
viding previously unpublished data. We are especially in-
debted to Paul M. Kirk for providing the raw data from
Index Fungorum, on which Figs. 1 to 3 were based.
Citation. Hawksworth DL, Lu
¨cking R. 2017. Fungal diversity
revisited: 2.2 to 3.8 million species. Microbiol Spectrum 5(4):
FUNK-0052-2016.
References
1. Locey KJ, Lennon JT. 2016. Scaling laws predict global
microbial diversity. Proc Natl Acad Sci USA 113:5970–
5975.
2. Mora C, Tittensor DP, Adl S, Simpson AGB, Worm B.
2011. How many species are there on Earth and in the
ocean? PLoS Biol 9:e1001127.
3. Hawksworth DL. 1991. The fungal dimension of bio-
diversity: magnitude, significance, and conservation.
Mycol Res 95:641–655.
4. Hawksworth DL. 2001. The magnitude of fungal diver-
sity: the 1.5 million species estimate revisited. Mycol
Res 105:1422–1432.
5. Erwin TL. 1982. Tropical forests: their richness in Cole-
optera and other arthropod species. Coleopt Bull 36:
74–75.
6. Lu
¨cking R, Dal-Forno M, Sikaroodi M, Gillevet PM,
Bungartz F, Moncada B, Ya´nez-Ayabaca A, Chaves JL,
Coca LF, Lawrey JD. 2014. A single macrolichen con-
stitutes hundreds of unrecognized species. Proc Natl
Acad Sci USA 111:11091–11096.
7. Lu
¨cking R, Johnston MK, Aptroot A, Kraichak E,
Lendemer JC, Boonpragob K, Ca´ ceres MES, Ertz D,
Ferraro LI, Jia ZF, Kalb K, Mangold A, Manoch L,
Mercado-Dı´az JA, Moncada B, Mongkolsuk P, Papong
K, Parnmen S, Pela´ ez RN, Poengsungnoen V, Rivas
Plata E, Saipunkaew W, Sipman HJM, Sutjaritturakan
J, Van den Broeck D, Von Konrat M, Weerakoon G,
Lumbsch HT. 2014. One hundred and seventy five
new species of Graphidaceae: closing the gap or a drop
in the bucket? Phytotaxa 189:7–38.
8. Aptroot A, Ca´ ceres MES, Johnston MK, Lu
¨cking R.
2016. How diverse is the lichenized fungal family Tryp-
etheliaceae (Ascomycota:Dothideomycetes): a quantita-
tive prediction of global species richness. Lichenologist
48:983–1011.
9. O’Brien HE, Parrent JL, Jackson JA, Moncalvo JM,
Vilgalys R. 2005. Fungal community analysis by large-
scale sequencing of environmental samples. Appl Envi-
ron Microbiol 71:5544–5550.
10. Blackwell M. 2011. The fungi: 1, 2, 3 ... 5.1 million
species? Am J Bot 98:426–438.
11. Hawksworth DL. 1992. The need for a more effective
biological nomenclature for the 21st century. Bot J Linn
Soc 109:543–567.
12. Costello MJ, Wilson SP. 2011. Predicting the number of
known and unknown species in European seas using
rates of description. Glob Ecol Biogeogr 20:319–330.
13. Richards RA. 2010. The Species Problem: a Philosophi-
cal Analysis. Cambridge University Press, Cambridge,
United Kingdom.
14. Kunz W. 2012. Do Species Exist? Principles of
Taxonomic Classification. Wiley-Blackwell, Weinheim,
Germany.
15. Hawksworth DL. 1996. Microbial collections as a tool
in biodiversity and biosystematic research, p 26–35. In
Samson RA, Stalpers JA, van de Mei D, Stouthamer
AH (ed), Culture Collections to Improve the Quality
of Life. Proceedings of the Eighth International Con-
gress. Centraalbureau voor Schimmelcultures and
World Federation for Culture Collections, Baarn, The
Netherlands.
16. Quaedvlieg W, Binder M, Groenewald JZ, Summerell
BA, Carnegie AJ, Burgess TI, Crous PW. 2014. Intro-
ducing the consolidated species concept to resolve spe-
cies in the Teratosphaericaeae.Persoonia 33:1–40.
17. Leavitt SD, Divakar PK, Crespo A, Lumbsch HT. 2016.
A matter of time: understanding the limits of the power
of molecular data for delimiting species boundaries.
Herzogia 29:479–492.
18. Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008.
Ainsworth & Bisby’s Dictionary of the Fungi, 10th ed.
CAB International, Wallingford, United Kingdom.
19. Schoch CL, et al, Fungal Barcoding Consortium, Fungal
Barcoding Consortium Author List. 2012. Nuclear ribo-
somal internal transcribed spacer (ITS) region as a uni-
versal DNA barcode marker for Fungi. Proc Natl Acad
Sci USA 109:6241–6246.
20. Geml J, Laursen GA, O’Neill K, Nusbaum HC, Taylor
DL. 2006. Beringian origins and cryptic speciation events
in the fly agaric (Amanita muscaria). Mol Ecol 15:225–
239.
21. Buyck B, Hofstetter V. 2011. The contribution of tef-1
sequences to species delimitation in the Cantharellus
cibarius complex in the southeastern USA. Fung Div 49:
35–46.
4. FUNGAL DIVERSITY REVISITED 91

22. Lu
¨cking R, et al. 2016. Turbo-taxonomy to assemble
a megadiverse lichen genus: seventy new species of
Cora (Basidiomycota: Agaricales: Hygrophoraceae),
honouring David Leslie Hawksworth’s seventieth birth-
day. Fungal Diversity 81:1–69.
23. Singh G, Dal Grande F, Divakar PK, Otte J, Leavitt SD,
Szczepanska K, Crespo A, Rico VJ, Aptroot A, Ca´ ceres
MES, Lumbsch HT, Schmitt I. 2015. Coalescent-based
species delimitation approach uncovers high cryptic di-
versity in the cosmopolitan lichen-forming fungal genus
Protoparmelia (Lecanorales, Ascomycota). PLoS One
10:e0124625.
24. Mark K, Saag L, Leavitt SD, Will-Wolf S, Nelsen
MP, To
˜rra T, Saag A, Randlane T, Lumbsch HT.
2016. Evaluation of traditionally circumscribed spe-
cies in the lichen-forming genus Usnea, section Usnea
(Parmeliaceae, Ascomycota) using a six-locus dataset.
Organ Div Evol 16:497–524. (Erratum, doi:10.1007/
s13127-016-0311-5.)
25. Nagy LG, Ha´zi J, Va´gvo
¨lgyi C, Papp T. 2012. Phyloge-
ny and species delimitation in the genus Coprinellus
with special emphasis on the haired species. Mycologia
104:254–275.
26. Dal Forno M. 2015. Evolution and diversity of the
Basidiolichen clade Dictyonema (Agaricales:Hygro-
phoraceae). PhD dissertation, College of Science, Envi-
ronmental Science and Public Policy, George Mason
University, Fairfax, VA.
27. Bensch K, Groenewald JZ, Dijksterhuis J, Starink-
Willemse M, Andersen B, Summerell BA, Shin H-D,
Dugan FM, Schroers H-J, Braun U, Crous PW. 2010.
Species and ecological diversity within the Clado-
sporium cladosporioides complex (Davidiellaceae,
Capnodiales). Stud Mycol 67:1–94.
28. Moncada B, Lu
¨cking R, Sua´ rez A. 2014. Molecular
phylogeny of the genus Sticta (lichenized Ascomycota:
Lobariaceae) in Colombia. Fung Div 64:205–231.
29. Le Gac M, Hood ME, Fournier E, Giraud T. 2007. Phy-
logenetic evidence of host-specific cryptic species in the
anther smut fungus. Evolution 61:15–26.
30. O’Donnell K, Kistler HC, Tacke BK, Casper HH.
2000. Gene genealogies reveal global phylogeographic
structure and reproductive isolation among lineages of
Fusarium graminearum, the fungus causing wheat scab.
Proc Natl Acad Sci USA 97:7905–7910.
31. Menkis A, Urbina H, James TY, Rosling A. 2014.
Archaeorhizomyces borealis sp. nov. and a sequence-
based classification of related soil fungal species. Fungal
Biol 118:943–955.
32. Spribille T, Tuovinen V, Resl P, Vanderpool D, Wolinski
H, Aime MC, Schneider K, Stabentheiner E, Toome-
Heller M, Thor G, Mayrhofer H, Johannesson H,
McCutcheon JP. 2016. Basidiomycete yeasts in the cor-
tex of ascomycete macrolichens. Science 353:488–492.
33. Lazarus KL, James TY. 2015. Surveying the biodiversity
of the Cryptomycota using a targeted PCR approach.
Fungal Ecol 14:62–70.
34. Livermore JA, Mattes TE. 2013. Phylogenetic detection
of novel Cryptomycota in an Iowa (United States) aqui-
fer and from previously collected marine and freshwater
targeted high-throughput sequencing sets. Environ
Microbiol 15:2333–2341.
35. Roy BA, Vogler DR, Bruns TD, Szaro TM. 1998. Cryp-
tic species in the Puccinia monoica complex. Mycologia
90:846–853.
36. Taylor DL, Herriott IC, Stone KE, McFarland JW,
Booth MG, Leigh MB. 2010. Structure and resilience of
fungal communities in Alaskan boreal forest soils. Can J
Res 40:1288–1301.
37. Tedersoo L, et al. 2014. Global diversity and geography
of soil fungi. Science 346:1256688.
38. Piepenbring M, Hofmann TA, Unterseher M, Kost G.
2012. Species richness of plants and fungi in western
Panama: towards a fungal inventory in the neotropics.
Biodivers Conserv 21:2181–2193.
39. Royal Botanic Gardens Kew. 2017. State of the World’s
Plants Report 2017. Royal Botanic Gardens Kew,
London, United Kingdom.
40. Geml J, Nouhra ER, Wicaksono CY, Pastor N,
Fernandez L, Becerra AG. 2012. Mycota of the Andean
Yungas forests: assessments of fungal diversity and habi-
tat partitioning in a threatened ecosystem. Inoculum
63:18.
41. Grantham NS, Reich BJ, Pacifici K, Laber EB,
Menninger HL, Henley JB, Barbera´ n A, Leff JW, Fierer
N, Dunn RR. 2015. Fungi identify the geographic origin
of dust samples. PLoS One 10:e0122605.
42. Leinonen R, Sugawara H, Shumway M, International
Nucleotide Sequence Database Collaboration. 2011.
The sequence read archive. Nucleic Acids Res 39(Data-
base):D19–D21.
43. Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi
I, Lipman DJ, Ostell J, Sayers EW. 2013. GenBank.
Nucleic Acids Res 41(D1):D36–D42.
44. Nagy LG, Petkovits T, Kova´ cs GM, Voigt K, Va´gvo
¨lgyi
C, Papp T. 2011. Where is the unseen fungal diversity
hidden? A study of Mortierella reveals a large contribu-
tion of reference collections to the identification of fun-
gal environmental sequences. New Phytol 191:789–794.
45. Lu
¨cking R, Lawrey JD, Gillevet PM, Sikaroodi M, Dal-
Forno M, Berger SA. 2014. Multiple ITS haplotypes
in the genome of the lichenized basidiomycete Cora
inversa (Hygrophoraceae): fact or artifact? J Mol Evol
78:148–162.
46. Edgar RC. 2010. Search and clustering orders of magni-
tude faster than BLAST. Bioinformatics 26:2460–2461.
47. Hibbett DS, Ohman A, Glotzer D, Nuhn M, Kirk P,
Nilsson RH. 2011. Progress in molecular and morpho-
logical taxon discovery in Fungi and options for formal
classification of environmental sequences. Fungal Biol
Rev 25:38–47.
48. Hibbett D. 2016. The invisible dimension of fungal di-
versity. Science 351:1150–1151.
49. de Beer ZW, Marincowitz S, Duong TA, Kim JJ,
Rodrigues A, Wingfield MJ. 2016. Hawksworthiomyces
gen. nov. (Ophiostomatales), illustrates the urgency for
a decision on how to name novel taxa known only from
environmental nucleic acid sequences (ENAS). Fungal
Biol 120:1323–1340.
92 FUNGAL BRANCHES ON THE EUKARYOTIC TREE OF LIFE

50. Hawksworth DL, Hibbett DS, Kirk PM, Lu
¨cking R.
2016. (308–310) Proposals to permit DNA sequence
data to serve as types of names of fungi. Taxon 65:899–
900.
51. Lu
¨cking R, Moncada M. 2017. Dismantling
Marchandiomphalina into Agonimia (Verrucariaceae)
and Lawreymyces gen. nov. (Corticiaceae): setting a
precedent to the formal recognition of thousands of
voucherless fungi based on type sequences. Fung Div
84:119–138.
52. Rosling A, Cox F, Cruz-Martinez K, Ihrmark K, Grelet
GA, Lindahl BD, Menkis A, James TY. 2011. Archaeo-
rhizomycetes: unearthing an ancient class of ubiquitous
soil fungi. Science 333:876–879.
53. Tedersoo L, Bahram M, Puusepp R, Nilsson RH, James
TY. 2017. Novel soil-inhabiting clades fill gaps in the
fungal tree of life. Microbiome 5:42.
54. Scheffers BR, Joppa LN, Pimm SL, Laurance WF. 2012.
What we know and don’t know about Earth’s missing
biodiversity. Trends Ecol Evol 27:501–510.
55. Henkel TW, Aime MC, Chin MML, Miller SL, Vilgalys
R, Smith ME. 2012. Ectomycorrhizal fungal sporocarp
diversity and discovery of new taxa in Dicymbe mono-
dominant forests of the Guiana Shield. Biodivers Con-
serv 21:2195–2220.
56. Lo´ pez-Quintero CA, Straatsma G, Franco-Molano AE,
Boekhoet T. 2012. Macrofungal diversity in Colombian
Amazon forests varies with regions and regimes of dis-
turbance. Biodivers Conserv 21:2221–2243.
57. Truong C, Mujic AB, Healy R, Kuhar F, Furci G, Torres
D, Niskanen T, Sandoval-Leiva PA, Ferna´ndez N,
Escobar JM, Moretto A, Palfner G, Pfister D, Nouhra E,
Swenie R, Sa´ nchez-Garcı´a M, Matheny PB, Smith ME.
2017. How to know the fungi: combining field invento-
ries and DNA-barcoding to document fungal diversity.
New Phytol 214:913–919.
58. Hawksworth DL, Minter DW, Kinsey GC, Cannon PF.
1997. Inventorying a tropical fungal biota: intensive
and extensive approaches, p 29–50. In Janardhanan
KK, Rajendran C, Natarajan K, Hawksworth DL (ed),
Tropical Mycology. Oxford & IBH Publishing, New
Dehli, India.
59. Clauzade G, Roux C. 1976. Les Champignons
Liche
´nicoles non Liche
´nise
´s. Univ. des Sciences et
Techn. du Languedoc, Laboratoire de Syste
´matique
et Ge
´obotanique Me
´diterrane
´enne, Inst. de Botanique,
Montpellier, France.
60. Hawksworth DL. 2003. The lichenicolous fungi of
Great Britain and Ireland: an overview and annotated
checklist. Lichenologist 35:191–232.
61. Etayo J, Sancho LG. 2008. Hongos Liquenicolas del Sur
de Sudamerica, Especialmente de Isla Navarino (Chile).
Bibliotheca Lichenologica series vol. 98. J. Cramer,
Berlin, Germany.
62. Fleischhacker A, Grube M, Kopun T, Hafellner J,
Muggia L. 2015. Community analyses uncover high di-
versity of lichenicolous fungi in alpine habitats. Microb
Ecol 70:348–360.
63. Zhang T, Wei XL, Wei YZ, Liu HY, Yu LY. 2016.
Diversity and distribution of cultured endolichenic
fungi in the Ny-A
˚lesund region, Svalbard (high Arctic).
Extremophiles 20:461–470.
64. Do
¨bbeler P, Hertel H. 2013. Bryophilous ascomycetes
everywhere: distribution maps of selected species on
liverworts, mosses and Polytrichaceae.Herzogia 26:
361–404.
65. Davey ML, Kauserud H, Ohlson M. 2014. Forestry
impacts on the hidden fungal biodiversity associated
with bryophytes. FEMS Microbiol Ecol 90:313–325.
66. Hirose D, Hobara S, Matsuoka S, Kato K, Tanabe Y,
Uchida M, Kudoh S, Osono T. 2016. Diversity and
community assembly of moss-associated fungi in ice-
free coastal outcrops of continental Antarctica. Fungal
Ecol 24:94–101.
67. Kohlmeyer J, Kohlmeyer E. 2013. Marine Mycology:
The Higher Fungi. Academic Press, San Diego, CA.
68. Furbino LE, Godinho VM, Santiago IF, Pellizari FM,
Alves TM, Zani CL, Junior PA, Romanha AJ, Carvalho
AG, Gil LH, Rosa CA, Minnis AM, Rosa LH. 2014.
Diversity patterns, ecology and biological activities of
fungal communities associated with the endemic macro-
algae across the Antarctic peninsula. Microb Ecol 67:
775–787.
69. Arnold AE. 2007. Understanding the diversity of foliar
endophytic fungi: progress, challenges, and frontiers.
Fungal Biol Rev 21:51–66.
70. Rodriguez RJ, White JF Jr, Arnold AE, Redman RS.
2009. Fungal endophytes: diversity and functional roles.
New Phytol 182:314–330.
71. Higgins KL, Arnold AE, Coley PD, Kursar TA. 2014.
Communities of fungal endophytes in tropical forest
grasses: highly diverse host-and habitat generalists char-
acterized by strong spatial structure. Fungal Ecol 8:
1–11.
72. Chomnunti P, Hongsanan S, Aguirre-Hudson B, Tian
Q, Persˇoh D, Dhami MK, Alias AS, Xu J, Liu X, Stadler
M, Hyde KD. 2014. The sooty moulds. Fung Div 66:
1–36.
73. Griffith GW, Baker S, Fliegerova K, Liggenstoffer A,
van der Giezen M, Voigt K, Beakes G. 2010. Anaerobic
fungi: Neocallimastigomycota.IMA Fungus 1:181–185.
74. Suh SO, McHugh JV, Pollock DD, Blackwell M. 2005.
The beetle gut: a hyperdiverse source of novel yeasts.
Mycol Res 109:261–265.
75. Lichtwardt RW. 2012. Trichomycete gut fungi from
tropical regions of the world. Biodiv Conserv 21:
2397–2402.
76. Gouba N, Raoult D, Drancourt M. 2013. Plant and
fungal diversity in gut microbiota as revealed by molec-
ular and culture investigations. PLoS One 8:e59474.
77. Wang Y, Tretter ED, Johnson EM, Kandel P,
Lichtwardt RW, Novak SJ, Smith JF, White MM.
2014. Using a five-gene phylogeny to test morphology-
based hypotheses of Smittium and allies, endosymbiotic
gut fungi (Harpellales) associated with arthropods. Mol
Phylogenet Evol 79:23–41.
78. Weir A, Hammond PM. 1997. Laboulbeniales on
beetles: host utilization patterns and species richness of
the parasites. Biodiv Conserv 6:701–719.
4. FUNGAL DIVERSITY REVISITED 93

79. Weir A. 2004. The Laboulbeniales: an enigmatic group
of arthropod-associated fungi. Symbiosis 4:611–620.
80. Ruibal C, Gueidan C, Selbmann L, Gorbushina AA,
Crous PW, Groenewald JZ, Muggia L, Grube M, Isola
D, Schoch CL, Staley JT, Lutzoni F, de Hoog GS. 2009.
Phylogeny of rock-inhabiting fungi related to Dothideo-
mycetes.Stud Mycol 64:123–133, S7.
81. Egidi E, de Hoog GS, Isola D, Onofri S, Quaedvlieg
W, de Vries M, Verkeley GJM, Stielow JB, Zucconi L,
Selbmann L. 2014. Phylogeny and taxonomy of meriste-
matic rock-inhabiting black fungi in the Dothideo-
mycetes based on multi-locus phylogenies. Fung Div 65:
127–165.
82. Le Calvez T, Burgaud G, Mahe´ S, Barbier G,
Vandenkoornhuyse P. 2009. Fungal diversity in deep-
sea hydrothermal ecosystems. Appl Environ Microbiol
75:6415–6421.
83. Hawksworth DL, Rossman AY. 1997. Where are all the
undescribed fungi? Phytopathology 87:888–891.
84. Bebber DP, Carine MA, Wood JR, Wortley AH, Harris
DJ, Prance GT, Davidse G, Paige J, Pennington TD,
Robson NKB, Scotland RW. 2010. Herbaria are a ma-
jor frontier for species discovery. Proc Natl Acad Sci
USA 107:22169–22171.
85. Brock PM, Do
¨ring H, Bidartondo MI. 2009. How to
know unknown fungi: the role of a herbarium. New
Phytol 181:719–724.
86. Larsson E, Jacobsson S. 2004. Controversy over
Hygrophorus cossus settled using ITS sequence data
from 200 year-old type material. Mycol Res 108:781–
786.
87. Begerow D, Nilsson H, Unterseher M, Maier W.
2010. Current state and perspectives of fungal DNA
barcoding and rapid identification procedures. Appl
Microbiol Biotechnol 87:99–108.
88. Staats M, Cuenca A, Richardson JE, Vrielink-van
Ginkel R, Petersen G, Seberg O, Bakker FT. 2011.
DNA damage in plant herbarium tissue. PLoS One 6:
e28448.
89. Sa
¨rkinen T, Staats M, Richardson JE, Cowan RS,
Bakker FT. 2012. How to open the treasure chest?
Optimising DNA extraction from herbarium specimens.
PLoS One 7:e43808.
90. Blaalid R, Kumar S, Nilsson RH, Abarenkov K, Kirk
PM, Kauserud H. 2013. ITS1 versus ITS2 as DNA
metabarcodes for fungi. Mol Ecol Resour 13:218–224.
91. Crous PW, Giraldo A, Hawksworth DL, Robert V,
Kirk PM, Guarro J, Robbertse B, Schoch CL, Damm
U, Trakunyingcharoen T, Groenewald JZ. 2014. The
genera of Fungi: fixing the application of type species of
generic names. IMA Fungus 5:141–160.
92. Ariyawansa HA, Hawksworth DL, Hyde KD,
Jones EBG, Maharachchikumbura SSN, Manamgoda
DS, Thambugala KM, Udayanga D, Camporesi E,
Daranagama A, Jayawardena R, Liu J-K, McKenzie
EHC, Phookamsak R, Senanayake IC, Shivas RG, Tian
Q, Xu J-C. 2014. Epitypification and neotypification:
guidelines with appropriate and inappropriate exam-
ples. Fung Div 69:57–91.
93. Bass D, Richards TA. 2011. Three reasons to re-evaluate
fungal diversity ‘on Earth and in the ocean’. Fungal Biol
Rev 25:159–164.
94. Schmit JP, Mueller GM. 2007. An estimate of the lower
limit of global fungal diversity. Biodivers Conserv 16:
99–111.
95. Hammond PM. 1995. Described and estimated species
numbers: an objective assessment of current knowledge,
p 29–71. In Allsopp D, Colwell RR, Hawksworth DL
(ed), Microbial Diversity and Ecosystem Function. CAB
International, Wallingford, United Kingdom.
96. Chapman AD. 2009. Numbers of Living Species in
Australia and the World, 2nd ed. Australian Biological
Resources Survey, Canberra, ACT, Australia.
97. Joppa LN, Roberts DL, Pimm SL. 2011. How many
species of flowering plants are there? Proc Biol Sci 278:
554–559.
98. May RM. 1994. Conceptual aspects of the quantifica-
tion of the extent of biological diversity. Philos Trans R
Soc Lond B Biol Sci 345:13–20.
99. Hawksworth DL. 2012. Global species numbers of
fungi: are tropical studies and molecular approaches
contributing to a more robust estimate? Biodivers
Conserv 21:2425–2433.
100. Dal-Forno M, Lu
¨cking R, Bungartz F, Ya´nez-Ayabaca
A, Marcelli MP, Spielmann AA, Coca LF, Chaves JL,
Aptroot A, Sipman HJM, Sikaroodi M, Gillevet P,
Lawrey JD. 2016. From one to six: unrecognized species
diversity in the genus Acantholichen (lichenized Basidio-
mycota:Hygrophoraceae). Mycologia 108:38–55.
101. Geiser DM, Pitt JI, Taylor JW. 1998. Cryptic speciation
and recombination in the aflatoxin-producing fungus
Aspergillus flavus.Proc Natl Acad Sci USA 95:388–393.
102. Pringle A, Baker DM, Platt JL, Wares JP, Latge´ JP,
Taylor JW. 2005. Cryptic speciation in the cosmo-
politan and clonal human pathogenic fungus Aspergil-
lus fumigatus.Evolution 59:1886–1899.
103. Koufopanou V, Burt A, Szaro T, Taylor JW. 2001.
Gene genealogies, cryptic species, and molecular evolu-
tion in the human pathogen Coccidioides immitis and
relatives (Ascomycota, Onygenales). Mol Biol Evol 18:
1246–1258.
104. Rehner SA, Buckley E. 2005. A Beauveria phylogeny in-
ferred from nuclear ITS and EF1-αsequences: evidence
for cryptic diversification and links to Cordyceps teleo-
morphs. Mycologia 97:84–98.
105. Brown EM, McTaggart LR, Zhang SX, Low DE,
Stevens DA, Richardson SE. 2013. Phylogenetic analysis
reveals a cryptic species Blastomyces gilchristii, sp.
nov. within the human pathogenic fungus Blastomyces
dermatitidis.PLoS One 8:e59237. (Erratum, 11:
e0168018. doi:10.1371/journal.pone.0168018.)
106. Walker AS, Gautier AL, Confais J, Martinho D, Viaud
M, Le Peˆcheur P, Dupont J, Fournier E. 2011. Botrytis
pseudocinerea, a new cryptic species causing gray mold
in French vineyards in sympatry with Botrytis cinerea.
Phytopathology 101:1433–1445.
107. Lombard L, Crous PW, Wingfield BD, Wingfield
MJ. 2010. Multigene phylogeny and mating tests
94 FUNGAL BRANCHES ON THE EUKARYOTIC TREE OF LIFE

reveal three cryptic species related to Calonectria
pauciramosa.Stud Mycol 66:15–30.
108. Douhan GW, Rizzo DM. 2005. Phylogenetic divergence
in a local population of the ectomycorrhizal fungus
Cenococcum geophilum.New Phytol 166:263–271.
109. Kauserud H, Svega
˚rden IB, Decock C, Hallenberg N.
2007. Hybridization among cryptic species of the cellar
fungus Coniophora puteana (Basidiomycota). Mol Ecol
16:389–399.
110. Alamouti SM, Wang V, Diguistini S, Six DL, Bohlmann
J, Hamelin RC, Feau N, Breuil C. 2011. Gene genealo-
gies reveal cryptic species and host preferences for the
pine fungal pathogen Grosmannia clavigera.Mol Ecol
20:2581–2602.
111. Queloz V, Gru
¨nig CR, Berndt R, Kowalski T, Sieber
TN, Holdenrieder O. 2011. Cryptic speciation in
Hymenoscyphus albidus.For Pathol 41:133–142.
112. Alves A, Crous PW, Correia A, Phillips AJL. 2008.
Morphological and molecular data reveal cryptic spe-
ciation in Lasiodiplodia theobromae.Fung Div 28:
1–13.
113. Kroken S, Taylor JW. 2001. A gene genealogical ap-
proach to recognize phylogenetic species boundaries
in the lichenized fungus Letharia.Mycologia 93:38–53.
114. Moncada B, Lu
¨cking R, Betancourt-Macuase L. 2013.
Phylogeny of the Lobariaceae (lichenized Ascomycota:
Peltigerales), with a reappraisal of the genus Lobariella.
Lichenologist 45:203–263.
115. Bidochka MJ, Small CLN, Spironello M. 2005. Recom-
bination within sympatric cryptic species of the insect
pathogenic fungus Metarhizium anisopliae.Environ
Microbiol 7:1361–1368.
116. Pavlic D, Slippers B, Coutinho TA, Wingfield MJ. 2009.
Multiple gene genealogies and phenotypic data reveal
cryptic species of the Botryosphaeriaceae: a case study
on the Neofusicoccum parvum/N. ribis complex. Mol
Phylogenet Evol 51:259–268.
117. Roberto TN, Rodrigues AM, Hahn RC, de Camargo
ZP. 2016. Identifying Paracoccidioides phylogenetic
species by PCR-RFLP of the alpha-tubulin gene. Med
Mycol 54:240–247.
118. Divakar PK, Leavitt SD, Molina MC, Del-Prado R,
Lumbsch HT, Crespo A. 2016. A DNA barcoding ap-
proach for identification of hidden diversity in Parmelia-
ceae (Ascomycota): Parmelia sensu stricto as a case
study. Bot J Linn Soc 180:21–29.
119. Molina MC, Del-Prado R, Divakar PK, Sa´ nchez-Mata
D, Crespo A. 2011. Another example of cryptic diver-
sity in lichen-forming fungi: the new species Parmelia
mayi (Ascomycota:Parmeliaceae). Organ Div Evol 11:
331–342.
120. Del-Prado R, Divakar PK, Lumbsch HT, Crespo AM.
2016. Hidden genetic diversity in an asexually repro-
ducing lichen forming fungal group. PLoS One 11:
e0161031.
121. Gru
¨nig CR, Duo` A, Sieber TN, Holdenrieder O.
2008. Assignment of species rank to six reproductively
isolated cryptic species of the Phialocephala fortinii
s.1.-Acephala applanata species complex. Mycologia
100:47–67.
122. Leavitt SD, Fankhauser JD, Leavitt DH, Porter LD,
Johnson LA, St Clair LL. 2011. Complex patterns of
speciation in cosmopolitan “rock posy” lichens: discov-
ering and delimiting cryptic fungal species in the lichen-
forming Rhizoplaca melanophthalma species-complex
(Lecanoraceae,Ascomycota). Mol Phylogenet Evol 59:
587–602.
123. Cruse M, Telerant R, Gallagher T, Lee T, Taylor JW.
2002. Cryptic species in Stachybotrys chartarum.
Mycologia 94:814–822.
124. Sato H, Yumoto T, Murakami N. 2007. Cryptic spe-
cies and host specificity in the ectomycorrhizal genus
Strobilomyces (Strobilomycetaceae). Am J Bot 94:
1630–1641.
125. Sato H, Murakami N. 2008. Reproductive isolation
among cryptic species in the ectomycorrhizal genus
Strobilomyces: population-level CAPS marker-based
genetic analysis. Mol Phylogenet Evol 48:326–334.
126. Carriconde F, Gardes M, Jargeat P, Heilmann-Clausen
J, Mouhamadou B, Gryta H. 2008. Population evi-
dence of cryptic species and geographical structure in
the cosmopolitan ectomycorrhizal fungus, Tricholoma
scalpturatum.Microb Ecol 56:513–524.
4. FUNGAL DIVERSITY REVISITED 95