Origin and evolution of carnivorism in the Ascomycota (fungi)
ABSTRACT Carnivorism is one of the basic life strategies of fungi. Carnivorous fungi possess the ability to trap and digest their preys
by sophisticated trapping devices. However, the origin and development of fungal carnivorism remains a gap in evolution biology.
In this study, five protein-encoding genes were used to construct the phylogeny of the carnivorous fungi in the phylum Ascomycota;
these fungi prey on nematodes by means of specialized trapping structures such as constricting rings and adhesive traps. Our
analysis revealed a definitive pattern of evolutionary development for these trapping structures. Molecular clock calibration
based on two fossil records revealed that fungal carnivorism diverged from saprophytism about 419 Mya, which was after the
origin of nematodes about 550–600 Mya. Active carnivorism (fungi with constricting rings) and passive carnivorism (fungi with
adhesive traps) diverged from each other around 246 Mya, shortly after the occurrence of the Permian–Triassic extinction event
about 251.4 Mya. The major adhesive traps evolved around 198–208 Mya, which was within the time frame of the Triassic–Jurassic
extinction event about 201.4 Mya. However, no major carnivorous ascomycetes divergence was correlated to the Cretaceous–Tertiary
extinction event, which occurred more recently (about 65.5 Mya). Therefore, a causal relationship between mass extinction
events and fungal carnivorism evolution is not validated in this study. More evidence including additional fossil records
is needed to establish if fungal carnivorism evolution was a response to mass extinction events.
- SourceAvailable from: Jinkui Yang[Show abstract] [Hide abstract]
ABSTRACT: Malate synthase (Mls), a key enzyme in the glyoxylate cycle, is required for virulence in microbial pathogens. In this study, we identified the AoMls gene from the nematode-trapping fungus Arthobotrys oligospora. The gene contains 4 introns and encodes a polypeptide of 540 amino acids. To characterize the function of AoMls in A. oligospora, we disrupted it by homologous recombination, and the ΔAoMls mutants were confirmed by PCR and Southern blot analyses. The growth rate and colony morphology of the ΔAoMls mutants showed no obvious difference from the wild-type strains on potato dextrose agar (PDA) plate. However, the disruption of gene AoMls led to a significant reduction in conidiation, failure to utilize fatty acids and sodium acetate for growth, and its conidia were unable to germinate on minimal medium supplemented with sodium oleate. In addition, the trap formation was retarded in the ΔAoMls mutants, which only produced immature traps containing one or two rings. Moreover, the nematicidal activity of the ΔAoMls mutants was significantly decreased. Our results suggest that the gene AoMls plays an important role in conidiation, trap formation and pathogenicity of A. oligospora.Applied Microbiology and Biotechnology 12/2013; · 3.69 Impact Factor
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ABSTRACT: Nematode-trapping fungi are a unique group of organisms that can capture nematodes using sophisticated trapping structures. The genome of Drechslerella stenobrocha, a constricting-ring-forming fungus, has been sequenced and reported, and provided new insights into the evolutionary origins of nematode predation in fungi, the trapping mechanisms, and the dual lifestyles of saprophagy and predation. The genome of the fungus Drechslerella stenobrocha, which mechanically traps nematodes using a constricting ring, was sequenced. The genome was 29.02 Mb in size and was found rare instances of transposons and repeat induced point mutations, than that of Arthrobotrys oligospora. The functional proteins involved in nematode-infection, such as chitinases, subtilisins, and adhesive proteins, underwent a significant expansion in the A. oligospora genome, while there were fewer lectin genes that mediate fungus-nematode recognition in the D. stenobrocha genome. The carbohydrate-degrading enzyme catalogs in both species were similar to those of efficient cellulolytic fungi, suggesting a saprophytic origin of nematode-trapping fungi. In D. stenobrocha, the down-regulation of saprophytic enzyme genes and the up-regulation of infection-related genes during the capture of nematodes indicated a transition between dual life strategies of saprophagy and predation. The transcriptional profiles also indicated that trap formation was related to the protein kinase C (PKC) signal pathway and regulated by Zn(2)-C6 type transcription factors. The genome of D. stenobrocha provides support for the hypothesis that nematode trapping fungi evolved from saprophytic fungi in a high carbon and low nitrogen environment. It reveals the transition between saprophagy and predation of these fungi and also proves new insights into the mechanisms of mechanical trapping.BMC Genomics 02/2014; 15(1):114. · 4.40 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Orbiliomycetes is one of the earliest diverging branches of the filamentous ascomycetes. The class contains nematode-trapping fungi that form unique infection structures, called traps, to capture and kill free-living nematodes. The traps have evolved differently along several lineages and include adhesive traps (knobs, nets or branches) and constricting rings. We show, by genome sequencing of the knob-forming species Monacrosporium haptotylum and comparison with the net-forming species Arthrobotrys oligospora, that two genomic mechanisms are likely to have been important for the adaptation to parasitism in these fungi. Firstly, the expansion of protein domain families and the large number of species-specific genes indicated that gene duplication followed by functional diversification had a major role in the evolution of the nematode-trapping fungi. Gene expression indicated that many of these genes are important for pathogenicity. Secondly, gene expression of orthologs between the two fungi during infection indicated that differential regulation was an important mechanism for the evolution of parasitism in nematode-trapping fungi. Many of the highly expressed and highly upregulated M. haptotylum transcripts during the early stages of nematode infection were species-specific and encoded small secreted proteins (SSPs) that were affected by repeat-induced point mutations (RIP). An active RIP mechanism was revealed by lack of repeats, dinucleotide bias in repeats and genes, low proportion of recent gene duplicates, and reduction of recent gene family expansions. The high expression and rapid divergence of SSPs indicate a striking similarity in the infection mechanisms of nematode-trapping fungi and plant and insect pathogens from the crown groups of the filamentous ascomycetes (Pezizomycotina). The patterns of gene family expansions in the nematode-trapping fungi were more similar to plant pathogens than to insect and animal pathogens. The observation of RIP activity in the Orbiliomycetes suggested that this mechanism was present early in the evolution of the filamentous ascomycetes.PLoS Genetics 11/2013; 9(11):e1003909. · 8.52 Impact Factor
Origin and evolution of carnivorism in the
Ence Yanga,1, Lingling Xua,b,1, Ying Yanga, Xinyu Zhanga, Meichun Xianga, Chengshu Wangc, Zhiqiang And,2,
and Xingzhong Liua,2
aState Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;bCollege of Biotechnology, Xi’an University
of Arts and Science, Xi’an 710065, China;cKey Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; anddBrown Foundation Institute of Molecular Medicine,
University of Texas Health Science Center at Houston, Houston, TX 77030
Edited* by Joan Wennstrom Bennett, Rutgers University, New Brunswick, NJ, and approved May 18, 2012 (received for review December 21, 2011)
Carnivorism is one of the basic life strategies of fungi. Carnivorous
fungi possess the ability to trap and digest their preys by sophisti-
cated trapping devices. However, the origin and development of
fungal carnivorism remains a gap in evolution biology. In this study,
five protein-encoding genes were used to construct the phylogeny
of the carnivorous fungi in the phylum Ascomycota; these fungi
prey on nematodes by means of specialized trapping structures
such as constricting rings and adhesive traps. Our analysis revealed
a definitive pattern of evolutionary development for these trap-
ping structures. Molecular clock calibration based on two fossil
records revealed that fungal carnivorism diverged from saprophy-
tism about 419 Mya, which was after the origin of nematodes
about 550–600 Mya. Active carnivorism (fungi with constricting
rings) and passive carnivorism (fungi with adhesive traps) diverged
from each other around 246 Mya, shortly after the occurrence of
the Permian–Triassic extinction event about 251.4 Mya. The major
adhesive traps evolved around 198–208 Mya, which was within the
time frame of the Triassic–Jurassic extinction event about 201.4
Mya. However, no major carnivorous ascomycetes divergence
was correlated to the Cretaceous–Tertiary extinction event, which
occurred more recently (about 65.5 Mya). Therefore, a causal re-
lationship between mass extinction events and fungal carnivorism
evolution is not validated in this study. More evidence including
additional fossil records is needed to establish if fungal carnivorism
evolution was a response to mass extinction events.
organic matter to inorganic molecules in most ecosystems
(1). The majority of fungi adopted saprophytic or symbiotic
(including parasitic) lifestyles (2). However, a small portion (less
than 0.5%) in the kingdom Fungi are carnivorous and possess
the ability to trap and digest nematodes, rhizopods, and rotifers
by specialized trapping devices (3–6). Carnivorous fungi, which
capture prey by trophic cells, contain species within the phyla
Zygomycota (Zoopagaceae), Basidiomycota (Nematoctonus),
and Ascomycota (Orbiliomycetes) (7, 8). Carnivorous fungi in
the Zygomycota produce adhesive hyphae or hyphal protuber-
ances that passively capture small animals (9); little is known
about the biology and evolution of those fungi because they
cannot be cultured on artificial media (10). Carnivorous fungi in
the Basidiomycota produce special adhesive knobs or adhesive
spores to capture victims (11, 12). All carnivorous fungi in the
Basidiomycota belong to Nematoctonus, an anamorphic genus of
Hohenbuehelia (Pleurotaceae family) that produces gilled
mushrooms (13, 14). Nematoctonus spp. evolved from an an-
cestor in Pleurotus that produced nematode-toxic droplets (15).
More than 90% of the carnivorous fungi belong to the Orbilio-
mycetes in the Ascomycota (7), where they form a mono-
phylogenetic group (16, 17). To capture nematodes and other
prey, carnivorous fungi in the Ascomycota produce sophisticated
trapping structures, including one type of active mechanical trap
(constricting rings, CR) and five types of passive adhesive traps:
sessile adhesive knobs (SSK), stalked adhesive knobs (SK),
s major decomposers, fungi play a critical role in degrading
adhesive nets (AN), adhesive columns (AC), and nonconstricting
rings (NCR). Nonconstricting rings are always associated with
stalked adhesive knobs and are referred to as NCR + SK in this
study (18, 19).
In part because carnivory is rare among fungi, researchers have
speculated on how such unusual behavior evolved. One hypothesis
is that carnivory was selected for in environments where dead
wood and soil are rich in carbon but poor in nitrogen. In nitrogen-
poor environments, direct capture of nitrogen from small animals
would give carnivorous fungi a competitive advantage over strictly
saprophytic fungi (20). However, this hypothesis does not explain
why carnivorous fungi are not more abundant and widespread in
many environmental niches where small animal prey are readily
available. In most environments where saprophytic fungi flourish,
it seems likely that the expenditure of nutrients and energy for
producing trapping devices may reduce the competitiveness of
carnivorous fungi, as producing traps costs additional energy in
carnivorous plants (21). A problem with this hypothesis is that,
unlike the symbiotic fungi, which independently evolved from
saprophytic fungi many times (22), carnivorous fungi evidently
evolved from saprophytic fungi only a few times, as indicated by
their close clustering in three phyla (Fig. S1). If the key to the
development of fungal carnivory is the presence of a carbon-rich,
nitrogen-poor environment, one would expect that carnivory have
arisen many times. Moreover, degeneration of carnivorous ability
seems to characterize the evolution of these fungi (15). These
intriguing questions surrounding the origin and evolution of
fungal carnivorism remain unsolved.
Estimating when carnivorous fungi diverged from other fungi
has been difficult due to the lack of fossil records (23). With the
recent discovery of fossils that provide multiple calibration
points (24–27), however, it is now possible to more accurately
estimate the divergence times of carnivorism in the Ascomycota,
in which the diversity of both species and trapping structures is
much higher than in the other two carnivorous fungal groups.
Author contributions: Z.A. and X.L. designed research; L.X., Y.Y., and M.X. performed
research; E.Y., L.X., and Y.Y. contributed new reagents/analytic tools; E.Y., L.X., X.Z.,
C.W., Z.A., and X.L. analyzed data; and E.Y., Z.A., and X.L. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Data deposition: The sequences reported in this paper have been deposited in the Gen-
Bank database (accession nos. AY773365, AY773366, AY773368, AY773370, AY773372,
AY773374, AY773375, AY773380-AY773385, AY773395, AY773396, AY773398,
AY773400, AY773402, AY773404, AY773405, AY773409-AY773414, AY773424,
AY773425, AY773427, AY773429, AY773431, AY773433, AY773434, AY773438-
AY773443, AY965822, DQ358227, DQ358229, DQ999800, DQ999810, DQ999840,
DQ999851, DQ999861, DQ999867, FJ687350, FJ687352-FJ687361, FJ687363-FJ687366,
FJ687368, FJ687370-FJ687379, FJ687381-FJ687384, JX124391, and JX124392).
1E.Y. and L.X. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: email@example.com or liuxz@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 3, 2012
| vol. 109
| no. 27www.pnas.org/cgi/doi/10.1073/pnas.1120915109
The evolution of ascomycetes that prey on nematodes was found
to have evolved along two lineages: that of fungi that capture
prey by constricting rings and that of fungi that capture prey by
adhesive structures (16).
In the present study, we investigated the origin of carnivorism
in the Ascomycota by three approaches. First, we conducted
a phylogenetic analysis based on five protein-coding genes from
16 carnivorous species in the Ascomycota representing a broad
diversity of trapping structures. Second, we used two fossil
records of carnivorous fungi in the Ascomycota as calibration
points to estimate when these fungi originated and diverged (24–
26). Third, we estimated when carnivorism diverged from sap-
rophytism in the Ascomycota by using the genome information
of Drechslerella stenobrocha, which produces constricting rings.
Evolution of the Carnivorous Structures in Ascomycota. Various
hypotheses on the evolution of trapping structures in the Asco-
mycota have been proposed, but conflicts exist between molec-
ular (28–30) and phenotypic (7, 31) phylogenies. We have
reported that the initial trapping structure evolved along two
lineages yielding two distinct trapping mechanisms—the con-
stricting rings and the adhesive traps—but the evolution of the
adhesive traps is still ambiguous, partly because the number of
taxa representing each type of trapping device was uneven (there
were more taxa with adhesive networks than with other adhesive
devices) (16). In the current study, we selected 16 taxa repre-
senting the six trapping structures: three species each for con-
stricting rings, sessile adhesive knobs, and adhesive nets; three
isolates within one species of adhesive columns; and two species
each for stalked adhesive knobs and nonconstricting rings (Table
1). As expected, individual nuclear genes did not provide a robust
analysis because of limited informative content (Fig. S2). How-
ever, the Bayesian inference phylogenetic tree, which was con-
structed with a data set consisting of 2,289 bp from five genes and
with a partitioned strategy, yielded a clear hypothesis regarding
the evolution of carnivorous fungi in Ascomycota (Fig. 1). To
minimize bias of the combined gene sequences, we constructed
phylogenetic trees by varying the order of the combined gene
sequences, and the same topology was obtained for the four trees
(Fig. S3). Taxa with the same trapping structure clustered to-
gether in the resulting consensus phylogenetic tree (Fig. 1 and Fig.
S3). The topology was also tested by a 1,000 bootstrap maximum-
likelihood analysis, and the bootstrap values for most of the major
clades were higher than 80%, except one clade between stalked
adhesive knobs and nonconstricting rings that is weakly supported
by a bootstrap value of 53% (Fig. 1 and Fig. S4). The results sug-
gest that the evolutionary relationship between SK and NCR + SK
is not fully resolved. One explanation is that the SKs are still in the
process of diverging from the NCR + SK.
The phylogenetic tree resulting from the five combined nuclear
genes not only confirmed our previous results of predicting evo-
lutionary trends of predacious cells in the Ascomycota (16), but
also provided insight into the evolution of the adhesive trapping
device lineage. The new tree resolved the adhesive lineage into
two major groups: one included the SSKs and AC clades, and the
other included the ANs, SKs, and NCR + SK clades (Fig. 1 and
Fig. S3). The high Bayesian posterior probabilities supported the
monophyly of the AC clade and the monophyly of the group
including the SSK clade and AC clade, suggesting that ACs were
derived from SSKs. In the other lineage, the divergence among
ANs, SK and nonconstricting rings plus stalked adhesive knobs
(NCR + SK) is not fully resolved in Fig. 1. However, SKs might
have derived from ANs and NCR + SK if Tuber melanosporum is
added as an outgroup (Fig. 2).
Estimation of the Divergence Times of Carnivorous Structures in
Ascomycota. Times of origination and divergence of the carniv-
orous structures were estimated by the combined gene sets. The
divergence time estimation may be unreliably relaxed if the root
range is unlimited. T. melanosporum (Pezizomycetes), whose
genome sequence has been published (32), was used as an out-
group because the base of Pezizomycetes and Orbiliomycetes
represents the crown of Pezizomycotina (33, 34). The root cali-
bration was set around 300–500 Mya (23). Two fossil records
were also included in the calibration (24–26). One of the fossils
was identified as an extinct trapping structure (unicellular ring,
or UCR) that was ancestral to ANs and NCRs (26) and was
Table 1. GenBank accession numbers for sequences used in the phylogenetic analysis of carnivorous Ascomycota
sp (543 bp)†
SpeciesTrapping structure* IsolateSYM + G‡
GTR + G‡
SYM + I + G‡
GTR + I + G‡
SYM + I + G‡
SK + NCR1
SK + NCR2
*AC, adhesive column; AN, adhesive net; CR, constricting ring; NCR, nonconstricting ring; OUT, outgroup; SSK, sessile adhesive knob; SK, stalked adhesive
†bt, β-tubulin; ef, elongation factor 1-α; mapk, mitogen-activated protein kinase; rpb2, RNA polymerase subunit II; sp, subtilisin-like serine protease.
‡Best nucleotide substitution model for each gene. SYM, symmetrical model; G, gamma distribution; GTR, general time reversible; I, proportion of invariable
Yang et al.PNAS
| July 3, 2012
| vol. 109
| no. 27
dated to 100 Mya (26). This fossil record enabled us to estimate
the divergence time of the last common ancestor (LCA) of ANs
and NCRs (Fig. 2, node A). The limits to node A were from 100
Mya (fossil) (25, 26) to 500 Mya (crown of Pezizomycotina) (23).
The second fossil record contained SKs and was dated to about
22.5–26 Mya (24), and this fossil record enabled us to estimate
the divergence time of the LCA of SKs (Fig. 2, node B). The
limits to node B were from 24 Mya (fossil) (24) to 500 Mya
(crown of Pezizomycotina) (23).
With these calibrations, we estimated that the divergence times
of major evolutionary events in carnivorous ascomycetes and
Pezizomycotina diverged about 419 Mya (95% highest posterior
density (HPD: 316–500 Mya). It took more than 150 million years
for the carnivorous fungi to further diverge, and the LCA for
carnivorous fungi in the Ascomycota centered on 246 Mya (95%
HPD: 158–349 Mya). The LCA of the mechanic trapping device
(constricting rings) and the adhesive trapping structures (adhe-
sive columns, adhesive nets, nonconstricting rings, stalked adhe-
sive knobs, and sessile adhesive knobs) centered on 148 Mya
(95% HPD: 82–229 Mya) and 208 Mya (95% HPD: 138–303
Mya), respectively. Within the adhesive trapping devices, the
SSKs and ACs originated 198 Mya (95% HPD: 125–286 Mya)
tioned strategy. All associated parameters were unlinked. The topology was also tested by a 1,000 bootstrap maximum-likelihood analysis; the bootstrap
value is marked on the branch. The branches with a bootstrap value lower than 70% are not shown. Trapping devices are drawn on the left. SSK, sessile
adhesive knob; SK, stalked adhesive knob; AN, adhesive net; AC, adhesive column; NCR, nonconstricting ring; CR, constricting ring.
Bayesian-inferred phylogenetic tree of carnivorous Orbiliomycetes by five protein-coding genes. The tree was constructed by MrBayes with a por-
Bayesian-inferred tree with three calibrations: root node (300–500 Mya), node A (100–500 Mya), and node B (24–500 Mya). Deep-branching phylogeny
illustrating the relationships among different lineages is shown by different colors. Error bars are shown and each represents the 95% highest posterior density
(HPD) for a node age. The red lines “a” and “b” represent the mass extinction events of the Permian–Triassic (251.4 Mya) and the Triassic–Jurassic (201.4 Mya),
respectively. The orange line “c” represents the mass extinction event of the Cretaceous–Tertiary (65.5 Mya). Nodes A and B indicate the two fossil calibration
nodes. SSK, sessile adhesive knob; SK, stalked adhesive knob; AN, adhesive net; AC, adhesive column; NCR, nonconstricting ring; CR, constricting ring.
Estimated divergence times of the major lineages in the carnivorous Orbiliomycetes. Chronogram was constructed using BEAST based on the
| www.pnas.org/cgi/doi/10.1073/pnas.1120915109Yang et al.
and differentiated adhesive columns 29 Mya (95% HPD: 11–55
Mya), respectively. The ANs, SKs, and nonconstricting rings
originated 200 Mya (95% HPD: 122–286 Mya) and differentiated
into adhesive nets 89 Mya (95% HPD: 49–136 Mya), NCR + SK
182 Mya (95% HPD: 115–268 Mya), stalked adhesive knobs 26
Mya (95% HPD: 24–34 Mya), and nonconstricting rings 162 Mya
(95% HPD: 97–245 Mya) (Table 2).
Divergence Time for the Pezizomycotina Crown by Phylogenomic
Analysis. In the analysis described in Fig. 2, the root node rep-
resents the origin of Pezizomycotina around 419 Mya (95%
HPD: 316–500 Mya). With genome sequences of D. stenobrocha,
which possesses CRs and seven other fungal genomes (Table S1),
we estimated divergence time for the Pezizomycotina crown at
three additional calibrated points independent of the two fossil
calibrations described in Fig. 2. On the basis of different iden-
tifications of taxonomy of a fossil record of Paleopyrenomycites
devonicus, the divergence time of Ascomycota and Basidiomy-
cota was estimated to be 452, 843, and 1,489 Mya, respectively
(23). Therefore, we set the origin of Dikaryotes between 452 and
1,489 Mya. The divergence times of Hypocreales, estimated by
the oldest fossil evidence of animals parasitized by fungi, were
also verified (23, 27), including the divergence time between
Clavicipitaceae and Hypocreaceae around 146–216 Mya and
among Nectriaceae, Clavicipitaceae, and Hypocreaceae around
150–213 Mya. The amino acid sequences of D. stenobrocha and
Trichoderma reesei were predicted by Augustus and Genemark,
and the predicted proteins were confirmed (35–38). For the six
other genomes, we collected the protein sequences on the basis
of previous annotations (32, 39–43). Calibration constraints used
in the phylogenomic dating analysis are summarized in Table S2.
The basal group of Pezizomycotina is ambiguous to date. One
study suggests that Orbiliomycetes is the basal group of Pezizo-
mycotina and that Pezizomycetes diverged afterward (33). The
other study proposes that Pezizomycetes is the basal group and
that Orbiliomycetes diverged afterward (34). On the basis of the
two conflicting hypotheses, we estimated the divergence time of
Pezizomycotina with two alternative topologies (Fig. 3 A and B).
We applied the same calibrations to the two topologies: root was
set around 452–1,489 Mya; the LCA of Clavicipitaceae and
Hypocreaceae (Hypocreales, Sordariomycetes) was centered on
150–213 Mya (27); and the LCA of Nectriaceae, Clavicipitaceae,
and Hypocreaceae (Hypocreales, Sordariomycetes) was centered
on 146–206 Mya (27). We identified 1,367 orthologous genes
from the eight genomes by Inparanoid 4.0 (44). However, 298
genes were excluded from analysis because they failed to apply to
at least one topology. Each gene was run in 600,000 generations
with tree collecting at every 100th generation. After discarding
the first 5,001 sampled trees, 1,000 trees remained for each gene.
We summarized 1,069,000 trees collected from the 1,069 genes
for each topology, and the results showed that time estimation
was similar in the two alternative topologies (Fig. 3 A and B).
The divergence time of Pezizomycotina was estimated at 261
Mya (Fig. 3A, 95% HPD: 151–525 Mya) and 260 Mya (Fig. 3B,
95% HPD: 151–511 Mya) in the alternative topologies. In Fig. 2,
we set the upper boundary of Pezizomycotina crown at 500 Mya,
and this is reasonable considering the upper ranges of 95% HPD
in Fig. 3 A and B are 525 and 511 Mya, respectively.
Evolution of Carnivorism in the Ascomycota. The differentiation
between trapping structures among major clades demonstrated
a clear evolutionary path of carnivorous fungi in the Ascomycota.
ibrations were applied to the two alternative topologies. The root node was
calibrated between 452 and 1,489 Mya. The last common ancestor (LCA) of
Nectriaceae, Clavicipitaceae, and Hypocreaceae was calibrated between 150
and 213 Mya. The divergence of Clavicipitaceae and Hypocreaceae was
calibrated between 146 and 206 Mya. Error bars are shown, each repre-
senting the 95% highest posterior density (95% HPD) for a node age.
Geological times are provided on the median axis. SSK, sessile adhesive
knob; SK, stalked adhesive knob; AN, adhesive net; AC, adhesive column;
NCR, nonconstricting ring; CR, constricting ring. (A) Orbiliomycetes is used as
the basal group of Pezizomycotima with Pezizomycetes diverging afterward.
(B) Pezizomycetes is used as the basal group with Orbiliomycetes diverging
The origin time of Pezizomycotina with 1,069 orthologs. Three cal-
Table 2. Divergence time estimation of major clades of carnivorous Ascomycota
95% HPD range
(Mya)Taxa Species 1Species 2
Ancestor of adhesive traps
Ancestor of CR
Ancestor of NCR
Ancestor of SK
Ancestor of NCR and SK
Ancestor of AN
Unicell ring (extinct)
Ancestor of SSK
Ancestor of AC
AC, adhesive column; AN, adhesive net; CR, constricting ring; NCR, nonconstricting ring; OUT, outgroup; SK, stalked adhesive knob;
SSK, sessile adhesive knob.
Yang et al.PNAS
| July 3, 2012
| vol. 109
| no. 27
an unknown ancestor. SSKs and UCRs originated from the adhe-
sive ancestor. ACs diverged from SSKs by the proliferation of ad-
hesive knobs into a column, which increased the adhesive surface
and predatory efficiency. the UCR probably became extinct due to
low predatory efficiency (the unicellular ring is morphologically
similar to NCRs, which have the lowest predatory efficiency of
existing trapping fungi). The unicellular ring evolved along two
lineages to increase predatory efficiency. One formed ANs by
proliferating the ring into a network of rings. The other formed
NCRs accompanied by SKs, in which the single-celled rings de-
veloped into three-celled nonconstricting rings, unicellular knobs,
and SKs. Our results suggest two potential evolutionary relation-
ships between SK and NCR + SK. One possibility is that NCRs
were addedin some specieswith stalked adhesiveknobsto increase
carnivorous efficiency. The other possibility is that fungi with
NCR + SK gradually degenerated into species with stalked adhe-
sive knobs only to reduce the high energy cost of producing non-
constricting rings. In our previous study, we hypothesized that the
evolutionary trend of carnivorous fungi in Orbiliomycetes (Asco-
mycota) is toward elongation of the stalk of trapping devices (16).
The results from this study not only confirmed that hypothesis, but
also demonstrated that the evolutionary trend of carnivorous
Orbiliomycetes is toward increased adhesive surface area or elon-
gation of the adhesive structures from the mycelium.
Origin of Fungal Carnivorism in the Orbiliomycetes. It is natural to
hypothesize that the nematode-trapping fungi evolved after the
appearance ofnematodes.The phylumNematodaandother major
invertebrate clades have been proposed to appear during the
Cambrian Explosion about 550–600 Mya (45), which is about 130–
180 million years ahead of the divergence of carnivorous ascomy-
cetes about 419 Mya as proposed in this study. Fossil records also
support the notion that nematodes evolved ahead of carnivorous
oldest carnivorous fungal fossil is dated about 100 Mya (26).
To shed light on the origin of fungal carnivorism, important
biological and geological events around the divergence times of
the trapping structures were surveyed. Although the 95% HPD
range of each node spans a large range, we found that the me-
dian timing of the major evolutionary events of these fungi
seemed to coincide with mass extinction events. The crown of
these carnivorous species arose around 246 Mya, which was 5
million years (Myr) after the Permian–Triassic extinction (251.4
Mya) (47). Major clades of the extant carnivorous species di-
verged around 208, 200, and 198 Mya, which centered on the
Triassic–Jurassic extinction (201.4 Mya).
Mass extinctions resulted in a sharp decrease in the diversity and
abundance of macroscopic creatures and the deposition of a huge
biomass of dead plants and animals. The dead organic materials
supported a significant increase in the diversity and biomass of
microorganisms (48). When ecosystems began to recover, these
decomposers likely encountered substantial competition for
nutrients because most of the dead organic materials had been
consumed. Geochronologic and biostratigraphic constraints based
on high-precision U-Pb dates of single zircons from south China
allow us to place the Early to Middle Triassic (Olenekian–Anisian)
boundary at 247.2 Mya (49). The new dates constrain the Early
Triassic interval characterized by delayed biotic recovery and car-
bon-cycle instability to 5 Mya (49). This time constraint must be
considered in any model for the end-Permian extinction and sub-
sequent recovery (50). Our data are consistent with a long recovery
time in that the crown of extant carnivorous Ascomycota arose
about 5 Myr later than the Permian–Triassic extinction event.
In addition to the Permian–Triassic and Triassic–Jurassic ex-
tinction events, another extinction event known as the Creta-
ceous–Tertiary extinction occurred more recently, about 65.5
Mya (51). In the current study, we did not find a similar re-
lationship between carnivorous ascomycetes and the Creta-
ceous–Tertiary extinction. As our data did not provide tight time
estimation for each of the major notes, the relationship between
fungal carnivorism evolution and a mass extinction event is not
validated in this study. More evidence, including additional fossil
records, is needed to establish if fungal carnivorism evolution
was a response to mass extinction events.
Materials and Methods
Fungal Isolates and Sequence Collection. Sixteen isolates of carnivorous
ascomycetes were used in this study (Table 1). Each of the six trapping
structures was represented by three isolates, except the stalked adhesive
knobs and nonconstricting rings, which were represented by two isolates.
Stalked adhesive knobs occurred without nonconstricting rings in two cases
and with nonconstricting rings in two other cases; nonconstricting rings al-
ways occurred with stalked adhesive knobs as nonconstricting rings plus
stalked adhesive knobs. In most cases, each isolate was selected from a dif-
ferent species. Only Gamsylella cionopaga produced adhesive columns traps;
however, we used three isolates of this species.
The sequences of bt, ef, and rpb2 were obtained from GenBank and from
our previous studies (Table 1), while mapk and sp were newly sequenced.
Primers and PCR conditions used in cloning and sequencing of the mapk and
sp genes are detailed in SI Materials and Methods. The five corresponding
gene sequences of the outgroup were collected from the genome of T.
melanosporum by BLAST (v.2.2.25) (52).
Phylogenetic Analysis of Carnivorous Fungi in the Ascomycota. DNA sequences
were assembled manually. Exons of the protein-coding genes were anno-
tated on the basis of the RefSeq database. Sequences used in phylogenetic
analysis were edited with BioEdit (ver. 220.127.116.11) (53, 54) and aligned with
ClustalX (ver. 2.0.12) (55). The protein-coding genes were aligned on the
basis of the translated amino acid sequences. The nucleotide substitution
models of each gene were selected by MrModeltest (ver. 2.3) (56) based on
the corrected Akaike Information Criterion. Bayesian phylogenetic analyses
and nodal support estimation are detailed in SI Materials and Methods.
Divergence Time Estimation of Carnivorous Structures in the Ascomycota. For
the molecular dating analysis of the data set, the Bayesian relaxed molecular
calibration points corresponding to the fossil records and evolutionary events
were used in the analysis as soft constraints following a uniform limitation.
These dates correspondedto the formation of stalked adhesive knobs (24–500
the rise of Pezizomycotina, was set at 300–500 Mya (23). We used as priors the
uncorrelated log-normal molecular clock model with a Yule process for the
model of speciation, whereas the SR06 model was used for protein-coding
Carlo calculation is detailed in SI Materials and Methods.
Divergence Time Estimation by Phylogenomic Data. We selected eight fungal
genomes to verify the root node in Fig. 2 (represented by the divergence
between D. stenobrocha and T. melanosporum) in the Ascomycota (Table 2).
Detailed parameters and methods are described in SI Materials and Methods.
ACKNOWLEDGMENTS. The authors thank Prof. Bruce A. Jaffee at the
University of California for valuable English editing of the manuscript. We
also thank the editor and anonymous reviewers. This work was supported by
the National Natural Scientific Foundation of China (Grant 30625001) and by
The Welch Foundation.
and radionuclides by fungi, bioweathering and bioremediation. Mycol Res 111:3–49.
2. Liu XZ, Xiang MC, Che YS (2009) The living strategy of nematophagous fungi.
3. Drechsler C (1941) Four Phycomycetes destructive to nematodes and rhizopods.
4. Pramer D (1964) Nematode-trapping fungi. Science 144:382–388.
5. Thorn RG, Barron GL (1984) Carnivorous mushrooms. Science 224:76–78.
| www.pnas.org/cgi/doi/10.1073/pnas.1120915109Yang et al.
6. McInnes SJ (2003) A predatory fungus (Hyphomycetes: Lecophagus) attacking Roti-
fera and Tardigrada in maritime Antarctic lakes. Polar Biol 26:79–82.
7. Li TF, Zhang KQ, Liu XZ (2000) Taxonomy of Nematophagous Fungi (Chinese Scientific
and Technological Publications, Beijing).
8. Kirk PM, Cannon PF, Minter DW, Stalpers JA (2008) Dictionary of the Fungi (CAB In-
ternational, Wallingford, Oxon, UK), 10th Ed.
9. Saikawa M (2011) Ultrastructural studies on zygomycotan fungi in the Zoopagaceae
and Cochlonemataceae. Mycoscience 52:83–90.
10. Duddington CL (1973) Zoopagales. The Fungi, eds Ainsworth GC, Sparrow FK, Suss-
man AS (Academic Press, New York), Vol 4B, pp 231–234.
11. Poloczek E, Webster J (1994) Conidial traps in Nematoctonus (nematophagous Basi-
diomycetes). Nova Hedwigia 59:201–205.
12. Durschnerpelz UU (1987) Traps of Nematoctonus leiosporus: An unusual feature of an
endoparasitic nematophagous fungus. Trans Br Mycol Soc 88:129–130.
13. Barron GL, Dierkes Y (1977) Nematophagous fungi: Hohenbuehelia, perfect state of
Nematoctonus. Can J Bot 55:3054–3062.
14. Thorn RG, Moncalvo JM, Reddy CA, Vilgalys R (2000) Phylogenetic analyses and the
distribution of nematophagy support a monophyletic Pleurotaceae within the poly-
phyletic pleurotoid-lentinoid fungi. Mycologia 92:241–252.
15. Koziak ATE, Cheng KC, Thorn RG (2007) Phylogenetic analyses of Nematoctonus and
Hohenbuehelia (Pleurotaceae). Can J Bot 85:762–773.
16. Yang Y, Yang E, An ZQ, Liu XZ (2007) Evolution of nematode-trapping cells of
predatory fungi of the Orbiliaceae based on evidence from rRNA-encoding DNA and
multiprotein sequences. Proc Natl Acad Sci USA 104:8379–8384.
17. Li J, et al. (2010) New insights into the evolution of subtilisin-like serine protease
genes in Pezizomycotina. BMC Evol Biol 10:68.
18. Barron GL (1977) The Nematode-Destroyiung Fungi (Canadian Biological Publications
Ltd., Guelph, ON).
19. Stirling GR (1991) Biological Control of Plant Parasitic Nematodes (CAB International,
20. Barron GL (2003) Predatory fungi, wood decay, and the carbon cycle. Biodiversity
21. Brewer JS (2003) Why don’t carnivorous pitcher plants compete with non-carnivorous
plants for nutrients? Ecology 84:451–462.
22. Stenroos S, et al. (2010) Multiple origins of symbioses between ascomycetes and
bryophytes suggested by a five-gene phylogeny. Cladistics 26:281–300.
23. Berbee ML, Taylor JW (2010) Dating the molecular clock in fungi: How close are we?
Fungal Biol Rev 24:1–16.
24. Jansson HB, Poinar GO (1986) Some possible fossil nematophagous fungi. Trans Br
Mycol Soc 87:471–474.
25. Schmidt AR, Dörfelt H, Perrichot V (2007) Carnivorous fungi from Cretaceous amber.
26. Schmidt AR, Dörfelt H, Perrichot V (2008) Palaeoanellus dimorphus gen. et sp. nov.
(Deuteromycotina): A Cretaceous predatory fungus. Am J Bot 95:1328–1334.
27. Sung GH, Poinar GO, Jr., Spatafora JW (2008) The oldest fossil evidence of animal
parasitism by fungi supports a Cretaceous diversification of fungal-arthropod sym-
bioses. Mol Phylogenet Evol 49:495–502.
28. Liou GY, Tzean SS (1997) Phylogeny of the genus Arthrobotyrs and allied nematode-
trapping fungi based on rDNA sequences. Mycologia 89:876–884.
29. Ahrén D, Ursing BM, Tunlid A (1998) Phylogeny of nematode-trapping fungi based on
18S rDNA sequences. FEMS Microbiol Lett 158:179–184.
30. Li Y, et al. (2005) Phylogenetics and evolution of nematode-trapping fungi (Orbiliales)
estimated from nuclear and protein coding genes. Mycologia 97:1034–1046.
31. Rubner A (1996) Revision of predacious hyphomycetes in the Dactylella-Mon-
acrosporium complex. Stud Mycol 39:1–129.
32. Martin F, et al. (2010) Périgord black truffle genome uncovers evolutionary origins
and mechanisms of symbiosis. Nature 464:1033–1038.
33. Spatafora JW, et al. (2006) A five-gene phylogeny of Pezizomycotina. Mycologia 98:
34. Schoch CL, et al. (2009) A class-wide phylogenetic assessment of Dothideomycetes.
Stud Mycol 64(1):1–15.
35. Stanke M, et al. (2006) AUGUSTUS: Ab initio prediction of alternative transcripts.
Nucleic Acids Res 34(Web Server issue):W435-W439.
36. Stanke M, Morgenstern B (2005) AUGUSTUS: A web server for gene prediction in
eukaryotes that allows user-defined constraints. Nucleic Acids Res 33(Web Server is-
37. Stanke M, Steinkamp R, Waack S, Morgenstern B (2004) AUGUSTUS: A web server for
gene finding in eukaryotes. Nucleic Acids Res 32(Web Server issue):W309-W312.
38. Besemer J, Borodovsky M (2005) GeneMark: Web software for gene finding in pro-
karyotes, eukaryotes and viruses. Nucleic Acids Res 33(Web Server issue):W451-W454.
39. Wood V, et al. (2002) The genome sequence of Schizosaccharomyces pombe. Nature
40. Gao QA, et al. (2011) Genome sequencing and comparative transcriptomics of the
model entomopathogenic fungi Metarhizium anisopliae and M. acridum. PLoS Genet
41. Coleman JJ, et al. (2009) The genome of Nectria haematococca: Contribution of su-
pernumerary chromosomes to gene expansion. PLoS Genet 5:e1000618.
42. Goffeau A, et al. (1996) Life with 6000 genes. Science 274:546, 563–567.
43. Loftus BJ, et al. (2005) The genome of the basidiomycetous yeast and human path-
ogen Cryptococcus neoformans. Science 307:1321–1324.
44. O’Brien KP, Remm M, Sonnhammer ELL (2005) Inparanoid: A comprehensive database
of eukaryotic orthologs. Nucleic Acids Res 33(Database issue):D476–D480.
45. van Megen H, et al. (2009) A phylogenetic tree of nematodes based on about 1200
full-length small subunit ribosomal DNA sequences. Nematology 11:927–950.
46. Poinar G, Kerp H, Hass H (2008) Palaeonema phyticum gen. n., sp. n. (Nematoda:
Palaeonematidae fam. n.), a Devonian nematode associated with early land plants.
47. Jin YG, et al. (2000) Pattern of marine mass extinction near the Permian-Triassic
boundary in South China. Science 289:432–436.
48. Visscher H, et al. (1996) The terminal Paleozoic fungal event: Evidence of terrestrial
ecosystem destabilization and collapse. Proc Natl Acad Sci USA 93:2155–2158.
49. Wang JA, et al. (2008) Chronology and geochemistry of the volcanic rocks in Woruo
Mountain region, northern Qiangtang depression: Implications to the Late Triassic
volcanic-sedimentary events. Sci China Ser D 51:194–205.
50. Lehrmann DJ, et al. (2006) Timing of recovery from the end-Permian extinction:
Geochronologic and biostratigraphic constraints from south China. Geology 34:
51. Schulte P, et al. (2010) The Chicxulub asteroid impact and mass extinction at the
Cretaceous-Paleogene boundary. Science 327:1214–1218.
52. Altschul SF, et al. (1997) Gapped BLAST and PSI-BLAST: A new generation of protein
database search programs. Nucleic Acids Res 25:3389–3402.
53. Hall T (2005) BioEdit: Biological Sequence Alignment Editor for Win95/98/NT/2K/XP.
(Ibis Therapeutics, Carlsbad, CA).
54. Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and
analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98.
55. Larkin MA, et al. (2007) Clustal W and clustal X version 2.0. Bioinformatics 23:
56. Nylander JAA (2004) MrModeltest v2. Program distributed by the author (Evolu-
tionary Biology Centre, Uppsala University, Uppsala, Sweden).
57. Drummond AJ, Rambaut A (2007) BEAST: Bayesian evolutionary analysis by sampling
trees. BMC Evol Biol 7:214.
Yang et al. PNAS
| July 3, 2012
| vol. 109
| no. 27