Phytotaxa 208 (4): 278–286 ISSN 1179-3155 (print edition) www.mapress.com/phytotaxa/ PHYTOTAXA Copyright © 2015 Magnolia Press Article ISSN 1179-3163 (online edition) http://dx.doi.org/10.11646/phytotaxa.208.4.3

Pochonia cordycepisociata, a new species associated with Chinese cordyceps in Tibet, China

HAI HUANG1, *, ZHANG LUO, HAI MEI YUE, WEN FENG GONG, LEI CAI2 & MU WANG1* 1Agriculture and Animal Husbandry College of Tibet University, Lyingchi, Tibet 860000, China 2State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 3 Park 1, Beichen West Road, Chaoy- ang District, Beijing 100101, China *Corresponding author’s email: [email protected]

Abstract

During a survey of fungi associated with the Chinese cordyceps (Ophiocordyceps sinensis) collected from Tibet, China, a new species, Pochonia cordycepisociata was isolated from the sclerotium of O. sinensis. It is characterized and differ- ent from other Pochonia species by its aggregated compact hyphae, slender phialides, global to ellipsoid conidia and the absence of dictyochlamydospores. A multi-locus (ITS, SSU, LSU, TEF, RPB1 and RPB2) phylogenetic analysis supported its independence from other species. A synopsis of the morphological characters of the new species and its related taxa is provided.

Key words: Metacordyceps, morphology, Ophiocordyceps sinensis, phylogeny, systematics

Introduction

The genus Pochonia Bat. & O.M. Fonseca was resurrected based on the phylogenetic investigation of Verticillium sec. Prostrata which was re-classified into Lecanicillium W. Gams & Zare, Pochonia, Haptocillium W. Gams & Zare and Simplicillium Zare & W. Gams (Zare et al. 2001). Pochonia was circumstantiated based on the type species of P. chlamydosporia (Goddard) Zare & W. Gams, and currently 13 taxa are recognized (Zare et al. 2001; Sung et al. 2007; Zare and Gams 2007; Nonaka et al. 2013). All species of Pochonia are characterized by dictyochlamydospores or swollen hyphae cells, and phialidic conidia (Zare and Gams 2001, 2004). Moreover, phylogenetic studies by Kepler et al. (2012) supported that the genus Pochonia belongs to Claviciptaceae, but did not constitute a monophyletic clade. It is well-known that Pochonia species usually isolated from soil are able to parasitize nematode cysts (Barron et al. 1996), such as P. chlamydosporia, which is a potential bio-control agent (Zare & Gams 2003, Esteves et al. 2009). Zare & Gams (2004) recognized six species in this genus, which were acquired from cysts and eggs of Heterodera spp., leaf litter and soil. In a survey of fungi associated with Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora collected from Miling, Tibet, China, several isolates had moderately slow growth, slender flask-shaper phialides clustering in regular terminal or intercalary whorls, fitting the generic concept of Pochonia. However, the isolates could be easily distinguished from other species of Pochonia by the shape of the mature conidia and aggregated compact hyphae submerged in the medium. A multi-locus phylogenetic analysis also showed that these isolates represent an independent species of Pochonia. Herewith we described and illustrated the new species.

Materials and Methods

Strains, media and morphological observation

278 Accepted by Jian-Kui Liu: 2 Apr. 2015; published: 25 May 2015 RPB2 DQ522417 EF469104 EF469104 DQ522424 JF415992 JF415993 EF468918 JF415995 JF415994 JF415996 JF415997 JF415998 DQ522434 AB758693 ...Continue on the next page EF468876 EF468877 RPB1 AY489648 DQ522366 EF469085 EF469085 JN049884 DQ522372 JN049886 JN049887 JN049889 JN049888 JN049890 JN049891 JN049892 JN049893 JN049894 DQ522383 AB758666 EF468770 EF468769 TEF AF543778 DQ522320 EF469056 EF469056 DQ522327 JF416010 JF416011 JF416013 JF416012 JF416014 EF468755 EF468756 JF416016 JF416015 JF416017 JF416018 AF543775 AB758463 EF468833 EF468834 LSU U17402 AF543789 DQ384937 EF469074 JF415966 DQ518758 JF415968 JF415969 JF415971 JF415970 JF415972 EF468814 EF468815 JF415974 JF415975 JF415973 JF415976 JF415977 JF415978 AF543787 AB709831 EF468979 SSU DQ522539 AF543765 EF469121 JF415950 DQ522544 JF415952 JF415953 JF415954 EF468961 EF468962 JF415956 JF415955 JF415957 AF543763 AB758255 JN049856 ITS JN049817 U57669 JN049840 JN049842 JN049882 JN049821 JN049874 JN049875 JN049868 JN049869 JN049873 JN049871 JN049881 JN049883 JN049870 JN049879 JN049829 AB709858 Lepidopteran pupa Lepidopteran pupa Substrate of isolation Pennisetum typhoideum - Nam Nao National Park Homoptera, scale insect Larva of Coleoptera Egg of slug Lepidoptera Lepidoptera Lepidoptera Lepidoptera Coleoptera pupa Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Nymph of cicada Coleoptera larva Hemiptera Soil and related species and related South Korea South Korea Locality Africa - Underside of dicotyledonous leaf Thailand Japan Yamagata, Brazil Japan Yamagata, Japan Yamagata, Thailand Thailand Japan Yamagata, Sichuan, China Sichuan, China - - - Japan Aomori, Japan - - Tokyo, Japan Tokyo, Pochonia EFCC 2131 EFCC 2135 Strain 26019 ATCC GAM 12885 BCC 8237 BCC 7869 TNM-F 10184 CBS 101244 TNS F18553 TNS F18554 BCC 12687 BCC 14290 TNS F18494 EFCC 1523 EFCC 1452 TTZ070716-04 EFCC 6863 HMAS 197472 (S) NBRC 33258 TNS F16380 HMAS 199601 ARSEF 5714 JCM 18597 sp. M. yongmunensis M. yongmunensis Species Claviceps fusiformis C. purpurea discoidea Hypocrella H. raciborskii atrovirens Metacordyceps M. chlamydosporia M. indigotica M. indigotica M. khaoyaiensis M. khaoyaiensis M. kusanagiensis M. liangshanensis M. liangshanensis M. martialis M. martialis M. martialis M. owariensis M. pseudoatrovirens M. M. taii Pochonia boninensis TABLE 1. Taxa used in phylogenetic analysis of Taxa 1. TABLE a new species associated with Chinese cordyceps Phytotaxa 208 (4) © 2015 Magnolia Press • 279 RPB2 EF468943 AB758690 EF469120 AB758686 AB758704 AB758699 AB758708 AB758695 AB758703 AB758696 AB758710 KM263579 KM263580 KM263581 DQ522458 EF468944 AB758685 RPB1 EF468902 AB758663 EF469098 AB758659 AB758677 AB758672 AB758681 AB758668 AB758676 AB758669 AB758683 KM263576 KM263578 KM263577 DQ522401 EF468903 AB758658 KJ398648 KJ398600 TEF EF468796 AB758460 EF469069 AB758456 AB758474 AB758469 AB758478 AB758465 AB758473 AB758466 AB758480 KM263584 KM263582 KM263583 DQ522354 EF468797 AB758455 KJ398789 KJ398790 LSU AF339542 AB709809 AF339544 AB709810 AB709812 AB709817 AB709819 AB709821 AB709823 AB709827 AB709830 KM263573 KM263574 KM263575 AF339550 AF339566 AB709832 SSU AF339591 AB758252 AF339593 AB758248 AB758266 AB758261 AB758270 AB758257 AB758265 AB758258 AB758272 KM263572 KM263570 KM263571 AF339599 AF339615 AB758247 ITS AJ292410 AB709836 AJ292398 AB709837 AB709839 AB709844 AB709846 AB709848 AB709850 AB709854 AB709857 KM263569 KM263567 KM263568 DQ516079 AJ292409 AJ292408 Z54112 AJ292400 AB709859 AJ292406 AJ292402 sp. i sp. sp. Phoenix Morus Alocasia odora Morus Cyathea Q. serrata

Heterodera Heterodera Picea abies menziesi P. Heterodera avenae Heterodera Heterodera avenae Heterodera Root of Soil under Soil Soil Soil under Soil Soil under Soil under Soil under Soil under lepifera of Chinese Sclerotium cordyceps of Chinese Sclerotium cordyceps of Chinese Sclerotium cordyceps Root of Pulcherricium caeruleum Rotifers in pine litter pile Manure Eggs of avenae Soil eggs of eggs of Substrate of isolation Soil Denmark Nagano, Japan Okinawa, Japan Japan Japan Tokyo, Okinawa, Japan Japan Tokyo, Japan Japan Tokyo, Okinawa, Japan China Tibet, China Tibet, China Tibet, Netherlands Germany Ibaraki, Japan Canada U. K. Kagoshima, Japan Sweden Sweden Locality Canada Strain CBS 145.70 JCM 18596 CBS 504.66 JCM 18598 JCM 18600 JCM 18605 JCM 18607 JCM 18609 JCM 18611 JCM 18613 JCM 18619 CGMCC 3.17365 CGMCC 3.17366 CGMCC 3.17367 CBS 203.86 CBS 891.72 CBS 101433 52203 ATCC CBS 464.88 JCM 18620 CBS 248.83 CBS 251.83

catenata var. var. var. (Continued)

catenulata suchlasporia catenulata chlamydosporia chlamydosporia ellipsopsora ellipsopsora spinulospora spinulospora var. var. var. var. var. var. var. var. var. var. var. TABLE 1. TABLE Species bulbillosa P. bulbillosa P. chlamydosporia P. catenulata c. P. c. P. c. P. c. P. c. P. c. P. c. P. c. P. cordycepisociata P. cordycepisociata P. cordycepisociata P. globispora P. gonioides P. microbactrospora P. parasitica P. rubescens P. rubescens P. suchlasporia P. s. P.

280 • Phytotaxa 208 (4) © 2015 Magnolia Press HUANG ET AL. The Chinese cordyceps (Ophiocordyceps sinensis) was collected from Miling County (E: 93°11'; N: 29°31''; h: 3080 m), Tibet, China, in April 2014. The fungus was isolated from a sclerotium tissue within dead insect part of the O. sinensis specimen. Isolation was made on a Potato Dextrose Agar (PDA) supplemented with 5 % wheat bran and 0.5 % peptone at 18 ºC. After the fungus grew out from the sclerotium tissue, individual conidium was picked up from phialide and inoculated onto agar plate for single spore isolation. The isolates were incubated on the agar plates containing different media such as potato dextrose agar (PDA), Malt extract agar (MEA), Cornmeal agar (CMA), and Oatmeal agar (OA) at 20 ºC. Colony morphology and microscopic characteristics were examined, measured and photographed after incubation for 15 days. All measurements were conducted from more than 50 individuals in water mounts. The ex-type living cultures were deposited in China General Microbiological Culture Collection Center (CGMCC) and the dried culture and microscope slide were deposited in Herbarium Mycologicum, Academica Sinica, Beijing, China (HMAS).

DNA extraction, PCR amplification and sequencing

Total genomic DNA was extracted from the fungal mycelia on PDA plates following the protocol described by Wu et al. (2001). PCR was performed in 25 μL reactions containing 1.0 μL DNA template, 1.0 μL of each forward and reverse primers, 12.5 μL 2 × MasterMix (TIANGEN Co. Ltd. Beijing, China) and 10.5 μL ddH2O by using the following parameters: 94 °C for 40 s; followed by 35 cycles at different temperatures for different primers (e.g. 52 °C for LSU, ITS and SSU) for 50 s and 72 °C for 60 s; and a final extension at 72 °C for 10 minutes. The amplified program of TEF, RPB1 and RPB2 were referred to Bischoff et al. (2006). The PCR products were sequenced by Invitrogen Biotechnology Co. Ltd. (Beijing, China). Sequences were compared with accessions in the GenBank database via BLASTn searching to obtain the most likely taxonomic designation.

Phylogenetic analysis

Sequence data of the six loci were aligned with Clustal X (Thompson et al. 1997). Reference sequences were retrieved from GenBank under the accession numbers indicated (Table 1). Further manual alignment was carried out with MEGA v. 5 (Tamura et al. 2011). Phylogenetic analysis of Maximum Parsimony (MP) and Bayesian were conducted by PAUP v. 4.0b10 (Swofford 2002) and MrBayes v. 3.1.2 (Altekar et al. 2004) respectively. For the MP analysis, ambiguously aligned regions were excluded from all analyses. An unweighted parsimony (UP) analysis was performed. Trees were inferred using the heuristic search option with TBR branch swapping and 1,000 random sequence additions. Branches of zero length were collapsed and all equally most parsimonious trees were saved. Descriptive tree statistics such as tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI), were calculated for trees generated. Clade stability was assessed using bootstrap analysis with 1,000 replicates, each with 10 replicates of random stepwise addition of taxa. For the Bayesian analyses, the models of evolution were estimated by using MrModeltest v. 2.3 (Nylander 2008). Posterior probabilities (PP) (Rannala & Yang 1996, Zhaxybayeva & Gogarten 2002) were determined by Markov Chain Monte Carlo sampling (MCMC), Six simultaneous Markov chains were run for 1,000,000 generations and trees were sampled every 100th generation (resulting in 10,000 total trees). The first 2,000 trees represented the burn-in phase of the analyses and were discarded and the remaining 8,000 trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree. Trees were visualised in TreeView v. 1.6.6 (Page 1996).

Results

Phylogenetic analysis

The dataset of combined 6 loci (ITS, SSU, LSU, TEF, RPB1 and RPB2) comprised 4,794 characters. The statistics for the parsimony analysis revealed that 3,383 characters were constant; 1,040 characters were parsimony informative, while 371 variable characters were parsimony-uninformative. Phylogenetic tree was constructed based on 45 taxa including Hypocrella raciborskii Zimm and Hypocrella discoidea (Berkeley & Broome) Saccardo as outgroup (Berkeley & Broome 1878, Zimm 1901). The terminal branch support value and additional internal branch support value of maximum parsimony (MP) (> 50%) and the Bayesian posterior probabilities (PP) (> 95%) were indicated in the tree (Fig. 2). a new species associated with Chinese cordyceps Phytotaxa 208 (4) © 2015 Magnolia Press • 281 Pochonia was well separated from Metacordyceps G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Sung et al. 2007). However, the species belonging to Pochonia clustered in two clades (Fig. 2), which indicated that Pochonia is not a monophylogenic group. Three isolates of the new species clustered with other six Pochonia species in clade I with a bootstrap support of 97% and Bayesian posterior probability of 100% (Fig. 2). The clade II included the type species, P. chlamydosporia var. chlamydosporia and other two species of Pochonia which produce conidial heads at the apex of the phialides and abuntant dictyochlamydosporia and is paralleled with Metacordyceps clade (Zare et al. 2001). Although the new species did not nest in the same clade with the type species of Pochonia (P. chlamydosporia), we still assigned the new species in Pochnoia before the new taxonomy system of Pochonia established.

FIGURE 1. Pochonia cordycepisociata (CGMCC 3.17366). a. Colonies on PDA after 15 days of incubation at 20 °C; b. reverse side of the colony; c–d. conidiophores; e. conidia; f–h. aggregated compact hyphae structures. Scale bars = 10 μm.

Taxonomy

Pochonia cordycepisociata H. Huang, M. Wang & L Cai, sp. nov. (Fig. 1) MycoBank MB 809869

Differs from other Pochonia species by aggregated compact hyphae submerged in the medium, and cordyceps habitat.

Type:—CHINA, Tibet, Miling County, on sclerotium of Ophiocordyceps sinensis, 23 April 2014, M. Wang, HMAS 245579 (holotype), ex-type culture CGMCC 3.17366.

Colonies slow growing, reaching 28 mm on CMA, 26 mm on MEA, 31 mm on PDA and 28 mm on OA at 20 ºC for 15 days, compact, floccose, white, reverse yellowish. Hyphae hyaline, smooth, mostly 1–4 μm ( x = 3 μm) wide. Phialides hyaline, produced from prostrate aerial hyphae, solitary or in whorls of 2–5, slender, tapering toward the tip hyphae, 16–64 × 0.5–2.4 μm ( x = 35 × 1.4 μm). Conidia formed in small globose heads at the apex of phialides, globose to ellipsoidal, smooth-walled, one-celled, 3–7 × 2–3 μm ( x = 5 × 2 μm), dictyochlamydospores absent, irregularly aggregated compact hyphae submerged in the agar present. Optimal growth temperature 20 ºC, maximum growth temperature 30 ºC. Etymology:—“cordycepisociata” = associated with Cordyceps. Distribution:—Miling County, Tibet, China.

282 • Phytotaxa 208 (4) © 2015 Magnolia Press HUANG ET AL. Habitat:—living in the sclerotium of Chinese cordyceps (Ophiocordyceps sinensis parasitized on larvae of Thitarodes sp. Other materials studied:—CHINA, Tibet, Miling County, on sclerotium of Chinese cordyceps, 23 April 2014, M. Wang (CGMCC 3.17365); ibid, CGMCC 3.17367.

FIGURE 2. Phylogenetic tree derived from maximum parsimony analysis based on the dataset of combined sequences of SSU+LSU+ ITS+RPB1+RPB2+TEF, depicting the relationships of the new species Pochonia cordycepisociata with closely related taxa. Bootstrap percentages over 50% and significant Bayesian posterior probability (≥ 95%) are shown on the respective branches. Hypocrella raciborskii BCC 7869 and Hypocrella discoidea BCC 8237 were used as outgroup.

Discussion

Morphologically, Pochonia cordycepisociata resembles P. boninensis Nonaka, Kaifuchi & Masuma and P. globispora Zare & W. Gams in the conidiophores and lack of typical dictyochlamydospores (Zare & Gams 2007, Nonaka et al. 2013). However, its slender phialides differ from those of P. globispora, and the agregated compact hyphae structures differ from P. boninensis. The differences were compared in Table 2. Other species such as P. bulbillosa (W. Gams & Malla) Zare & W. Gams, P. suchlasporia (W. Gams & Malla) Zare & W. Gams and P. gonioides (Drechsler) Zare & W. Gams (Zare & Gams 2001, 2007) are also morphologically similar to the new species (Table 2) but they are phylogenetically distant and produce typical dictyochlamydospores. Both morphology and phylogenic analysis supported P. cordycepisociata to be a distinct new species. a new species associated with Chinese cordyceps Phytotaxa 208 (4) © 2015 Magnolia Press • 283 . 2013 .2001 . 2001 . 2001 . 2001 . 2007 et al et al et al et al et al et al in this study Zare Zare et al. 2001 Zare Zare Zare Zare Literature cited Literature Nonaka 6–18 29–30 20–35 14–16 25–29 14–21 10–12 30–31 Colonies C) º 24 21 25 25 15–20 21–24 18–21 18 or 21 Optimal growth Optimal growth temperature ( temperature Absent scanty(in) 16–28×14–25 15–25×10–20 more or less scanty Irregularly swollen hyphae Irregular, mostly submerged Irregular, Dictyochlamydopores (μm) Dictyochlamydopores Aggregated compact hyphae to fusiform chromophilic chromophilic Conidia (μm) to ellipsoidal Smooth Falcate with blunt ends 2.3–4×1.5–2.5 Subglobose, with the related taxa with the related 4–6.3×1.5–2.0,2.2–3.0×1.3–2.0 2–3.8 diam lobose to subglobose 1.8–2.5 Polyhedral to tuberculate 2.8–4.5×(2.2–)2.8–4.0 Subglobose 2–3.7×1.7–2.3 Subglobose to ovoid, 2.5–3.5×2.0–3.0 Globose to Subglobose 3–7×2.0–3.0 solitary, globose, ellipsoidal 3–7×2.0–3.0 solitary, P. insectivorus P. - - 13–30×1–2 16–64×0.5–2.4 18–25×0.7–1.0 15–30×1.2–2.5 12–25×1.2–2.2 10–30×0.8–1.8 Phialides (μm) . var Species . suchlasporia var P. cordycepisociata P. bulbillosa P. gonidides P. rubescens P. suchlasporia. P. catenate s. P. globispora P. boninensis P. TABLE 2. Morphological comparison of TABLE

284 • Phytotaxa 208 (4) © 2015 Magnolia Press HUANG ET AL. Pochonia species, including the type species (P. chlamydosporia) are nematode parasites (Zare & Gams 2007). Zare & Gams (2004) recognized six species in this genus, which produce dictyochlamydospores and extended habitats to the cysts and eggs of Heterodera spp., soil and leaf litter, but rarely insects. However, P. cordycepisociata was obtained from the sclerotia of O. sinensis in Tibet, without dictyochlamydosores and produce abundant aggregated compact hyphae. It is still unknown whether this species could parasitize or colonize on the nematode as other Pochonia species. Further study needs to be conducted for a better understanding of its ecological significance on the Chinese cordyceps. Although the genus Pochonia has been well recognized and accepted in Clavicipitaceae, multi-locus phylogenetic reconstructions failed to support the monophyly of Pochonia clade (Kepler et al. 2012). Moreover, Nonaka et al. also (2013) confirmed that Pochonia was not a monophyletic group based on analysis of SSU, LSU, TEF, RPB1 and RPB2 loci. Our phylogenetic tree based on 6 loci also indicated that the species of Pochonia were separated into two clades, in agreement with the results of Kepler et al. (2012). The type species clustered with P. boninensis and P. globispora into Clade II which was phylogenetically close to Metacordyceps, while most of Pochonia species were clustered into Clade I. The results also supported that the currently circumscribed genus Pochonia is a paraphyletic group. Hence, before the further clarification of the taxonomy of Pochonia, we prefer to deal with the new species in Pochonia at current situation.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 31270068). We are grateful for the help of Xing Zhong Liu for kindly improving the manuscript and helpful comments for the name of the new taxon.

References

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