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

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.


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 min-ute 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.

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. (2018Mongkolsamrit et al. ( , 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 burnin 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.
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.