Taxonomic and phylogenetic characterizations reveal four new species of Simplicillium (Cordycipitaceae, Hypocreales) from Guizhou, China

Simplicillium species are commonly found from soil, seawater, rock surface, decayed wood, air and as symbiotic, endophytic, entomopathogenic and mycoparasitic fungi. Minority insect-associated species was reported. Simplicillium coccinellidae, S. hymenopterorum, S. neolepidopterorum and S. scarabaeoidea were introduced as the newly insect-associated species. The phylogenetic analyses of two combined datasets (LSU + RPB1 + TEF and SSU + ITS + LSU) revealed that S. coccinellidae and S. hymenopterorum were both nested in an independent clade. S. neolepidopterorum and S. scarabaeoidea have a close relationship with S. formicidae and S. lepidopterorum, respectively. S. neolepidopterorum can be easily distinguished from S. formicidae by ellipsoidal to cylindrical, solitary conidia which occasionally gather in short imbricate chains. S. scarabaeoidea could be easily distinguished from S. lepodopterorum by having longer phialides and larger conidia. Based on the morphological and phylogenetic conclusion, we determine the four newly generated isolates as new species of Simplicillium and a new combination is proposed in the genus Leptobacillium.

Notes: Based on the analysis of the combined dataset SSU + ITS + LSU, S. scarabaeoidea is phylogenetically close to S. lepodopterorum. However, the pairwise dissimilarities of RPB1 sequences show 31 bp difference within 760 bp between S. scarabaeoidea and S. lepodopterorum. When comparing with the typical characteristics of S. lepodopterorum (Table 1), S. scarabaeoidea could be easily distinguished from S. lepodopterorum by having longer phialides and larger conidia. Thus, molecular phylogenetic results and morphologically based conclusion were supported S. scarabaeoidea was a new species in the genus Simplicillium.  Notes: Okane et al. 3 transferred Simplicillium chinense and S. coffeanum to the genus Leptobacillium. In the present study, S. chinense, S. coffeanum and S. filiform were clustered into an independent clade (Fig. 2), and supported by Crous et al. 7 , Chen et al. 20 and Wei et al. 28 . Thus, L. filiform is proposed as a new combination.

Discussion
Sung et al. 40 refined the classification of Cordyceps and the Clavicipitaceae; the genus Simplicillium thus belongs to the Cordycipitaceae sensu stricto. The result of phylogenetic analysis of the combined dataset (SSU, LSU, RPB1, RPB2 and TEF) showed that Simplicillium species were all clustered in an independent group and as the most ancient lineage in the phylogenetic tree 41 . In this study, all Simplicillium species were also clustered into a clade at the end of the tree (Fig. 1) based on the analysis of the concentrated dataset (LSU, RPB1 and TEF). The four newly identified species, S. scarabaeoidea, S. hymenopterorum, S. neolepidopterorum and S. coccinellidae, were all clustered in a separate subclade. Liu & Cai 9 reported a new species based on the morphological comparison and phylogenetic analysis of ITS and LSU sequences, which was the earliest application for the identification of Simplicillium species. Kondo et al. 29 added the loci SSU in the analysis of Simplicillum species. Thus, three loci (ITS, LSU and SSU) were applied in the analysis of the relationship among Simplicillium species in this study.
The nutritional mode from plant to animals and fungi is the evolutionary characteristics of Hypocreales 42 . Plants associated fungi, which including living plants and plant residues were the common ancestor in the families Hypocreaceae and Clavicipitaceae 41 . The animal pathogenic fungi are likely inherited from the plant associated fungi by a series of interkingdom host jumps 42 . In the phylogenetic tree of analysis 2 (Fig. 2), S. chinense, S. filiforme and S. coffeanum were nested in a clade and at the end of the tree. The substrates of S. chinense, S. coffeanum and S. filiforme were decaying wood, branches of Coffea arabica and leaves of Citrullus lanatus 6,7,9 . All of them were belongs to plants associated fungi, and might reflect the initial state of Simplicillium species, which then underwent a host jump or transferred their nutritional preference. Simplicillium species have rich diversity www.nature.com/scientificreports/ in substrates and life modes, such as soil, seawater, air, and isolated as symbiotic, endophytic, entomopathogenic and mycoparasitic fungi. Simplicillium species associated with predatory insects or animals, like spiders, will likely soon be reported. Thus, the genus Simplicillium will completely fit with the nutritional model of Hypocreales fungi and could be used as a model to study the evolutionary relationship. Numerous new secondary metabolites were found from Simplicillium species, such as alkaloids 43 , diketopiperazine 44 and anthraquinones 45 , especially aogacillin A, B and Simpotentin, which have antibacterial and antifungal activities and shown great potential applications in medicine 46,47 . In addition, some Simplicillium species were isolated as symbiotic, entomopathogenic and mycoparasitic fungi, and could be used to biocontrol of insect pest, nematode and microbial diseases [48][49][50] . Thus, it is expected that useful novel compounds will be discovered from the newly-reported Simplicillium species described here and be a natural resource for the application in biocontrol, medicine and health.

Materials and methods
Specimen collection and identification. Four infected insect specimens (DY10139, DY10169, DY10175 and DY10179) were collected from Duyun City (26°21′24.71″ N, 107°22′48.22″ E), Guizhou Province, on 1 October, 2019. Isolation of strains was conducted as described by Chen et al. 20 . 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. 21 . Accordingly, strains were obtained. The 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 PDA 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. 22 . The extracted DNA was stored at − 20 °C. The amplification of internal transcribed spacer (ITS) region, small subunit ribosomal RNA (SSU), large subunit ribosomal RNA (LSU) gene, RNA polymerase II largest subunit 1 (RPB1) and translation elongation factor 1 alpha (TEF) were amplified by PCR as described by White et al. 23 , Rakotonirainy et al. 24 , Castlebury et al. 25 and van den Brink et al. 26 , respectively. PCR products were purified and sequenced at Sangon Biotech (Shanghai) Co. The generated sequences were submitted to GenBank.  29 and others selected on the basis of BLAST algorithm-based searches in GenBank ( Table 2). The Multiple datasets of ITS, LSU, SSU, RPB1 and TEF were aligned and edited by MAFFT v7.037b 30 and MEGA6 31 . Assembling of the combined datasets (LSU + RPB1 + TEF and SSU + ITS + LSU) were performed by SequenceMatrix v.1.7.8 32 . The partition homogeneity test was conducted in PAUP4.0b10 33 by using the command "hompart".

Sequence alignment and phylogenetic analyses.
The datasets (LSU + RPB1 + TEF and SSU + ITS + LSU) were analysis by Bayesian inference (BI) and maximum likelihood (ML) methods and aimed to analysis of the relationship among Simplicillium species and its related species in the family Cordycipitaceae (analysis 1) and the relationship among Simplicillium spp. (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 34 for the combined sequence datasets. The model for BI analysis was selected by ModelFinder 35 in the software PhyloSuite 36 . The Bayesian analysis resulted in 20,001 trees after 10,000,000 generations. The first 4000 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 37 to determine burn-in and confirm that both runs had converged. ML analyses were constructed with RAxMLGUI 38 . The GTRGAMMA model was used for all partitions, in accordance with recommendations in the RAxML manual against the use of invariant sites. The final alignment is available from TreeBASE under submission ID: 26290 (http:// www. treeb ase. org).