Multi-gene phylogenetic evidence indicates that Pleurodesmospora belongs in Cordycipitaceae (Hypocreales, Hypocreomycetidae) and Pleurodesmospora lepidopterorum sp. nov. on pupa from China.

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

Introduction

The genus was established for the type species (Petch) Samson, W. Gams & H.C. Evans (Samson et al. 1980). The typical characteristic of 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).

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 as a newly recorded genus in China and confirmed for the first time that has strong pathogenicity to black whitefly. According to Index Fungorum, the taxonomic status of 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 . In our phylogenetic analyses of combined RPB1, RPB2 and TEF sequences, clustered in (, ) with strong statistical support and was closely related to Vuill. and Lebert. Thus, we propose that belongs to family and introduce 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 specimen (DY1050) was collected from Duyun City (), 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 and related genera in the order (analysis 1) and the relationship among and related genera in the family (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

(Link) Schroers, Samuels, Seifert & W. Gams isolates (AFTOL ID.187 and GJS 90227) were used as the outgroup in analysis 1 (Fig. 1), and (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 and related genera in the order 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.

Figure 2.

Phylogenetic relationships among and related genera in the family 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.

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 , , and , and belonged to family .

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 ; therefore, these strains represent a new species described as .

Taxonomy

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

10F80ED8-145D-5185-B29C-D112B174E23E

839148

Figure 3

Figure 3.

A infected pupa () 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).

Diagnosis.

Differs from 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 (), 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.

Host.

Pupa, order .

Distribution.

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

Etymology.

Referring to its insect host, which belongs to order .

Remarks.

was readily identified as belonging to in the family-level phylogenetic tree (Fig. 2). When compared with the typical characteristics of , 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 sp. (isolate ICMP:20146); RPB1, 88.55% similar to Bissett & Widden (isolate ARSEF 7117); RPB2, 86.53% similar to sp. (isolate A12116); TEF, 95.33% similar to (Bals.-Criv.) Vuill. (isolate CHE-CNRCB 82). In the family-level phylogenetic tree, strains DY10501 and DY10502 formed an independent branch and clustered with in a subclade.

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