Lecanicilliumcauligalbarum sp. nov. (Cordycipitaceae, Hypocreales), a novel fungus isolated from a stemborer in the Yao Ren National Forest Mountain Park, Guizhou

Abstract A new species of entomopathogenic fungi, Lecanicilliumcauligalbarum, was discovered from a survey of invertebrate-associated fungi in the Yao Ren National Forest Mountain Park in China. The synnemata of this species emerged from the corpse of a stemborer (Lepidoptera), which was hidden amongst pieces of wood on the forest floor. It differs from morphologically similar Lecanicillium species mainly in its short conidiogenous cells and ellipsoid to ovoid and aseptate conidia. Phylogenetic analysis of a combined data set comprising ITS, SSU, LSU, TEF, RPB1 and RPB2 sequence data supported the inclusion of L.cauligalbarum in the Lecanicillium genus and its recognition as a distinct species.


Introduction
The entomopathogenic fungal genus Lecanicillium W. Gams & Zare belongs to Ophiocordycipitaceae. It is typified by Lecanicillium lecanii with Torrubiella confragosa as the sexual morph Gams 2001, Wijayawardene et al. 2017). Lecanicillium lecanii was first named as Cephalosporium lecanii Zimm. by Zimmermann in 1898. Viegas incorporated the species in Verticillium Nees in 1939 . The genus Verticillium has a wide host range, including arthropods, nematodes, plants and fungi (Goettel et al. 2008). Zare and Gams (2001) recircumscribed the genus following analyses of morphological data and sequence data for the internal transcribed spacer (ITS) rDNA region (which comprises the ITS1 spacer, 5.8S coding region and ITS2 spacer). All insect pathogens formerly included in Verticillium were reclassified in a newly established genus, Lecanicillium. In more recent studies, a multilocus nuclear DNA dataset combining sequence data for the nuclear small subunit rDNA (SSU), nuclear large subunit rDNA (LSU), translation elongation factor 1α (TEF), DNAdependent RNA polymerase II largest subunit (RPB1) and DNA-dependent RNA polymerase II second largest subunit (RPB2) genes suggests that the genus Lecanicillium is paraphyletic (Sung et al. 2007). Phylogenetic analysis of ITS sequence data also supports this conclusion (Sukarno et al. 2009). Kepler et al. (2017) revisited the taxonomic affinities of the Cordycipitaceae (Hypocreales) and proposed that Lecanicillium should be rejected because L. lecanii is included within the Akanthomyces clade and the name Akanthomyces Lebert has nomenclatural priority over Lecanicillium (Kepler et al. 2017). However, Kepler et al. (2017) transferred to Akanthomyces only several species for which sufficient information was available. The phylogenetic affinities of the majority of species in the original circumscription of Lecanicillium remain uncertain. Given that there remain unresolved phylogenetic and taxonomic matters concerning Lecanicillium, Huang et al. (2018) and Crous et al. (2018) chose to describe new taxa in Lecanicillium to avoid creating further confusion in the taxonomy (Crous et al. 2018;Huang et al. 2018).
Presently, 29 Lecanicillium species have been formally described and are listed in the Index Fungorum (http://www.indexfungorum.org). Zare and Gams (2001) recognised 14 Lecanicillium species based primarily on morphology and ITS sequence data . Subsequently, an additional five new Lecanicillium species, based on ITS sequence data, were described (Kope and Leal 2006, Sukarno et al. 2009. In order to add more sequence information with ITS, Zare and Gams (2008) reassessed the genus Verticillium and transferred four species to Lecanicillium based on ITS and SSU sequence data (Zare and Gams 2008). Except for the SSU and ITS gene, more and more researchers have labelled the Lecanicillium genus by TEF gene. Based on this, two new Lecanicillium species were confirmed based on combined with ITS and TEF sequence data (Crous et al. 2018). With combined multigene identification of species gradually becoming the convention, two new Lecanicillium species were identified based on multilocus (TEF, RPB1, RPB2, LSU and SSU) sequence data (Park et al. 2016. Lecanicillium sabanense was identified based on phylogenetic analysis of combined multilocus and ITS sequences (Chiriví-Salomón et al. 2015). Lecanicillium subprimulinum was identified based on combined analysis of LSU, SSU, TEF and ITS sequence data (Huang et al. 2018).
We carried out a survey of invertebrate-associated fungi in the Yao Ren National Forest Mountain Park near Sandu county in Guizhou province, China. A parasitic fungus was found on a stemborer (Lepidoptera) hiding amongst pieces of wood. Attempting to identify the fungus, we determined it to be a member of Lecanicillium but its morphological traits and gene sequences did not correspond with those of any known Lecanicillium species. On the basis of its morphology and molecular phylogenetic analysis of multilocus nuclear genes (TEF, RPB1, RPB2, LSU and SSU) and ITS sequence data, this fungus was suggested to be an unnamed species of Lecanicillium and is here described and named Lecanicillium cauligalbarum sp. nov.

Specimen collection and fungus isolation
The specimen was collected from Yao Ren National Forest Mountain Park, Sandu county, Guizhou, China (107°53', 107°58'E; 24°54', 25°59'N, approximately 560-1365 m above sea level), in September 2015 by Yeming Zhou and Xiao Zou. The synnemata of this species emerged from a dead stemborer (Lepidoptera) hidden amongst pieces of wood on the forest floor. The specimen GZUIFR-2015ZHJ and two isolated strains of the fungal asexual stage, GZUIFRZHJ01 and GZUIFRZHJ02, were deposited at the Institute of Fungal Resources of Guizhou University (GZUIFR). The fungal strains were isolated on potato dextrose agar (PDA) medium; one strain was isolated from part of the body and the second strain was isolated from the synnemata.

Strain culture and identification
The isolated strains were inoculated on PDA at 25 °C for 14 d under 12-h light/12-h dark conditions. The fresh hyphae were observed with an optical microscope (OM, BK5000, OPTEC, USA) following pretreatment with lactophenol cotton blue solution or normal saline.

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted using a previously described method (Chiriví-Salomón et al. 2015, Zou et al. 2016. The primers used for PCR amplification of the ITS region, SSU, LSU, TEF, RPB1 and RPB2 are listed in Table 1. The PCR reaction conditions employed for each genetic region followed those used in the references listed in Table 1. To conduct phylogenetic analysis of the sequences obtained, sequences for selected taxa based on recent phylogenetic studies of Lecanicillium , Huang et al. 2018 and Cordycipitaceae (Sung et al. 2007, Kepler et al. 2017, Mongkolsamrit et al. 2018 were downloaded from the National Center for Biotechnology Infor-mation GenBank database (https://www.ncbi.nlm.nih.gov/genbank/). A total of 79 accessions of Cordycipitaceae were selected for this study. The sequences used in the study are listed in Table 2.

Sequence alignment and phylogenetic analyses
The DNA sequences used in this study were edited using the LASERGENE software (version 6.0; DNASTAR, Madison, WI, USA). Multiple sequence alignments for TEF, RPB1 and RPB2 were performed in MAFFT (Katoh and Standley 2013) with the default settings. Multiple sequence alignments for ITS, LSU and SSU were conducted using MUSCLE algorithm (Edgar 2004) from MEGA 6 (Tamura et al. 2013). The sequences were edited manually. A multiple alignment of the combined partial ITS+SSU+LSU+TEF+RPB1+RPB2 sequences were assembled with MEGA 6 (Tamura et al. 2013) and SEQUENCEMATRIX 1.7.8 (Vaidya et al. 2011). The command 'hompart' in PAUP* 4.0b10 was used for assessment of concordance amongst the genes and the ITS region (Swofford 2001). Bayesian inference (BI) was performed using MRBAYES 3.2 (Ronquist et al. 2012) and maximum likelihood (ML) analysis was performed using RAxML (Alexandros 2014) to analyse the combined data which were divided into twelve separate partitions (Kepler et al. 2017;Mongkolsamrit et al. 2018). Two maximum likelihood (ML) analysis and Bayesian inference (BI) analysis were performed. The first analysis was performed as reported by Huang et al. (2018), using the Simplicillium lanosoniveum as the outgroup. The second analysis was performed with Akanthomyces, Samsoniella, Blackwellomyces, Hevansia, Simplicillium, all the lecanicillium and use of Beauveria as outgroup (Mongkolsamrit et al. 2018). Nucleotide substitution models were determined by MrModeltest 2.3 (Nylander 2004). For BI, 10 000 000 generations were performed with one tree selected every 500th generation and the GTR+I+G evolutionary model was used. For ML, the model GTRGAMMA was used and a bootstrap analysis with 500 replicates was performed to assess statistical support for the tree topology. Phylogenetic trees were viewed with TREEGRAPH.

Results
Sequencing and phylogenetic analysis The first tree formed with almost all the Lecanicillium species (only Lecanicillium evansii could not be found in the NCBI) and one Simplicillium species (Simplicillium lanosoniveum). The phylogeny was resolved into 4 clades obviously. Lecanicillium cauligalbarum formed an independent branch in a polytomy together with a clade containing L. flavidum and L. fungicola and a major clade consisting of 27 accessions. The L. cauligalbarum lineage received maximum statistical support (BI posterior probabilities 1, ML boostrap 100%), which still remains unnamed (Figure 1). In the second tree, the four Lecanicillium clades were also be supported. Lecanicillium cauligalbarum formed an independent branch in a polytomy together with a clade containing Blackwellomyces cardinalis and Blackwellomyces pseudomilitaris (BI posterior probabilities 1, ML boostrap 85%) (Figure 2). Description. Colony on PDA 15 mm in diameter after 7 days, 33 mm in diameter after 14 days at 25 °C, colony circular, white, cottony, umbonate, with radiating surface texture from above, with clear radial crack and primrose-yellow from reverse. Mycelium 0.9-1.8 µm wide, hyaline, smooth, septated, branched. Conidiophores usually arising from aerial hyphae, sporulate abundant. Phialides gradually tapering towards the apex, solitary or 2-3 whorls, 9-14.4 × 1.4-1.8 µm. Conidia cylindric, aseptate, 3.6-6.3 × 0.9-1.8 µm. In culture, both phialides and conidia are of similar general shape and size to those found on the host stemborer.

Host. Stemborer (Lepidoptera) hidden amongst wooden sticks.
Habitat and distribution. Hidden amongst pieces of wood in humid forests of southwest China.
Remarks. With regard to phylogenetic relationships, L. cauligalbarum is closely related to the L. fungicola clade and L. fusisporum. The two strains (GZUIFRZHJ01 and GZUIFRZHJ02) formed a distinct lineage. All Lecanicillium species were included in the phylogenetic analysis except for L. evansii for which sequence data could not be located in public databases, although Zare and Gams (2001) published ITS sequences. The morphological features of L. evansii include brownish-cream to brown reverse, phialides solitary or up to 3-4 per node and two types of the conidia, slightly falcate with a pointed end macroconidia 4.5-7.5 × 0.8-1.2 µm and slightly curved microconidia 2.0-3.0 × 0.8-1.2 µm . L. evansii is distinct from L. cauligalbarum, which has conidia of 3.6-6.3 × 0.9-1.8 µm and 9-14.4 × 1.4-1.8 µm phialides.
In morphology L. cauligalbarumis is similar to L. aphanocladii, L. attenuatum and L. nodulosum with regard to the short conidiogenous cell (Table 3). However, L. cauligalbarum is distinguished by the pattern of spore production and the frequency of the wheel structure.

Discussion
The genera Lecanicillium and Simplicillium belong to the Cordycipitaceae (Sung et al. 2007). The two genera are indistinguishable in morphological traits (Sung et al. 2001;Zare and Gams 2001). However, Lecanicillium and Simplicillium are clearly separated in molecular phylogenetic analyses (Kouvelis et al. 2008;Maharachchikumbura et al. 2015;Nonaka et al. 2013). As an insect pathogen, Lecanicillium spp. has potential for development as effective biological control agents against a number of plant diseases, insect pests and plant-parasitic nematodes (Goettel et al. 2008). Fifteen commercial preparations based on Lecanicillium spp. have been developed or are in the process of being developed (Faria and Wraight 2007). Kepler et al. (2017) concluded that Lecanicillium should be incorporated into Akanthomyces and formally transferred a number of Lecanicillium species. However, the compatibility of Lecanicillium was not so good in this study. Species that have been transferred to Akanthomyces were all assembled in the L. lecanii clade in the present study. The remaining species included in the present analyses were divided into multiple clades similar to those retrieved by Kepler et al. (2017). Relationships amongst Lecanicillium species thus appear to be more complicated than expected. Thus, we also prefer to describe the new taxon as a Lecanicillium species, consistent with Huang et al. (2018), owing to the uncertainty in generic boundaries.
In a comparison of all Lecanicillium species included in the present study, we were unable to identify morphological synapomorphies that characterise the phylogenetic groups. However, the species that show a close phylogenetic relationship are more similar in morphology than those that are phylogenetically distant. For example, the L. lecanii clade, which has globose heads with a higher number of conidia, are distinguishable from those clades that usually have one conidium visible at the top of the phialide in the phylogenetic tree presented here. In our phylogeny study, the node connecting L. antillanum and L. tenuipes is the basal node for the major clade. So the relationships of all of the lineages involved may change with more data or a different dataset. Therefore, more species are needed to enrich the phylogenetic study of Lecanicillium spp.
We know that Lecanicillium has a different origin into the Cordycipitaceae. We consider that the ones 'L. lecanii clade' in pig.1 form a strong clade inside of Akanthomyces. Maybe all these should be moved to the Akanthomyces including Lecanicillium longisporum. In addition, the elimination of the genus may create more chaos considering the unsolved other clades.
Blackwellomyces Spatafora & Luangsa-ard is diagnosed by the unique characters of the ascospore, which have irregularly spaced septa and do not disarticulate into partspores at maturity as advised by Kepler et al. (2017). It includes Blackwellomyces cardinalis and Blackwellomyces pseudomilitaris. Asexual morphs have been described as similar to species in Clonostachys, Hirsutella, Isaria and Mariannaea (Hywel-Jones 1994; Sung and Spatafora 2004). Although the new species are close to the Blackwellomyces in the phylogenetic tree, we think they are clearly distinguished from Blackwellomyces by the morphology. We also treat the new species as Lecanicillium considering the small sample and the unknown teleomorph.Thus, based on the present molecular phylogeny, derived from nuclear and ribosomal DNA sequence data, together with morphological evidence, a distinct new Lecanicillium species, L. cauligalbarum, is proposed.