The phylogenetic placement of hypocrealean insect pathogens in the genus Polycephalomyces: An application of One Fungus One Name

Abstract

Understanding the systematics and evolution of clavicipitoid fungi has been greatly aided by the application of molecular phylogenetics. They are now classified in three families, largely driven by reevaluation of the morphologically and ecologically diverse genus Cordyceps. Although reevaluation of morphological features of both sexual and asexual states were often found to reflect the structure of phylogenies based on molecular data, many species remain of uncertain placement due to a lack of reliable data or conflicting morphological characters. A rigid, darkly pigmented stipe and the production of a Hirsutella-like anamorph in culture were taken as evidence for the transfer of the species Cordyceps cuboidea, Cordyceps prolifica, and Cordyceps ryogamiensis to the genus Ophiocordyceps. Data from ribosomal DNA supported these species as a single group, but were unable to infer deeper relationships in Hypocreales. Here, molecular data for ribosomal and protein coding DNA from specimens of Ophiocordyceps cuboidea, Ophiocordyceps ryogamiensis, Ophiocordyceps paracuboidea, Ophiocordyceps prolifica, Cordyceps ramosopulvinata, Cordyceps nipponica, and isolates of Polycephalomyces were combined with a broadly sampled dataset of Hypocreales. Phylogenetic analyses of these data revealed that these species represent a clade distinct from the other clavicipitoid genera. Applying the recently adopted single system of nomenclature, new taxonomic combinations are proposed for these species in the genus Polycephalomyces, which has been historically reserved for asexual or anamorphic taxa.

Full text

The phylogenetic placement of hypocrealean insect
pathogens in the genus Polycephalomyces: An application
of One Fungus One Name
Ryan KEPLER
a,
*, Sayaka BAN
b
, Akira NAKAGIRI
c
, Joseph BISCHOFF
d
,
Nigel HYWEL-JONES
e
, Catherine Alisha OWENSBY
a
, Joseph W. SPATAFORA
a
a
Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA
b
Department of Biotechnology, National Institute of Technology and Evaluation, 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan
c
Division of Genetic Resource Preservation and Evaluation, Fungus/Mushroom Resource and Research Center, Tottori University, 101, Minami
4-chome, Koyama-cho, Tottori-shi, Tottori 680-8553, Japan
d
Animal and Plant Health Inspection Service, USDA, Beltsville, MD 20705, USA
e
Bhutan Pharmaceuticals Private Limited, Upper Motithang, Thimphu, Bhutan
article info
Article history:
Received 27 August 2012
Received in revised form
28 May 2013
Accepted 12 June 2013
Corresponding Editor: Kentaro Hosaka
Keywords:
Anamorph-teleomorph connection
Cordyceps
Hirsutella
Molecular phylogenetics
Morphological plasticity
abstract
Understanding the systematics and evolution of clavicipitoid fungi has been greatly aided by
the application of molecular phylogenetics. They are now classified in three families, largely
driven by reevaluation of the morphologically and ecologically diverse genus Cordyceps.
Although reevaluation of morphological features of both sexual and asexual states were
often found to reflect the structure of phylogenies based on molecular data, many species
remain of uncertain placement due to a lack of reliable data or conflicting morphological
characters. A rigid, darkly pigmented stipe and the production of a Hirsutella-like anamorph
in culture were taken as evidence for the transfer of the species Cordyceps cuboidea,Cordyceps
prolifica, and Cordyceps ryogamiensis to the genus Ophiocordyceps. Data from ribosomal DNA
supported these species as a single group, but were unable to infer deeper relationships in
Hypocreales. Here, molecular data for ribosomal and protein coding DNA from specimens
of Ophiocordyceps cuboidea,Ophiocordyceps ryogamiensis,Ophiocordyceps paracuboidea,Ophiocor-
dyceps prolifica,Cordyceps ramosopulvinata,Cordyceps nipponica, and isolates of Polycephalomy-
ces were combined with a broadly sampled dataset of Hypocreales. Phylogenetic analyses
of these data revealed that these species represent a clade distinct from the other clavicipi-
toid genera. Applying the recently adopted single system of nomenclature, new taxonomic
combinations are proposed for these species in the genus Polycephalomyces, which has
been historically reserved for asexual or anamorphic taxa.
Published by Elsevier Ltd on behalf of The British Mycological Society.
Introduction
Molecular phylogenetic investigation of the family Clavicipita-
ceae sensu Rogerson, particularly for the genus Cordyceps, has
revealed significant phylogenetic diversity best represented by
unique family level taxa (Sung et al. 2007a). The reevaluation of
Cordyceps showed that characters historically used to define
genera and subgenera did not corroborate with results from
molecular data. Current taxonomic concepts for the clavicipi-
toid fungi now recognize three families: Clavicipitaceae sensu
*Corresponding author. Current address: Systematic Mycology and Microbiology Laboratory, USDA-ARS, Beltsville, MD 20705, USA.
journal homepage: www.elsevier.com/locate/funbio
fungal biology xxx (2013) 1e12
1878-6146/$ esee front matter Published by Elsevier Ltd on behalf of The British Mycological Society.
http://dx.doi.org/10.1016/j.funbio.2013.06.002
Please cite this article in press as: Kepler R, et al., The phylogenetic placement of hypocrealean insect pathogens in the genus
Polycephalomyces: An application of One Fungus One Name, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.06.002

stricto, Cordycipitaceae and Ophiocordycipitaceae. Traits
such as orientation of perithecia and morphology of asco-
spores, which previously were used to define subgeneric
boundaries, were distributed across all three families. Mor-
phological characters that were most consistent with the
resulting molecular phylogeny and that correlated with ge-
neric boundaries included texture and colour of the stroma
as well as ecological niche (Sung et al. 2007a). The genus Cor-
dyceps s.s. in Cordycipitaceae contains species with a fleshy
texture and brightly coloured stromata and tends to attack
hosts in leaf litter or shallow soil. The genus Metacordyceps
in Clavicipitaceae contains species producing a stroma with a
firm, fibrous texture and predominantly green, pallid or lilac
coloration, which darkens to a purple or black upon bruising
or drying (Sung et al. 2007a;Kepler et al. 2012a). Ophiocordyceps
in Ophiocordycipitaceae comprises fungi producing a rigid,
pliant or wiry stipe that is darkly coloured and are typically
found on hosts buried in soil or in rotting wood. Although
these characters correspond well to clades, exceptions occur
due to the homoplasious distributions of several character
states (e.g., brightly pigmented clava of Ophiocordyceps nutans
attacking adult hemipterans).
Anamorph morphologies were also demonstrated to have
varying degrees of phylogenetic informativeness (Sung et al.
2007a). Species in the genus Metacordyceps produce ana-
morphs in the genera Pochonia and Metarhizium, as well as
green-spored forms of Nomuraea, whereas pink or lilac forms
of Nomuraea can be found in Ophiocordycipitaceae (Sung
et al. 2007a;Kepler et al. 2012a). Ophiocordycipitaceae are
also associated with Hymenostilbe and Hirsutella anamorphs,
which are produced on stromata that often concurrently or
subsequently give rise to perithecia. Anamorphic forms re-
stricted to Cordycipitaceae include Lecanicillium,Isaria and
Beauveria associated with Cordyceps s.s. and Gibellula, associ-
ated with Torrubiella (Sung et al. 2007a). Although these ana-
morphic forms are fairly indicative of family and genus level
associations, examples do exist of broadly distributed ana-
morph genera (e.g., residual Verticillium,Zare et al. 2000;Paeci-
lomyces,Luangsa-ard et al. 2004), which can complicate the
placement of asexually reproductive taxa in the modern phy-
logenetic classification.
Recent hypotheses concerning evolution of host affiliation
support arthropod pathogens as being an ancestral ecology for
many lineages of clavicipitoid fungi with dynamic host shifts
among diverse insect groups and repeated jumping onto
plants and other fungi (Spatafora et al. 2007; Sung et al. 2008;
Kepler et al. 2012b). For example, although pathogens of other
fungi can be found throughout clavicipitoid fungi, those
attacking false truffles in the genus Elaphomyces are restricted
to Elaphocordyceps of Ophiocordycipitaceae (Sung et al. 2007a)
and species infecting the sclerotia of Claviceps are restricted
to Tyrannicordyceps (Kepler et al. 2012b) of Clavicipitaceae.
Pathogens of spiders are most commonly encountered in Cor-
dycipitaceae in the genera Torrubiella and Cordyceps, but insect
hosts tend be more broadly distributed with Coleoptera, Lepi-
doptera and Hemiptera found in all three families.
Polycephalomyces Kobayasi is an anamorph genus with an
unconfirmed phylogenetic placement and teleomorph affinity
that has proven difficult to incorporate into evolutionary hy-
potheses of clavicipitoid fungi. This confusion stems from
a long history of conflicting hypotheses regarding host sub-
strate and teleomorph affinities. Species of Polycephalomyces
have often been found associated with the stromata of ento-
mopathogenic Cordyceps (Massee 1895; Kobayasi 1941). How-
ever, it has remained unclear whether Polycephalomyces spp.
represent anamorphic expressions of Cordyceps spp. or are hy-
perparasites of the latter (Seifert 1985). The type species, Poly-
cephalomyces formosus, is synnematous, determinate, and
produces small obovoid to ellipsoidal conidia (A-conidia) in
a mucous-like matrix (Seifert 1985).
The convergent nature of some characters across all
three families of clavicipitoid fungi leaves a considerable
number of taxa of uncertain placement, resulting in the re-
sidual Cordyceps s.l. of Sung et al. (2007a). Morphological and
ecological character states for these taxa were either lacking
or inconclusive and no molecular sequence data were avail-
able to test character state homologies. Ban et al. (2009) used
the large subunit of nuclear ribosomal RNA (LSU) and the
complete span of the internal transcribed spacer region
(ITS) to address the phylogenetic placement of four species
of residual Cordyceps s.l., including Cordyceps alboperitheciata
Kobayasi & Shimizu, Ophiocordyceps cuboidea (Kobayasi &
Shimizu) S. Ban, Sakane & Nakagiri, Ophiocordyceps ryoga-
miensis (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-
Jones & Spatafora, and Oryogamiensis prolifica (Kobayasi)
S. Ban, Sakane & Nakagiri. They also successfully cultured
the anamorphic forms from fresh material. The molecular
data showed these species formed a well-supported clade
sister to the species Cordyceps ramosopulvinata Kobayasi &
Shimizu and Cordyceps kanzashiana Kobayasi & Shimizu. Fur-
thermore, a cryptic species was uncovered (Ophiocordyceps
paracuboidea S. Ban, Sakane & Nakagiri) and the name
C. alboperitheciata was found to be synonymous with
O. cuboidea. However, genus and family level relationships
for this group remained unsupported and classification of
these species within the phylogenetic framework for clavici-
pitoid fungi was not possible using molecular data alone.
The anamorphic forms were described as Hirsutella-like
and this was used as justification to move these taxa into
the genus Ophiocordyceps.
In this paper we expand sampling of molecular data for of
O. cuboidea,O. ryogamiensis,O. paracuboidea and O. prolifica
sampled by Ban et al. (2009). We also expand the sampling to
include Cordyceps nipponica Kobayasi, Cordyceps pleuricapitata
Kobayasi and Shimizu, and the anamorph species P. formosus
and Blistum tomentosum, a species previously included in Poly-
cephalomyces. When incorporated into a multigene dataset
including representatives from six hypocrealean families,
including all clavicipitoid lineages (Sung et al. 2007a), we find
these taxa are not supported as members of Ophiocordyceps,
but rather represent a unique taxon that is not placed in any
existing genus or family of teleomorphs. Consistent with
the application of a single system of nomenclature to a clade
of fungi regardless of life history states (Taylor 2011;
Hawksworth et al. 2011), we emend the genus Polycephalomyces
to include teleomorphs C. kanzashiana,C. ramosopulvinata,
C. nipponica,O. cuboidea,O. ryogamiensis,O. paracuboidea and
O. prolifica, and discuss its phylogenetic relationship to other
hypocrealean fungi. We also conclude B. tomentosum belongs
in Polycephalomyces.
2 R. Kepler et al.
Please cite this article in press as: Kepler R, et al., The phylogenetic placement of hypocrealean insect pathogens in the genus
Polycephalomyces: An application of One Fungus One Name, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.06.002

Materials and methods
Specimen collection
Tissue from cultures of Ophiocordyceps cuboidea,Ophiocordyceps
ryogamiensis,Ophiocordyceps paracuboidea and Oryogamiensis
prolifica sampled in the Ban et al. (2009) paper were resampled
from stocks maintained at the National Institute of Technology
and Evaluation Biological Resource Center (NBRC). Material
from cultures of Cordyceps pleuricaptiata, also maintained at
NBRC, was included. In addition fresh material was collected
from field sites in Japan during the months of JuneeAug. in
the years 2007 and 2008. Upon collection, specimens were
cleaned of dirt and placed in a wax paper bag, a piece of tissue
was removed and placed in CTAB buffer for DNA extraction
and later air dried for herbarium storage.
Ascospores of Thai material were discharged onto PDA
and germinated in 24e36 h at ambient field temperatures
(20e32 C). Part-spores of Cordyceps ramosopulvinata were
discharged onto Sabouraud Dextrose Agar and germinated
at w25 C. These isolations were then sent to Rutgers Univer-
sity where they were subcultured onto PDA and kept at
24e27 C in continuous light (fluorescent lights). (See Table 1
for further details on isolates used in this study.)
DNA extraction, PCR and sequencing
Tissue received an initial grinding by power drill with an Eppen-
dorf pestle in 50 ml CLS-VF buffer from the FastDNA Spin Kit (MP
Biomedicals, Salon, OH). Four hundred microlitres of CLS-VF
was then added to the ground tissue in a FastDNA lysing matrix
A tube and groundfurther with thefast prep machine for twocy-
cles, 20 s each. Cell lysis was enhanced by soaking for 20 min in
a water bath at 60 C. Tissue was then separated from the super-
natant by centrifugation for 10 min at 14 000 rpm. Four hundred
microlitres of supernatant was then removed for further clean-
ing with centrifugation at 14 000 rpm for 20 min in 500 ml chlor-
oform:isoamyl alcohol (24:1). Cleaned and concentrated DNA
was then obtained from 300 ml of the top layer of liquid after
chloroform:isoamyl centrifugation with the GeneCleanIII Kit
following the manufacturers protocol and eluting from glass
milk in the final step with 30 ml of water.
PCR methods were used to amplify a total of six nuclear loci
for each specimen. As an initial qualitycontrol step, and toserve
as a voucher for barcoding efforts, the complete span of the
internal transcribed spacer region of ribosomal DNA (ITS1-
5.8s-ITS2) was amplified and sequenced. A BLAST search of
the GenBank database was performed to ensure that DNAs
obtained were not from contaminants outside of Hypocreales.
After passing quality control measures, five nuclear loci were
amplified and sequenced for phylogenetic analysis: SSU and
LSU, elongation factor 1a(TEF),and the largest and second larg-
est subunitsof RNA polymerase II (RPB1 and RPB2, respectively).
Primer information and PCR cycle parameters are described
in Kepler et al. (2012a) unless otherwise noted. PCR reactions
were performed in a MyCycler thermocycler (BioRad, Hercules,
CA) using either MasterAmp 2X PCR premix E (Epicenter, Madi-
son WI) and GenScript Taq polymerase or PuReTaq Ready-To-
Go PCR Beads (GE Healthcare, Little Chalfont, Buckinghamshire,
UK). Sequencing reactions were performed at University of
Washington High-Throughput Sequencing Solutions (Seattle,
WA) with the primers used for the initial amplifications.
Phylogenetic analyses
Processing of raw sequence reads and construction of contigs
was performed using CodonCode Aligner, version 2.0.6 (Ded-
ham, MA). A dataset was then assembled with representative
species throughout Hypocreales in the families Hypocreaceae,
Nectriaceae and Bionectriaceae in Hypocreales, as well as the
outgroup taxa Glomerella cingulata and Verticillium dahliae. For
nearly all specimens examined, at least three of the five genes
sought were obtained. Data for Cordyceps kanzashiana included
only SSU and LSU. GenBank and specimen voucher informa-
tion is provided in Table 1. MAFFT version 6 (Katoh et al. 2002;
Katoh & Toh 2008) was used to obtain an initial alignment
that was then improved by visual examination with the pro-
gram BioEdit version 7.05 (Hall 1999). Ambiguously aligned re-
gions were identified with the default settings of Gblocks server
(http://molevol.cmima.csic.es/castresana/Gblocks_ser-
ver.html)(Castresana 2000;Talavera & Castresana 2007) and
excluded from phylogenetic analyses and gaps were treated
as missing data. The final dataset contained sequences from
153 specimens. After exclusion of ambiguously aligned sites
there were 1021 nucleotides for SSU, 814 for LSU, 920 for TEF,
641 for RPB1 and 1111 for RPB2. The total length of aligned se-
quences was 4507 bp. Conflict between loci was examined
with the program compat.py (Kauff & Lutzoni 2002).
Maximum Likelihood (ML) estimation of phylogeny was
performed with RAxML version 7.0.4 (Stamatakis 2006) with
500 rapid bootstrap replicates on a concatenated dataset con-
taining all five genes. Eleven data partitions were defined for
the final combined dataset, one each for SSU and LSU plus
nine for each of the three codon positions for the protein cod-
ing genes TEF, RPB1, and RPB2. The CAT-GAMMA model of nu-
cleotide substitution was applied to each partition during the
rapid bootstrapping phase and the GTR-GAMMA model of nu-
cleotide substitution was specified for the final likelihood tree
as suggested by the program manual for large datasets. Bayes-
ian estimation of phylogenetic relationships was conducted
with the program Mr. Bayes v3.1 (Ronquist & Huelsenbeck
2003). The same eleven data partitions were applied with the
GTR þIþG nucleotide substitution model used for all gene
partitions. Two runs were conducted simultaneously, each
with four chains for ten million generations. Each chain was
sampled every 100 generations, and trees saved with branch
length information every 500 generations. After the analysis
finished, each run was examined with the program Tracer
v1.5 (Drummond & Rambaut 2007) to determine burn-in and
confirm that both runs had converged. Summary of the model
parameters was determined with the sump command. A strict
consensus tree with branch lengths and posterior probabili-
ties was then obtained with the sumt command. The same
burnin value was used for both sump and sumt commands.
Results
ML and Bayesian analyses confirmed Cordyceps ramosopulvi-
nata,Cordyceps nipponica,Cordyceps kanzashiana Ophiocordyceps
The phylogenetic placement of hypocrealean insect pathogens in the genus Polycephalomyces 3
Please cite this article in press as: Kepler R, et al., The phylogenetic placement of hypocrealean insect pathogens in the genus
Polycephalomyces: An application of One Fungus One Name, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.06.002

Table 1 eSpecimen information for materials used in this study.
Taxon Host Voucher# GenBank accession numbers
ITS SSU LSU TEF RPB1 RPB2
Akanthomyces novoguineensis Araneae NHJ 11923 EU369095 EU369032 EU369013 EU369052 EU369072
Aphysiostroma stercorarium Cow dung ATCC 62321 AF543769 AF543792 AF543782 AY489633 EF469103
Aschersonia cf. badia Hemiptera BCC 7016 JN049839 DQ372091 DQ384941 DQ384969 DQ385009 DQ452460
Aschersonia confluence Hemiptera BCC 7961 JN049841 DQ372100 DQ384947 DQ384976 DQ384998 DQ452465
Aschersonia placenta Hemiptera BCC 7869 JN049842 EF469121 EF469074 EF469056 EF469085 EF469104
Balansia epichlo€
ePoaceae AEG 96-15a JN049848 EF468949 EF468743 EF468851 EF468908
Balansia henningsiana Poaceae GAM 16112 JN049815 AY545723 AY545727 AY489610 AY489643 DQ522413
Balansia pilulaeformis Poaceae AEG 94-2 JN049816 AF543764 AF543788 DQ522319 DQ522365 DQ522414
Bionectria ochroleuca Bark CBS 114056 AY489684 AY489716 AY489611 DQ842031 DQ522415
Claviceps fusiformis Poaceae ATCC 26019 JN049817 DQ522539 U17402 DQ522320 DQ522366
Claviceps paspali Poaceae ATCC 13892 JN049818 U32401 U47826 DQ522321 DQ522367 DQ522416
Claviceps purpurea Poaceae GAM 12885 U57669 AF543765 AF543789 AF543778 AY489648 DQ522417
Claviceps purpurea Poaceae SA cp11 EF469122 EF469075 EF469058 EF469087 EF469105
Conoideocrella luteorostrata Hemiptera NHJ 11343 JN049859 EF468995 EF468850 EF468801 EF468906
Conoideocrella luteorostrata Hemiptera NHJ 12516 JN049860 EF468994 EF468849 EF468800 EF468905 EF468946
Conoideocrella tenuis Hemiptera NHJ 345.01 EU369111 EU369045 EU369030 EU369088
Conoideocrella tenuis Hemiptera NHJ 6293 JN049862 EU369112 EU369044 EU369029 EU369068 EU369087
Conoideocrella tenuis Hemiptera NHJ 6791 JN049863 EU369113 EU369046 EU369028 EU369069 EU369089
Cordyceps bifusispora Lepidoptera EFCC 5690 EF468952 EF468806 EF468746 EF468854 EF468909
Cordyceps brongniartii Lepidoptera BCC 16585 JN049867 JF415951 JF415967 JF416009 JN049885 JF415991
Cordyceps cardinalis Lepidoptera OSC 93610 JN049843 AY184974 AY184963 EF469059 EF469088 EF469106
Cordyceps coccidioperitheciata Araneae NHJ 6709 JN049865 EU369110 EU369042 EU369025 EU369067 EU369086
Cordyceps confragosa Hemiptera CBS 101247 JN049836 AF339604 AF339555 DQ522359 DQ522407 DQ522466
Cordyceps gunnii Lepidoptera OSC 76404 JN049822 AF339572 AF339522 AY489616 AY489650 DQ522426
Cordyceps kyusyu€
ensis Lepidoptera EFCC 5886 EF468960 EF468813 EF468754 EF468863 EF468917
Cordyceps militaris Lepidoptera OSC 93623 JN049825 AY184977 AY184966 DQ522332 DQ522377 AY545732
Cordyceps cf. ochraceostromata Lepidoptera ARSEF 5691 JN049849 EF468964 EF468819 EF468759 EF468867 EF468921
Cordyceps pluricapitata Hemiptera NBRC 100745 KF049606 KF049624 KF049679 KF049642 KF049667
Cordyceps pluricapitata Hemiptera NBRC 100746 KF049607 KF049625 KF049680 KF049643 KF049668
Cordyceps scarabaeicola Coleoptera ARSEF 5689 JN049827 AF339574 AF339524 DQ522335 DQ522380 DQ522431
Cordyceps tuberculata Lepidoptera OSC 111002 JN049830 DQ522553 DQ518767 DQ522338 DQ522384 DQ522435
Cosmospora coccinea Hymenochaetales CBS 114050 JN049831 AY489702 AY489734 AY489629 AY489667 DQ522438
Elaphocordyceps japonica Eurotiales OSC 110991 JN049824 DQ522547 DQ518761 DQ522330 DQ522375 DQ522428
Elaphocordyceps ophioglossoides Eurotiales OSC 106405 AY489691 AY489723 AY489618 AY489652 DQ522429
Elaphocordyceps subsessilis Eurotiales OSC 71235 JN049844 EF469124 EF469077 EF469061 EF469090 EF469108
Engyodontium aranearum Araneae CBS 309.85 AJ292391 AF339576 AF339526 DQ522341 DQ522387 DQ522439
Epichlo€
e typhina Poaceae ATCC 56429 JN049832 U32405 U17396 AF543777 AY489653 DQ522440
Gibellula sp. Araneae NHJ 13158 JN049864 EU369100 EU369037 EU369020 EU369057 EU369077
Glomerella cingulata Rosaceae FAU 513 U48427 U48428 AF543772 DQ858454 DQ858455
Glomerella cingulata Rosaceae CBS 114054 DQ286202 AF543762 AF543786 AF543773 AY489659 DQ522441
Haptocillium sinense Nematoda CBS 567.95 AJ292417 AF339594 AF339545 DQ522343 DQ522389 DQ522443
Hirsutella sp. Hemiptera OSC 128575 JN049845 EF469126 EF469079 EF469064 EF469093 EF469110
Hirsutella sp. Hemiptera NHJ 12525 EF469125 EF469078 EF469063 EF469092 EF469111
Hydropisphaera peziza Bark CBS 102038 AY489698 AY489730 AY489625 AY489661 DQ522444
Hypocrella discoidea Hemiptera BCC 8237 JN049840 DQ384937 DQ384977 DQ385000 DQ452461
Hypocrea lutea Wood ATCC 208838 AF543768 AF543791 AF543781 AY489662 DQ522446
Hypocrea rufa Bark CBS 114374 AY489694 AY489726 AY489621 AY489656 EF692510
Isaria coleopterorum Coleoptera CBS 110.73 AY624177 JF415965 JF415988 JF416028 JN049903 JF416006
Isaria farinosa Lepidoptera CBS 240.32 AY624178 JF415958 JF415979 JF416019 JN049895 JF415999
Isaria tenuipes Lepidoptera ARSEF 5135 AY624196 JF415980 JF416020 JN049896 JF416000
Lecanicillium attenuatum Leaf litter CBS 402.78 AJ292434 AF339614 AF339565 EF468782 EF468888 EF468935
Lecanicillium psalliotae Soil CBS 532.81 JN049846 AF339609 AF339560 EF469067 EF469096 EF469112
Mariannaea elegans var.punicea Soil CBS 239.56 AY624201 AY526489 JF415981 JF416021 JN049897 JF416001
Mariannaea pruinosa Lepidoptera ARSEF 5413 JN049826 AY184979 AY184968 DQ522351 DQ522397 DQ522451
Metacordyceps atrovirens Coleoptera TNM F10184 JN049882 JF415950 JF415966 JN049884
Metacordyceps chlamydosporia Nematoda CBS 101244 JN049821 DQ522544 DQ518758 DQ522327 DQ522372 DQ522424
Metacordyceps indigotica Lepidoptera TNS F18553 JN049874 JF415953 JF415968 JF416010 JN049886 JF415992
Metacordyceps indigotica Lepidoptera TNS F18554 JN049875 JF415952 JF415969 JF416011 JN049887 JF415993
Metacordyceps khaoyaiensis Lepidoptera BCC 12687 JN049868 JF415970 JF416012 JN049888
Metacordyceps khaoyaiensis Lepidoptera BCC 14290 JN049869 JF415971 JF416013 JN049889
Metacordyceps kusanagiensis Coleoptera TNS F18494 JN049873 JF415954 JF415972 JF416014 JN049890
Metacordyceps liangshanensis Lepidoptera EFCC 1523 EF468961 EF468814 EF468755 EF468918
4 R. Kepler et al.
Please cite this article in press as: Kepler R, et al., The phylogenetic placement of hypocrealean insect pathogens in the genus
Polycephalomyces: An application of One Fungus One Name, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.06.002

Table 1 e(continued)
Taxon Host Voucher# GenBank accession numbers
ITS SSU LSU TEF RPB1 RPB2
Metacordyceps liangshanensis Lepidoptera EFCC 1452 EF468962 EF468815 EF468756
Metacordyceps martialis Lepidoptera TTZ070716-04 JN049871 JF415955 JF415973 JN049891
Metacordyceps martialis Lepidoptera EFCC 6863 JF415974 JF416015 JF415994
Metacordyceps martialis Lepidoptera HMAS 197472(S) JN049881 JF415956 JF415975 JF416016 JN049892 JF415995
Metacordyceps owariensis Hemiptera NBRC 33258 JN049883 JF415976 JF416017 JF415996
Metacordyceps pseudoatrovirens Coleoptera TNSF 16380 JN049870 JF415977 JN049893 JF415997
Metacordyceps sp. Coleoptera HMAS 199601 JN049879 JF415957 JF415978 JF416018 JN049894 JF415998
Metacordyceps taii Lepidoptera ARSEF 5714 JN049829 AF543763 AF543787 AF543775 DQ522383 DQ522434
Metacordyceps yongmunensis Lepidoptera EFCC 2131 JN049856 EF468977 EF468833 EF468770 EF468876
Metacordyceps yongmunensis Lepidoptera EFCC 2135 EF468979 EF468834 EF468769 EF468877
Metarhizium album Hemiptera ARSEF 2082 AY375446 DQ522560 DQ518775 DQ522352 DQ522398 DQ522452
Metarhizium anisopliae Coleoptera ARSEF 3145 JN049834 AF339579 AF339530 AF543774 DQ522399 DQ522453
Metarhizium flavoviride Hemiptera ARSEF 2037 AF138271 AF339580 AF339531 DQ522353 DQ522400 DQ522454
Metarhizium sp. Coleoptera HMAS 199590 JN049876 JF415960 JF415983 JF416023 JN049898 JF416002
Metarhizium sp. Coleoptera HMAS 199592 JN049877 JF415961 JF415984 JF416024 JN049899 JF416003
Metarhizium sp. Coleoptera HMAS 199596 JN049878 JF415962 JF415985 JF416025 JN049900 JF416004
Metarhizium sp. Coleoptera HMAS 199603 JN049880 JF415963 JF415986 JF416026 JN049901 JF416005
Moelleriella mollii Hemiptera BCC 7963 DQ372087 DQ384964 DQ385004 DQ452466
Moelleriella schizostachyi Hemiptera BCC 1985 DQ372105 DQ384939 DQ384959 DQ385012 DQ452471
Myriogenospora atramentosa Poaceae AEG 96-32 JN049835 AY489701 AY489733 AY489628 AY489665 DQ522455
Nectria cinnabarina Betulaceae CBS 114055 U32412 U00748 AF543785 AY489666 DQ522456
Nectria sp. Plant CBS 478.75 U47842 U17404 EF469068 EF469097 EF469115
Nomuraea cylindrosporae Hemiptera RCEF 3632 JN049872 JF415959 JF415982 JF416022
Nomuraea cylindrosporae Hemiptera TNS 16371 JF415964 JF415987 JF416027 JN049902
Nomuraea rileyi Lepidoptera CBS 806.71 AY624205 AY624205 AY624250 EF468787 EF468893 EF468937
Ophiocordyceps acicularis Coleoptera OSC 128580 JN049820 DQ522543 DQ518757 DQ522326 DQ522371 DQ522423
Ophiocordyceps agriotidis Coleoptera ARSEF 5692 JN049819 DQ522540 DQ518754 DQ522322 DQ522368 DQ522418
Ophiocordyceps aphodii Coleoptera ARSEF 5498 DQ522541 DQ518755 DQ522323 DQ522419
Ophiocordyceps brunneipunctata Coleoptera OSC 128576 DQ522542 DQ518756 DQ522324 DQ522369 DQ522420
Ophiocordyceps entomorrhiza Coleoptera KEW 53484 JN049850 EF468954 EF468809 EF468749 EF468857 EF468911
Ophiocordyceps gracilis Lepidoptera EFCC 8572 JN049851 EF468956 EF468811 EF468751 EF468859 EF468912
Ophiocordyceps heteropoda Hemiptera EFCC 10125 JN049852 EF468957 EF468812 EF468752 EF468860 EF468914
Ophiocordyceps longissima Hemiptera EFCC 6814 EF468817 EF468757 EF468865
Ophiocordyceps nigrella Lepidoptera EFCC 9247 JN049853 EF468963 EF468818 EF468758 EF468866 EF468920
Ophiocordyceps ravenelii Lepidoptera OSC 110995 DQ522550 DQ518764 DQ522334 DQ522379 DQ522430
Ophiocordyceps rhizoidea Isoptera NHJ 12522 JN049857 EF468970 EF468825 EF468764 EF468873 EF468923
Ophiocordyceps sinensis Lepidoptera EFCC 7287 JN049854 EF468971 EF468827 EF468767 EF468874 EF468924
Ophiocordyceps sobolifera Hemiptera KEW 78842 JN049855 EF468972 EF468828 EF468875 EF468925
Ophiocordyceps longissima Hemiptera EFCC 6814 EF468817 EF468757 EF468865
Ophiocordyceps stylophora Coleoptera OSC 111000 JN049828 DQ522552 DQ518766 DQ522337 DQ522382 DQ522433
Ophiocordyceps unilateralis Hymenoptera OSC 128574 DQ522554 DQ518768 DQ522339 DQ522385 DQ522436
Ophiocordyceps variabilis Diptera ARSEF 5365 DQ522555 DQ518769 DQ522340 DQ522386 DQ522437
Ophionectria trichospora Plant CBS 109876 AF543766 AF543790 AF543779 AY489669 DQ522457
Orbiocrella petchii Hemiptera NHJ 5318 EU369105 EU369040 EU369021 EU369062 EU369080
Orbiocrella petchii Hemiptera NHJ 6209 JN049861 EU369104 EU369039 EU369023 EU369061 EU369081
Paecilomyces carneus Sand dune CBS 239.32 AY624171 EF468988 EF468843 EF468789 EF468894 EF468938
Paecilomyces lilacinus Nematoda CBS 431.87 AY624188 AY624188 EF468844 EF468791 EF468897 EF468940
Paecilomyces marquandii Soil CBS 182.27 AY624193 EF468990 EF468845 EF468793 EF468899 EF468942
Paecilomyces carneus Soil CBS 239.32 AY624171 EF468988 EF468843 EF468789 EF468894 EF468938
Pochonia bulbillosa Plant CBS 145.70 AJ292410 AF339591 AF339542 EF468796 EF468902 EF468943
Pochonia chlamydosporia Nematoda CBS 504.66 AJ292398 AF339593 AF339544 EF469069 EF469098 EF469120
Pochonia gonioides Nematoda CBS 891.72 AJ292409 AF339599 AF339550 DQ522354 DQ522401 DQ522458
Pochonia parasiticum Rotifera ARSEF 3436 FJ973068 EF468993 EF468848 EF468799 EF468904 EF468945
Pochonia rubescens Nematoda CBS 464.88 AJ292400 AF339615 AF339566 EF468797 EF468903 EF468944
Polycephalomyces cuboidea Coleoptera TNS-F-18487 KF049609 KF049628 KF049683
Polycephalomyces cuboidea Coleoptera NBRC 101740 KF049610 KF049629 KF049684 KF049646
Polycephalomyces formosus Coleoptera ARSEF 1424 KF049661 KF049615 AY259544 DQ118754 DQ127245 KF049671
Polycephalomyces kanzashiana Hemiptera AB027326 AB027372
Polycephalomyces nipponica Neuroptera BCC 18108 KF049657 KF049608 KF049626 KF049681 KF049644
Polycephalomyces nipponica Neuroptera BCC 1881 KF049618 KF049636 KF049692 KF049674
Polycephalomyces nipponica Neuroptera BCC 1682 KF049664 KF049620 KF049638 KF049694
(continued on next page)
The phylogenetic placement of hypocrealean insect pathogens in the genus Polycephalomyces 5
Please cite this article in press as: Kepler R, et al., The phylogenetic placement of hypocrealean insect pathogens in the genus
Polycephalomyces: An application of One Fungus One Name, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.06.002

cuboidea,O. ryogamiensis,Ophiocordyceps paracuboidea,Ophiocor-
dyceps prolifica and Polycephalomyces formosus form a well-sup-
ported clade not associated with any existing teleomorph
genus of clavicipitoid fungi. We refer to this clade as the Poly-
cephalomyces clade. The placement of C. kanzashiana as mono-
phyletic with C. nipponica is suspect, likely resulting from only
having nuclear ribosomal gene data available. This relation-
ship should be investigated further with more appropriate
data before making a definitive statement of relatedness.
Cordyceps pleuricaptiata was recovered as sister to the Poly-
cephalomyces clade, but it was not well supported in these
analyses (bootstrap proportions <75 %; Fig 1). We therefore
continue to regard Cordyceps pleuricapitata as residual Cordy-
ceps sensu lato (i.e., incertae sedis) until further work is able
to clarify its relationship with statistical confidence. Bayesian
analyses and ML resolve placement of C. pleuricapitata plus the
Polycephalomyces clade as sister to Ophiocordycipitaceae, al-
though this placement was not well supported by bootstrap
proportions (<75 %, Fig 1). In addition the placement of the Pol-
ycephalomyces clade was found to be sensitive to taxon sam-
pling. For example, when C. pleuricapitata was excluded from
the analyses the Polycephalomyces clade was inferred as sister
group to Clavicipitaceae in RAxML analyses but sister to
Ophiocordycipitaceae in Bayesian analyses. At no time, how-
ever, was a topology recovered supporting an association
within Ophiocordyceps. The topology recovered here for rela-
tionships between families of Hypocreales is consistent with
the results of previous analyses (Sung et al. 2007a;Johnson
Table 1 e(continued)
Taxon Host Voucher# GenBank accession numbers
ITS SSU LSU TEF RPB1 RPB2
Polycephalomyces nipponica Neuroptera NHJ4286 KF049621 KF049639 KF049695 KF049654 KF049676
Polycephalomyces nipponica Neuroptera BCC 2325 KF049665 KF049622 KF049640 KF049696 KF049655 KF049677
Polycephalomyces paracuboidea Coleoptera NBRC 101742 KF049611 KF049630 KF049685 KF049647 KF049669
Polycephalomyces prolifica Hemiptera TNS-F-18481 KF049659 KF049612 KF049631 KF049686 KF049648
Polycephalomyces prolifica Hemiptera TNS-F-18547 KF049660 KF049613 KF049632 KF049687 KF049649 KF049670
Polycephalomyces
ramosopulvinata
Hemiptera SU-65 DQ118742 DQ118753 DQ127244
Polycephalomyces
ramosopulvinata
Hemiptera EFCC 5566 KF049658 KF049627 KF049682 KF049645
Polycephalomyces
ryogamiensis
Coleoptera NBRC 101751 KF049614 KF049633 KF049688 KF049650
Polycephalomyces
tomentosus
Trichiales BL4 KF049666 KF049623 AY259545 KF049697 KF049656 KF049678
Polycephalomyces sp. 1 Neuroptera BCC 2637 KF049663 KF049619 KF049637 KF049693 KF049675
Polycephalomyces sp. 2 Unknown JB07.08.16_08 KF049662 KF049616 KF049635 KF049690 KF049652 KF049672
Polycephalomyces sp. 2 Unknown JB07.08.17_07b KF049617 KF049691 KF049653 KF049673
Pseudonectria rousseliana Buxaceae CBS 114049 AF543767 U17416 AF543780 AY489670 DQ522459
Regiocrella camerunensis Hemiptera ARSEF 7682 DQ118735 DQ118743 DQ127234
Rotiferophthora
angustispora
Rotifera CBS 101437 AJ292412 AF339584 AF339535 AF543776 DQ522402 DQ522460
Roumegueriella rufula Nematoda CBS 346.85 DQ522561 DQ518776 DQ522355 DQ522403 DQ522461
Samulesia rufobrunnea Hemiptera P.C. 613 AY986918 AY986944 DQ000345
Septofusidium herbarum Plant root CBS 265.58 JN049866 JF415990 JF416030 JN049905 JF416008
Shimizuomyces paradoxus Smilacaceae EFCC 6279 JN049847 EF469131 EF469084 EF469071 EF469100 EF469117
Shimizuomyces paradoxus Smilacaceae EFCC 6564 EF469130 EF469083 EF469072 EF469101 EF469118
Simplicillium lamellicola Agaricales CBS 116.25 AJ292393 AF339601 AF339552 DQ522356 DQ522404 DQ522462
Simplicillium lanosoniveum Uredinales CBS 101267 AJ292395 AF339603 AF339554 DQ522357 DQ522405 DQ522463
Sphaerostilbella berkeleyana Polyporales CBS 102308 AF543770 U00756 AF543783 AY489671 DQ522465
Torrubiella ratticaudata Araneae ARSEF 1915 JN049837 DQ522562 DQ518777 DQ522360 DQ522408 DQ522467
Torrubiella wallacei Araneae CBS 101237 EF513022 AY184978 AY184967 EF469073 EF469102 EF469119
Verticillium dahliae Solanaceae ATCC 16535 AY489705 AY489737 AY489632 AY489673 DQ522468
Verticillium epiphytum Uredinales CBS 384.81 AF339596 AF339547 DQ522361 DQ522409 DQ522469
Verticillium epiphytum Uredinales CBS 154.61 AJ292404 AF339596 AF339547 EF468802 EF468947
Verticillium incurvum Polyporales CBS 460.88 AF339600 AF339551 DQ522362 DQ522410 DQ522470
Verticillium sp. Araneae CBS 101284 JN049858 AF339613 AF339564 EF468803 EF468907 EF468948
Viridispora diparietispora Galls on Crataegus CBS 102797 JN049838 AY489703 AY489735 AY489630 AY489668 DQ522471
Herbarium Codes: AEG, A. E. Glenn personal collection; ARSEF, USDA-ARS Collection of Entomopathogenic Fungal cultures, Ithaca, NY; ATCC,
American Type Culture Collection, Manassa, VA; BCC, BIOTEC Culture Collection, Klong Luang, Thailand; BCC, BIOTEC Culture Collection, Pa-
thum Thani, Thailand; CBS, Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; CUP, Cornell University Plant Pathology Herbar-
ium; EFCC, Entomopathogenic Fungal Culture Collection, Chuncheon, Korea; FAU, F. A. Uecker personal collection; GAM, Julian H. Miller
Mycological Herbarium Athens, GA; HMAS, Chinese Academy of Sciences, Beijing, China; JB, Joseph Bischoff, personal collection; KEW, mycol-
ogy collection of Royal Botanical Garden, KEW, Surrey, UK; NBRC, National Institute of Technology and Evaluation, Chiba, Japan; NHJ, Nigel
Hywel-Jones personal collection; OSC, Oregon State University Herbarium, Corvallis, OR; SA, S. Alderman personal collection; TNS, National Mu-
seum of Science and Nature, Tsukuba, Japan.
6 R. Kepler et al.
Please cite this article in press as: Kepler R, et al., The phylogenetic placement of hypocrealean insect pathogens in the genus
Polycephalomyces: An application of One Fungus One Name, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.06.002

Fig 1 eML tree obtained from analysis in RAxML of a concatenated five gene dataset (SSU, LSU, TEF, RPB1, RPB2) showing
placement of Polycephalomyces. Values above branches represent ML bootstrap proportions greater than 70% from 500 rep-
licates. Branches in bold denote strongly supported nodes in Bayesian analyses (‡0.95 posterior probability). Type species of
Polycephalomyces is shown in larger font.
The phylogenetic placement of hypocrealean insect pathogens in the genus Polycephalomyces 7
Please cite this article in press as: Kepler R, et al., The phylogenetic placement of hypocrealean insect pathogens in the genus
Polycephalomyces: An application of One Fungus One Name, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.06.002

et al. 2009), however, support values for the relationship be-
tween Hypocreaceae and Cordycipitaceae were weakened,
as was the branch subtending Nectriaceae and the rest of
Hypocreales (Supplementary Fig S1).
We apply the name Polycephalomyces to both the anamorph
and teleomorph forms present in this clade based on the in-
clusion of the type species P. formosus (Kobayasi 1941) and
emend the definition accordingly. Although authenticated
ex-type material is unavailable for P. formosus, the isolate ex-
amined in this study (ARSEF 1424, collected in Poland) has un-
dergone extensive morphological and molecular investigation
in previous work (Bischoff et al. 2003; Chaverri et al. 2005). We
also transfer Blistum tomentosum back into Polycephalomyces.
Taxonomy
Polycephalomyces Kobayasi emend. Kepler and Spatafora
Genus accepted as circumscribed by Seifert (1985), but includ-
ing teleomorphic taxa with the following general characteris-
tics: Stromata firm but pliant, white to yellow or brown, often
multifurcating or with several stipes representing several cy-
cles of growth; rhizomorphs present or absent. Perithecia im-
mersed in an apical or subapical pulvinate cushion, or
superficial, scattered to gregarious, often concentrated on
the upper stroma but the stroma tip bare. Asci long, asco-
spores forming many small partspores of nearly equal length.
Hosts include nymphs of Cicadidae (Hemiptera), Neuroptera
and larvae of Coleoptera.
Type: Polycephalomyces formosus Kobayasi
MB#9494
Anamorphic states: Acremonium-like, Hirsutella-like,
Polycephalomyces
A full list of species in Polycephalomyces is given below, with
new combinations made as appropriate. We continue to rec-
ognize P. cuboidea and P. ryogamiensis as separate taxa based
on ITS data presented in Ban et al. (2009).
Polycephalomyces cylindrosporus Samson & H.C. Evans, Pro-
ceedings van de Koninklijke Nederlandse Akademie van
Wetenschappen Section C, 84(3): 297 (1981).
MB#111844
Polycephalomyces cuboideus (Kobayasi & Shimizu) Kepler &
J.W. Spatafora comb. nov.
hCordyceps cuboidea Kobayasi & Shimizu. Bull. natn. Sci. Mus.,
Tokyo, Bot. 6(4): 131 (1980).
hOphiocordyceps cuboidea (Kobayasi & Shimizu) S. Ban, Sakane
& Nakagiri. Mycoscience 50(4): 268 (2009).
Host: Larvae of Coleoptera; Habitat: Rotten wood; Anamorph:
Hirsutella-like.
MB#804387
Polycephalomyces ditmarii Van Vooren & Audibert, Bulletin
Mensuel de la Soci
et
e Linn
eenne de Lyon, 74(7e8): 231 (2005).
MB#511222
Polycephalomyces formosus Kobayasi, Science Reports of the
Tokyo Bunrika Daigaku, 5: 245 (1941).
MB#289806
Polycephalomyces kanzashianus (Kobayasi & Shimizu) Kep-
ler & Spatafora comb. nov.
hCordyceps kanzashiana Kobayasi & Shimizu. Bull. nat. Sci.
Mus., Tokyo, Bot. 8(3): 86 (1982).
Host: Nymph of Cicadidae (Hemiptera); Habitat: Buried in soil;
Anamorph: unknown.
MB#804388
Polycephalomyces nipponicus (Kobayasi) Kepler & J.W. Spata-
fora comb. nov.
hCordyceps nipponica Kobayasi. Bull. of the Biogeogr. Soc. Jap.
9: 151 (1939).
Host: Nymph of Cicadidae (Hemiptera), Neuroptera; Habitat:
Buried in soil; Anamorph: unknown.
MB#804389
Polycephalomyces paracuboideus (S. Ban, Sakane & Nakagiri)
Kepler & J.W. Spatafora comb. nov.
hOphiocordyceps paracuboidea S. Ban, Sakane & Nakagiri.
Mycoscience 50(4): 268 (2009)
Host: Larvae of Coleoptera; Habitat: Rotten wood; Anamorph:
Hirsutella-like.
MB#804390
Polycephalomyces prolificus (Kobayasi) Kepler & J.W. Spata-
fora comb. nov.
hCordyceps prolifica Kobayasi in Kobayasi, Y.; Shimizu, D., Bull.
nat. Sci. Mus., Tokyo, Bot. 6: 289 (1963).
hOphiocordyceps prolifica (Kobayasi & Shimizu) S. Ban, Sakane
& Nakagiri. Mycoscience 50(4): 270 (2009).
Host: Nymph of Cicadidae (Hemiptera); Habitat: Buried in soil;
Anamorph: Unknown.
MB#804391
Polycephalomyces ramosopulvinatus (Kobayasi & Shimizu)
Kepler & J.W. Spatafora comb. nov.
hCordyceps ramosopulvinata Kobayasi & Shimizu. Bull. natn.
Sci. Mus., Tokyo, Bot. 9(1): 2 (1983).
Host: Nymph of Cicadidae (Hemiptera); Habitat: Buried in soil;
Anamorph: Unknown.
MB#804392
Polycephalomyces ramosus (Peck) Mains, Mycologia, 40 (4):
414 (1948).
hStilbum ramosum Peck, Bulletin of the Buffalo Society of
Natural Sciences, 1: 69, 1872 hBotryonipha ramosa (Pers.)
Kuntze, Revisio generum plantarum, 2: 845, 1891
hStilbella ramosa (Peck) Petch, Transactions of the British
Mycological Society, 21 (1-2): 53, 1938
MB#289808
Polycephalomyces ryogamiensis (Kobayasi & Shimizu) Kepler
& J.W. Spatafora comb. nov.
hCordyceps ryogamiensis Kobayasi & Shimizu. Bull. natn. Sci.
Mus., Tokyo, Bot. 9(1): 4 (1983).
hOphiocordyceps ryogamiensis (Kobayasi & Shimizu) G.H. Sung,
J.M. Sung, Hywel-Jones & Spatafora in Sung, Hywel-Jones,
Sung, Luangsa-ard, Shrestha & Spatafora, Stud. Mycol. 57:45
(2007).
Host: Larvae of Coleoptera; Habitat: Rotten wood; Anamorph:
Hirsutella-like.
MB#804393
Polycephalomyces tomentosus (Schrad.) Seifert, Studies in
Mycology, 27: 175, 1985
hStilbum tomentosum Schrad., Journal f€
ur die Botanik, 2: 65,
t. 3:1, 1799
hStilbella tomentosa (Schrad.) Bres., Annales Mycologici, 1
(2): 129, 1903
8 R. Kepler et al.
Please cite this article in press as: Kepler R, et al., The phylogenetic placement of hypocrealean insect pathogens in the genus
Polycephalomyces: An application of One Fungus One Name, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.06.002

hTilachlidium tomentosum (Schrad.) Lindau, Raben-
horst’s Kryptogamen-Flora, Pilze eFungi imperfecti, 1(9):
306, 1908
hBlistum tomentosum (Schrad.) B. Sutton, Mycological Papers,
132: 19, 1973
MB#104650
Discussion
Article 59 of the International Code of Botanical Nomenclature
allowed the naming of teleomorph and anamorph states of
nonlichenized species of Ascomycota and Basidiomycota
with unique Latin binomials, a practice referred to as the
dual system of fungal nomenclature. At the 2011 meeting of
the Nomenclature Session of the XVIII Botanical Congress
(Melbourne, Australia), Article 59 was abandoned, effective
Jan. 1, 2013, in favour of a single system of nomenclature, or
One Fungus One Name (1F1N; Hawksworth et al. 2011; Taylor
2011). This change in the code allows for teleomorph and ana-
morph names to be considered equally for the purposes of
naming clades (e.g., genera) of fungi. The recognition that sev-
eral teleomorphs of Cordyceps s.l. are phylogenetically distinct
from all other clades of cordycipioid fungi (e.g., Cordyceps,
Ophiocordyceps, etc.), and are members of a clade containing
the type species of Polycephalomyces, provides an example of
applying 1F1N to a pleomorphic taxon regardless of the known
reproductive life history stages of the species that are mem-
bers of the clade.
The phylogenetic classification of Hypocreales is develop-
ing rapidly with the application of multigene phylogenies
(Castlebury et al. 2004;Sung et al. 2007a;Chaverri et al. 2008).
The recognition of Polycephalomyces as separate from the
known genera of clavicipitoid fungi continues this work and
provides additional support for phylogenetic associations
among teleomorphs and anamorphs, which were noted previ-
ously by Ban et al. (2009). However, neither the depth of taxon
sampling nor the nature of the molecular data was sufficient
to support placement in relation to established genera. Ban
et al. (2009) used a 504 base-pair fragment of large subunit nu-
clear ribosomal RNA (LSU), which did not adequately resolve
deep fungal nodes (Hofstetter et al. 2007). Previously,
Chaverri et al. (2005) analyzed LSU, TEF and RBP1 data for Poly-
cephalomyces ramosopulvinatus and Polycephalomyces formosus,
however taxon sampling in this analysis was focused on scale
insect pathogens in Clavicipitaceae s.s. and did not include
other members of Polycephalomyces. The dataset examined
here includes additional ribosomal data, as well as protein
coding genes, which have shown to be better suited for
addressing divergences of genera and families of Hypocreales
(Zhang et al. 2006; Hofstetter et al. 2007; Sung et al. 2007b;
Schoch et al. 2009), and we also greatly expand the taxonomic
scope by including species throughout the order. These data
and analyses supported the Polycephalomyces clade as being
a unique genus among hypocrealean fungi. Although the Poly-
cephalomyces clade may represent a family level clade, due to
conflicting support values between RAxML and Bayesian anal-
yses, and different resolutions of this relationship to other
families of Hypocreales based on different taxon sampling,
we refrain from describing a new family at this time. Bayesian
analyses place Polycephalomyces sister to Ophiocordycipita-
ceae, however this placement was unsupported in ML analy-
ses (Fig 1). Therefore, we feel it is prudent to consider
Polycephalomyces as an incertae sedis member of Hypocreales
until taxonomic and phylogenetic concepts of clavicipitoid
fungi are resolved further.
Polycephalomyces is morphologically distinct from the other
animal pathogens of Clavicipitaceae. Species in the core Meta-
cordyceps are typically pigmented green to yellow or red, and
perithecia are usually oblique and embedded in a fibrous
stroma. Species outside of the core clade are typically pallid
with variable presentation of perithecia (Kepler et al. 2012a).
Teleomorphs of Polycephalomyces produce tough, wiry, long-
lasting stromata, usually with a darkened colour, which are
common character traits among Ophiocordyceps (Fig 2). Sung
et al. (2007) included P. ryogamiensis in Ophiocordyceps based
on morphological characters found to be consistent with the
relationships inferred from molecular data. Ban et al. (2009)
presented additional data on the anamorph morphology for
P. ryogamiensis, as well as other closely related species. All spe-
cies produce Hirsutella-like anamorphic forms in culture, with
Acremonium-like morphologies developing near the edge of the
colony. Hirsutella s.s. was identified by Sung et al. (2007a) as be-
ing associated only with the species of Ophiocordyceps and is
distinguished from other anamorphs of clavicipitoid fungi by
having phialides swollen at the base then tapering to a tip
where conidia are produced in a slimy mass. When naturally
occurring on a host, hirsutelloid-anamorphs of Ophiocordyceps
produce grey or brown stromata that often give rise to perithe-
cia. Phialides are typically solitary, although they may be
whorled or verticillate. Acremonium is a simple or reduced
form genus associated with Hypocreales and several species
associated with grass-endophytic species in Clavicipitaceae
were moved from Acremonium to Neotyphodium (Glenn et al.
1996). Species of Acremonium also produce conidia in slimy
heads, however the phialides are awl shaped, tapering at the
tip without swollen bases. Phialides of both Acremonium and
Hirsutella share a similarity to Verticillium, a polyphyletic
form genus with multiple occurrences throughout Hypo-
creales (Zare et al. 2000; Gams & Zare 2001; Sung et al. 2001;
Zare & Gams 2001; Zare et al. 2001). The range of anamorphic
form genera reported by Ban et al. (2009) for the species of Poly-
cephalomyces is therefore polyphyletic. Historical uses of Hirsu-
tella have been broad with the name applied to species
occurring outside of Ophiocordyceps that were later reclassified,
e.g., Simplicillium (Hywel-Jones 1994;Zare & Gams 2001). It is
therefore possible that the Hirsutella-like anamorph types pro-
duced in culture by Polycephalomyces reflect variation on an
Acremonium or Verticillium-like anamorph commonly pro-
duced by other clavicipitoid fungi in culture.
Anamorphic forms of Polycephalomyces produce extremely
small conidia (often 2 mm or smaller in width) in a slimy
mass at the tip of a prominent synnema. These anamorphic
forms are similar to Hirsutella, which also produces conidia
in slime at the tips of phialides, however phialides lack the
swollen base and are concentrated at the tips of synnemata.
Seifert (1985) reported the teleomorphic state for Polycephalo-
myces tomentosus as Byssostilbe stilbigera (Berk. & Br.) Petch
and was later found to be phylogenetically distinct from other
species of Polycephalomyces (Bischoff et al. 2003), a distinction
The phylogenetic placement of hypocrealean insect pathogens in the genus Polycephalomyces 9
Please cite this article in press as: Kepler R, et al., The phylogenetic placement of hypocrealean insect pathogens in the genus
Polycephalomyces: An application of One Fungus One Name, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.06.002

not supported by these analyses. A phylogenetic connection
between P. formosus and P. ramosopulvinatus was recovered
by Chaverri et al. (2005), however the taxon sampling was
not sufficient for further inferences. Many different names
have been proposed for anamorphic forms associated with
this genus, and the taxonomic status has been somewhat un-
stable. A thorough evaluation of material from anamorphic
states is necessary to fully determine relationships within
this genus, including material from type localities.
The host associations for Polycephalomyces sensu Kobaysi
are complicated and specimens are reported as hyperparasites
of other clavicipitoid insect pathogens and myxomycetes, as
well as insect cadavers (Seifert 1985). The invertebrate host as-
sociations for the emended Polycephalomyces include cicada
nymphs (Hemiptera: Cicadidae), as well as larvae of Coleoptera
and Lepidoptera. Specimens of Polycephalomyces nipponicus
have also been collected from larvae of Neuroptera (Isaka &
Tanticharoen 2001), which share a similar below-ground hab-
itat that could facilitate host switching. Pathogens of cicada
nymphs have evolved multiple times in Ophiocordycipitaceae,
as well as in Clavicipitaceae (Sung et al. 2007a;Kepler et al.
2012a). These hosts occur buried in the soil, whereas the beetle
pathogens in Polycephalomyces were excavated from decaying
wood. This pairing of habitats is possibly another convergent
character between fungi of Ophiocordyceps and Polycephalomy-
ces (Sung et al. 2007a), or symplesiomorphic with respect to
the most recent common ancestor of Polycephalomyces and
Ophiocordycipitaceae.
Conclusions
Introduction of molecular phylogenetic methods to the study
of clavicipitoid fungi has enabled major advances in under-
standing the evolution of species relationships, key life-
history traits, morphologies and ecologies. Morphological
characteristics such as texture and colour of teleomorphs
and anamorph morphologies are phylogenetically informa-
tive for many taxa (e.g., Cordyceps s.s. and Beauveria,Ophiocor-
dyceps and Hymenostilbe), but exceptions and problematic taxa
do exist and Polycephalomyces is indicative of such a taxon.
The character states associated with teleomorphs are either
products of convergent evolution or they are symplesiomor-
phic for the genus and Ophiocordycipitaceae. This research
Fig 2 ePolycephalomyces prolifica and Cordyceps pleuricapitata.(AeC) Polycephalomyces prolifica. A. Stroma emerging from soil.
B. Ascus. C. Partspores. D-F Cordyceps pleuricapitata. D. Dried specimens. E. Ascus. F. Partspore.
10 R. Kepler et al.
Please cite this article in press as: Kepler R, et al., The phylogenetic placement of hypocrealean insect pathogens in the genus
Polycephalomyces: An application of One Fungus One Name, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.06.002

also highlights the need for additional work to refine character
state homologies between Hirsutella synnemata of Ophiocordy-
ceps and the Hirsutella-like anamorphs of Polycephalomyces.
To date no Hirsutella anamorph typical of the brown or grey
synnematous forms encountered in Ophiocordyceps have
been recovered for Polycephalomyces, although Hirsutella-like
anamorphs are observed in other species outside of Ophiocor-
dycipitaceae when growing in culture (Hywel-Jones 1994).
Regardless, we provide strong evidence for the placement of
teleomorphs with isolates of Polycephalomyces formosus and
we apply the name Polycephalomyces to all life history states
of the clade based on priority as allowed by the recently adop-
ted single system of nomenclature for fungi.
Acknowledgements
We thank the Japan Society for Vegetable Wasps and Plant
Worms, Yasumasa Okuzawa and Takeshi Sakane for assis-
tance with field surveys in Japan and for contributing speci-
mens from their personal collections. Ryan Woolverton
assisted with lab work.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.funbio.2013.06.002.
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