Two new species of Hirsutella (Ophiocordycipitaceae, Sordariomycetes) that are parasitic on lepidopteran insects from China

Abstract Hirsutella are globally distributed entomopathogenic fungi that offer important economic applications in biological control and biomedicine. Hirsutella was suppressed in favour of Ophiocordyceps affected by the ending of dual nomenclature for pleomorphic fungi in 2011. Currently, Hirsutella has been resurrected as a genus under Ophiocordycipitaceae. In this study, we introduce two new species of Hirsutella, based on morphological and phylogenetic analyses. Hirsutellaflava and H.kuankuoshuiensis are pathogenic on different species of larval Lepidoptera in China. Hirsutellaflava primarily differs from related species by its awl-shaped base; long and narrow neck, 24–40.8 × 2.2–2.5 μm; long and narrow cymbiform or fusoid conidia, 6.5–10 × 2.1–4.3 μm. Hirsutellakuankuoshuiensis has two types of phialides and distinctive 9.9–12.6 × 2.7–4.5 μm, clavate or botuliform conidia. The distinctions amongst the new species and phylogenetic relationships with other Hirsutella species are discussed.


Introduction
The entomopathogenic fungal genus Hirsutella Pat. was erected by Patouillard (1892) based on the type species H. entomophila. The genus was introduced to the family Ophiocordycipitaceae and its sexual morph was linked to Ophiocordyceps (Sung et al. 2007a; Simmons et al. 2015). In Hirsutella sensu stricto, conidiation is synnematous and phialides typically have a swollen base that tapers abruptly into a long neck producing either a single conidium or 2-3 conidia coated with mucus. The colour of the synnemata ranges from ash-grey or brown to dark brown. The size and shape of the hyaline conidia vary from citriform to oblong, subcylindric, globose, rhombic, or reniform (Luangsa-ard et al. 2017;Quandt et al. 2014). These taxa are important pathogens of agricultural pests and are used as popular traditional medicine and a nutritious food in many Asian countries (Evans 1974;Quandt et al. 2014;Hyde et al. 2019). Several common species of Hirsutella, such as H. thompsonii and H. rhossiliensis, are potentially important biological control agents for nematodes and mites (Jaffee 1992; Van der Geest 2010;Hyde et al. 2019). Further uses involve the development and application of several effective bioactive secondary metabolites (Mazet and Vey 1995;Lang et al. 2005;Qu et al. 2017).
Research on Hirsutella originated in the 1920s. Through the 1950s, Speare (1920), Petch (1924) and subsequent researchers reported 25 new species of the genus. However, many of these species were not described in detail and lacked adequate drawings, as well as holotypes. In addition, many specimens were damaged or lost during wartime (Zou et al. 2016a). In the 1970s and 1980s, Miner, Samson and Evans re-examined the status of Hirsutella and established the modern scientific definition for the genus (Minter and Brady 1980;Evans andSamson 1982, 1984). Since the beginning of the 21 st century, the taxonomy, molecular evolution and phylogeny of Hirsutella have been addressed by a small number of Chinese and international studies, with sporadic reports of new species (Seifert 2004;Xiang et al. 2006;Zou et al. 2010). However, it is likely that further new species remain to be discovered, and specific information on insect hosts, pathogenicity and habitats are lacking (Sung et al. 2007a;Hoyos-Carvajal et al. 2009). Quandt et al. (2014) proposed that Hirsutella should be suppressed in favour of Ophiocordyceps affected by the ending of dual nomenclature for pleomorphic fungi in 2011 (McNeill et al. 2012). Ophiocordyceps is the type genus in the family Ophiocordycipitaceae (Hypocreales, Sordariomycetes) (Sung et al. 2007a). The main characteristics of the sexual morphs of Ophiocordyceps are fibrous, hard, pliant-to-wiry, dark stromata with superficial to immersed perithecia (Sung et al. 2007a;Xiao et al. 2019). Most of the sexual species of Ophiocordyceps were transferred from the genus Cordyceps (Cordycipitaceae) by Sung et al. (2007a). Since many species of Hirsutella are closely related to Cordyceps, the asexual morphs in most of the species in Ophiocordyceps have hirsutella-like features (Kepler et al. 2013;Quandt et al. 2014;Maharachchikumbura et al. 2015Maharachchikumbura et al. , 2016. Therefore, Hirsutella was treated as a separate genus from Ophiocordyceps before the taxonomic revision (Sung et al. 2007a;McNeill et al. 2012;Quandt et al. 2014). For example, some new species only known from a Hirsutella morph have been accepted into Ophiocordyceps (Simmons et al. 2015a;Qu et al. 2018b).
In recent years, the taxonomic transitions of Ophiocordycipitaceae changed rapidly under the new rules. Quandt et al. (2014) included Ophiocordyceps, Tolypocladium, Polycephalomyces, Purpureocillium, Drechmeria and Harposporium in Ophiocordycipi-taceae based on morphological and phylogenetic analyses. In the paper "Outline of Ascomycota: 2017", the genus Hymenostilbe was added into the Ophiocordycipitaceae families (Wijayawardene et al. 2018). According to the latest taxonomic report, the number of genera included in Ophiocordycipitaceae has increased to ten, and among them, Hirsutella, Paraisaria and Perennicordyceps are new additions (Hyde et al. 2020). The taxonomic revision of Ascomycota is continuing. Further research into the phylogeny of these organisms is needed. Examples include investigating the new resources to supplement the available taxonomic information and perform phylogenetic research.
During an investigation of the genetic resources of entomopathogenic fungi in southwest China, we collected two specimens of Lepidoptera insects that were infected by fungi. Two hirsutella-like species were isolated and their gene sequences and morphological traits were shown to be related to Hirsutella sensu stricto. In this study, two new species of Hirsutella are introduced.

Specimens
The specimens HKAS112884 and HKAS112885 were deposited at the Kunming Institute of Botany, Chinese Academy of Sciences (KIB), Kunming, China. The isolated strains of their asexual stage were deposited at the Institute of Fungal Resources of Guizhou University (GZAC), Guiyang, China. More information about these specimens is shown in Suppl. material 1: Table S1.

Fungal isolation and culture
The fungi were isolated as described by Qu et al. (2018). The surface of specimens was rinsed with sterile water, followed by surface sterilisation with 75% ethanol for 3-5 s. Parts of the insect body were cut off and a piece of tissue was inoculated in haemocoel on a PDA plate for 20 days at 16 °C.

LM and SEM observation
For light microscopy (LM) observations and imaging, the morphological characteristics of mycelia were observed using an optical microscope (OM, BK5000, OPTEC, Chicago, IL, USA) after staining with a lactic acid/phenol cotton blue solution. The captured images of new species were edited and digitally contrasted using Paint Shop Pro v. 5.0.1 (Corel, Ottawa, Canada).
Electron microscopy was performed as described by Qu et al. (2018). Briefly, 1 cubic cm of hyphae with conidia were cut from the fungus on PDA cultures, fixed with 4% glutaraldehyde at 4 °C overnight, and then washed three times with phosphate buffer saline (PBS) (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 and 1.5 mM KH 2 PO 4 , pH 7.4) for 10 min each time. Fixed hyphae and conidia were dehydrated using 50%, 70%, 90% and 100% ethanol, 10 min for each concentration, and were finally dehydrated with super-critical carbon dioxide. After being sprayed with gold, the conidia and mucilage were examined by scanning electron microscopy (SEM) (S-3400N, Hitachi, Tokyo, Japan) and photographed.

Molecular phylogeny
To construct a phylogeny of major lineages, 71 representative species were chosen to represent the ecological diversity of Hirsutella and Ophiocordyceps based on previous phylogenetic studies (Simmons et al. 2015b;Xiao et al. 2017;Qu et al. 2018;Xiao et al. 2019). Tolypocladium inflatum and T. ophioglossoides were selected as the outgroup taxa and are classified within Ophiocordycipitaceae (Xiao et al. 2019). The sequences used in this study were combined with published data on hirsutella-like species and Ophiocordycipitaceae. All the other sequences were collected from GenBank and the accession numbers are shown in Table 1.
All the sequences were edited for multi-alignment using the BioEdit Sequence Alignment Editor v.7.0.5.3 (Hall 1999) with the Clustal X v.1.83 software package (Thompson et al. 1999). Gaps were excluded from the phylogenetic analysis based on previous research (Qu et al. 2018). The ITS, SSU, LSU, TEF1α and RPB1 regions were aligned in combined datasets using MAFFT v.7 (Katoh and Standley 2013, http://mafft.cbrc.jp/alignment/server/). The Akaike Information Criterion (AIC) in jModeltest 0.1.1 (Guindon and Gascuel 2003;Posada 2008) was used to select the nucleotide substitution model for each region. The combined data included a 4778 bp character set of the five regions and were analysed. Maximum likelihood phylogenetic analyses were conducted in RAxML (Stamatakis et al. 2008) with the recommended partition parameters to determine the best tree topology. The bootstrap support values were achieved after 500 search replicates and summarised in TreeGraph. Bayesian Posterior Probabilities (BPP) were estimated in MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) with the same partition parameters. In this analysis, two runs of four chains each were executed simultaneously for 5,000,000 generations, with sampling every 500 generations. TreeGraph was used to compute the BPP from a summary of 7,501 trees retained after a burn-in of the first 2,500 trees collected.
Host. Larva of a species of Lepidoptera. Habitat and distribution. On decaying leaves in broadleaved forests, Zhejiang Province, China.
Etymology. Refers to the yellow colour (Lat. 'flava') of the holotype and colony.

Teleomorph. Unknown.
Remarks. This species is allied with the H. sinensis and H. strigosa clade. The phialides of H. flava are subulate, and the necks are slenderer. In particular, the colony  Description. Synnemata are single, extending from the head of insect; 8.6 cm long, dark brown and changing to brown towards the apex; no conidiation was observed (Fig. 3A). The fungus spreads slowly on PDA agar at 20-22 °C and grows to a diam. of 22-30 mm after 14 d; the colony is round, centre of surface with brown dense bulges and grey-white sparse flocculent aerial hyphae. Colony margin is flat with radial groove; a large amount of brown pigment secreted into the medium causes the back of colony to appear dark brown; thickness 10-12 mm (Fig. 3B, C). Mycelium hyaline, smooth, septate, 1.5-3.0 μm wide. Conidiogenous cells monophialidic, hyaline, borne perpendicular or at an acute angle to the subtending hyphae. Phialides subulate or slender columnar, tapering gradually to a long and narrow neck, 30-45 × 1-3 μm long. Conidia clavate, narrow fusiform or botuliform without a diaphragm, 9.9-12.6 × 2.7-4.5 μm, single-or double-enveloped in a hyaline mucus, thickness 2.0-3.0 μm (Fig. 3D-J).
Habitat and distribution. On the decaying leaves of broadleaved forests, Guizhou Province, China.
Remarks. This species possesses two types of conidiogenous cells and long fusiform or clavate without diaphragm conidia (9.9-12.6 × 2.7-4.5 μm), which is extremely rare in Hirsutella species. In addition, H. kuankuoshuiensis could produce long thin synnemata on the culture media that contain few or no conidia.

Discussion
Previous taxonomic studies have shown that the Hirsutella species are reconstructed in five main groups, and clustering taxa shared the same phialide structures (Simmons et al. 2015b;Qu et al. 2017;Qu et al. 2018). In general, the H. nodulosa lineage possesses phialides with apical helical twists. The H. citriformis clade is primarily represented by a squat ovoid base and a single slender neck. The H. thompsonii clade, the most widely studied hirsutella-like species and a potential biocontrol agent for mite pests, has a small cylindrical or round phialide, usually less than 25 μm, while the H. sinensis clade includes isolates that originate from a variety of taxa, including nematodes, mites and both hemi (Hemiptera) and holometabolous (Coleoptera, Lepidoptera) insect hosts (Simmons et al. 2015b). The majority of these species share a cylindrical base and an average phialide length greater than 40 μm. In our phylogenetic tree, these five typical branches of Hirsutella were more dispersed owing to the addition of more Ophiocordyceps species. Hirsutella flava and H. kuankuoshuiensis formed a separate clade that is represented by the subulate phialides and narrow fusiform conidia and have a close relationship with the H. sinensis and H. strigosa clades. In addition, this separate clade is distant from the H. thompsonii and H. citriformis clades. Species in these clades primarily share similarly large phialides and long fusiform conidia (Qu et al. 2018).
The phylogenetic tree confirmed the distinction between two new species and extant species. Among the species with an awl-shaped base and a long narrow neck, H. flava differs in its subulate phialides (e.g. H. danubiensis Balazy et al., 2008;H. tunicate Ciancio et al., 2013), cylindrical phialides (e.g. H. changbeisanensis Liang, 1991;H. strigosa Petch, 1939) and two types of conidiogenous cells (e.g. H. stilbelliformis Evans & Samson, 1982;H. shennongjiaensis Zou et al., 2016b) (Suppl. material 1: Table S2). In addition, H. flava is unique in the colony morphology of isolated strains. The fungus spreads more quickly than other hirsutella-like species on PDA media, and the colony surface appears very rough, owing to the hyphae being gathered into outwardly radiating filamentous bundles of varying sizes. H. flava could be distinguished from similar species by the shape and size of the conidiogenous cells. Morphological comparisons of relevant taxa are shown in Suppl. material 1: Table S2.
Hirsutella kuankuoshuiensis possesses two types of conidiogenous cells and long fusiform or clavate conidia, which are unique to Hirsutella. Furthermore, this species can readily produce long thin synnemata on culture media, but it produces few or no conidia. There are five other species similar to this species: H. shennongjiaensis (Zou et al. 2016), H. stilbelliformis var. stilbelliformis (Evans and Samson 1982), H. sporodochialis (Evans and Samson 1984), H. subramanianii (Samson and Evans 1985), and H. zhangjiajiensis (Liang et al. 2005). Among them, the conidia of H. shennongjiaensis are primarily rod-like and slender; H. stilbelliformis var. stilbelliformis has a larger base with thorny phialides, greater than 50 μm long; H. sporodochialis has longer conidia; H. subramanianii has hymenopteran hosts and thinner stick-shaped conidia, 10-13.5 × 1.8-2.5 μm; and H. zhangjiajiensis conidia are lanceolate or resemble an orange segment (Suppl. material 1: Table S3). Within the framework of the available data for the genus, the phylogenetic tree and the morphological analysis confirmed the status of Hirsutella flava and H. kuankuoshuiensis as new species.