Research Article |
Corresponding author: Bo Huang ( bhuang@ahau.edu.cn ) Academic editor: Sajeewa Maharachchikumbura
© 2022 MingJun Chen, Ting Wang, Yan Lin, Bo Huang.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Chen M, 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
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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 (
Araneogenous fungi, Cordycipitaceae, spider, Taxonomy
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 (
In 1894, the genus Gibellula was proposed by
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.
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
Total genomic DNA was extracted from fresh synnema with a modified CTAB method (
We constructed a phylogenetic tree using the five loci (SSU, LSU, TEF, RPB1 and RPB2) from 50 taxa (Table
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 (
Latin “penicillioides” referring to the fungus with penicillate conidiophores.
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.
Occurring on spider attached to the underside of unidentified leaves nearby rivers.
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.
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
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 |
Latin “longispora” referring to the fungus with slender long conidia.
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.
Occurring on spider attached to the underside of leaf nearby the river.
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.
The new species G. longispora is similar to five Gibellula species in having multi-synnemum and aspergillate, distinctly roughened conidiophores (Table
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 |
We constructed phylogenetic trees of the five concatenated loci from 11 newly-collected samples and 39 closely-related taxa from GenBank (Table
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.
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 (
In China, spider-pathogenic fungi have been investigated for a long time, but until the 1980s, only one species (G. pulchra) was reported (
Gibellula is characterised by its specialised growth requirements; it is very hard to establish in culture (
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 |
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.