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69
New species and new combinations in the genus Paraisaria
(Hypocreales, Ophiocordycipitaceae) from the U.S.A., supported
by polyphasic analysis
Richard M. Tehan1,2 , Connor B. Dooley1,3 , Edward G. Barge4, Kerry L. McPhail1, Joseph W. Spatafora3
1 Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon 97331, USA
2 Department of Chemistry and Biochemistry, Utica University, Utica, New York 13502, USA
3 Department of Botany and Plant Pathology, College of Agricultural and Life Sciences, Oregon State University, Corvallis, Oregon 97331, USA
4 Seed Testing Laboratory, Idaho State Department of Agriculture, Boise, ID 83712, USA
Corresponding author: Richard M. Tehan (rmtehan@utica.edu)
Copyright: © Richard M. Tehan et al.
This is an open access article distributed under
terms of the Creative Commons Attribution
License (Attribution 4.0 International –
CC BY 4.0).
Research Article
Abstract
Molecular phylogenetic and chemical analyses, and morphological characterization
of collections of North American Paraisaria specimens support the description of two
new species and two new combinations for known species. P. cascadensis sp. nov. is a
pathogen of CyphoderrisP. pseudohet-
eropoda sp. nov.
combinations are made for Ophiocordyceps insignis and O. monticola based on mor-
phological, ecological, and chemical study. A new cyclopeptide family proved indispens-
able in providing chemotaxonomic markers for resolving species in degraded herbarium
specimens for which DNA sequencing is intractable. This approach enabled the critical
linkage of a 142-year-old type specimen to a phylogenetic clade. The diversity of Parais-
aria in North America and the utility of chemotaxonomy for the genus are discussed.
Key words: Ascomycota, chemotaxonomy, Cicada, Cordyceps, Cyphoderris, entomo-
pathogen, Ophiocordyceps, Prionus
Introduction
Paraisaria-
nally described by Samson and Brady in 1983, characterized by synnemata with
-
ing phialides (Samson and Brady 1983). These asexual morphs were derived
from larvae (Delacroix 1893) and from cultured isolates of the sexual morphs
of species in the genus Cordyceps (Samson and Brady 1983; Li et al. 2004),
which were later transferred to Ophiocordyceps (Sung et al. 2007). Paraisaria
was later proposed for suppression, along with four other genera then in use, in
favor of recognizing a broad concept of Ophiocordyceps (Quandt et al. 2014).
This limited the number of new combinations required to accommodate 1F1N
rules following the abolition of the dual system of nomenclature in which sex-
-
lar analyses, Paraisaria has been recovered as a distinct monophyletic clade,
Academic editor: Huzefa Raja
Received:
19 August 2023
Accepted:
12 October 2023
Published:
13 November 2023
Citation: Tehan RM, Dooley CB, Barge
EG, McPhail KL, Spatafora JW (2023)
New species and new combinations
in the genus Paraisaria (Hypocreales,
Ophiocordycipitaceae) from the U.S.A.,
supported by polyphasic analysis.
MycoKeys 100: 69–94. https://doi.
org/10.3897/mycokeys.100.110959
MycoKeys 100: 69–94 (2023)
DOI: 10.3897/mycokeys.100.110959
70
MycoKeys 100: 69–94 (2023), DOI: 10.3897/mycokeys.100.110959
Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
being referred to as the “gracilis subclade” within the “ravenelii subclade” of
Ophiocordyceps by Sanjuan et al. (2015). Paraisaria was ultimately resurrected
in 2019, segregated from Ophiocordyceps, and amended to include sexual mor-
phology (Mongkolsamrit et al. 2019). Paraisaria species possess distinctive
sexual morphs characterized by a globose fertile terminal portion of the stroma
with immersed perithecia. Thus, Paraisaria constitutes a distinct, and robustly
-
gation from Ophiocordyceps rendered Ophiocordyceps into several paraphyletic
Ophiocordyceps sensu Sung et
al. (2007), is needed to establish robust generic concepts and restore global
monophyly. A major sticking point for this action is the uncertain placement
of the type of Ophiocordyceps, O. blattae, among the paraphyletic subclades
of Ophiocordyceps.
Paraisaria species are unique among most Cordyceps
sensu lato in that they form fruiting bodies in the spring, whereas most other
insect pathogens fruit in the summer, fall, or winter months, which is evident in
herbarium records on MycoPortal (MycoPortal 2023) and observations on the
community science platform iNaturalist (https://www.inaturalist.org/projects/
north-american-cordyceps-sensu-lato). Most Paraisaria species, and thus far,
all known Paraisaria species occurring in North America, form fruiting bodies
on subterranean insect hosts.
Some of the insect hosts of Paraisaria species are sought as food and their
contamination by Paraisaria species could pose a human health concern. Doan
et al. (2017) reported a series of poisonings and one fatality in Southern Viet-
nam, among people who had consumed cicadae infected with a fungus identi-
Paraisaria heteropoda (=Cordyceps heteropoda, Ophiocordyceps hetero-
poda), between 2008 and 2015. The toxicity was attributed to the presence of
mycotoxins in the otherwise edible cicadae, and the toxic agent was putative-
causing food-borne mycotoxin poisonings underscores the need to describe
the biological and chemical diversity present in this group of fungi.
-
ucts can be highly useful phenotypic characters for taxonomic purposes.
a generic chemotype for a taxonomic group, and also unique suites of com-
pounds within a chemical family can be used to resolve species. For example,
-
acterize and distinguish species and genera in the family Hypoxylaceae.
Paraisaria species, both of which investi-
Paraisaria heteropoda. A third
study reports leucinostatin analogs from an organism reported as Ophiocordy-
ceps heteropoda (=Parasiara heteropoda) (Kil et al. 2020), but which is evident-
ly a Purpureocillium
Doan et al. (2017) also report the amino acid, ibotenic acid from this spe-
currently no published genome sequences available to mine the specialized
metabolic potential of Paraisaria species, although the sequenced genomes
71
MycoKeys 100: 69–94 (2023), DOI: 10.3897/mycokeys.100.110959
Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
of other Ophiocordycipitaceae species display a familial trend of high biosyn-
-
ber of this genus resulted in the discovery of the new 8-residue antimicrobial
-
utive 4-hydroxyproline residues at the N-terminus (Krasnoff et al. 2005). The
known antifungal and immunosuppressant sphingosine analog, myriocin was
also isolated in this study. Heteropodamides A and B are N-methylated cy-
clic heptapeptides reported as cytotoxins from P. heteropoda
2011). Their absolute structures are yet to be determined. The further discov-
ery of Paraisaria species and their natural products presents fertile grounds
for investigation.
-
tive natural products from Paraisaria species (Tehan 2022), it became critical
to perform a taxonomic analysis of North American Paraisaria to better un-
-
ined 29 recent collections of Paraisaria to investigate the diversity of North
American Paraisaria. We also analyzed the type collections of Ophiocordyceps
insignis and O. monticola, both of which were anticipated to belong in Paraisaria
based on morphological description, ecology, and phenology. One phylogenet-
ically informative DNA sequence was afforded from the 87-year old O. mon-
ticola specimen. The 142-year old O. insignis type did not permit successful
DNA sequencing, however, chemical analysis of the newly characterized para-
isariamide family of compounds by LC-HRMS provided robust support for the
combination of both species into Paraisaria
of a species of importance to human health, as P. insignis. This study provides
a novel framework for the use of minimally destructive chemical analysis in
taxonomic assessment of type specimens where DNA sequencing is not possi-
ble. The combined analysis of molecular data, morphology, ecology, phenology,
and chemical data support the circumscription of two new species and two
new combinations, and provides an initial overview of the diversity of American
Paraisaria species.
Materials and methods
Specimens and isolates
Twenty nine new collections of Paraisaria specimens and their insect hosts
were examined. Macroscopic characters were examined from fresh stroma-
ta, and microscopic characters were examined from fresh and dried stroma-
ta, including ascospores discharged from fresh stromata when possible and
sections of dried specimens. Colors are in general terms of the senior author.
-
logical collection. Culture isolates of fungi were made from tissue dissected
from the context of stromata, placed on PDA with 50 µg/ml ampicillin and
100 µg/ml streptomycin, or from ascospores germinated on PDA. Agar plugs
were taken from outgrowth of stromatic tissue and subcultured onto PDA and
-
pathogenic Fungal Cultures (ARSEF).
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MycoKeys 100: 69–94 (2023), DOI: 10.3897/mycokeys.100.110959
Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
Morphological observations
Fruiting bodies were examined for morphological measurements using a Vernier
caliper (Fowler). Sections of ascogenous tissue were mounted in lactophenol cot-
ton blue, 5% KOH, or distilled water, and microanatomical characters were exam-
ined with light microscopy using a Leica DM2500. Twenty each, perithecia, asci,
DNA extraction and sequencing
DNA was extracted from the ascogenous portion of dried stromata, ground
with mortar and pestle in CTAB buffer (1.4 M NaCl, 100 mM Tris–HCl pH 8.0,
20 mM EDTA pH 8.0, 2% CTAB w/v) and processed following the method of Ke-
pler et al. (2012). Samples were extracted with 25:24:1 phenol:chloroform:iso-
amyl alcohol, (affymetrix), and DNA was precipitated with 3 M sodium acetate
-
-
(RPB1) using RPB1-Af and RPB
CRPB-1 (Castlebury et al. 2004) was used as a forward primer for samples
where RPB1-Af
983F and 2218R (Castlebury et al. 2004). PCR was performed with an iCycler
2,
0.5 µM each forward and reverse primers, 200 µM of each of the four dNTPs,
consisted of an initial 1 min denaturation at 95 °C; 34 cycles of 30 s at 94 °C,
1 min at 52 °C, 1.5 min at 72 °C, and a termination with an elongation 7 min at
72 °C. For RPB1 and RPB2, the PCR thermal cycle consisted of an initial 1.5 min
denaturation at 95 °C; 39 cycles of 30 s at 94 °C, 1 min at 47 °C, 2 min at
72 °C, and a termination with an elongation 4 min at 72 °C. Sequencing was
performed by the Sanger method at the Center for Quantitative Life Sciences at
to GenBank (Table 1).
Data analysis
regions were manually removed and the trimmed alignments were concatenat-
ed for analysis using Geneious Prime® 2023.0.4. A Maximum Likelihood Tree
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MycoKeys 100: 69–94 (2023), DOI: 10.3897/mycokeys.100.110959
Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
Table 1. Sequences used in phylogenetic tree construction.
Species Code Host ITS SSU LSU EF1a RPB1 RPB2 Reference
Cordyceps
kyushuensis
Lepidoptera — Sung et al.
2007
Cordyceps militaris Lepidoptera JN049825 AY184977 DQ522332 DQ522377 —Kepler et al.
2017
Drechmeria balanoides CBS 250.82 Nematoda AF339588 AF339539 DQ522342 DQ522388 DQ522442 Vu et al. 2019
Drechmeria sinensis Nematoda AF339594 AF339545 DQ522343 DQ522389 DQ522443 Spatafora et al.
2007
Harposporium
anguillulae
ARSEF 5407 Nematoda — — ———Chaverri et al.
2005
Harposporium
helicoides
ARSEF 5354 Nematoda —AF339577 AF339527 ———Sung et al.
2001
Ophiocordyceps
australis
Hymenoptera — —Sanjuan et al.
2015
Ophiocordyceps
australis
Hymenoptera — —Sanjuan et al.
2015
Ophiocordyceps
curculionum
OSC 151910 Coleoptera —KJ878918 KJ878885 —KJ878999 —Quandt et al.
2014
Ophiocordyceps
irangiensis
NBRC101400 Hymenoptera JN943335 JN941714 —JN992449 —Schoch et al.
2012
Ophiocordyceps
kimemingiae
SC30 Hymenoptera — KX713727 —Araújo et al.
2018
Ophiocordyceps
konnoana
EFCC 7315 Coleoptera — — Mongkolsamrit
et al. 2019
Ophiocordyceps
longissima
TNS F18448 Hemiptera —KJ878925 KJ878892 KJ878971 KJ879005 —Quandt et al.
2014
Ophiocordyceps
melolonthae
OSC.110993 Coleoptera —DQ522548 DQ522331 —Mongkolsamrit
et al. 2019
Ophiocordyceps
monticola
BPI 634610 Orthoptera OQ709246 —————This Study
Ophiocordyceps
nigrella
EFCC 9247 Coleoptera JN049853 Mongkolsamrit
et al. 2019
Ophiocordyceps
nutans
OSC 110994 Hemiptera —DQ522549 DQ522333 DQ522378 —Quandt et al.
2014
Ophiocordyceps
pulvinata
TNS-F 30044 Hymenoptera — — —Kepler et al.
2011
Ophiocordyceps
ravenelii
OSC 151914 Coleoptera —KJ878932 —KJ878978 KJ879012 KJ878950 Quandt et al.
2014
Ophiocordyceps
sinensis
EFCC 7287 Lepidoptera JN049854 Quandt et al.
2014
Ophiocordyceps
stylophora
OSC_111000 Coleoptera JN049828 DQ522552 DQ522337 DQ522382 DQ522433 Quandt et al.
2014
Ophiocordyceps
variabilis
OSC 111003 Diptera — Mongkolsamrit
et al. 2019
Ophiocordyceps
variabilis
Diptera —DQ522555 DQ522340 DQ522437 Mongkolsamrit
et al. 2019
Paraisaria alba HKAS_102484 Orthoptera MN947219 MN943843 MN943839 MN929085 MN929078 MN929082 Wei et al. 2021
Paraisaria amazonica Orthoptera — KJ917571 KM411989 KP212902 KM411982 Sanjuan et al.
2015
Paraisaria amazonica Orthoptera — KJ917572 —KP212903 KM411980 Sanjuan et al.
2015
Paraisaria arcta HKAS_102553 Lepidoptera MN947221 MN943845 MN943841 MN929087 MN929080 —Wei et al. 2021
Paraisaria arcta HKAS 102552 Lepidoptera MN947220 MN943844 MN943840 MN929079 MN929083 Wei et al. 2021
Paraisaria blattarioides Blattodea —KJ917559 KJ917570 KM411992 KP212910 —Sanjuan et al.
2015
Paraisaria blattarioides Blattodea —KJ917558 —KP212912 KM411984 Sanjuan et al.
2015
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MycoKeys 100: 69–94 (2023), DOI: 10.3897/mycokeys.100.110959
Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
Species Code Host ITS SSU LSU EF1a RPB1 RPB2 Reference
Paraisaria
cascadensis
OSC-M-052010 Orthoptera OQ709237 OQ800918 OQ708931 OR199814 OR199828 OR199838 This Study
Paraisaria
cascadensis
OSC-M-052012 Orthoptera OQ709239 OQ800920 OQ708933 OR199816 OR199830 —This Study
Paraisaria
cascadensis
OSC-M-052017 Orthoptera OQ709240 OQ800921 OQ708934 OR199817 OR199831 —This Study
Paraisaria coenomyia Diptera — Ban et al. 2015
Paraisaria coenomyia NBRC 108993 Diptera — Ban et al. 2015
Paraisaria gracilioides Coleoptera — —KM411994 KP212914 —Sanjuan et al.
2015
Paraisaria gracilioides Coleoptera —KJ917555 KJ130992 —KP212915 —Sanjuan et al.
2015
Paraisaria gracilis EFCC 3101 Lepidoptera — Sung et al.
2007
Paraisaria gracilis EFCC 8572 Lepidoptera JN049851 Ban et al. 2015
Paraisaria heteropoda Hemiptera — AY489722 —Quandt et al.
2014
Paraisaria heteropoda EFCC 10125 Hemiptera JN049852 Quandt et al.
2014
Paraisaria heteropoda Hemiptera —JN941719 JN941422 JN992453 Ban et al. 2015
Paraisaria heteropoda BCC 18235 Hemiptera —JN941720 JN941421 JN992454 Ban et al. 2015
Paraisaria heteropoda Hemiptera AB113352 —MK214083 MK214087 —Ban et al. 2015
Paraisaria insignis OSC.164134 Coleoptera OQ709231 OQ800911 OQ708924 OR199807 OR199822 —This Study
Paraisaria insignis OSC.164135 Coleoptera OQ709232 OQ800912 OQ708925 OR199808 OR199823 —This Study
Paraisaria insignis OSC.164137 Coleoptera OQ709233 OQ800913 OQ708926 OR199809 OR199824 —This Study
Paraisaria insignis OSC-M-052004 Coleoptera OQ709234 OQ800914 OQ708927 OR199810 — — This Study
Paraisaria insignis OSC-M-052008 Coleoptera OQ709236 OQ800917 OQ708930 OR199813 OR199827 —This Study
Paraisaria insignis OSC-M-052013 Coleoptera OQ709244 OQ800924 OQ708938 OR199820 OR199834 —This Study
Paraisaria
orthopterorum
BBC 88305 Orthoptera MH754742 —MK332583 MK214080 MK214084 —Mongkolsamrit
et al. 2019
Paraisaria
orthopterorum
TBRC 9710 Orthoptera MH754743 —MK332582 MK214081 MK214085 —Mongkolsamrit
et al. 2019
Paraisaria
phuwiangensis
TBRC 9709 Coleoptera MK192015 —MK192057 MK214082 —Mongkolsamrit
et al. 2019
Paraisaria
phuwiangensis
BBH 43492 Coleoptera MH188541 — MH211355 MH211352 —Mongkolsamrit
et al. 2019
Paraisaria
pseudoheteropoda
OSC-M-052005 Hemiptera —OQ800915 OQ708928 OR199811 OR199825 OR199836 This Study
Paraisaria
pseudoheteropoda
OSC-M-052007 Hemiptera OQ709235 OQ800916 OQ708929 OR199812 OR199826 OR199837 This Study
Paraisaria
pseudoheteropoda
OSC-M-052022 Hemiptera OQ709245 OQ800925 OQ708939 OR199821 OR199835 OR199841 This Study
Paraisaria
pseudoheteropoda
OSC-M-052020 Hemiptera OQ709243 OQ800923 OQ708937 OR199819 OR199833 —This Study
Paraisaria
pseudoheteropoda
OSC-M-052009 Hemiptera OQ709241 OQ800922 OQ708935 OR199818 OR199832 OR199840 This Study
Paraisaria rosea Coleoptera MN947222 MN943842 MN929088 MN929081 MN929084 Wei et al. 2021
Paraisaria sp. OSC-M-052011 Insecta OQ709238 OQ800919 OQ708932 OR199815 OR199829 OR199839 This Study
Paraisaria sp. OSC-M-052026 Insecta OQ709242 —OQ708936 ———This Study
Paraisaria tettigonia
Orthoptera KT345954 KT345955 —KT375440 KT375441 —
Paraisaria yodhathaii Coleoptera MH188539 —MK332584 MH211353 MH211349 —Mongkolsamrit
et al. 2019
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MycoKeys 100: 69–94 (2023), DOI: 10.3897/mycokeys.100.110959
Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
Species Code Host ITS SSU LSU EF1a RPB1 RPB2 Reference
Paraisaria yodhathaii TBRC 8502 Coleoptera MH188540 — MH211354 MH211350 —Mongkolsamrit
et al. 2019
Perennicordyceps
cuboideus
CEM 1514 Coleoptera — — — Kepler et al.
2013
Perennicordyceps
prolica
TNS-F-18547 Hemiptera Kepler et al.
2013
Pleurocordyceps
nipponicus
BCC_2325 Neuroptera Kepler et al.
2013
Pleurocordyceps
sinensis
ARSEF_1424 Coleoptera AY259544 DQ118754 DQ127245 Kepler et al.
2013
Pleurocordyceps
yunnanensis
Hemiptera MN598051 MN598042 Wang et al.
2021
Polycephalomyces
formosus
CGMCC_5.2204 Coleoptera MN598054 MN598045 Wang et al.
2021
Polycephalomyces
formosus
CGMCC_5.2208 Coleoptera MN598058 MN598049 Wang et al.
2021
Purpureocillium
atypicola
CEM 1185 Araneae —KJ878907 KJ878872 KJ878955 — — Quandt et al.
2014
Purpureocillium
atypicola
OSC 151901 Araneae —KJ878914 KJ878880 KJ878994 —Quandt et al.
2014
Purpureocillium
takamizusanensis
NHJ_3497 Hemiptera — Johnson et al.
2009
Tolypocladium
capitatum
OSC 71233 Fungi
(Eurotiales)
— AY489721 DQ522421 Spatafora et al.
2007
Tolypocladium
inatum
OSC 71235 Coleoptera JN049844 Kepler et al.
2012
Tolypocladium
ophioglossoides
Fungi
(Eurotiales)
— AY489723 DQ522429 Castlebury et
al. 2004
Tolypocladiumn
japonicum
OSC 110991 Fungi
(Eurotiales)
JN049824 DQ522547 DQ522330 DQ522375 DQ522428 Quandt et al.
2014
Torrubiellomyces
zombiae
NY04434801 Fungi
(Hypocreales)
—ON493543 ON513398 ON513402 Araújo et al.
2022
Chemical extraction and LCMS analysis
Paraisaria collections, individually placed in MeOH (1 ml, HPLC-grade), sonicat-
ed for 5 min, and extracted for 1 hr at 35 °C, then 24 h at ambient temperature.
and dried in vacuo before dissolution in MeOH (0.1 mg/ml, LC-MS-grade) for
2O + 0.1% Formic Acid (A) MeCN + 0.1% Formic
Acid (B) as mobile phase solvents at 0.4 ml/min. The LC method was as thus:
0.5 mins at 20% B, a linear gradient from 20–90% B over 14 mins, 4 min at 90%
B, a linear gradient from 90–100% B over 0.5 mins, 4.5 mins at 100% B, followed
by a linear return to 20% B over 3 mins, and re-equilibration at 20% B for 5 mins,
m/z 100–3200, with MS/MS spectra obtained using
-
cluding precursor ions with m/z less than 210, and fragmenting with collision
ion chromatograms were produced for m/z
mass range for the paraisariamide peptide family (Tehan 2022).
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MycoKeys 100: 69–94 (2023), DOI: 10.3897/mycokeys.100.110959
Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
Molecular networking
-
ing the default network settings but with minimum peak intensity set to 3000.
-
sualized using (Ctyoscape ver. 3.9.1). The GNPS job is accessible at https://
-
.
Principal component analysis
LC-MS data were processed in MZmine v2.53 (Pluskal et al. 2010). Feature
4. Chromatograms were
2,
4, and m/z tolerance was set to m/z
0.001 or 10 ppm. Chromatogram deconvolution was performed with minimum
4, peak duration was set to 0.1–10 mins, and the base-
2m/z
0.001 or 15 ppm, RT tolerance was set to 1, with the most intense ion taken
as the representative, and max charge was set to 2. Peaks were aligned with
mass tolerance m/z 0.001 or 12 ppm, RT tolerance set to 0.8 mins, with m/z
falling within the range m/z
was performed with an intensity tolerance of 10%, mass tolerance m/z 0.001 or
Principal Component Analysis (PCA).
Results
Molecular phylogeny
http://purl.
org/phylo/treebase/phylows/study/TB2:S30820
tree (Fig. 1), ten genera in the family Ophiocordycipitaceae are represented.
Cordyceps kyushuensis and C. militaris (Cordycipitaceae) were designated as
outgroup taxa. All genera, with the exception of Ophiocordyceps, are supported
as monophyletic clades. A clade comprising several species morphologically
similar to the well-known cicada pathogen, P. heteropoda, referred to here as
as additional samples revealing cryptic diversity. Two new species within the
P. heteropoda complex, Paraisaria cascadensis and Paraisaria pseudoheteropo-
da, are supported as monophyletic clades, and are described below. Ophiocordy-
ceps insignis samples produced a monophyletic clade within the P. heteropoda
complex supporting its combination into Paraisaria, and is redescribed based
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MycoKeys 100: 69–94 (2023), DOI: 10.3897/mycokeys.100.110959
Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
on a fresh collection, which is designated here as an epitype. The type collec-
tion of Ophiocordyceps monticola also occurred within the genus Paraisaria,
grouping closely with P. yodhathaii and P. alba
Paraisaria species analyzed in this study which did not fall within the P. hetero-
poda complex.
Figure 1.
displaying the relationship of Paraisaria species within family Ophiocordycipitaceae.
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MycoKeys 100: 69–94 (2023), DOI: 10.3897/mycokeys.100.110959
Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
LC-MS analysis
Molecular Network Analysis of nineteen Paraisaria endosclerotium extracts
-
clopeptides, with constituent molecular ion masses ([M+H]+) ranging from m/z
to possess a subset of paraisariamide congeners with partial overlap between
species. Production of paraisariamide cyclopeptides in host/endosclerotium
Figure 2. Chemical comparison of paraisariamide content in the endosclerotia of Paraisaria
A molecular network of the paraisariamide molecular family of cyclic peptides detected in methanol extracts of endosclerotia
of Paraisaria specimens. Nodes are displayed as pie charts conveying the relative abundance of paraisariamide mass ion
features in each Paraisaria species (Orange = P. cascadensis, Purple = P. pseudoheteropoda, Green = P. insignis, Yellow = “Para-
isaria sp. 1”, Red = P. monticola) B extracted ion chromatograms of m/z
Paraisaria specimens C principal component analysis of mass features m/z-
tia of Paraisaria specimens, color-coded by phylogenetic clade.
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MycoKeys 100: 69–94 (2023), DOI: 10.3897/mycokeys.100.110959
Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
is thus supported as a conserved chemotype for Paraisaria. Paraisariamides
can thus potentially be used as a generic diagnostic character. Chromatograms
generated from the extracted ion range m/z
mass range for the peptide family of paraisariamides, were unique to and con-
sistent within each species (Fig. 2B). From the processed mass data, a feature
list was produced comprising 59 LC-MS ion features (Suppl. material 1). A PCA
plot generated from this feature list afforded three major clusters (Fig. 2C).
Samples derived from P. insignis and P. pseudoheteropoda were resolved in
distinct clusters. Samples derived from P. cascadensis together with samples
from its sister clade, “Paraisaria sp. 1”, grouped apart from other samples.
Ophiocordyceps monticola afforded two prominent ion peaks with quasimolec-
ular ions, m/z 708.502 and 722.518 eluting at 8.0 and 8.7 min respectively, and
grouped most closely with P. insignis in the PCA plot. Qualitatively, the general
shape of ion chromatograms was highly conserved within each species and
distinct between species. The resolution of species by LC-MS analysis overall
accorded very well with the phylogenetic analysis.
Taxonomy
Paraisaria cascadensis Tehan, Dooley & Spatafora, sp. nov.
MycoBank No: 849757
Fig. 3
Type material. Holotype. . Skamania County, Gifford Pin-
1,042 m alt., 9 June 2021, on adult Cyphoderris monstrosa buried in the ground,
in mixed coniferous forest comprising Pinus contorta, Pseudotsuga menziesii,
and Abies sp., collected by R. Tehan, C. Dooley (RMT-2021-072, OSC-M-052017,
Etymology. cascadensis occurring in the Cascade Mountain range in the Pa-
Description. Stroma capitate, solitary, rhizoids solitary arising from heads
of adult Cyphoderris monstrosa buried in soil. Ascogenous portion globose
ostioles of perithecia. Perithecia obclavate, immersed, ordinally arranged,
-
Culture characteristics.
to yellow, reverse reddish brown to orange. Mycelium septate, smooth-walled
hyaline. No conidial state was observed.
Host. Cyphoderris monstrosa (Prophalangopsidae, Orthoptera).
Habitat. Specimens occur on hypogeous adult hump-winged grigs, Cypho-
derris monstrosa, in coniferous forest.
Additional materials examined.: Skamania County, at
Cypho-
derris monstrosa
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Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
2020, on adult Cyphoderris monstrosa, buried in soil, collected by Daniel Win-
: Skamania County,
Figure 3. Paraisaria cascadensis A OSC-M-052017 B fertile head C cross section of fertile head showing arrangement of
perithecia D perithecia E ascus F Ascus apex G–I ascospores J part-spores K, LK obverse, L reverse).
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Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
cf. Cyphoderris monstrosa, collected by Ben McCormick (OSC-M-052012).
: Skamania County, Gifford Pinchot National Forest, Mt.
adult Cyphoderris monstrosa buried in soil, in mixed coniferous forest compris-
ing Pinus contorta, Pseudotsuga menziesii, and Abies sp., collected by Richard
Notes. This species is uncommon and has thus far only been collected in the
Cascade Mountains of Washington State in the vicinity of Mount St. Helens at ele-
the range of its host, Cyphoderris monstrosa, which is known to occur in coniferous
Paraisaria pseudoheteropoda Tehan & Spatafora, sp. nov.
MycoBank No: 849758
Fig. 4
Type material. Holotype. : Searcy County, Grinder's Ferry,
35.985, -92.732, elevation: 252 m, 15 May 2022, on nymphs of cicadidae (He-
miptera) buried in soil, in near Quercus sp., Carya sp., and Juniperus virginiana,
Etymology. pseudoheteropoda resembling another cicada-pathogenic spe-
cies, Paraisaria heteropoda.
Description. Stromata capitate or subclavate, unbranched, growing singly or
up to two stromata attached by rhizoids to hypogeous nymphs of Cicadidae (He-
long, 4–5 mm wide, papillate with ostioles of perithecia. Perithecia obclavate,
-
Culture characteristics.-
verse yellow to orange. Mycelium septate, smooth-walled hyaline. No conidial
state was observed.
Host. Nymphs of Cicadidae (Hemiptera).
Habitat. Specimens occur on hypogeous nymphs of cicadae at the base of
coniferous and deciduous trees, especially oaks.
Additional materials examined.: Barry County, Cassville, at
-
: Barry County, Cassville, at approxi-
-
da buried in soil, collected by Aaron Peters (OSC-M-052009, living culture: AR-
: Lincoln County, Crab Orchard, at approximately
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Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
Figure 4. Paraisaria pseudoheteropoda A OSC-M-052022 B fertile head C, D cross section of fertile head showing ar-
rangement of perithecia E perithecia F ascus G ascus apex H, I ascospores J ascospore tip K part-spores L, K colonies
L obverse, M reverse).
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Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
: Putnam
2022, on nymph of cicada buried in soil in mixed hardwood forest comprising
Quercus sp., Fagus sp., Populus sp. and Arundinaria gigantea, collected by Ja-
: Putnam County, Silver Point,
of cicada buried in soil among Acer negundo, Carpinus caroliniana, Carya sp.,
Quercus rubra, Lindera sp., Amphicarpaea bracteata, Phlox divaricata, Salvia ly-
rata: Searcy Coun-
ty, Grinder's Ferry, at approximately 35.983, -92.719, elevation: 222 m, 14 May
2022, on nymphs of cicadae buried in soil, in near Quercus sp., Carya sp., and
Juniperus virginiana -
-
: Albemarle County, Charlottesville, at ap-
Neotibicen sp. (Cicadidae, Hemiptera) buried in soil near Acer rubrum, collected
: Barry County, Roaring River,
-
tion: 198 m, 12 March 2023, on nymphs of cicadae buried in soil, collected by
: Jefferson County, Birming-
Notes. This species is the only Paraisaria species known to occur on cicadas
P. insignis but that species is distinguished by its strict occurrence on Cole-
optera. P. pseudoheteropoda sometimes has a pallid stroma which is not ob-
served in P. insignis.
Paraisaria insignis (Cooke & Ravenel) Tehan & Spatafora, comb. nov.
Fig. 5
Cordyceps insignis Cooke & Ravenel, Grevillea
Ophiocordyceps insignis (Cooke & Ravenel) G.H. Sung, J.M. Sung, Hywel-Jones
& Spatafora, Stud. Mycol. 57: 43 (2007). Synonym.
Type., “seaboard”, 4 January 1881, on larva coleoptera,
Epitype: Saline County, Avilla, at approxi-
Prionus imbri-
cornis (Cerambycidae, Coleoptera) buried in soil near Quercus sp., collected by
Description. Stromata capitate, unbranched, growing singly to gregarious, in
groups of up to four stromata on a single host. Stromata 20–52.5 mm long. As-
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Figure 5. Paraisaria insignis A OSC-M-052013 Epitype B fertile head C, D cross section of fertile head showing arrange-
ment of perithecia E rhizomorphs F perithecia G ascus H, I asci apices J–L ascospores M part-spores N, O colony on
PDA 70 d (N obverse, O reverse).
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papillate with ostioles of perithecia. Stipe golden yellow to reddish orange,
rhizomorphs, extending 25–45 mm. Mycelial growth occurring between, and
sometimes over, larval segments, forming a thin membrane. Perithecia em-
-
-
Growing on larvae of Prionus cf. imbricornis. (Cerambycidae, Coleoptera).
Culture characteristics. Colonies on PDA 70 days at 20 °C, 37.5 mm, white,
reverse reddish brown to yellow. Mycelium septate, smooth-walled hyaline. No
conidial state was observed.
Host. larvae of Prionus cf. imbricornis. (Cerambycidae, Coleoptera)
Habitat. Specimens occur on hypogeous larvae of coleoptera typically at the
base of oak trees.
Additional materials examined. : Saline County, Avilla, at
Prionus imbricornis (Cerambycidae, Coleoptera) buried in soil near Quercus sp.,
: Saline County, Avil-
of Prionus imbricornis (Cerambycidae, Coleoptera) buried in soil near Quercus
: Saline County, Avilla, at approximately 34.713, -92.587, elevation:
Prionus imbricornis (Cerambycidae, Coleop-
tera) buried in soil near Quercus
: Pulaski County, North Little Rock, at approximately 34.7989,
-92.312, elevation: 99 m, 17 April 2018, on larva of Prionus imbricornis (Ceram-
bycidae, Coleoptera) buried in soil near Quercus sp., and Ulmus sp., collected by
: Barry County, Cassville, at approx-
Prionus
imbricornis (Cerambycidae, Coleoptera) buried in soil, collected by Aaron Peters
29.5501, -95.1972, 19 m, 15 February 2020, on larva of Coleoptera, cf. Prionus im-
bricornis-
29 March 2021, on larva of Coleoptera buried in soil near Quercus sp., collected
: Saline County, Avilla, at
Pri-
onus imbricornis (Cerambycidae, Coleoptera) buried in soil near Quercus sp.,
-
tion 152 m, 25 March 2023, on larva of coleoptera, buried in soil, collected by
: Bibb County, Musella, at approxi-
buried in soil near Quercus phellos
Notes. Recent collections of this species were initially determined to not
match any described species and were given the provisional name Paraisaria
tortuosa, which was used in a doctoral dissertation (Tehan 2022), and in confer-
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Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
Ophiocordyceps insignis (=Cordy-
ceps insignis-
tion of the stroma as “livid purple”. However, that species was described from
a dried specimen and the true colors of the fresh specimen were evidently not
observed by the authority. Petch (1935) cast doubt on the accurate description
of the color of C. insignis and though the original host is not able to be precisely
that the host is one that pupates in wood, which accords with the host of recent
Prionus imbricornis
fresh collections, and strongly supports the combination into Paraisaria.
Paraisaria monticola (Mains) Tehan & Spatafora, comb. nov.
Cordyceps monticola Mains, Mycologia 32(3): 310 (1940). Basionym.
Ophiocordyceps monticola (Mains) G.H. Sung, J.M. Sung, Hywel-Jones & Spata-
fora, Stud. Mycol. 57: 45 (2007). Synonym.
Materials examined. Type: , Monroe County, Vonore, June
Neocurtilla hexadactyla
Notes. P. monticola is known to occur on adult Northern mole cricket, Neocurtil-
la hexadactyla (= Gryllotalpa hexadactyla, Orthoprtera, Gryllotalpidae). Other patho-
gens of mole crickets, Gryllotalpidae include Beauveria gryllotalpidicola, Beauveria
sinensis, Cordyceps neogryllotalpae, Ophiocordyceps gryllotalpae, Ophiocordyceps
krachonicola, and Polycephalomycs albiramus, all of which are only known from
east Asia. Lloyd (1920) reported C. gryllotalpae from a mole cricket collected in
-
mature stromata with no ascogenous tissue. Owing to the absence of microana-
tomical character data available for C. gryllotalpae, and the lack of genetic data
available for either species, future studies could compare P. monticola to C. gry-
llotalpae by chemical means, focusing on paraisariamide content of the fungal
endosclerotium. P. monticola is only known from the type collection.
Additional Paraisaria specimens examined
Two additional collections were examined which were phylogenetically closest
to P. cascadensis but occurring on undetermined insect hosts, outside of the
known geographic distribution of Cyphoderris monstrosa, the host of P. casca-
densis. Together they form a clade which is sister to P. cascadensis. We do not
P. cascadensis, but their formal
description was not within the scope of the present study owing to lack of ad-
equate sampling and host data. We anticipate that they represent two distinct
-
-
tion: 352 m, 5 April 2019, on undetermined insect host buried in soil, collected
: Johnson County, Solon, at
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Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
approximately 41.7572, -91.5457, elevation: 238 m, 30 June 2022, on undeter-
Discussion
Paraisaria species are described and two known species
are combined into Paraisaria. The entomopathogenic fungal genus Paraisaria
thus currently comprises 18 formally described species which occur on six con-
tinents, as deduced from a combination of herbarium records (MycoPortal 2023)
and citizen science observations (iNaturalist 2023). The extent of Paraisaria diver-
study, which warrants future studies of this group. The results of our phylogenetic
Figure 6. Paraisaria monticola AB fertile head C ascus D ascus apex E portion of ascospore F part spores.
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Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
and chemical analyses support the presence of additional cryptic diversity yet
to be elucidated. For such a geographically widespread genus, there has been a
relative paucity of sampling and analyses of Paraisaria specimens globally. Con-
tinued study of this group promises to reveal additional new Paraisaria species,
Paraisaria populations in North America prove to be enriched in species falling
within the Paraisaria heteropoda complex. Species in this clade are characterized
by fruiting bodies with yellow, brown, and reddish hues and prodigious orange to
brown rhizomorphs attaching to hypogeous insect hosts. Aboveground portions
Tolypocla-
dium capitatum, with which they have been compared (Cooke 1883), and with
which they are frequently confused. Numerous host shifts have accompanied
speciation in the P. heteropoda complex with species occurring on insect hosts in
-
Paraisaria species. P. insignis and
P. pseudoheteropoda overlap extensively in fruiting body morphology and geo-
graphic distribution but are easily distinguished by their respective distinct hosts.
P. insignis occurs strictly on coleopteran hosts and P. pseudoheteropoda is the
only known Paraisaria species to occur on cicadas in North America. P. cascaden-
sis and P. monticola both occur on orthopteran hosts, but the geographic distribu-
tion of P. cascadensis
Northwest, which accords with the distribution of its host, Cyphoderris monstrosa.
P. monticola is only known from the type specimen collected in Vonore, TN. Re-col-
lection efforts for this species would be valuable and could focus on records of
its host Neocurtilla hexadactyla, in the vicinity of the type locality. Notably, N. hexa-
dactyla is widely distributed, and may support a wide distribution of P. monticola.
The life cycles of Paraisaria species, including mode of infection of their in-
sect hosts, their possible occurrence in soil, as endophytes, saprophytic, and
nematophagous nutritional modes, are not well characterized. Owing to the
observation that Paraisaria species produce fruiting bodies in spring months
in North America, we hypothesize that they colonize their insect hosts in the
prior season and overwinter as endosclerotia which are observed to possess
high concentrations of cyclopeptide specialized metabolites. The molecular
structures, biological activities, and chemical ecology of Paraisaria specialized
metabolites are the focus of ongoing studies (Tehan 2022).
The targeted LC-MS analysis of specialized metabolites from fungi that are
only partially represented in phylogenetic analyses represents a robust applica-
tion of chemotaxonomy to resolve species. Fungi that produce cyclopeptides
-
clopeptides are particularly resistant to degradation by oxidation, heating, or
stable metabolites also provides a framework for the analysis of fungal groups
lacking genetic data for type specimens, whereby type specimens that afford
only chemical data can be linked to samples for which both chemical and ge-
netic data are available, if both types of data resolve species groups. The lack of
genetic data for type material is especially challenging when type specimens are
very old and possess degraded, highly-fragmented DNA, and for which no suit-
able neotype has been designated. Micromorphological characters lack robust-
ly distinct differences between Paraisaria species for use in reliable species di-
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Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
of P. insignis-
vation of the general paraisariamide chemotype also supports paraisariamides
as chemotaxonomic markers for genus Paraisaria, as these compounds were
detected in the endosclerotia of all Paraisaria specimens analyzed. These mark-
ers are substantially more durable than DNA over long periods of time as is evi-
-
type of P. insignis. Notably, the shape of chromatograms was visually identical
between old and new specimens, indicating that even the relative abundance
surveys should be conducted across Paraisaria species and related groups of
the utility of these metabolites as chemotaxonomic markers.
Other specialized metabolite families may offer promise as critical chemo-
taxonomic markers, depending on the relative stability of their biosynthetic
genes over time, and whether or not they are reliably expressed. For example,
genomic analyses show that the cyclosporin genotype is highly conserved
within the insect pathogen, Tolypocladium inatum (Ophiocordycipitaceae),
whereas peptaibiotics have evolved rapidly (Olarte et al. 2019) though neither
cyclosporins nor peptaibiotics are detected by LCMS in every Tolypocladium
strain exhibiting those genotypes (Blount 2018; Tehan et al. 2022).
Ophiocordyceps blattae, the type species of the large genus Ophiocordyceps,
presents another system for potential chemotyping to compare with the various
paraphyletic clades of Ophiocordyceps. Grounding of genus Ophiocordyceps in
Ophiocordyceps clade and circumscribe
other clades, has remained a longstanding problem owing to the rarity of the type
by high resolution LC-MS and metabolomics analysis applied to the characteriza-
tion of fungi in taxonomic studies adds an additional layer of phenotypic assess-
useful data for taxonomists but is critical for understanding fungal ecology and
may also guide pharmaceutical drug discovery efforts. These pursuits are highly
complementary, as demonstrated here and in ongoing research. The isolation,
structure elucidation, organic synthesis, biosynthesis, biological characterization,
and chemical ecology of the paraisariamides are the focus of ongoing research.
Acknowledgements
and accessioning specimens, Lee Davies at the Fungarium of Royal Botanic
-
assistance with culture curation. We thank Dr. Chris Marshall at Oregon State
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Richard M. Tehan et al.: New species and new combinations in the genus Paraisaria from the U.S.A.
Additional information
Conict of interest
The authors have declared that no competing interests exist.
Ethical statement
No ethical statement was reported.
Funding
Foundation (DEB-135944 to JWS, KLM), The Sonoma County Mycological Society, The
Oregon Mycological Society, and The Cascade Mycological Society.
Author contributions
Conceptualization: RMT, JWS, KLM. Methodology: RMT, JWS, KLM. Formal analysis:
Writing - Original draft: RMT. Writing - Review and Editing: RMT, KLM, JWS. Visualization:
RMT, EGB. Supervision: JWS, KLM. Project administration: RMT. Funding Acquisition:
RMT, KLM, JWS.
Author ORCIDs
Richard M. Tehan
Connor B. Dooley
Edward G. Barge
Kerry L. McPhail
Joseph W. Spatafora https://orcid.org/0000-0002-7183-1384
Data availability
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Supplementary material 1
Endosclerotia LCMS feature list
Author: Richard M. Tehan
Data type: csv
Explanation note: This table comprises processed LCMS data for methanol extracts of
the endosclerotia of 19 vouchered specimens.
Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/mycokeys.100.110959.suppl1
Available via license: CC BY 4.0
Content may be subject to copyright.
