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 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.

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).

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.

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.