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Research Article
Multi-gene phylogenetic evidence indicates that Pleurodesmospora belongs in Cordycipitaceae (Hypocreales, Hypocreomycetidae) and Pleurodesmospora lepidopterorum sp. nov. on pupa from China
expand article infoWan-Hao Chen, Yan-Feng Han§, Jian-Dong Liang, Wei-Yi Tian, Zong-Qi Liang§
‡ Guizhou University of Traditional Chinese Medicine, Guiyang, China
§ Guizhou University, Guiyang, China
Open Access

Abstract

A new species, Pleurodesmospora lepidopterorum, isolated from a pupa, is introduced. Morphological comparisons and phylogenetic analyses based on multigene datasets (ITS+RPB1+RPB2+TEF) support the establishment of the new species. Pleurodesmospora lepidopterorum is distinguished from P. coccorum by its longer conidiogenous pegs located in the terminal or lateral conidiophores, and smaller subglobose or ellipsoidal conidia. A combined dataset of RPB1, RPB2, and TEF confirmed the taxonomic placement of Pleurodesmospora in Cordycipitaceae for the first time.

Keywords

Insect, morphological characteristic, new species, phylogenetic analysis, taxonomic placement

Introduction

The genus Pleurodesmospora was established for the type species P. coccorum (Petch) Samson, W. Gams & H.C. Evans (Samson et al. 1980). The typical characteristic of Pleurodesmospora is its erect or procumbent conidiophores, which bear numerous minute phialidic conidiogenous pegs in the terminal or mostly intercalary position, often in whorls below the septa. Conidiogenous pegs are short-cylindrical and give rise to short chains of conidia. Conidia are ellipsoid to dacryoid with a slightly truncate base (Samson et al. 1980).

Pleurodesmospora species have diverse ecological characteristics, and have been found on scale insects, whitefly, aphids, leaf-hoppers, spider and scavenger mites (Petch 1931; Samson and McCoy 1982; Samson et al. 1980). Li et al. (1991) reported Pleurodesmospora as a newly recorded genus in China and confirmed for the first time that P. coccorum has strong pathogenicity to black whitefly. According to Index Fungorum, the taxonomic status of Pleurodesmospora is incertae sedis.

During a survey of entomopathogenic fungi from Southwest China, a new insect-associated species was found. The morphological characteristics of the new species resembled Pleurodesmospora. In our phylogenetic analyses of combined RPB1, RPB2 and TEF sequences, Pleurodesmospora clustered in Cordycipitaceae (Hypocreales, Hypocreomycetidae) with strong statistical support and was closely related to Beauveria Vuill. and Akanthomyces Lebert. Thus, we propose that Pleurodesmospora belongs to family Cordycipitaceae and introduce Pleurodesmospora lepidopterorum sp. nov. as a new insect-associated species on the basis of morphological comparison and molecular phylogenetic analyses.

Materials and methods

Specimen collection and identification

An infected pupa of Lepidoptera specimen (DY1050) was collected from Duyun City (26°21'24.71"N, 107°22'48.22"E), Qiannan Buyi and Miao Autonomous Prefecture, Guizhou Province, on 1 October 2019. Isolation of strains was conducted as described by Chen et al. (2019). Fungal colonies emerging from 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). Specimens and the isolated strains were deposited in the Institute of Fungus Resources, Guizhou University (formally Herbarium of Guizhou Agricultural College; code, GZAC), Guiyang City, Guizhou, China.

Macroscopic and microscopic morphological characteristics of the fungi were examined and the growth rates were determined from potato dextrose agar (PDA) and oatmeal agar (OA) cultures incubated at 25 °C for 14 days. Hyphae and conidiogenous structures were mounted in lactophenol cotton blue or 20% lactate solution and observed with an optical microscope (OM, DM4 B, Leica, Germany).

DNA extraction, polymerase chain reaction amplification and nucleotide sequencing

DNA extraction was carried out by Fungal genomic DNA Extraction Kit (DP2033, BioTeke Corporation) in accordance with Liang et al. (2011). The extracted DNA was stored at −20 °C. The internal transcribed spacer (ITS) region, RNA polymerase II largest subunit 1 (RPB1), RNA polymerase II largest subunit 2 (RPB2) and translation elongation factor 1 alpha (TEF) were amplified by PCR as described by White et al. (1990), Castlebury et al. (2004) and van den Brink et al. (2004), respectively. PCR products were purified and sequenced at Sangon Biotech (Shanghai) Co. The resulting sequences were submitted to GenBank.

Sequence alignment and phylogenetic analyses

Lasergene software (version 6.0, DNASTAR) was applied for the assembling and editing of DNA sequence. The ITS, RPB1, RPB2 and TEF sequences were downloaded from GenBank, based on Mongkolsamrit et al. (2018, 2020) and others selected on the basis of BLAST algorithm-based searches in GenBank (Table 1). The multiple datasets of ITS, RPB1, RPB2 and TEF were aligned and edited by MAFFT v7.037b (Katoh and Standley 2013) and MEGA6 (Tamura et al. 2013). Assembling of the combined datasets (RPB1+RPB2+TEF and ITS+RPB1+RPB2+TEF) was performed by SequenceMatrix v.1.7.8 (Vaidya et al. 2011). The model was selected for Bayesian analysis by ModelFinder (Kalyaanamoorthy et al. 2017) in the software PhyloSuite (Zhang et al. 2020).

Table 1.

Taxa included in the phylogenetic analyses.

Species Strain No. GenBank accession No.
ITS RPB1 RPB2 TEF
Akanthomyces aculeatus HUA 186145 MF416465
HUA 772 KC519371 KC519366
Akanthomyces attenuates CBS 402.78 EF468888 EF468935 EF468782
Akanthomyces lecanii CBS 101247 DQ522407 DQ522466 DQ522359
Akanthomyces waltergamsii TBRC 7251 MF140781 MF140805 MF140833
TBRC 7252 MF140782 MF140806 MF140834
Ascopolyporus polychrous P.C. 546 DQ127236 DQ118745
Ascopolyporus villosus ARSEF 6355 AY886544 DQ127241 DQ118750
Beauveria bassiana ARSEF 1564 HQ880761 HQ880833 HQ880905 HQ880974
ARSEF 7518 HQ880762 HQ880834 HQ880906 HQ880975
Beauveria brongniartii ARSEF 617 HQ880854 HQ880926 HQ880991
Beauveria caledonica ARSEF 2567 HQ880889 HQ880961 EF469057
Blackwellomyces cardinalis OSC 93609 DQ522370 DQ522370 DQ522325
OSC 93610 JN049843 EF469088 EF469106 EF469059
Claviceps purpurea S.A. cp11 EF469087 EF469105 EF469058
Clonostachys rosea AFTOL ID.187 DQ862029
GJS 90227 AY489611
Conoideocrella luteorostrata NHJ 11343 EF468906 EF468801
NHJ 12516 EF468905 EF468946 EF468800
Cordyceps kyusyuensis EFCC 5886 EF468863 EF468754
Cordyceps militaris OSC 93623 JN049825 DQ522377 DQ522332
Cordyceps ninchukispora E.G.S.38.165 EF468900 EF468795
E.G.S.38.166 EF468901 EF468794
Cordyceps piperis CBS 116719 DQ127240 EU369083 DQ118749
Gibellula gamsii BCC 25798 MH152532 EU369056 EU369076 EU369018
BCC 27968 MH152529 MH152547 MH152560
Hevansia novoguineensis CBS 610.80 MH532831 MH521844 MH521885
NHJ 11923 EU369052 EU369072 EU369013
Hyperdermium pulvinatum P.C. 602 DQ127237 DQ118746
Lecanicillium antillanum CBS 350.85 MH861888 DQ522396 DQ522450 DQ522350
Lecanicillium psalliotae CBS 101270 EF469096 EF469112 EF469067
CBS 532.81 EF469095 EF469113 EF469066
Lecanicllium tenuipes CBS 309.85 DQ522387 DQ522439 DQ522341
Metarhizium anisopliae ARSEF 7487 DQ468355 DQ468370 DQ463996
CBS 130.71 MT078861 MT078918 MT078845
Metarhizium flavoviride CBS 125.65 MT078862 MT078919 MT078846
CBS 700.74 MT078863 MT078920 MT078847
Neotorrubiella chinghridicola BCC 39684 MK632071 MK632181 MK632148
BCC 80733 MK632072 MK632176 MK632149
Ophiocordyceps gracilis EFCC 8572 EF468859 EF468912 EF468751
Ophiocordyceps sinensis EFCC 7287 EF468874 EF468924 EF468767
Orbiocrella petchii NHJ 6209 EU369061 EU369081 EU369023
Pleurodesmospora coccorum CBS 458.73 MH860741
CBS 459.73 MH860742
CBS 460.73 MH860743
Pleurodesmospora lepidopterorum DY10501 MW826576 MW834315 MW834316 MW834317
DY10502 MW826577 MW834318 MW834319
Polycephalomyces formosus ARSEF 1424 DQ127245 KF049671 DQ118754
Polycephalomyces paracuboideus NBRC 101742 KF049647 KF049669 KF049685
Purpureocillium lilacinum ARSEF 2181 EF468896 EF468790
CBS 431.87 EF468897 EF468940 EF468791
Purpureocillium lilacinum CBS 284.36 MH855800 EF468898 EF468941 EF468792
Samsoniella aurantia TBRC 7271 MF140791 MF140846
TBRC 7272 MF140763 MF140817 MF140845
Simplicillium lanosoniveum CBS 101267 DQ522405 DQ522463 DQ522357
CBS 704.86 AJ292396 DQ522406 DQ522464 DQ522358
Yosiokobayasia kusanagiensis TNS–F18494 JN049890 JF416014

The datasets (RPB1+RPB2+TEF and ITS+RPB1+RPB2+TEF) were analyzed by Bayesian inference (BI) and maximum likelihood (ML) methods to determine the relationship among Pleurodesmospora and related genera in the order Hypocreales (analysis 1) and the relationship among Pleurodesmospora and related genera in the family Cordycipitaceae (analysis 2), respectively. 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 analyses, were discarded, while the remaining 16,001 trees were used for calculating posterior probabilities in the majority rule consensus tree. After the analysis was finished, each run was examined using the program Tracer v1.5 (Drummond and Rambaut 2007) to determine burn-in and confirm that both runs had converged. ML analyses were constructed with RAxMLGUI (Silvestro et al. 2012). The GTRGAMMA model was used for all partitions, in accordance with recommendations in the RAxML manual against the use of invariant sites.

Results

Phylogenetic analyses

Clonostachys rosea (Link) Schroers, Samuels, Seifert & W. Gams isolates (AFTOL ID.187 and GJS 90227) were used as the outgroup in analysis 1 (Fig. 1), and Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones & Samson isolates (CBS 284.36 and CBS 431.87) were used as the outgroup in analysis 2 (Fig. 2). The concatenated sequences of analysis 1 and 2 included 23 and 21 taxa, respectively, and consisted of 2,262 (RPB1, 561; RPB2, 821; and TEF, 880) and 2,711 (ITS, 597; RPB1, 508; RPB2, 852; and TEF, 754) characters with gaps, respectively.

Figure 1. 

Phylogenetic relationships among Pleurodesmospora and related genera in the order Hypocreales based on a multigene dataset (RPB1, RPB2, and TEF). Statistical support values (≥ 50%/0.5) are shown at the nodes for maximum likelihood bootstrap support/ Bayesian inference posterior probabilities.

Analysis 1: The final value of the highest scoring tree was –18,860.236896, which was obtained from the ML analysis of the dataset (RPB1+RPB2+TEF). The parameters of GTR model to analysis of the dataset were estimated base frequencies; A = 0.240138, C = 0.290732, G = 0.262224, T = 0.206905; substitution rates AC = 1.004710, AG = 3.103423, AT = 0.837508, CG = 0.886482, CT = 5.821155, GT = 1.000000; gamma distribution shape parameter α = 0.309925. The selected model for BI analysis were K2P+G4 (RPB2) and GTR+F+I+G4 (RPB1+TEF). In the order-level phylogenetic tree (Fig. 1), the maximum likelihood and Bayesian inference trees were generally congruent, and most branches were strongly supported. The new strains clustered with the genera Cordyceps, Akanthomyces, and Beauveria, and belonged to family Cordycipitaceae.

Analysis 2: The final value of the highest scoring tree was –19,321.404482, which was obtained from the ML analysis of the dataset (ITS+RPB1+RPB2+TEF). The parameters of GTR model to analysis of the dataset were estimated base frequencies; A = 0.238334, C = 0.298168, G = 0.261443, T = 0.202055; substitution rates AC = 0.963749, AG = 2.807654, AT = 0.822463, CG = 0.766574, CT = 5.738062, GT = 1.000000; gamma distribution shape parameter α = 0.339059. The selected model for BI analysis were HKY+F+G4 (ITS) and GTR+F+I+G4 (RPB1+RPB2+TEF). In the family-level phylogenetic tree (Fig. 2), the maximum likelihood and Bayesian inference trees were generally congruent, and most branches were strongly supported. The new strains formed an independent branch but clustered with Pleurodesmospora coccorum; therefore, these strains represent a new species described as P. lepidopterorum.

Figure 2. 

Phylogenetic relationships among Pleurodesmospora and related genera in the family Cordycipitaceae based on a multigene dataset (ITS, RPB1, RPB2 and TEF). Statistical support values (≥ 50%/0.5) are shown at the nodes for maximum likelihood bootstrap support/Bayesian inference posterior probabilities.

Taxonomy

Pleurodesmospora lepidopterorum W.H. Chen, Y.F. Han & Z.Q. Liang, sp. nov.

MycoBank No: 839148
Figure 3

Diagnosis

Differs from P. coccorum by having longer conidiogenous pegs located in the terminal or lateral conidiophores, and smaller subglobose or ellipsoidal conidia.

Type

China, Guizhou Province, Qiannan Buyi and Miao Autonomous Prefecture, Duyun City (26°21'24.71"N, 107°22'48.22"E), 1 October 2019, Wanhao Chen, holotype GZAC DY1050, ex-type culture GZAC DY10501. Sequences from isolated strain DY10501 have been deposited in GenBank with accession numbers: ITS = MW826576, RPB1 = MW834315, RPB2 = MW834316 and TEF = MW834317.

Description

Colonies on PDA, 3.9–4.1 cm diam. in 14 d at 25 °C, white, consisting of a basal felt and cottony, floccose hyphal overgrowth, reverse pale yellowish. Prostrate hyphae smooth, septate, hyaline, 1.3–1.9 μm diam. Erect or procumbent conidiophores usually arising from aerial hyphae, barely differentiated from vegetative hyphae, usually branched. Conidiogenous cells polyphialidic, terminal and intercalary, bearing numerous short-cylindrical, 1.8–3.5 μm long and 0.7–1.3 μm wide conidiogenous pegs, in whorls often below the septa. The terminal or lateral conidiogenous cells cylindrical, 5.9–12.0 × 1.8–2.2 μm. Conidia in chains, hyaline, smooth-walled, subglobose or ellipsoidal, one-celled, 2.3–3.6 × 1.7–3.3 μm. Chlamydospores and synnemata not observed. Size and shape of phialides and conidia similar in culture on PDA, OA agar and on natural substrate. Sexual state not observed.

Figure 3. 

Pleurodesmospora lepidopterorum A infected pupa (Lepidoptera) B, C top (B) and underside (C) of a colony cultured on PDA medium at 14 d D–J conidiogenous pegs and conidia K conidia in chains. Scale bars: 10 mm (B, C) 10 μm (D–K).

Host

Pupa, order Lepidoptera.

Distribution

Duyun City, Qiannan Buyi and Miao Autonomous Prefecture, Guizhou Province, China.

Etymology

Referring to its insect host, which belongs to order Lepidoptera.

Remarks

Pleurodesmospora lepidopterorum was readily identified as belonging to Pleurodesmospora in the family-level phylogenetic tree (Fig. 2). When compared with the typical characteristics of P. coccorum, P. lepidopterorum was easily distinguished by its longer conidiogenous pegs located in the terminal or lateral conidiophores, and smaller subglobose or ellipsoidal conidia.

Discussion

BLAST results of ITS, RPB1, RPB2, and TEF sequence data revealed that the strain DY10501 was similar to several taxa in GenBank: ITS, 98.62% similar to Lecanicillium sp. (isolate ICMP:20146); RPB1, 88.55% similar to Beauveria caledonica Bissett & Widden (isolate ARSEF 7117); RPB2, 86.53% similar to Cordyceps sp. (isolate A12116); TEF, 95.33% similar to Beauveria bassiana (Bals.-Criv.) Vuill. (isolate CHE-CNRCB 82). In the family-level phylogenetic tree, strains DY10501 and DY10502 formed an independent branch and clustered with P. coccorum in a subclade.

Samson et al. (1980) introduced the genus Pleurodesmospora with P. coccorum, but the taxonomic status of the genus was unclear. Unfortunately, P. coccorum lacked RPB1, RPB2, and TEF sequences in GenBank. Therefore, P. lepidopterorum was used for multigene analysis of Pleurodesmospora and related genera in the order Hypocreales. In the order-level phylogenetic tree, P. lepidopterorum clustered into Cordycipitaceae (Hypocreales, Hypocreomycetidae, Sordariomycetes). Thus, the combined dataset of RPB1, RPB2, and TEF confirmed the taxonomic placement of Pleurodesmospora in Cordycipitaceae for the first time.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 31860002), High-level Innovative Talents Training Object in Guizhou Province (No. Qiankehepingtairencai [2020]6005), Science and Technology Foundation of Guizhou Province (No. Qiankehejichu [2020]1Y060), Program of Innovative Scientific and Technological Talent Team of Guizhou Province(2020-5010) and Construction Program of Guizhou Engineering Research Center (Qian Fa Gai Gao Ji 2020-896) and National Survey of Traditional Chinese Medicine Resources (No. Caishe [2017]66, 216). We thank Mallory Eckstut, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

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Supplementary materials

Supplementary material 1 

Dataset for Figure 1

Wan-Hao Chen, Yan-Feng Han, Jian-Dong Liang, Wei-Yi Tian, Zong-Qi Liang

Data type: molecular data

Explanation note: A dataset of RPB1, RPB2 and TEF for Figure 1.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (91.02 kb)
Supplementary material 2 

Dataset for Figure 2

Wan-Hao Chen, Yan-Feng Han, Jian-Dong Liang, Wei-Yi Tian, Zong-Qi Liang

Data type: molecular data

Explanation note: A dataset of ITS, RPB1, RPB2 and TEF for Figure 2

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (119.21 kb)
Supplementary material 3 

Table S1. Taxa included in the phylogenetic analyses

Wan-Hao Chen, Yan-Feng Han, Jian-Dong Liang, Wei-Yi Tian, Zong-Qi Liang

Data type: molecular data

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (61.08 kb)