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Research Article
Morphological and molecular analyses reveal two new species of Gibellula (Cordycipitaceae, Hypocreales) from China
expand article infoMingJun Chen, Ting Wang, Yan Lin, Bo Huang
‡ Anhui Agricultural University, Hefei, China
Open Access

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) × 2.5–3.5 μm). G. longispora sp. nov. is sister to G. pigmentosinum, but has slender long conidia (5–7 × 1–2 μm); teleomorph and Granulomanus-synanamorphic conidiogenous cells are absent in these two species. Type specimens of G. penicillioides sp. nov. and G. longispora sp. nov. were deposited in the 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 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 identified 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 conidia. 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.

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 observations 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 observed 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 Agricultural University (RCEF).

DNA extraction, PCR amplification and sequencing

Total genomic DNA was extracted from fresh synnema with a modified CTAB method (Spatafora et al. 1998). Two gene portions from cell nuclei and three protein coding genes were used in this study: small subunit ribosomal RNA (SSU), large subunit ribosomal RNA (LSU), elongation factor-1a (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 LR0R and LR5 (Rehner and Samuels 1994), TEF-1 with TEF1–983F and TEF1–2218R (Rehner and Buckley 2005), RPB1 with CRPB1and 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, TEF, 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).

Table 1.

Accession numbers, strain numbers, and origins of Gibellula and related taxa used in this study, new sequences were shown in bold.

Taxon Specimen vouchera GenBank accession nos
SSU LSU TEF RPB1 RPB2
Akanthomyces aculeatus TS772 EU369110 KC519370
A. aculeatus HUA 186145T MF416572 MF416520 MF416465
Beauveria bassiana ARSEF 7518 HQ880975 HQ880834 HQ880906
B. bassiana ARSEF 1564T HQ880974 HQ880833 HQ880905
Cordyceps militaris OSC 93623 AY184977 AY184966 DQ522332 DQ522377 AY545732
C. nidus TS903C KY360300 KY360293 KY360296
C. caloceroides MCA 2249 MF416578 MF416578 MF416525 MF416470 MF416632
Blackwellomyces cardinalis OSC 93609T AY184973 AY184962 DQ522325 DQ522370 DQ522422
B. cardinalis OSC 93610 AY184974 AY184963 EF469059 EF469088 EF469106
Engyodontium aranearum CBS 309.85 AF339576 AF339526 DQ522341 DQ522387 DQ522439
E. aranearum CBS 658.80 LC092916
Gibellula cebrennini BCC 39705 MH394673 MH521895 MH521822 MH521859
G. cebrennini BCC 53605T MT477062 MT503328 MT503321 MT503336
G. clavulifera var. alba ARSEF 1915T DQ522562 DQ518777 DQ522360 DQ522408 DQ522467
G. flava WFS09061701 GU827389
G. flava WFS20190625-25 MW036749 MW084343 MW091325 MW384883
G. fusiformispora BCC 56802T MT477063 MT503329 MT503322 MT503337
G. fusiformispora BCC 45076 MH521823 MH521860
G. gamsii BCC 27968T MH152539 MH152560 MH152547
G. gamsii BCC 28797 MH152541 MH152562 MH152549 MH152557
G. leiopus BCC 16025 MF416602 MF416548 MF416492 MF416649
G. longispora NHJ 12014 EU369098 EU369017 EU369055 EU369075
G. longispora GNJ20200813–16 MW961414 MW980145
G. longispora GNJ20210710-02 OL854201 OL854212 OL981628 OL981635
G. longispora SZ20210904-02 OL981630
G. longispora SZ20210915-01 OL981631
G. pigmentosinum NHJ 11679 EU369016 EU369054
G. pulchra GNHJ 10808 EU369099 EU369035 EU369018 EU369056 EU369076
G. pigmentosinum BCC 41203T MT503330 MT503323
G. pigmentosinum BCC 39707 MH394674 MH521894 MH521801 MH521856
G. scorpioides BCC 47976T MT477066 MT503335 MT503325 MT503339
G. scorpioides BCC 47530 MT477065 MT503334 MT503338
G. scorpioides BCC 47514 MT503333
G. scorpioides BCC 43298 MH394677 MH521900 MH521816 MH521858
G. scorpioides BCC 13020 MH394686 MH521901 MH521814
Gibellula sp. NHJ 7859 EU369107 EU369064 EU369085
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

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 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 (iTOL) (https://itol.embl.de) online tool (Letunic and Bork 2016).

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 Reserve, 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) × (4–) 4.5–6 μm (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) × 3.5–5 (–5.5) μm, 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) × (2.5–) 3–4 (–5) μm. Conidia fusiform, (7–) 7.5–9 (–10) × 2.5–3.5 μm, in chains, borne on each phialide (Figs 1i–j). Teleomorph and granulomanus synanamorphs not observed.

Figure 1. 

Gibellula penicillioides sp. nov. a–b fungus on spider c synnema solitary d–f Penicillate conidiophores g conidiophore head bearing conidia h conidia i conidia in chains. Scale bars: 50 μm (d, e, f); 10 μm (g, h, i).

Habitat

Occurring on spider attached to the underside of unidentified leaves nearby rivers.

Additional materials examined

China. Anhui Province: Shitai County, Guniujiang National Nature Reserve, on a spider, 1 August 2020, Mingjun Chen & Ting Wang, GNJ20200814–11, GNJ20200814–17 and GNJ20200812–05. China. 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 & W.J. 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.

Table 2.

Comparison of Gibellula clavulifera, G. dabieshanensis, G. scorpioides and G. penicillioides sp. nov. with penicillate conidiophores.

Species Conidiophore(μm) metulae (μm) Phialide (μm) Conidia (μm)
Gibellula penicillioides sp. nov.1 penicillate, smooth, mostly biverticillate or terverticillate, (40–) 52.5–92 (115) × (4–) 4.5–6 obovoid to cylindrical, (11–) 13–17.5 (21.5) × 3.5–5 (–5.5) broadly cylindrical, (10–) 12.5–15.5 (–17) × (2.5–) 3–4 (–5) (7–) 7.5–9 (–10) × 2.5–3.5
Gibellula clavulifera var. major2 penicillate, Smooth‐walled, mostly bi- or terverticillate, occasionally monoverticillate 140 × 4.8-7.1 clavate to cylindrical, 12.7-19.8 × 4.0-5.6 ampulliform to cylindrical, 12.7–19.8 × 3.6–4.8 (-5.3) 7.1–12.0 (–13.9) × 2.4–4.0 (–5.6)
Gibellula scorpioides3 penicillate, smooth, mostly biverticillate, 20–29 (–30) × 4 obovoid, slightly broadening toward the base, (7–) 9.5–12.5 (–15) × (2–) 3–5 (–7) broadly cylindrical, (9–) 10–12.5 (–14) × (2–) 2.5–3.5 (–4) 5–7 (–9) × (1.5–) 2–3
Gibellula clavulifera var. clavulifera4 penicillate, Smooth-walled, 45–50 clavate cylindrical, with short neck 15–17.3 × 3.2–4.3 5.4–7.6 × 2.1–3.2
Gibellula clavulifera var. alba5 penicillate, smooth, mono-or biverticillate, up to 100 cylindricrical, 9–15 × 3–4 cylindrical or slightly swollen near the middle 10–12.4 × 1.5–2.5 5–7.5 × 1.5–2
Gibellula dabieshanensis penicillate with swollen vesicle, smooth 27–44 Obovoid to cylindricrical 8.6–11.5 × 5–6 cylindrical, 7.9-10.8 × 1.8-2.9 3.2-4.0 × 1.1-1.8

Gibellula longispora Ming J. Chen & B. Huang, sp. nov.

MycoBank No: 843175
Fig. 2

Etymology

Latin “longispora” referring to the fungus with slender long conidia.

Type

China. Anhui Province: Shitai County, Guniujiang National Nature Reserve, 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) × 8–10 μm (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) × (5–) 5.5–8μm (Fig. 2c, f). Spherical conidial heads consisting of vesicle, metulae and phialide, (25.5–) 38.5–49 (–50) × (24.5) 36–46.5 (–49) μm. A number of broadly obovate to oval metulae, 6.5–9.5 × (4.5–)5–7 μm (Fig. 2c), borne on vesicle, each metulae bearing several clavate phialides, (6.5–) 7–9.5 (–11) × (1.5–) 2–3 μm (Fig. 2c, f). Conidia, 5–7 × 1–2 μm (Fig. 2g), narrowly fusiform. Teleomorph and granulomanus synanamorphs not observed. (Fig. 2f).

Figure 2. 

Gibellula longispora sp. nov. a, b fungus on a spider c, d conidiophores showing conidial head e part of conidiophore showing rough walls f, g conidial head h conidia. Scale bars: 50 μm (c, d); 20 μm (e), 10 μm (f, g, h).

Habitat

Occurring on spider attached to the underside of leaf nearby the river.

Additional materials examined

China. Anhui Province: Shitai County, Guniujiang 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, SZ20210904-02, and SZ20210915-01.

Note

The new species G. longispora is similar to five Gibellula species in having multi-synnemum and aspergillate, distinctly roughened conidiophores (Table 3), namely G. pigmentosinum, G. flava, G. pulchra, G. clavispora Z.Q. Liang, Wan H. Chen & Y.F. Han and G. shennongjiaensis X. Zou, Wan H. Chen, Y.F. 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.

Table 3.

Comparison of the morphological characters of Gibellula longispora sp. nov. and related species.

Species Conidiophore (μm) Metulae (μm) Phialide (μm) Conidia (μm)
Gibellula longispora sp. nov.1 verrucose, (19–) 60–153.5 (–170) × 8–10 obovoid to cylindrical, 6.5–9.5 × (4.5–) 5–7 clavate to broadly cylindrical, (6.5–) 7–9.5 (–11) × (1.5–) 2–3 fusiform, 5–7 × 1–2
Gibellula pigmentosinum2 smooth to verrucose, (55–) 97.5–170 (–226) × (5–) 7–10 (–12.5) broadly obovoid, (5.5–) 6–8 (–10) × (3–) 4–6 (–7.5) obovoid to clavate, (5-) 5.5-8 (-9) × 2-3 (-4.5) obovoid with an acute apex (2.5-) 3.5-5 (-5.5) × 1-2 (-3)
Gibellula flava3 verrucose, 33.5–123.5(–182.5) × (3–) 4–9.5 (–11.5) obovoid to broadly obovoid, (4.5–) 5.5–7 × 3.5–5.5 narrowly obovate to clavate, 5.5–7 × 1.5–2.5 fusiform, (2.5–) 3–4 (–5.5) × 1–2(–3)
Gibellula pulchra4 verrucose, 155–170 × (6–) 7.5–10 cylindrical, 6.2–7.5 × 5 clavate, 7.5–8 × 1.5–2.5 fusiform to fusiform-ellipsoid, 3–5 × 1.5–2.5
Gibellula clavispora5 smooth or occasionally roughened 96–113 long obovoid, 8.6–10.8 × 2.2 clavate 5.4–6.5 × 1.1–2.2 clavate, single, 5.4–6.5 × 1.1–2.2
Gibellula shennongjiaensis6 verrucose, 77–107 long elliptical, 5.4–7.6 × 2.1–4.3 clavate,5.4–10.8 × 1.1–2.2 cylindrical or fusiform, 3.2–6.5 × 1.1–1.6

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 TEF, 720 bases from RPB1 and 1056 bases from RPB2. The ML and BI phylogenic topologies were generally congruent (Fig. 3).

Figure 3. 

Phylogenetic relationships amongst Gibellula and related genera in Cordycipitaceae obtained from analyses of Maximum Likelihood (ML) analysis of five loci (SSU, LSU, TEF, 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.

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.

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) × 2.5–3.5 μm) and that G. longispora is sister to G. pigmentosinum, but has slender long conidia (5–7 × 1–2 μm).

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. longispora 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 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 & W.J. Wu. G. pulchra 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 interested in exploiting Gibellula fungi for bioactive compounds. For example, EPF083CE extracted from G. pulchra EPF083 was shown to be a new effective antimicrobial compound (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 Kuephadungphan, Tasan. & Luangsa-ard (Kuephadungphan et al. 2019). Interestingly, pigmentosin B and gibellamines are specific to G. pigmentosinum and G. gamsii, respectively 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 2
Conidiophores typically rough-walled, mostly synnematous 8
2 Conidiophores strictly mononematous, with abruptly narrowing apex and vesicle 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 ratticaudata G. clavulifera var. alba
4 Conidiophores > 90 μm long; conidia large 5
Conidiophores < 50 μm long; conidia small 6
5 Granulomanus synanamorph present G. clavulifera var. major
Granulomanus synanamorph absent G. penicillioides
6 Conidial heads purple, teleomorph absent G. clavulifera var. clavulifera
Conidial heads colourless, teleomorph present 7
7 Vesicle swollen; conidia 3.2–4.0 × 1.1–1.8 μm G. dabieshanensis
Vesicles absent or hardly developed; conidia 5–7(–9) × (1.5–)2–3 μm G. scorpioides
8 Synnemata single or double 9
Synnemata multiple 16
9 Synnemata terminating in a bulbous outgrowth from which a number of conidiophores and a typical wing-like structure arise G. alata
Synnemata not terminating in a bulbous outgrowth with a wing-like structure, but cylindrical, clavate or bulb-shaped 10
10 Synnemata typically club-shaped or clavate with a cylindrical sterile apical projection 11
Synnemata cylindrical without a sterile apical projection 13
11 Synnemata typically club-shaped; conidiophores > 80 μm long G. mirabilis
Synnemata clavate; conidiophores < 80 μm long 12
12 Granulomanus synanamorph present G. clavata
Granulomanus synanamorph absentG. gamsii
13 Granulomanus synanamorph present 14
Granulomanus synanamorph absent or occasionally present 15
14 Granulomanus synanamorph with well-differentiated conidiophore and polyblastic conidiogenous cells G. dimorpha
Granulomanus synanamorph with polyblastic conidiogenous cells G. cebrennini
15 Conidiophore 97–170 μm long; conidia obovoid with an acute apex G. pigmentosinum
Conidiophore 31–53 μm long; conidia fusiform to broadly fusiform G. fusiformispora
16 Synnemata with a stout yellowish-tan stipe, broadening into globose to pyriform fertile area and narrowed into a pale brown compact acuminate sterile tip G. brunnea
Synnemata cylindrical 17
17 Granulomanus synanamorph present 18
Granulomanus synanamorph absent 19
18 Granulomanus synanamorph with well-differentiated conidiophore and polyblastic conidiogenous cells G. unica
Granulomanus synanamorph with polyblastic conidiogenous cells in culture G. shennongjiaensis
19 Conidia clavate or botuliform 20
Conidia fusiform 21
20 Conidia 4.7–11 μm long, botuliform; Phialide globose in base G. curvispora
Conidia 3.2–6.5 μm long, clavate; Phialide clavate G. clavispora
21 Conidia > 5 μm long G. longispora
Conidia < 5 μm long 22
22 Conidiophores long, with radiate and often loose conidial heads 23
Conidiophores short, with compact conidial heads G. leiopus
23 Conidiophores up to 600 μm; conidia 3–5 μm in size G. pulchra
Conidiophores up to 120 μm; conidia 3–4 μm in size 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 Shenzhen, for providing some specimens. We also thank Dr. Ian Gilman at Yale University 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.

References

  • Castlebury LA, Rossman AY, Sung GH, Hyten AS, Spatafora JW (2004) Multigene phylogeny reveals new lineage for Stachybotrys chartarum, the indoor air fungus. Mycological Research 108(8): 864–872. https://doi.org/10.1017/S0953756204000607
  • Cavara F (1894) Ulteriore contribuzione alla Micologia Lombarda. Atti dell’Ist Bot Univ e Lab Crittogamico Pavia 3: 313–350.
  • Chen WH, Han YF, Liang ZQ, Wang YR, Zou X (2014) Classification of Gibellula spp. by DELTA system. Microbiology China 41(2): 399–407.
  • Chen WH, Han YF, Liang ZQ, Zou X (2016) Morphological traits, DELTA system, and molecular analysis for Gibellula clavispora sp. nov. from China. Mycotaxon 131(1): 111–121. https://doi.org/10.5248/131.111
  • Faruk S, Elşad H, Muhammet G (2004) Occurrence of the araneogenous fungus Gibellula pulchra in Turkey. Mycologia Balcanica 1: 61–62.
  • Gao RX (1981) Description of a parasitic fungus Gibellula suffulta on spiders in Fujian. Acta Microbiologica Sinica 21: 308–310.
  • Helaly SE, Kuephadungphan W, Phongpaichit S, Luangsa-Ard JJ, Rukachaisirikul V, Stadler M (2017) Five unprecedented secondary metabolites from the spider parasitic fungus Akanthomyces novoguineensis. Molecules (Basel, Switzerland) 22(6): e991. https://doi.org/10.3390/molecules22060991
  • Helaly SE, Kuephadungphan W, Phainuphong P, Ibrahim MAA, Tasanathai K, Mongkolsamrit S, Luangsa-Ard JJ, Phongpaichit S, Rukachaisirikul V, Stadler M (2019) Pigmentosins from Gibellula sp. as antibiofilm agents and a new glycosylated asperfuran from Cordyceps javanica. Beilstein Journal of Organic Chemistry 15: 2968–2981. https://doi.org/10.3762/bjoc.15.293
  • Huang B, Ding DG, Fan MZ, Li ZZ (1998) A new entomopathogenic fungus on spiders. Junwu Xuebao 17(2): 109–113.
  • Johnson D, Sung GH, Hywel-Jones NL, Luangsa-Ard JJ, Bischoff JF, Kepler RM, Spatafora JW (2009) Systematics and evolution of the genus Torrubiella (Hypocreales, Ascomycota). Mycological Research 113(Pt 3): 279–289. https://doi.org/10.1016/j.mycres.2008.09.008
  • Kepler RM, Sung GH, Ban S, Nakagiri A, Chen MJ, Huang B, Li Z, Spatafora JW (2012) New teleomorph combinations in the entomopathogenic genus Metacordyceps. Mycologia 104(1): 182–197. https://doi.org/10.3852/11-070
  • Kepler RM, Luangsa-Ard JJ, Hywel-Jones NL, Quandt CA, Sung GH, Rehner SA, Aime MC, Henkel TW, Sanjuan T, Zare R, Chen M, Li Z, Rossman AY, Spatafora JW, Shrestha B (2017) A phylogenetically-based nomenclature for Cordycipitaceae (Hypocreales). IMA Fungus 8(2): 335–353. https://doi.org/10.5598/imafungus.2017.08.02.08
  • Kobayasi Y, Shimizu D (1976) The genus Cordyceps and its allies from New Guinea. Bulletin of the National Science Museum, Tokyo 2: 133–152.
  • Kobayasi Y, Shimizu D (1982) Monograph of the genus Torrubiella. Bulletin of the National Science Museum, Tokyo 8: 43–78.
  • Kuephadungphan W, Phongpaichit S, Luangsa-ard JJ, Rukachaisirikul V (2013) Antimicrobial activity of invertebrate-pathogenic fungi in the genera Akanthomyces and Gibellula. Mycoscience 55(2): 127–133. https://doi.org/10.1016/j.myc.2013.06.007
  • Kuephadungphan W, Macabeo APG, Luangsa-ard JJ, Tasanathai K, Thanakitpipattana D, Phongpaichit S, Yuyama K, Stadler M (2019) Studies on the biologically active secondary metabolites of the new spider parasitic fungus Gibellula gamsii. Mycological Progress 18(1–2): 135–146. https://doi.org/10.1007/s11557-018-1431-4
  • Kuephadungphan W, Tasanathai K, Petcharad B, Khonsanit A, Stadler M, Luangsa-Ard JJ (2020) Phylogeny- and morphology-based recognition of new species in the spider-parasitic genus Gibellula (Hypocreales, Cordycipitaceae) from Thailand. MycoKeys 72: 17–42. https://doi.org/10.3897/mycokeys.72.55088
  • Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics (Oxford, England) 23(21): 2947–2948. https://doi.org/10.1093/bioinformatics/btm404
  • Letunic I, Bork P (2016) Interactive tree of life (iTOL) v3: An online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Research 44(W1): W242–W245. https://doi.org/10.1093/nar/gkw290
  • Nylander JAA (2004) MrModeltest V2. Program Distributed by the Author. Evolutionary Biology Centre, Uppsala University.
  • Petch T (1932) Gibellula. Annales Mycologici 30: 386–393.
  • Rehner SA, Buckley E (2005) A Beauveria phylogeny inferred from nuclear ITS and EF1-α sequences: Evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 97(1): 84–98. https://doi.org/10.3852/mycologia.97.1.84
  • Rehner SA, Minnis AM, Sung GH, Luangsa-ard JJ, Devotto L, Humber RA (2011) Phylogeny and systematics of the anamorphic, entomopathogenic genus Beauveria. Mycologia 103(5): 1055–1073. https://doi.org/10.3852/10-302
  • Sanjuan T, Tabima J, Restrepo S, Læssøe T, Spatafora JW, Franco-Molano AE (2014) Entomopathogens of Amazonian stick insects and locusts are members of the Beauveria species complex (Cordyceps sensu stricto). Mycologia 106(2): 260–275. https://doi.org/10.3852/13-020
  • Shrestha B, Kubátová A, Tanaka E, Oh J, Yoon DH, Sung JM, Sung GH (2019) Spider-pathogenic fungi within Hypocreales (Ascomycota): Their current nomenclature, diversity, and distribution. Mycological Progress 18(8): 983–1003. https://doi.org/10.1007/s11557-019-01512-3
  • Spatafora JW, Volkmann-Kohlmeyer B, Kohlmeyer J (1998) Independent terrestrial origins of the Halosphaeriales (Marine Ascomycota). American Journal of Botany 85(11): 1569–1580. https://doi.org/10.2307/2446483
  • Spatafora JW, Sung GH, Sung JM, Hywel-Jones NL, White Jr JF (2007) Phylogenetic evidence for an animal pathogen origin of ergot and the grass endophytes. Molecular Ecology 16(8): 1701–1711. https://doi.org/10.1111/j.1365-294X.2007.03225.x
  • Tsang CC, Chan JFW, Pong WM, Chen JHK, Ngan AHY, Cheung M, Lai CKC, Tsang DNC, Lau SKP, Woo PCY (2016) Cutaneous hyalohyphomycosis due to Parengyodontium album gen. et comb. nov. Medical Mycology 54(7): 699–713. https://doi.org/10.1093/mmy/myw025
  • White TJ, Bruns TD, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
  • Zou X, Chen WH, Han YF, Liang ZQ (2016) A new species of the genus Gibellula. Junwu Xuebao 35(10): 1161–1168.
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