MycoKeys 90: 53-69 (2022) er-reviewed open-access journal doi: 10.3897/mycokeys.90.8380 | < Mycokeys https://mycokeys.pensoft. net Launched to accelerate biodiversity research Morphological and molecular analyses reveal two new species of Gibellula (Cordycipitaceae, Hypocreales) from China MingJun Chen', Ting Wang', Yan Lin', Bo Huang! | Anhui Provincial Key Laboratory for Microbial Pest Control, Anhui Agricultural University, Hefei 230036, China Corresponding author: Bo Huang (bhuang@ahau.edu.cn) Academic editor: S. Maharachchikumbura | Received 15 March 2022 | Accepted 13 May 2022 | Published 2 June 2022 Citation: Chen MJ, Wang T, Lin Y, Huang B (2022) Morphological and molecular analyses reveal two new species of Gibellula (Cordycipitaceae, Hypocreales) from China. MycoKeys 90: 53-69. https://doi.org/10.3897/ mycokeys.90.83801 Abstract Gibellula penicillioides sp. nov. and G. longispora sp. nov., two new species parasitising spiders collected in China, are illustrated and described, based on morphological features and multiloci phylogenetic analysis. The G. penicillioides sp. nov. group is sister to the G. scorpioides group, but form long penicilloid conidiophore producing enlarged fusiform conidia ((7—) 7.5—9 (-10) x 2.5—3.5 um). G. longispora sp. nov. is sister to G. pigmentosinum, but has slender long conidia (5—7 x 1—2 um); teleomorph and Granulomanus- synanamorphic conidiogenous cells are absent in these two species. Type specimens of G. penicillioides sp. nov. and G. /ongispora sp. nov. were deposited in the Research Center for Entomogenous Fungi of Anhui Agricultural University (RCEF). In addition, a key to all known species of Gibellula is illustrated. Keywords Araneogenous fungi, Cordycipitaceae, spider, Taxonomy Introduction Spider—pathogenic fungi, also called araneogenous or araneopathogenic fungi, are the group that infect spiders (phylum Arthropoda, class Arachnida, order Araneae) and belong to the Hypocreales (Evans and Samson 1987). About 91 Hypocrealean spider- and harvestman-pathogenic fungi were recognised to accommodate the genera Copyright MingJun Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 54 MingJun Chen et al. / MycoKeys 90: 53-69 (2022) Akanthomyces Lebert, Beauveria Vuill., Clonostachys Corda, Cordyceps Fr., Engyodontium de Hoog, Gibellula Cavara, Hevansia Luangsa-ard, Hywel-Jones & Spatafora, Hirsutella Pat., Hymenostilbe Petch, Lecanicillium W. Gams & Zare, Ophiocordyceps Petch, Purpureocillium Luangsa-ard, Hywel-Jones, Houbraken & Samson and Torrubiella Boud. (Shrestha et al. 2019). Of the above genera, only Gibellula and Hevansia are exclusively spider—pathogenic and present host specificity (Shrestha et al. 2019; Kuephadungphan et al. 2020). Gibellula species are amongst the most common spider pathogens in the world and are distributed from temperate to subtropical and tropical regions. Morphologically, the group can produce cylindrical synnemata from the outer loose hyphae covering spider cadavers with conidiophores abruptly narrowing to a short distinct neck and forming a subsphaeroidal vesical (Mains 1950; Samson and Evans 1992; Kuephadungphan et al. 2019). In 1894, the genus Gibellula was proposed by Cavara (1894), based on Gibellula pulchra (Sacc.) Cavara (Corethropsis pulchra Sacc.). Since then, many new taxa of parasitic Gibellula (mostly on spiders) have been described. Petch (1932) and Mains (1949, 1950) treated a number of Gibellula species as synonyms of G. pulchra and recognised only four species in the genus Gibellula. Kobayasi and Shimizu (1976, 1982) revised some of the existing species of Gibellula and described two new taxa. In a phylogenetically-based nomenclature for Cordycipitaceae (Hypocreales), all Gibellula samples fell into a single clade in the Cordycipitaceae; therefore, the genus Gibellula was revised and recognised as spider pathogens that produce synnemata with swollen conidiophores reminiscent of Aspergillus (Kepler et al. 2017). Recently, current nomenclature, diversity and distributions of Gibellula were reviewed and seventeen Gibellula species were recognised (Shrestha et al. 2019). Since then, five new species were described (Kuephadungphan et al. 2020; Chen et al. 2021): G. cebrennini Tasan., Kuephadungphan & Luangsa-ard, G. fusiformispora Tasan., Kuephadungphan & Luangsa-ard, G. pigmentosinum Tasan., Kuephadungphan & Luangsa-ard, G. scorpioides Tasan., Khons., Kuephadungphan & Luangsa-ard and G. flava Ming J. Chen & B. Huang. In all, we consider the genus Gibellula to include 22 species. We carried out a series of collection trips for insect and spider pathogenic fungi in the Guniujiang National Forest Park in Anhui Province, China beginning in 2020. A total of seven spider cadavers infected by Gibellula were collected. One was identi- fied as G. flava and four were similar to G. scorpioides in having solitary whip-like synnemata arising from host abdomens and penicillately-arranged conidiogenous cells. However, the four differed from G. scorpioides in having much longer synnemata and conidiophores and, thus, are here described as a new species, G. penicillioides. Three specimens from Nanling Nature Reserve, Guangdong Province were also identified as this new species through combined morphological and sequence data. We also found two collections similar to G. pigmentosinum, but with long and thin fusiform conid- ia. Due to these differences, we also describe them as a new species, G. longispora. Two additional specimens from Shenzheng, Guangdong Province were recognised as G. longispora. Multi-gene phylogenetic trees from these sampled fungi confirm their taxonomic placements. Here, we describe these two new species, distinguish them morphologically and phylogenetically and compare them with closely-related species. Gibellula new taxa 55 Materials and methods Sample collection and morphology We collected five Gibellula samples from Guniujiang National Forest Park, Anhui Province, two samples from Shenzhen City, Guangdong Province and three samples from Nanling National Nature Reserve, Guangdong Province. The collections were carefully deposited in plastic boxes and returned to the laboratory. Microscopic ob- servations were made from squash mounts and sections made from fresh material. The fresh structures were mounted in water for measurements and lactophenol cotton blue solution for microphotography, following Kuephadungphan et al. (2020). We ob- served microscopic characteristics, such as size and shape of conidia, phialide, vesicles, metulae and conidiophores using a ZEISS Axiolab 5 microscope. All samples studied here were deposited in the Research Center for Enotomogenous Fungi of Anhui Agri- cultural University (RCEF). DNA extraction, PCR amplification and sequencing Total genomic DNA was extracted from fresh synnema with a modified CTAB meth- od (Spatafora et al. 1998). Two gene portions from cell nuclei and three protein cod- ing genes were used in this study: small subunit ribosomal RNA (SSU), large subunit ribosomal RNA (LSU), elongation factor-la (TEF) and the largest and second largest subunits of RNA polymerase II (RPB1 and RPB2). SSU with NS1 and NS2 (White et al. 1990), LSU was amplified with primers LROR and LR5 (Rehner and Samu- els 1994), TEF-1 with TEF1—983F and TEF1—2218R (Rehner and Buckley 2005), RPB1 with CRPBland RPB1—Cr (Castlebury et al. 2004) and RPB2 with fRPB2- 7CR and fRPB2-5 (Liu et al. 1999). PCR amplification of the five nuclear loci was performed according to Kuephadungphan et al. (2019). PCR products were purified and sequenced by Sangon Company (Shanghai, China). The resulting sequences were checked manually before submission to GenBank. Sequence alignment and phylogenetic analysis We constructed a phylogenetic tree using the five loci (SSU, LSU, TEE RPB1 and RPB2) from 50 taxa (Table 1) within the Cordycipitaceae (Hypocreales). Multiple sequence alignment was performed with Clustal X (version 2.0) (Larkin et al. 2007) and manual adjustments of sequences were done using BioEdit, adjusted to maximise homology. All loci were subsequently concatenated using PhyloSuite v1.2.1 (https:// github.com/dongzhang0725/PhyloSuite). The alignment was deposited at TreeBase (No. S29496). Phylogenetic inference was done according to Maximum Likelihood (ML) using RAxML 7.2.8 (Stamatakis 2006) and Bayesian Inference (BI) using MrBayes 3.3.7 (Ronquist and Huelsenbeck 2003). For the ML analysis, we used the GTRCAT model for all partitions, in accordance with recommendations in the RAxML manual against 56 MingJun Chen et al. / MycoKeys 90: 53-69 (2022) the use of invariant sites and 1000 rapid bootstrap replicates. The GTR+I+G model was selected by MrModeltest 2.2 (Nylander 2004) as the best nucleotide substitution model for the Bayesian analysis. Four MCMC chains were executed simultaneously for 2000,000 generations, sampling every 100 generations. Finally, phylogenetic trees were visualised using the Interactive Tree of Life ((TOL) (https://itol.embl.de) online tool (Letunic and Bork 2016). Table |. Accession numbers, strain numbers, and origins of Gibellula and related taxa used in this study, new sequences were shown in bold. Taxon Akanthomyces aculeatus A. aculeatus Beauveria bassiana B. bassiana Cordyceps militaris C. nidus C. caloceroides Blackwellomyces cardinalis B. cardinalis Engyodontium aranearum E. aranearum Gibellula cebrennini G. cebrennini G. clavulifera var. alba G. flava G. flava G. fusiformispora G. fusiformispora G. gamsii G. gamsii G. leiopus G. longispora Specimen vouchera TS772 HUA 186145T ARSEF 7518 ARSEF 1564T OSC 93623 TS903C MCA 2249 OSC 93609T OSC 93610 CBS 309.85 CBS 658.80 BCC 39705 BCC 53605T ARSEF19T51° WFS09061701 WFS20190625-25 BCC 56802T BCC 45076 BCC 27968T BCC 28797 BCC 16025 NHJ 12014 GenBank accession nos G. longispora G. longispora G. longispora G. longispora G. pigmentosinum G. pulchra G. pigmentosinum G. pigmentosinum G. scorpioides G. scorpioides G. scorpioides G. scorpioides G. scorpioides Gibellula sp. GNJ20200813-16 GNJ20210710-02 $Z20210904-02 $Z20210915-01 NH] 11679 GNH] 10808 BCC 41203T BCC 39707 BCC 47976T BCC 47530 BCC 47514 BCC 43298 BCC 13020 NH] 7859 SSU EU369110 MF416572 AY 184977 KY360300 MF416578 AY184973 AY184974 AF339576 DQ522562 MW036749 MF416602 EU369098 OL854201 EU369099 EU369107 LSU KC519370 MF416520 AY184966 KY360293 MF416578 AY184962 AY184963 AF339526 LC092916 MH394673 MT477062 DQ518777 GU827389 MW084343 MT477063 MH152539 MH152541 MF416548 OL854212 EU369035 MH394674 MT477066 MT477065 MH394677 MH394686 TEF MP416465 HQ880975 HQ880974 DQ522332 MF416525 DQ522325 EF469059 DQ522341 MH521895 MT503328 DQS522360 MW091325 MT503329 MH152560 MH152562 MF416492 EU369017 MW961414 OL981628 OL981630 OL981631 EU369016 EU369018 MT503330 MH521894 MT503335 MT503334 MT503333 MH521900 MH521901 RPB1 HQ880834 HQ880833 DQ522377 KY360296 MF416470 DQ522370 EF469088 DQ522387 MHS521822 MT503321 DQ522408 MW384883 MT503322 MHS521823 MH152547 MH152549 MF416649 EU369055 MW980145 EU369054 EU369056 MT503323 MH521801 MT503325 MH521816 MH521814 EU369064 HQ880906 HQ880905 AY545732 MF416632 DQ522422 EF469106 DQ522439 MHS521859 MT503336 DQ522467 MT503337 MH521860 MH152557 EU369075 OL981635 EU369076 MH521856 MT503339 MT503338 MH521858 EU369085 Gibellula new taxa 57 Taxon Specimen GenBank accession nos vouchera SSU LSU TEF RPB1 RPB2 Gibellula sp. NHJ 10788 EU369101 EU369036 EU369019 EU369058 EU369078 Gibellula sp. NHJ 5401 EU369102 - - EU369059 EU369079 G. penicillioides GNJ20200814-11 MW969669 MW969661 MW961415 MZ215998 - G. penicillioides GNJ20200814-14 MW969670 MW969662 MW961416 MZ215999 - G. penicillioides GNJ20200814-17 MW969671 MW969663 MW961417 - - G. penicillioides GNJ20200812-05 MW969672 MW969664 MW961418 - - G. penicillioides NL20210822-01 - - OL981632 - - G. penicillioides NL20210822-09 - - OL981633 - = G. penicillioides NL20210822-20 - - OL981634 - - Hevansia cinerea NHJ 3510 EU369091 - EU369009 EU369048 EU369070 H.. novoguineensis CBS 610.80T — MH394646 MH521885 — MH521844 H. novoguineensis NHJ 11923 EU369095 EU369032 EU369013 EU369052 EU369072 H. novoguineensis BCC 47881 - MH394650 MH521886 MH521807 MH521845 References: (Sanjuan et al. 2014; Kepler et al. 2017; Rehner et al. 2011; Spatafora et al. 2007; Luangsa-ard et al. 2005; Helaly et al. 2019; Sung et al. 2007; Sung et al. 2001; Johnson et al. 2009; Kuephadungphan et al. 2020; Chirivi- Salomon et al. 2015; Kepler et al. 2012; Sung and Spatafora 2004; Tsang et al. 2016; Kuephadungphan et al. 2019; Helaly et al. 2017) Results Taxonomy Gibellula penicillioides Ming J. Chen & B. Huang, sp. nov. MycoBank No: 843174 Fig. 1 Etymology. Latin “penicillioides” referring to the fungus with penicillate conidiophores. Type. China. Anhui Province: Shitai County, Guniujiang National Nature Re- serve, on a spider, on unidentified leaf, 1 August 2020, Mingjun Chen & Bo Huang, holotype GNJ20200814-14. GenBank sequence data for GNJ20200814-14: SSU = MW969670; LSU = MW96966; TEF = MW961416; RPB1 = MZ215999. Description. Mycelium covering the host, brownish—white cream—yellow to light-brown mycelial mat. Light greyish-brown to violaceous-brown when dried. Synnema solitary, white to yellowish, arising from the tip of the host’s abdomen, slender, cylindrical, 6.8 mm long, 0.6 mm wide at base and 0.1 mm at tip. Conidiophores rising from mycelial mat and synnema, smooth, septate, cylindrical, mostly biverticillate, (40—) 52.5-92 (115) x (4—) 4.5-6 um (Fig. 1d, e), vesicles rarely developed. Several metulae are borne on the apex of conidiophore. Metulae clavate (slightly broadening towards the base) to cylindrical, (11—) 13-17.5 (21.5) x 3.5-5 (-5.5) um, with a number of phialides in whorls. Phialides broadly cylindrical, with the apex tapering abruptly to a short neck (10—) 12.5—15.5 (-17) x (2.5—) 3-4 (—5) um. Conidia fusiform, (7—) 7.5—9 (—10) x 2.5—3.5 um, in chains, borne on each phialide (Figs 1i-j). Teleomorph and granulomanus synanamorphs not observed. 58 MingJun Chen et al. / MycoKeys 90: 53-69 (2022) => < ) £ ge d * e om f @ © > = = ~~ ¢ . “© & Vu © # g oti | Figure |. Gibellula penicillioides sp. nov. a—b fungus on spider € synnema solitary d-f Penicillate conidi- ophores g conidiophore head bearing conidia h conidia i conidia in chains. Scale bars: 50 um (d, e, f); 10 um (g, h, i). Habitat. Occurring on spider attached to the underside of unidentified leaves nearby rivers. Additional materials examined. Cuina. Anhui Province: Shitai County, Gu- niujiang National Nature Reserve, on a spider, 1 August 2020, Mingjun Chen & Ting Wang, GNJ20200814—11, GNJ20200814-17 and GNJ20200812-05. China. Gibellula new taxa 59 Guangdong Province: Nanling Nature Reserve, August 2021, on a spider, Qianle Lu, NL20210822-01, NL20210822-09, and NL20210822-20. Notes. In its morphological characters, G. penicillioides resembles G. scorpioides, G. dabieshanensis B. Huang, M.Z. Fan & Z.Z. Li, G. clavulifera var. clavulifera (Petch) Samson & H.C. Evans, G. clavulifera var. major Tzean, L.S. Hsieh, J.Y. Liou & WJ. Wu and G. clavulifera var. alba Humber & Rombach by single synnema producing smooth penicillate conidiophores. Table 2 provides a comparative summary of the main characters of G. penicillioides and the other four species. Microscopically, G. penicillioides can be distinguished from G. scorpioides, G. dabieshanensis and G. clavulifera var. clavulifera by having longer conidiophores and slightly larger conidia. Furthermore, G. penicillioides differs from G. clavulifera var. alba by forming larger metulae, phialides and conidia, while G. clavulifera var. major produces the largest conidia and the longest conidiophore. Gibellula longispora Ming J. Chen & B. Huang, sp. nov. MycoBank No: 843175 Fig. 2 Etymology. Latin “/ongispora” referring to the fungus with slender long conidia. Type. China. Anhui Province: Shitai County, Guniujiang National Nature Re- serve, on a spider, on unidentified leaf, 1 August 2020, Mingjun Chen & Bo Huang, holotype GNJ20200813-16. GenBank sequence data for GNJ20200813-16: TEF = MW961414; RPB1 = MW980145. Description. Mycelium covering the host, white to cream fluffy, light greyish- brown to violaceous-brown when dried. Synnema multiple, cylindrical, growing from abdomen of host spider, cream to yellowish—white. Conidiophores, (19-) 60-153.5 (-170) x 8-10 pm (Fig. 2d), crowded, lately arising from hyphae loosely attached to the surface of the synnema, verrucose, multiseptate, suddenly narrowing to a tip, then forming a globose vesicle, (5.5—) 6—-8.5 (—9.5) x (5—) 5.5-8um (Fig. 2c, f). Spherical co- nidial heads consisting of vesicle, metulae and phialide, (25.5—) 38.5—49 (—50) x (24.5) 36-46.5 (—49) um. A number of broadly obovate to oval metulae, 6.5—9.5 x (4.5—)5— 7 um (Fig. 2c), borne on vesicle, each metulae bearing several clavate phialides, (6.5—) 7-9.5 (-11) x (1.5—) 2-3 pm (Fig. 2c, f). Conidia, 5—7 x 1-2 um (Fig. 2g), narrowly fusiform. Teleomorph and granulomanus synanamorphs not observed. (Fig. 2f). Habitat. Occurring on spider attached to the underside of leaf nearby the river. Additional materials examined. Cutna. Anhui Province: Shitai County, Guniu- jiang National Nature Reserve, on a spider, 10 July 2020, Mingjun Chen & Ting Wang, GNJ20210710-02. China. Guangdong Province: Shenzhen, 10 October 2021, on spiders, Qianle Lu, $Z20210904-02, and SZ20210915-01. Note. The new species G. longispora is similar to five Gibellula species in hav- ing multi-synnemum and aspergillate, distinctly roughened conidiophores (Table 3), namely G. pigmentosinum, G. flava, G. pulchra, G. clavispora Z.Q. Liang, Wan H. 60 MingJun Chen et al. / MycoKeys 90: 53-69 (2022) Figure 2. Gibellula longispora sp. nov. a, b fungus on a spider ¢, d conidiophores showing conidial head e part of conidiophore showing rough walls f, g conidial head h conidia. Scale bars: 50 um (¢, d); 20 um (e), 10 pum (f, g h). Chen & Y.F. Han and G. shennongjiaensis X. Zou, Wan H. Chen, Y.E Han & Z.Q. Liang. However, G. longispora differs from G. pigmentosinum, G. flava and G. pulchra by its longer, slender conidia. Furthermore, compared to G. longispora, the species G. shennongjiaensis has shorter conidiophores with smaller phialide and metulae and slightly smaller conidia, while G. clavispora bears clavate conidia. Gibellula new taxa 61 Table 2. Comparison of Gibellula clavulifera, G. dabieshanensis, G. scorpioides and G. penicillioides sp. nov. with penicillate conidiophores. Species Conidiophore(um) metulae (um) Phialide (um) Conidia (um) Gibellula penicillate, smooth, obovoid to cylindrical, broadly cylindrical, | (7—) 7.5—9 (-10) penicillioides mostly biverticillate or | (11-) 13-17.5 (21.5) x | (10—-) 12.5-15.5 (-17) x 2.5-3.5 sp. nov.! terverticillate, (40—) 52.5— 3.5—5 (5.5) x (2.5—) 3-4 (—5) 92 (115) x (4-) 4.5-6 Gibellula penicillate, Smooth- clavate to cylindrical, ampulliform to 7.1-12.0 (-13.9) clavulifera vat. walled, mostly bi- or 12.7-19.8 x 4.0-5.6 | cylindrical, 12.7-19.8 x | x 2.4-4.0 (5.6) major” terverticillate, occasionally 3.6-4.8 (-5.3) monoverticillate 140 x 4.8-7.1 Gibellula penicillate, smooth, obovoid, slightly broadly cylindrical, (9-) 5-7 (-9) x scorpioides* mostly biverticillate, broadening toward the | 10—12.5 (14) x (2-) (1.5—-) 2-3 20-29 (—30) x 4 base, (7—) 9.5-12.5 2.5—3.5 (—4) (-15) x (2-) 3-5 (-7) Gibellula penicillate, Smooth- clavate cylindrical, with short 5.4-7.6 x clavulifera walled, 45—50 neck 15—17.3 x 3.2-4.3 D352; var. clavulifera* Gibellula penicillate, smooth, cylindricrical, 9-15 x cylindrical or slightly | 5-7.5 x 1.5-2 clavulifera mono-or biverticillate, up 3-4 swollen near the middle var. alba? to 100 LOAN 2:4. 1.522 5 Gibellula penicillate with swollen | Obovoid to cylindricrical} cylindrical, 7.9-10.8 x |3.2-4.0 x 1.1-1.8 dabieshanensis vesicle, smooth 27—44 8.6-11.5 x 5-6 1.8-2.9 Note: ‘Current study, *Tzean et al. 1997, *Kuephadungphan et al. 2020, *Chen et al. 2014, *Humber and Rombach 1987, ° Huang et al. 1998. Phylogenetic analysis We constructed phylogenetic trees of the five concatenated loci from 11 newly-collected samples and 39 closely-related taxa from GenBank (Table 1). Our sampling included seven genera belonging to Cordycipitaceae, including Akanthomyces, Beauveria, Blackwellomyces, Cordyceps, Engyodontium, Gibellula and Hevansia, with Engyodontium aranearum being used as the outgroup. The concatenated alignment was 4581 bases long, with 525 bases from SSU, 838 bases from LSU, 924 bases from TEE, 720 bases from RPB1 and 1056 bas- es from RPB2. ‘The ML and BI phylogenic topologies were generally congruent (Fig. 3). All Gibellula species, including the 11 new specimens, formed a monophyletic group with high support that was sister to Hevansia. Moreover, the seven samples (GNJ20200814-11, 20200814-14, 20200814—-17, 20200812—05; NL20210822- 01, 20210822-09, 20210822-20), newly described as G. penicillioides, formed a clade sister to G. scorpioiodes. The four Gibellula specimens, newly described as G. longispora (GNJ20200813-16, 20210710-02; SZ20210904-02, 20210915-01), formed a clade with two previous Gibellula collections (NHJ 12014, 7859) with posterior probability of 1% and 71% bootstrap support, respectively; this lineage was sister to G. pigmentosinum. Furthermore, a BLASTn search for homologues showed that the Gibellula GNJ20200813—16 TEF sequence had highest similarity to the corresponding sequence of Gibellula sp. (NHJ 12014) (99.33%), further supporting that all members of this lineage belong to G. longispora. 62 MingJun Chen et al. / MycoKeys 90: 53-69 (2022) Table 3. Comparison of the morphological characters of Gibellula longispora sp. nov. and related species. Spee Phialide (um) | Conidia (um) Gibellula verrucose, (19—) 60— | obovoid to cylindrical,| clavate to broadly fusiform, 5—7 x 1-2 longispora sp. 153.5 (-170) x 8-10 | 6.5-9.5 x (4.5—) 5-7 | cylindrical, (6.5-) nov.! 7-9.5 (-11) x (1.5-) 2-3 Gibellula smooth to verrucose, broadly obovoid, obovoid to clavate, | obovoid with an acute pigmentosinum? | (55—) 97.5-170 (226) | (5.5—) 6-8 (-10) x (5-) 5.5-8 (-9) x 2-3 | apex (2.5-) 3.5-5 (-5.5) x (5-) 7-10 (-12.5) (3-) 4—G (-7.5) (-4.5) x 1-2 (-3) Gibellula flava’ verrucose, 33.5— obovoid to broadly narrowly obovate fusiform, (2.5—) 3-4 123.5(—182.5) x (3-) | obovoid, (4.5—) 5.5-7 | to clavate, 5.5—7 x (—5.5) x 1-2(-3) 4—9.5 (—11.5) x 3.5-5.5 1.5-2.5 Gibellula verrucose, 155-170 x | cylindrical, 6.2-7.5 clavate, 7.5-8 x fusiform to fusiform- pulchra* (6—-) 7.5-10 Sen) 1.5—2.5 ellipsoid, 3-5 x 1.5-2.5 Gibellula smooth or occasionally | obovoid, 8.6-10.8 clavate 5.4—6.5 x clavate, single, 5.4—6.5 clavispora® roughened 96-113 % 2.2 1.1-2.2 x 1.1-2.2 long Gibellula verrucose, 77-107 long] elliptical, 5.4—7.6 x clavate,5.4-10.8 x | cylindrical or fusiform, shennongjiaensis® 2.1-4.3 1.1-2.2 3.2-6.5 x 1.1-1.6 Note: 'Current study, *>Kuephadungphan et al. 2020, *Chen et al. 2021, * Chen et al. 2016, *Faruk et al. 2004, Zou et al. 2016. Discussion Our combined morphological and multilocus phylogenetic analyses distinguish Gibellula penicillioides and G. longispora as new species, which we described and illustrated. We showed that G. penicillioides is sister to G. scorpioides, but forms long penicilloid conidiophores producing enlarged fusiform conidia ((7—) 7.5—9 (-10) x 2.5-3.5 um) and that G. longispora is sister to G. pigmentosinum, but has slender long conidia (5—7 x 1-2 um). The fungal name Gibellula longispora for isolate NHJ12014 was first proposed, based on phylogenetic analysis with SSU, TEF, RPB1 and RPB2 sequences, but without morphological description (Johnson et al. 2009). In GenBank, sequences of isolate NHJ12014 were recorded as an unidentified Gibellula isolate. Furthermore, the name G. longispora has not been recorded in the global fungal databases Index Fungorum (www.indexfungorum.org) or MycoBank (www.mycobank.org) (Kuephadungphan et al. 2020). Therefore, due to the lack of formal description of isolate NHJ12014, the species name G. /ongispora was an invalid publication in 2009. Our molecular phylogeny showed that the five specimens from China (GNJ20200813-16, GNJ20210710-02, NL20210822-20, SZ20210904-02 and SZ20210915-01) formed a clade with isolates NHJ12014 and NHJ 7859. ‘The close phylogenetic relationship of these specimens suggests that they are conspecific despite the lack of morphological data for isolates NHJ12014 and NHJ 7859. Here, we described and illustrated the type specimen GNJ20200813-16 as a new species under the name Gibellula longispora. In China, spider-pathogenic fungi have been investigated for a long time, but until the 1980s, only one species (G. pulchra) was reported (Gao 1981). However, the first Gibellula new taxa 63 100/1 Cordyceps nidus TS903C 100/1 Cordyceps caloceroides MCA 2249 | Cordyceps 98/ Cordyceps militaris OSC 93623 Beauveria bassiana ARSEF 7518 . 100/1 eauveria bassiana | es Beauveria bassiana ARSEF 1564T 92/1 100/17-— Akanthomyces aculeatus HUA 186145T | Akanthomyces Akanthomyces coccidioperitheciatus NHJ 6709 Blackwellomyces cardinalis OSC 93609T Blackwellomyces cardinalis OSC 93610 Gibellula pigmentosinum NHJ 11679 Gibellula pigmentosinum BCC 41203T §0/0.9% Gibellula pigmentosinum BCC 39707 98/0.99 Gibellula longispora GNJ20200813-16 Gibellula longispora GNJ20210710-02 Gibellula sp. NHJ 7859 Gibellula sp. NHJ 12014 81/1 Gibellula longispora SZ20210904-02 Gibellula longispora $Z20210915-01 1oo/1p Gibellula sp. NHJ 5401 1004| “ Gibellula sp. NHJ 10788 api} Gibellula pulchra GNHJ 10808 Gibellula flava WFS20190625-25 100/1! Gibellula flava WFS09061701 100/1 Gibellula gamsii BCC 27968T Gibellula gamsii BCC 28797 Gibellula leiopus BCC 16025 100/1) Gibellula cebrennini BCC 53605T Gibellula cebrennini BCC 39705 Gibellula fusiformispora BCC 45076 760.291 Gipeltula fusiformispora BCC 56802T Gibellula scorpioides BCC 47514 Gibellula scorpioides BCC 47530 Gibellula scorpioides BCC 45127 Gibellula scorpioides BCC 47976T Gibellula scorpioides BCC 43298 Gibellula scorpioides BCC 13020 99/1 Gibellula penicillioides GNJ20200812-05 Gibellula penicillioides GNJ20200814-17 Gibellula penicillioides GNJ20200814-14 Gibellula penicillioides NL20210822-20 71 Gibellula penicillioides NL20210822-09 Gibellula penicillioides NL20210822-01 Gibellula penicillioides GNJ20200814-11 Gibellula clavulifera vat. alba ARSEF 1915T Hevansia novoguineensis CBS 610.80T | Blackwellomyces 100/1 83/0.68 Dynegy 98/1 Hevansia novoguineensis BCC 47881 H : Hevansia novoguineensis NHJ 11923 i alin Hevansia cinerea NHJ 3510 Engyodontium aranearum CBS 309.85 Engyodontium aranearum CBS 658.80 — 0.05 Outgroup Figure 3. Phylogenetic relationships amongst Gibellula and related genera in Cordycipitaceae obtained from analyses of Maximum Likelihood (ML) analysis of five loci (SSU, LSU, TEK RPB1 and RPB2). ML and BI topologies were generally congruent; therefore, we show only the ML analysis for brevity. At each node with support < 100%, we show ML bootstrap support / BI posterior probabilities; thick branches indicate 100% ML and BI support. The newly-proposed stains are highlighted in bold. 64 MingJun Chen et al. / MycoKeys 90: 53-69 (2022) Gibellula species in China was misidentified and is actually G. leiopus (Vuill. ex Maubl.), mainly based on its very short conidiophore, which imparts a compact appearance. In the 1990s, three new Gibellula species and a new variety were described from Taiwan and Anhui Province. During the past decade, Zongqi Liang’s research group have carried out a comprehensive study of the taxonomy of Gibellula in China and proposed three new species and two Chinese new records. Recently, we also found and published a new Gibellula species with Torrubiella-like sexual morph. Overall, ten species or varieties have been reported in China (Kuephadungphan et al. 2020; Chen et al. 2021): G. clavispora, G. clavulifera, G. clavulifera var. major, G. curvispora Y.F. Han, Wan H. Chen, X. Zou & Z.Q. Liang, G. dabieshanensis, G. dimorpha Tzean, L.S. Hsieh & W.J. Wu, G. flava, G. leiopus, G. pulchra, G. shennongjiaensis and G. unica L.S. Hsieh, Tzean & WJ. Wu. G. pul- chra and G. leiopus are commonly distributed spider pathogenic fungi in southern China. The specimens used in this study were collected from Anhui and Guangdong Provinces, which suggests that the two new species may be widely distributed in southern China. Kuephadungphan et al. (2020) indicated that host specificity can be used to assess the virulence and potential of biocontrol agents. Mycologists are increasingly inter- ested in exploiting Gibellula fungi for bioactive compounds. For example, EPFO83CE extracted from G. pulchra EPF083 was shown to be a new effective antimicrobial com- pound (Kuephadungphan et al. 2013). Pigmentosins A and B have been isolated from the spider—associated fungus G. pigmentosinum (Helaly et al. 2019) and two secondary metabolites, named gibellamines A and B, have been extracted from G. gamsii Kue- phadungphan, Tasan. & Luangsa-ard (Kuephadungphan et al. 2019). Interestingly, pigmentosin B and gibellamines are specific to G. pigmentosinum and G. gamsii, re- spectively and these specialised compounds may be used as markers for the species’ chemical taxonomy (Kuephadungphan et al. 2020). Gibellula is characterised by its specialised growth requirements; it is very hard to establish in culture (Samson and Evans 1973). Fortunately, the new taxon G. penicillioides was successfully isolated from conidia on the standard medium of potato dextrose agar (PDA), although the isolates grew slowly. In the future, we may be able to take advantage of Gibellula culture to explore more useful bio-active secondary metabolites or chemotaxonomic markers. Key to the species of Gibellula 1 Conidiophores smooth-walled, mononematous or synnematouS..........eeeeeeeeee 2 — Conidiophores typically rough-walled, mostly synnematous ......... cesses 8 2. Conidiophores strictly mononematous, with abruptly narrowing apex and vesi- (Re ae Pr ae CRs Sao ee SE G. mainsii Conidiophores mononematous or synnematous; typically penicillate................ 3 3 Conidiophores mononematous or synnematous, teleomorph absent or present 4 Conidiophores strictly mononematous, hyaline; teleomorph Torrubiella ratticau- DADA oc Poh ASS te TEAS ba eet BES ire SRE a IVER d Ec ctats G. clavulifera var. alba A? ec@onidiophores->-90-umelone conidia large... bee suse thea nce sentioceedeis edaeantheisea 5 == ":Conidiophores<:50 ain lone rcomidiarsialll, css. eas ssaoeeiugelonssvnunesunededsipuonstanene 6 NN N Gibellula new taxa 65 Granulomanus synanamorph present..........seeeeeeseeeee G. clavulifera var. major Granulomanus synanamorph absent ..........eseeseeeseeseeeseeeneeeeeees G. penicillioides Conidial heads purple, teleomorph absent .......... G. clavulifera var. clavulifera Gonidial heads-coloupless; teleomOnp iapreseint 12.24). bb stig cance seneselnsntee eetcreede Zs Vesicle swollen; conidia 3.2—4.0 x 1.1-1.8 Um... eee G. dabieshanensis Vesicles absent or hardly developed; conidia 5—7(—9) x (1.5—)2-3 um wee Soccer See ae Pes PAY i, Pe teal Na ee eh na Ae ra AE Fs G. scorpioides SYNC Matar Sie ean COU Le a ac medesneears SM ent Sdeoe case Net 2, Seaeattaiadhaateaee dite chenit 9 Sh iagU as] 00vz (7 0 | Ua] 0) ok mage Ne Rah Rio MER eo ce eae 16 Synnemata terminating in a bulbous outgrowth from which a number of conidi- ophores-and a:typical wirig-like:- structure arise. 02... si.cssoceeissedasssbeeseies G. alata Synnemata not terminating in a bulbous outgrowth with a wing-like structure, bift-eylindrical,gclavateror bulb-shaped s2i.555..2e sae et eee 10 Synnemata typically club-shaped or clavate with a cylindrical sterile apical projec- THOM fh ut tnlugeapeenteshsabens Vonetsdisea'cs sBiaens sVsnohe (fagaht Ap eacec ts eEeite dunes BonwheAcuawe dliteuces dee) 11 Synnemata cylindrical without a sterile apical projection... eeeeeeeeeeeeeeeeeees 13 Synnemata typically club-shaped; conidiophores > 80 um long....... G. mirabilis Syiniemata, clavate;-conidiophores 4:80pm long & 425s. tesa ects Bosnshas cacesdasteets 12 Granulomanus synanamorph present 1.2.0 0eter needa G. clavata Granulomanus synatiamnorp ly ADSENt ss. .ndesedonaceeeaeEeretwnssdasypouniien ole G. gamsii Grahulomatiussynahamonp hapresentss 2.00. celcbueacvecceecttar et atria hare eernneae 14 Granulomanus synanamorph absent or occasionally present ..........eeseeeeeeeees 15 Granulomanus synanamorph with well-differentiated conidiophore and poly- blastigicomidiogen@us Cells Fuh. .u00. Stas eeontss Siac veal S imitate G. dimorpha Granulomanus synanamorph with polyblastic conidiogenous cells... G. cebrennini Conidiophore 97-170 um long; conidia obovoid with an acute apex... a sn atd AME 8 She orl edemsbatsesh teas Fac shellse Rana see ecg atsbah cba aalsatehe G. pigmentosinum Conidiophore 31—53 um long; conidia fusiform to broadly fusiform................ ede Sea cets hal Re aS Laat lad scans Saat ctedndane Rais gna E Saad SU, SSE auld G. fusiformispora Synnemata with a stout yellowish-tan stipe, broadening into globose to pyriform fer- tile area and narrowed into a pale brown compact acuminate sterile tip....G. brunnea Slave Hira tats yl Mal Pi Cal. 8 Ronn Lares hole acces Wie Mceacal Acct REE Crees eee eae 17 Gianulomativs-syiatariotp i: presen tls igre OE ahh hatch ede tancciSng del. 18 Granulomatiussynanamotph Absentee. a .as.0 0s eeiest tea eteteennen Smee hate oeesccot 19 Granulomanus synanamorph with well-differentiated conidiophore and poly- BlAStieCOMMCORERGUSACEIISs Lz pous i gict hu sli songiiontaslonr te eidetrotdaelenctueteselael’ G. unica Granulomanus synanamorph with polyblastic conidiogenous cells in culture...... waht wicle oithe ehh Baahos aoe Heh re cn cps Mancoars eee Seaccdebe ontedteces ities G. shennongjiaensis CS OMiIGiaG Avate Ol MOTO MORI cae. steve Aee es talk 8A, ome, eae. 20 On HD TSO FIN Aa hore Nenana aall saca ns Mack MM adasbesiet aust stead asso halo tistads 21 Conidia 4.7—11 um long, botuliform; Phialide globose in base.....G. curvispora Conidia 3.2—6.5 um long, clavate; Phialide clavate oie G. clavispora Dela oir Weel ecell evaluat eye in Ravirot ise cr ROOM Hae ee arn Nr ere.) G. longispora SOM Ha SONU ONG aelceakesceninuremindsra it ne vetnpvedcanbndar cee Gunebeings dnlivamicdixad 22 66 MingJun Chen et al. / MycoKeys 90: 53-69 (2022) 22 Conidiophores long, with radiate and often loose conidial heads ................ 23 — Conidiophores short, with compact conidial heads... eects eeeeeeeeeee G. leiopus 23 Conidiophores up to 600 um; conidia 3—5 pm in Size... eee G. pulchra — Conidiophores up to 120 um; conidia 3—4 um in Size... eee G. flava Acknowledgements The authors would like to thank to Deshui Yu and Cheng Zhao in our laboratory for their help during field investigations and Qianle Lu, a lover of arachnology in Shen- zhen, for providing some specimens. We also thank Dr. Ian Gilman at Yale Univer- sity for his assistance with English language and grammatical editing. This study was conducted under research projects (Nos. 32172473 and 31972332) of the National Natural Science Foundation of China. 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