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Research Article
Study on species diversity of Akanthomyces (Cordycipitaceae, Hypocreales) in the Jinyun Mountains, Chongqing, China
expand article infoWan-Hao Chen‡, Jian-Dong Liang‡, Xiu-Xiu Ren‡, Jie-Hong Zhao‡, Yan-Feng Han§
‡ Guizhou University of Traditional Chinese Medicine, Guiyang, China
§ Guizhou University, Guiyang, China

Corresponding author: Yan-Feng Han ( swallow1128@126.com )

Academic editor: Rungtiwa Phookamsak
© 2023 Wan-Hao Chen, Jian-Dong Liang, Xiu-Xiu Ren, Jie-Hong Zhao, Yan-Feng Han.
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.
Citation: Chen W-H, Liang J-D, Ren X-X, Zhao J-H, Han Y-F (2023) Study on species diversity of Akanthomyces (Cordycipitaceae, Hypocreales) in the Jinyun Mountains, Chongqing, China. MycoKeys 98: 299-315. https://doi.org/10.3897/mycokeys.98.106415
Open Access

Abstract

Akanthomyces species have only been reported from Guizhou and Qinghai Province, with few reports from other regions in China. In this research, the species diversity of Akanthomyces in the Jinyun Mountains, Chongqing was investigated. Fourteen infected spider specimens were collected and two new species (A. bashanensis and A. beibeiensis) and a known species (A. tiankengensis) were established and described according to a multi-locus phylogenetic analysis and the morphological characteristics. Our results reveal abundant Akanthomyces specimens and three species were found at Jinyun Mountain. Due to its being an important kind of entomopathogenic fungi, further attention needs to be paid to the diversity of other entomopathogenic fungi in Chongqing, China.

Key words

entomopathogenic fungi, morphology, phylogenetic analysis, Sordariomycetes, spider-pathogenic fungi

Introduction

Akanthomyces Lebert was established by Lebert for the species, A. aculeatus Lebert in 1858. Mains (1950) reported three additional species: A. ampullifer (Petch) Mains, A. angustisporus Mains and A. aranearum (Petch) Mains. Subsequently, several other species have been reported (Samson and Evans 1974; Koval 1977; Vincent et al. 1988; Hywel-Jones 1996; Hsieh et al. 1997; Huang et al. 2000; Mongkolsamrit et al. 2018; Chen et al. 2018, 2019c, 2020b, 2020c, 2022a; Shrestha et al. 2019; Aini et al. 2020; Wang et al. 2023).

Based mainly on phylogenetic analyses, several Akanthomyces species (A. arachnophilus (Petch) Samson & H.C. Evans, A. cinereus Hywel-Jones, A. koratensis Hywel-Jones, A. longisporus B. Huang et al., A. novoguineensis Samson & B.L. Brady, A. ovalongatus L.S. Hsieh et al. and A. websteri Hywel-Jones) were transferred to the new genus Hevansia Luangsa-ard et al. (Kepler et al. 2017). In addition, Lecancillium attenuatum Zare & W. Gams, L. lecanii (Zimm.) Zare & W. Gams, L. longisporum (Petch) Zare & W. Gams, L. muscarium (Petch) Zare & W. Gams, L. pissodis Kope & I. Leal and L. sabanense Chir.-Salom., S. Restrepo & T.I. Sanjuan were transferred to Akanthomyces. As a result, the genus Akanthomyces currently consists of 34 species.

Akanthomyces is an important genus in entomopathogenic fungi and its diverse bioactive substances have attracted widespread attention (Lee et al. 2005; Madla et al. 2005; Kuephadungphan et al. 2014; Putri et al. 2014; Kinoshita et al. 2017). However, Akanthomyces species have only been reported from Guizhou and Qinghai Province and there have been few reports from other regions in China (Chen et al. 2018, 2019c, 2020b, 2020c, 2022a; Wang et al. 2023). In this research, the species diversity of Akanthomyces in the Jinyun Mountains, Chongqing was investigated. Several spider-associated specimens were found and a few new Akanthomyces strains were isolated and purified. The goal of this research was to identify these new strains by multigene phylogeny as well as by morphological characteristics.

Materials and methods

Specimen collection and identification

Fourteen infected spider specimens were collected from Jinyun Mountain (29°50'22.14959"N, 106°23'18.0744"E), Beibei District, Chongqing, in May 2021. The surface of each spider body was rinsed with sterile water, followed by surface sterilisation with 75% ethanol for 3–5 s and rinsing 3 times with sterilised water. After drying on sterilised filter paper, the mycelium or a part of the sclerotium was removed from the specimen and inoculated on potato dextrose agar (PDA) and improved potato dextrose agar (PDA, 1% w/v peptone) plates (Chen et al. 2019b). Fungal colonies emerging from the specimens were isolated and cultured at 25 °C for 14 days under 12 h light/12 h dark conditions following protocols described by Zou et al. (2010). The specimens and axenic cultures were deposited at the Institute of Fungus Resources, Guizhou University (formally Herbarium of Guizhou Agricultural College; code, GZAC), Guiyang City, Guizhou, China.

Macroscopic characterisation was determined from PDA cultures incubated at 25 °C for 14 days, including the growth rate of the colony, the presence of octahedral crystals, and the colours of the colony (surface and reverse) were observed. To investigate the microscopic characteristics, a small amount of the colony was removed and mounted in lactophenol cotton blue or 20% lactate acid solution and observed using an optical microscope (OM, DM4 B, Leica, Germany).

DNA extraction, polymerase chain reaction amplification and nucleotide sequencing

DNA extraction was carried out using a fungal genomic DNA extraction kit (DP2033, BioTeke Corporation) according to Liang et al. (2011). The extracted DNA was stored at −20 °C. Polymerase chain reaction (PCR) was used to amplify genetic markers using the following primer pairs: ITS4/ITS5 for the internal transcribed spacer (ITS) region (White et al. 1990), LR0R/LR5 for 28s large subunit ribosomal (LSU) (Vilgalys and Hester 1990), fRPB2-7cR/fRPB2-5F for RNA polymerase II second largest subunit (RPB2) (Liu et al. 1999) and 2218R/983F for translation elongation factor 1 alpha (TEF) (Castlebury et al. 2004). The thermal cycle of PCR amplification for these phylogenetic markers was set up following the procedure described by Chen et al. (2021). PCR products were purified and sequenced at Sangon Biotech (Shanghai) Co. The resulting sequences were submitted to GenBank (Table 1).

Table 1.
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List of strains and GenBank accession numbers of sequences used in this study.

Species Strain GenBank accession numbers
ITS LSU RPB2 TEF
Akanthomyces aculeatus HUA 186145 - MF416520 - MF416465
A. aculeatus TS772 KC519371 KC519370 - KC519366
A. araneicola GY29011 MK942430 - MK955947 MK955950
A. araneicola GY29012 MK942435 - MK955948 MK955951
A. araneicola GY29013 MK942436 - MK955949 -
A. araneogenus GZUIFDX2 KU893153 - MH978185 MH978187
A. araneogenus GZUIFDX1 KU893152 - MH978184 -
A. araneogenus GZUIFSN1 MH978177 - MH978186 MH978188
A. araneosus KY11341 ON502826 ON502832 ON525442 ON525443
A. araneosus KY11342 ON502844 ON502837 ON525444 ON525445
A. attenuatus CBS 402.78 AJ292434 AF339565 EF468935 EF468782
A. bashanensis CQ05621 OQ300412 OQ300420 OQ349684 OQ325024
A. bashanensis CQ05622 OQ300411 OQ300421 OQ349685 OQ325025
A. beibeiensis CQ05921 OQ300415 OQ300424 OQ349688 OQ325028
A. beibeiensis CQ05922 OQ300416 OQ300427 OQ349689 OQ325029
A. coccidioperitheciatus NHJ 6709 JN049865 EU369042 EU369086 EU369025
A. kanyawimiae TBRC 7242 MF140751 MF140718 MF140808 MF140838
A. kanyawimiae TBRC 7243 MF140750 MF140717 MF140807 MF140837
A. kanyawimiae TBRC 7244 MF140752 MF140716 - MF140836
A. lecanii CBS 101247 JN049836 AF339555 DQ522466 DQ522359
A. lepidopterorum SD05151 MT705971 MT705973 MT727044 -
A. lepidopterorum SD05152 MT705972 MT705974 MT727045 -
A. neoaraneogenus GZU1031Lea - KX845703 KX845701 KX845697
A. neoaraneogenus GZU1032Lea - KX845704 KX845702 KX845698
A. neocoleopterorum GY11241 MN093296 - MN097812 MN097813
A. neocoleopterorum GY11242 MN093297 - MN097814 MN097815
A. noctuidarum BCC36265 MT356072 MT356084 MT477987 MT477978
A. noctuidarum BBH16595 MT356073 MT356085 MT478005 MT477979
A. noctuidarum BCC47498 MT356074 MT356086 MT477988 MT477980
A. noctuidarum BCC28571 MT356075 MT356087 MT478006 MT477981
A. pissodis CBS 118231 - KM283799 KM283864 KM283822
A. pyralidarum BCC28816 MT356080 MT356091 MT478007 MT477982
A. pyralidarum BCC32191 MT356081 MT356092 MT477989 MT477983
A. pyralidarum BCC40869 MT356082 MT356093 MT477990 MT477984
A. pyralidarum BCC29197 MT356083 MT356094 MT477991 MT508840
A. sabanensis JCh041 - - KC875224 KC633274
A. sulphureus TBRC 7247 MF140756 MF140720 MF140811 MF140841
A. sulphureus TBRC 7248 MF140758 MF140722 MF140812 MF140843
A. sulphureus TBRC 7249 MF140757 MF140721 MF140734 MF140842
A. thailandicus TBRC 7245 MF140754 - MF140809 MF140839
A. thailandicus TBRC 7246 MF140755 MF140719 MF140810 MF140840
A. tiankengensis KY11571 ON502848 ON502825 ON525446 ON525447
A. tiankengensis KY11572 ON502821 ON502827 ON525448 ON525449
A. tiankengensis CQ05171 OQ300407 OQ300417 OQ349683 OQ325022
A. tiankengensis CQ05172 OQ300408 OQ300419 OQ349690 OQ325023
A. tiankengensis CQ05811 OQ300413 OQ300423 OQ349686 OQ325026
A. tiankengensis CQ05812 OQ300414 OQ300425 OQ349687 OQ325027
A. tortricidarum BCC72638 MT356076 MT356088 MT477992 MT478004
A. tortricidarum BCC41868 MT356077 MT356089 MT478008 MT477985
A. tortricidarum BCC28583 MT356079 MT356090 MT477993 MT477986
A. tuberculatus HUA186131 - MF416521 - MF416466
A. waltergamsii TBRC7250 MF140749 MF140715 - MF140835
A. waltergamsii TBRC7251 MF140747 MF140713 MF140805 MF140833
A. zaquensis HMAS 246917 MT789698 MT789696 - MT797811
A. zaquensis HMAS 246915 MT789699 MT789697 - MT797812
Samsoniella aurantia TBRC7271 MF140764 MF140728 MF140818 MF140846
S. aurantia TBRC7272 MF140763 MF140727 MF140817 MF140845

Sequence alignment and phylogenetic analyses

DNASTAR Lasergene (version 6.0) was used to edit the DNA sequences. The ITS, LSU, RPB2 and TEF sequences were downloaded from GenBank, based on Sung et al. (2007), Kepler et al. (2017), Mongkolsamrit et al. (2018), Chen et al. (2018, 2019c, 2020b, 2020c, 2022a), Aini et al. (2020) and Wang et al. (2023) and others selected on the basis of BLAST searches in GenBank. ITS sequences and other loci were aligned and edited by MAFFT online service (Katoh et al. 2019) and MEGA6 (Tamura et al. 2013). Combined sequences of ITS, LSU, RPB2 and TEF were obtained using SequenceMatrix v.1.7.8 (Vaidya et al. 2011). The model was selected for Bayesian analysis by ModelFinder (Kalyaanamoorthy et al. 2017) in PhyloSuite software (Zhang et al. 2020).

The combined loci were analysed using Bayesian inference (BI) and maximum likelihood (ML) methods. For BI, a Markov chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities using MrBayes v.3.2 (Ronquist et al. 2012) for the combined sequence datasets. The Bayesian analysis resulted in 20,001 trees after 10,000,000 generations. The first 4,000 trees, representing the burn-in phase of the analysis, were discarded, while the remaining 16,001 trees were used to calculate posterior probabilities in the majority rule consensus tree. After the analysis was finished, each run was examined using the programme Tracer v.1.5 (Drummond and Rambaut 2007) to determine burn-in and confirm that both runs had converged. ML analyses were performed with IQ-TREE (Trifinopoulos et al. 2016), using an automatic selection of the model. The final alignment and the original phylogenetic tree are available from TreeBASE under submission ID 30378.

Genealogical Concordance Phylogenetic Species Recognition (GCPSR) analysis

The Genealogical Concordance Phylogenetic Species Recognition model was applied to analyse the related species. The pairwise homoplasy index (PHI) (Bruen et al. 2006) is a model test based on the fact that multiple gene phylogenies will be concordant between species and discordant due to recombination and mutations within a species. The test was performed in SplitsTree4 (Huson and Bryant 2006) as described by Quaedvlieg et al. (2014) to determine the recombination level within phylogenetically closely-related species using a four-locus concatenated dataset. The new species and their closely-related species were analysed using this model. The relationships between closely-related species were visualised by constructing a split graph, using both the LogDet transformation and splits decomposition options.

Result

Phylogenetic analyses

In the phylogenetic tree, Samsoniella aurantia Mongkols., et al. (TBRC 7271 and TBRC 7272) was used as the outgroup. The concatenated sequences (ITS, LSU, RPB2 and TEF) included 23 species (49 strains) and consisted of 2,620 characters with gaps (ITS, 478; LSU, 745; RPB2, 717; and TEF, 680).

The final value of the highest scoring tree was –11,790.345, which was obtained from the ML analysis of the dataset (ITS+LSU+RPB2+TEF). The parameters of the GTR model used to analyse the dataset were estimated, based on the following frequencies: A = 0.236, C = 0.283, G = 0.272, T = 0.209; substitution rates AC = 1.00000, AG = 2.12340, AT = 1.00000, CG = 1.00000, CT = 5.43884 and GT = 1.00000, as well as the gamma distribution shape parameter α = 0.557. The selected model for BI analysis was GTR+F+I+G4 (ITS+LSU+TEF) and K2P+G4 (RPB2). The phylogenetic trees (Fig. 1) constructed using ML and BI analyses were largely congruent and strongly supported in most branches. Phylogenetic analyses demonstrated that eight new strains formed a subclade with Akanthomyces tiankengensis (KY11571 and KY11572) with high statistical support in ML analysis (92% ML). Strains CQ05171, CQ05172, CQ05811 and CQ05812 clustered with A. tiankengensis into a subclade, while the new species A. beibeiensis (CQ05921 and CQ05922) and A. bashanensis (CQ05621 and CQ05622) clustered in a subclade with high statistical support (96% ML/0.98 PP; Fig. 1).

Figure 1. 

Phylogenetic relationships amongst the new strains and their allies based on multigene dataset (ITS, LSU, RPB2 and TEF). Statistical support values (≥70%/0.95) are shown at the nodes for ML bootstrap support/BI posterior probabilities.

GCPSR analysis

A four-locus concatenated dataset (ITS, LSU, RPB2 and TEF) was used to determine the recombination level within Akanthomyces bashanensis (CQ05621), A. beibeiensis (CQ05921) and A. tiankengensis (KY11571, CQ05171, CQ05811). Chaiwan et al. (2022) noted that, if the PHI is below the 0.05 threshold (Φw < 0.05), it indicates that there is significant recombination in the dataset, meaning that related species in a group and recombination level are not different. If the PHI is above the 0.05 threshold (Φw > 0.05), it indicates that it is not significant, which means the related species in a group level are different. The result of the pairwise homoplasy index (PHI) test of A. bashanensis, A. beibeiensis and A. tiankengensis was 0.333 and revealed that the three species were different (Fig. 2).

Figure 2. 

Results of the pairwise homoplasy index (PHI) test of closely-related species using both LogDet transformation and splits decomposition. PHI test results (Φw) < 0.05 indicate significant recombination within the dataset. The new taxon is in bold type.

Taxonomy

Akanthomyces bashanensis W.H. Chen, Y.F. Han & J.D. Liang, sp. nov.

MycoBank No: 847339
Fig. 3

Type

China, Chongqing, Beibei District, Jinyun Mountain (29°50'22.14959"N, 106°23'18.0744"E). On a dead spider (Araneae), 1 May 2021, Wanhao Chen, GZAC CQ0562 (holotype), ex-type living culture, CQ05621.

Description

Spider host completely covered by white mycelium. Conidiophores mononematous, arising from the lateral hyphae. Colonies on PDA, attaining a diameter of 26–27 mm after 14 days at 25 °C, white, consisting of a basal felt, floccose hyphal overgrowth; reverse yellowish. Hyphae septate, hyaline, smooth-walled, 1.5–1.9 μm wide. Conidiophores mononematous, hyaline, smooth-walled, with single phialide or whorls of 2–4 phialides or verticillium-like from hyphae directly, 12.1–20.5 × 1.5–2.1 μm. Phialides consisting of a cylindrical, somewhat inflated base, 11.8–12.9 × 1.3–1.6 μm, tapering to a thin neck. Conidia hyaline, smooth-walled, fusiform to ellipsoidal, 1.7–2.6 × 1.6–1.8 μm, forming divergent and basipetal chains. Sexual state not observed.

Figure 3. 

Akanthomyces bashanensis A infected spider (Araneae) B, C PDA culture plate showing top (B) and reverse (C) sides of the colony D–H phialides and conidia. Scale bars: 10 mm (B, C); 10 μm (D–H).

Etymology

Referring to its location in Jinyun Mountain, which was formerly known as Bashan.

Additional strain examined

China, Chongqing, Beibei District, Jinyun Mountain (29°50'22.14959"N, 106°23'18.0744"E). On a dead spider (Araneae), 1 May 2021, Wanhao Chen, CQ05622.

Remarks

Akanthomyces bashanensis was easily identified as Akanthomyces, based on the BLASTn result in NCBI and the phylogenetic analysis of combined datasets (ITS, LSU, RPB2, TEF) (Fig. 1) and it has a close relationship with another new species, A. beibeiensis. A. bashanensis was easily distinguished from A. beibeiensis by its longer phialides and smaller conidia. Jeewon and Hyde (2016) recommended that a minimum of > 1.5% nucleotide differences in the ITS regions and protein coding genes may be indicative of a new species. The pairwise dissimilarities of ITS, LSU, RPB2 and TEF sequences show 11 bp differences within 569 bp (1.93%), 19 bp differences within 881 bp (2.15%), 13 bp differences within 1070 bp (1.21%) and 4 bp differences within 973 bp (0.41%) between A. bashanensis and A. beibeiensis, respectively. The pairwise dissimilarities of ITS, LSU, RPB2 and TEF sequences show 10 bp differences within 569 bp (1.75%), 20 bp differences within 881 bp (2.27%), 19 bp differences within 1070 bp (1.77%) and 4 bp differences within 973 bp (0.41%) between A. bashanensis and A. tiankengensis, respectively. Furthermore, A. aranearum (Petch) Mains and A. ryukyuensis (Kobayasi & Shimizu) Mongkols., Noisrip., Thanakitp., Spatafora & Luangsa-ard were both absent from the available sequence in NCBI and having a spider host. Comparing with the typical characteristics (Table 2), A. bashanensis was easily distinguished from A. aranearum by its cylindrical phialide, smaller fusiform to ellipsoidal conidia and absence of synnemata and distinguished from A. ryukyuensis by absence of teleomorphs. Thus, the morphological characteristics and molecular phylogenetic results support A. bashanensis as a new species.

Table 2.
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Morphological comparison of two new species with other related Akanthomyces species.

Species Synnemata (mm)/ Perithecia (μm) Conidiophores (μm) Phialides (μm)/ Asci (μm) Conidia (μm)/ Part-spores (μm) Reference
Akanthomyces aranearum Cylindrical to clavate, 0.8–10 × 0.1–0.2, brown Obovoid or ellipsoid 6–12 × 4–8 Narrowly obclavate, 8–14 × 1.5–3 Mains (1950)
Akanthomyces bashanensis Synnemata not observed Mononematous, 12.1–20.5 × 1.5–2.1. cylindrical, somewhat inflated base, 11.8–12.9 × 1.3–1.6 fusiform to ellipsoidal, 1.7–2.6 × 1.6–1.8 This study
Akanthomyces beibeiensis Synnemata not observed Mononematous, 14.2–19.4 × 1.0–2.1 cylindrical, somewhat inflated base, 7.0–9.2 × 2.1–2.5 fusiform to ellipsoidal, 2.0–3.3 × 2.0–2.6 This study
Akanthomyces ryukyuensis Synnemata not observed/ Pyriformia, 570–630 × 170–250 Conidiophores not observed Phialides not observed/5 wide, cap 3 wide Conidia not observed/ 1 × 1–4 Kobayasi and Shimizu (1982)
Akanthomyces tiankengensis Synnemata not observed Erect, usually arising from the aerial hyphae 13.9–17.1 × 1.1–1.6 with a cylindrical basal portion Fusiform, 2.3–3.0 × 1.5–2.3 Chen et al. (2022a)

Akanthomyces beibeiensis W.H. Chen, Y.F. Han & J.D. Liang, sp. nov.

MycoBank No: 847340
Fig. 4

Type

China, Chongqing, Beibei District, Jinyun Mountain (29°50'22.14959"N, 106°23'18.0744"E). On a dead spider (Araneae), 1 May 2021, Wanhao Chen, GZAC CQ0592 (holotype), ex-type living cultures, CQ05921.

Description

Spider host completely covered by white mycelium. Colonies on PDA, attaining a diameter of 34–37 mm after 14 days at 25 °C, white, consisting of a basal felt, floccose hyphal overgrowth; reverse yellowish. Hyphae septate, hyaline, smooth-walled, 1.4–1.9 μm wide. Conidiophores mononematous, hyaline, smooth-walled, with single phialide or whorls of 2–6 phialides or verticillium-like from hyphae directly, 14.2–19.4 × 1.0–2.1 μm. Phialides consisting of a cylindrical, somewhat inflated base, 7.0–9.2 × 2.1–2.5 μm, tapering to a thin neck. Conidia hyaline, smooth-walled, fusiform to ellipsoidal, 2.0–3.3 × 2.0–2.6 μm, forming divergent and basipetal chains. Sexual state not observed.

Figure 4. 

Akanthomyces beibeiensis A infected spider (Araneae) B, C PDA culture plate showing top (B) and reverse (C) sides of the colony D–J phialides and conidia. Scale bars: 10 mm (B, C); 10 μm (D–J).

Etymology

Referring to its location in Beibei District.

Additional strain examined

China, Chongqing, Beibei District, Jinyun Mountain (29°50'22.14959"N, 106°23'18.0744"E). On a dead spider (Araneae), 1 May 2021, Wanhao Chen, CQ05692.

Remarks

Akanthomyces beibeiensis was easily identified as Akanthomyces according to the blast result in NCBI and the phylogenetic analysis of combined datasets (ITS, LSU, RPB2, TEF) (Fig. 1) and it has a close relationship with another new species, A. bashanensis. A. beibeiensis is easily distinguished from A. bashanensis by its shorter phialide and larger conidia. The pairwise dissimilarities of ITS, LSU, RPB2 and TEF sequences show 4 bp differences within 569 bp (0.7%), 8 bp differences within 881 bp (0.9%), 20 bp differences within 1070 bp (1.86%) and 2 bp differences within 973 bp (0.2%) between A. beibeiensis and A. tiankengensis, respectively. Furthermore, A. aranearum and A. ryukyuensis were both absent from the available sequences in NCBI and had spider hosts. Comparing with the typical characteristics (Table 2), A. beibeiensis was easily distinguished from A. aranearum by its cylindrical phialide, smaller fusiform to ellipsoidal conidia and absence of synnemata, and distinguished from A. ryukyuensis by absence of teleomorphs. Thus, the morphological characteristics and molecular phylogenetic results support A. beibeiensis as a new species.

Akanthomyces tiankengensis W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, Microbiology Spectrum 10(5): e01975-22

Fig. 5

Description

Spider host completely covered by white mycelium. Colonies on PDA, attaining a diameter of 27–28 mm after 14 days at 25 °C, white, consisting of a basal felt, floccose hyphal overgrowth; reverse yellowish. Hyphae septate, hyaline, smooth-walled, 2.4–2.6 μm wide. Conidiophores mononematous, hyaline, smooth-walled, with single phialide or whorls of 2 phialides. Phialides consisting of a cylindrical, somewhat inflated base, 16.2–25.3 × 2.1–2.9 μm, tapering to a thin neck. Conidia hyaline, smooth-walled, subglobose to ellipsoidal, 2.4–3.8 × 2.1–3.0 μm, forming divergent and basipetal chains. Sexual state not observed.

Figure 5. 

Akanthomyces tiankengensis A infected spider (Araneae) B, C PDA culture plate showing reverse (B) and top (C) sides of the colony D–J phialides and conidia. Scale bars: 10 mm (B, C); 10 μm (D–J).

Strains and specimen examined

China, Chongqing, Beibei District, Jinyun Mountain (29°50'22.14959"N, 106°23'18.0744"E). On a dead spider (Araneae), 1 May 2021, Wanhao Chen, GZAC CQ0581, living cultures, CQ05811 and CQ05812; GZAC CQ0517, living cultures, CQ05171 and CQ05172; GZAC CQ0501, CQ0509, CQ0510, CQ0512, CQ0516, CQ0558, CQ0582, CQ0586, CQ0598, CQ0590.

Remarks

Strains CQ05811 and CQ05812 were identified as belonging to Akanthomyces, based on the phylogenetic analyses (Fig. 1) and clustered with A. tiankengensis in a subclade. The characteristics of CQ05811 and CQ05812 were very closely linked with A. tiankengensis, which had fusiform conidia (2.3–3.0 × 1.5–2.3 μm) and shorter phialide (13.9–17.1 × 1.1–1.6 μm). Furthermore, the pairwise dissimilarities of ITS sequences show 3 bp difference within 569 bp between CQ05811 and A. tiankengensis (0.52%) and no difference within 569 bp between CQ05171, CQ05172 and A. tiankengensis. Thus, molecular phylogenetic results and morphologically based conclusions supported the idea that strains CQ05811, CQ05812, CQ05171 and CQ05172 were A. tiankengensis.

Discussion

Akanthomyces species are widely distributed and commonly isolated from soil, insects and spiders (Chen et al. 2018, 2019c; Shrestha et al. 2019). Amongst the 33 species, A. aranearum (Petch) Mains, A. araneicola W.H. Chen, et al., A. araneogenus Z.Q. Liang, W.H. Chen & Y.F. Han, A. araneosus W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, A. coccidioperitheciatus (Kobayasi & Shimizu) Spatafora, Kepler & B. Shrestha, A. kanyawimiae Mongkols., Noisrip., Thanakitp., Spatafora & Luangsa-ard, A. neoaraneogenus (W.H. Chen et al.) W.H. Chen et al., A. lecanii (Zimm.) Spatafora, Kepler & B. Shrestha, A. ryukyuensis (Kobayasi & Shimizu) Mongkols., Noisrip., Thanakitp., Spatafora & Luangsa-ard, A. sulphureus Mongkols., Noisrip., Thanakitp., Spatafora & Luangsa-ard, A. thailandicus Mongkols., Spatafora & Luangsa-ard, A. tiankengensis W.H. Chen, et al. and A. waltergamsii Mongkols., Noisrip., Thanakitp., Spatafora & Luangsa-ard have been reported to be spider-associated fungi.

In the present study, the new strains differed from other spider-pathogenic species and had a close relationship with Akanthomyces tiankengensis, based on the phylogenetic analysis. Two new species were established by combining phylogenetic analysis and morphological characteristics. Interestingly, A. tiankengensis was located at Monkey-Ear Tiankeng and found in November, indicating that it had adapted to the cold environment. Whether these new species can adapt to their environment and have special metabolic processes is worthy of further research.

The hosts of Akanthomyces species cover Hemiptera, Coleoptera, Lepidoptera, Orthoptera and Araneae (Hodge 2003; Mongkolsamrit et al. 2018; Chen et al. 2020b, 2022c). Chen et al. (2022b) noted that a host jump may be common in Simplicillium species, the spider-associated species may have originated from insects and then jumped to a spider host. An abundant diversity in insects and spiders has been discovered at Jinyun Mountain (Huang and Zhang 1991; Li et al. 2009a, 2009b; Wang et al. 2012; Huang et al. 2021; Yan et al. 2021). Whether the new species originally came from an insect host or other substrates and then jumped to a spider host, is also worthy of further research.

Mains (1950) and Vincent et al. (1988) surmised that cylindrical synnemata covered by a hymenium-like layer of phialides producing one-celled catenulate conidia were the typical characteristics of Akanthomyces. However, Chen et al. (2020b, 2020c) reported two new Akanthomyces species with mononematous conidiophores. In the present study, the two new species had mononematous conidiophores. Akanthomyces species with mononematous conidiophores are often present on the surface of moss or in open places, from which their conidia can easily be spread by airflow diffusion or other methods. Those Akanthomyces species with synnematous conidiophores often appear in the shrubbery of the original forest, litter layer or shallow soil (Hywel-Jones 1996), where air flow under the forest canopy is slow and humidity is high and where dispersal of conidia through airflow diffusion is difficult. Therefore, the presence of synnematous conidiophores may be the result of convergent evolution, which could help them to fit in their niche (Abbott 2002). Thus, the Akanthomyces species may change their type of conidiophores to increase their adaptability to different environmental conditions.

The taxonomic delimitation of Akanthomyces was originally based on morphological characteristics. Kepler et al. (2017) proposed the rejection of Torrubiella Boud. and Lecanicillium W. Gams & Zare in favour of Akanthomyces and transferred Torrubiella and Lecanicillium species into Akanthomyces, which has resulted in a combined analysis of morphological characteristics and phylogenetic analysis for the taxonomy of Akanthomyces. In this research, a PHI test and base difference rate were added, which could solve the taxonomic delimitation of cryptic species. Amongst the four loci (ITS, LSU, RPB2 and TEF), the locus TEF could not be used to distinguish A. bashanensis, A. beibeiensis and A. tiankengensis. However, any two of the three loci could easily be used to distinguish these three species. Thus, we recommend that at least two loci should be provided for the cryptic Akanthomyces species and analysis of the cryptic species with its related species should be done using the multiple methods. Furthermore, the genomics data, phylogenetic networks and haplotype analysis should be applied to cryptic species and the taxonomy of Akanthomyces made it closer to the natural taxonomy system.

Currently, the diversity of entomopathogenic fungi in some Natural Reserves and Forest Parks in different regions of China has shown that the abundant diversity of entomopathogenic fungi is present in the study areas, and there is a high species diversity in specific areas (Chen et al. 2019a, 2020a; Fan et al. 2020; Zhao et al. 2020, 2021; Zhang et al. 2021). Jiyun Mountain is located in Chongqing City and has varied altitudes, abundant plant and animal resources, which have nurtured abundant fungal resources (Zhou et al. 2012a, b). In this research, abundant Akanthomyces specimens were found at Jinyun Mountain and further attention needs to be paid to the diversity of other entomopathogenic fungi in Chongqing, China.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was funded by National Natural Science Foundation of China (31860002, 81960692), “Hundred” Talent Projects of Guizhou Province (Qian Ke He [2020] 6005), Science and Technology Foundation of Guizhou Province (Qiankehejichu [2020]1Y060), Program of Innovative Scientific and technological Talent Team of Guizhou Province (2020-5010) and the Construction Program of Guizhou Engineering Research Center (Qian Fa Gai Gao Ji 2020-896).

Author contributions

W.H. Chen: Writing-original draft, Data curation, Formal analysis, Funding acquisition, Resources; Jiandong Liang: Writing-review and editing, Funding acquisition, Resources; Xiuxiu Ren: Writing-review and editing, Resources; Jiehong Zhao: Writing-review and editing, Funding acquisition; Yanfeng Han: Writing-review and editing, Funding acquisition.

Author ORCIDs

Wan-Hao Chen https://orcid.org/0000-0001-7240-6841

Jian-Dong Liang https://orcid.org/0000-0002-3939-3900

Yan-Feng Han https://orcid.org/0000-0002-8646-3975

Data availability

All of the data that support the findings of this study are available in the main text.

References

  • Aini AN, Mongkolsamrit S, Wijanarka W, Thanakitpipattana D, Luangsa-ard JJ, Budiharjo A (2020) Diversity of Akanthomyces on moths (Lepidoptera) in Thailand. MycoKeys 71: 1–22. https://doi.org/10.3897/mycokeys.71.55126
  • 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
  • Chaiwan N, Jeewon R, Pem D, Jayawardena RS, Nazurally N, Mapook A, Promputtha I, Hyde KD (2022) Fungal species from Rhododendron sp.: Discosia rhododendricola sp. nov., Neopestalotiopsis rhododendricola sp. nov. and Diaporthe nobilis as a new host record. Journal of Fungi (Basel, Switzerland) 8(9): 907. https://doi.org/10.3390/jof8090907
  • Chen ZH, Wang YB, Dai YD, Chen K, Xu L, He QC (2019a) Species diversity and seasonal fluctuation of entomogenous fungi of Ascomycota in Taibaoshan Forest Park in western Yunnan. Shengwu Duoyangxing 27(9): 993–1001. https://doi.org/10.17520/biods.2019135
  • Chen WH, Liu C, Han YF, Liang JD, Tian WY, Liang ZQ (2019b) Three novel insect-associated species of Simplicillium (Cordycipitaceae, Hypocreales) from Southwest China. MycoKeys 58: 83–102. https://doi.org/10.3897/mycokeys.58.37176
  • Chen W, Liang J, Ren X, Zhao J, Han Y, Liang Z (2021) Cryptic diversity of isaria-like species in Guizhou, China. Life (Chicago, Ill. ) 11(10): 1093. https://doi.org/10.3390/life11101093
  • Chen WH, Liang JD, Ren XX, Zhao JH, Han YF, Liang ZQ (2022a) Species diversity of Cordyceps-like fungi in the Tiankeng karst region of China. Microbiology Spectrum 10(5): e01975-22. https://doi.org/10.1128/spectrum.01975-22
  • Chen W, Liang J, Ren X, Zhao J, Han Y, Liang Z (2022b) Multigene phylogeny, phylogenetic network, and morphological characterizations reveal four new arthropod-associated Simplicillium species and their evolutional relationship. Frontiers in Microbiology 13: 950773. https://doi.org/10.3389/fmicb.2022.950773
  • Fan Q, Wang YB, Tang DX, Wang Y, Xu ZH, Yu H (2020) Species diversity of Cordyceps sensu lato in Kunming Wild Duck Lake Forest Park. Acta Edulis Fungi 27(2): 101–108.
  • Hodge KT (2003) Clavicipitaceous anamorphs. In: White Jr JF, Bacon CW, Hywel-Jones NL, Spatafora JW (Eds) Clavicipitalean Fungi. CRC Press, Boca Raton, 82–129. https://doi.org/10.1201/9780203912706
  • Huang TL, Zhang J (1991) Key to the family Calypidae (Coleoptera) in Jinyun Mountain. Sichuan Journal of Zoology 10(1): 24–27.
  • Huang Y, Bian DJ, Tong YF, Zhang ZS, Li SQ (2021) Two new species of the genus Trilacuna (Araneae: Oonopidae) from Jinyun Mountain of Chongqing, China. European Journal of Taxonomy 748: 1–14. https://doi.org/10.5852/ejt.2021.748.1337
  • Jeewon R, Hyde KD (2016) Establishing species boundaries and new taxa among fungi: Recommendations to resolve taxonomic ambiguities. Mycosphere : Journal of Fungal Biology 7(11): 1669–1677. https://doi.org/10.5943/mycosphere/7/11/4
  • Kalyaanamoorthy S, Minh BQ, Wong TK, Von Haeseler A, Jermiin LS (2017) ModelFinder: Fast model selection for accurate phylogenetic estimates. Nature Methods 14(6): 587–589. https://doi.org/10.1038/nmeth.4285
  • Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics 20(4): 1160–1166. https://doi.org/10.1093/bib/bbx108
  • Kepler RM, Luangsa-ard JJ, Hywel-Jones NL, Quandt CA, Sung GH, Rehner SA, Aime MC, Henkel TW, Samjuan T, Zare R, Chen MJ, Li ZZ, 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
  • Kinoshita H, Wongsuntornpoj S, Ihara F, Nihira T (2017) Anti‐Rhodotorula activity of mycophenolic acid enhanced in the presence of polyene antibiotic nystatin. Letters in Applied Microbiology 64(2): 144–149. https://doi.org/10.1111/lam.12692
  • Kobayasi Y, Shimizu D (1982) Monograph of the genus Torrubiella. Bulletin of the National Science Museum, Tokyo, Series B 8: 43–78.
  • Koval EZ (1977) Species novae fungorum entomophilorum hyphomycetalum. Novosti Sis tematihi vysskhikh i nizshikh Rastenii 1976: 203–208.
  • Kuephadungphan W, Phongpaichit S, Luangsa-ard JJ, Rukachaisirikul V (2014) 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
  • Lee SY, Nakajima I, Ihara F, Kinoshita H, Nihira T (2005) Cultivation of entomopathogenic fungi for the search of antibacterial compounds. Mycopathologia 160(4): 321–325. https://doi.org/10.1007/s11046-005-0179-y
  • Li X, Liu YA, Li C, Diao S (2009a) Effect of forest succession on arthropod diversity in Jinyun Mountain, Chongqing. Dongbei Linye Daxue Xuebao 37(10): 35–38.
  • Li Z, Chen L, Mu HL (2009b) A preliminary analysis of longicorn fauna in Jinyun Mountain Nature Reserve in Chongqing. Journal of Southwest China Normal University 34(5): 102–106. https://doi.org/10.13718/j.cnki.xsxb.2009.05.036 [Natural Science Edition]
  • Liang JD, Han YF, Zhang JW, Du W, Liang ZQ, Li ZZ (2011) Optimal culture conditions for keratinase production by a novel thermophilic Myceliophthora thermophila strain GZUIFR-H49-1. Journal of Applied Microbiology 110(4): 871–880. https://doi.org/10.1111/j.1365-2672.2011.04949.x
  • Madla S, Methacanon P, Prasitsil M, Kirtikara K (2005) Characterization of biocompatible fungi-derived polymers that induce IL-8 production. Carbohydrate Polymers 59(3): 275–280. https://doi.org/10.1016/j.carbpol.2004.07.002
  • Mongkolsamrit S, Noisripoom W, Thanakitpipattana D, Wuthikun T, Spatafora JW, Luangsaard JJ (2018) Disentangling cryptic species with Isaria-like morphs in Cordycipitaceae. Mycologia 110(1): 230–257. https://doi.org/10.1080/00275514.2018.1446651
  • Putri SP, Ishido KI, Kinoshita H, Kitani S, Ihara F, Sakihama Y, Igarashi Y, Nihira T (2014) Production of antioomycete compounds active against the phytopathogens Phytophthora sojae and Aphanomyces cochlioides by clavicipitoid entomopathogenic fungi. Journal of Bioscience and Bioengineering 117(5): 557–562. https://doi.org/10.1016/j.jbiosc.2013.10.014
  • Quaedvlieg W, Binder M, Groenewald JZ, Summerell BA, Carnegie AJ, Burgess TI, Crous W (2014) Introducing the consolidated species concept to resolve species in the Teratosphaeriaceae. Persoonia 33(1): 1–40. https://doi.org/10.3767/003158514X681981
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542. https://doi.org/10.1093/sysbio/sys029
  • 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
  • Sung GH, Hywel-Jones NL, Sung JM, Luangsa-ard JJ, Shrestha B, Spatafora JW (2007) Phylogenetic classification of Cordyceps and the clavicipitaceous fungi. Studies in Mycology 57: 1–64. https://doi.org/10.3114/sim.2007.57.01
  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30(12): 2725–2729. https://doi.org/10.1093/molbev/mst197
  • Trifinopoulos J, Nguyen LT, von Haeseler A, Minh BQ (2016) W-IQ-TREE: A fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Research 44(W1): W232–W235. https://doi.org/10.1093/nar/gkw256
  • Vaidya G, Lohman DJ, Meier R (2011) SequenceMatrix: Concatenation software for the fast assembly of multi‐gene datasets with character set and codon information. Cladistics 27(2): 171–180. https://doi.org/10.1111/j.1096-0031.2010.00329.x
  • Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172(8): 4238–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990
  • Wang YH, Wang WJ, Wang K, Dong CH, Hao JR, Kirk PM, Yao YJ (2023) Akanthomyces zaquensis (Cordycipitaceae, Hypocreales), a new species isolated from both the stroma and the sclerotium of Ophiocordyceps sinensis in Qinghai, China. Phytotaxa 579(3): 198–208. https://doi.org/10.11646/phytotaxa.579.3.5
  • White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR protocols: a guide to methods and applications. Academic Press, New York, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
  • Yan ZT, Lu MJ, Li TJ, Fu Q, Fu WB, Chen B (2021) Investigation and fauna analysis of butterflies in Jinyun Mounta in National Nature Reserve in Chongqing. Journal of Ecology and Rural Environment 37(2): 194–200.
  • Zhang D, Gao F, Jakovlić I, Zou H, Zhang J, Li WX, Wang GT (2020) PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular Ecology Resources 20(1): 348–355. https://doi.org/10.1111/1755-0998.13096
  • Zhao ZY, Wang YB, Wang ZQ, Tang DX, Geng YP, Yu H (2020) Species diversity of Cordyceps sensu lato in Weishan, Yunnan. Redai Yaredai Zhiwu Xuebao 28(5): 455–462.
  • Zhou GL, Deng HP, Zhang JH (2012a) The macrofungi resources diversity of Jinyunshan Natural Reserve. Guizhou Agricultural Sciences 40(12): 126–130.
  • Zou X, Liu AY, Liang ZQ, Han YF, Yang M (2010) Hirsutella liboensis, a new entomopathogenic species affecting Cossidae (Lepidoptera) in China. Mycotaxon 111(1): 39–44. https://doi.org/10.5248/111.39
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