Novel taxa and species diversity of Cordyceps sensu lato (Hypocreales, Ascomycota) developing on wireworms (Elateroidea and Tenebrionoidea, Coleoptera)

Abstract Species of Cordyceps sensu lato (Hypocreales, Sordariomycetes) have always attracted much scientific attention for their abundant species diversity, important medicinal values and biological control applications. The insect superfamilies Elateroidea and Tenebrionoidea are two large groups of Coleoptera and their larvae are generally called wireworms. Most wireworms inhabit humid soil or fallen wood and are often infected with Cordyceps s.l. However, the species diversity of Cordyceps s.l. on Elateroidea and Tenebrionoidea is poorly known. In the present work, we summarise taxonomic information of 63 Cordyceps s.l. species that have been reported as pathogens of wireworms. We review their hosts and geographic distributions and provide taxonomic notes for species. Of those, 60 fungal species are accepted as natural pathogens of wireworms and three species (Cordyceps militaris, Ophiocordyceps ferruginosa and O. variabilis) are excluded. Two new species, O. borealis from Russia (Primorsky Krai) and O. spicatus from China (Guizhou), are described and compared with their closest allies. Polycephalomyces formosus is also described because it is reported as a pathogen of wireworms for the first time. Phylogeny was reconstructed from a combined dataset, comprising SSU, LSU and TEF1-α gene sequences. The results, presented in this study, support the establishment of the new species and confirm the identification of P. formosus.


Introduction
The superfamilies Elateroidea and Tenebrionoidea are two large groups of Coleoptera. Species within these superfamilies are phytophagous, xylophagous, saprophagous or omnivorous and most of them are important agricultural pests (Gullan and Cranston 2010;Ren et al. 2016). Elateroidea larvae are the well-known wireworms, closely resembling Tenebrionoidea larvae which are known as mealworms or pseudo-wireworms (Ren et al. 2016). As a result, in practice, larvae of both Elateroidea and Tenebrionoidea are generally referred to as wireworms. Most wireworms inhabit humid soil, humus layer or decayed wood and are, thus, easily encountered and infected with entomopathogenic fungi (Kabaluk et al. 2017;Rogge et al. 2017).
Cordyceps sensu lato (Hypocreales, Sordariomycetes) is a well-known group of entomopathogenic fungi. Previously, most species of this group were assigned to the previous Cordyceps Fr. genus, so they had commonly been called 'Cordyceps'. It was not until 2007 that Sung et al. revised the classification system of this group, based on substantial molecular and morphological data. In the new classification system, all these fungi are assigned to three families (Cordycipitaceae, Ophiocordycipitaceae and, in part, Clavicipitaceae) and only a few species were retained in the revised Cordyceps Fr. emend. G.H. Sung et al. genus  . As a result, the concept of 'Cordyceps' has been extended from the previous genus Cordyceps Fr. to Cordyceps s.l. So far, more than 1000 Cordyceps s.l. species have been reported (Wei et al. 2020) and these entomopathogenic hypocrealean fungi are widely distributed in all terrestrial regions (except Antarctica), especially tropics and subtropics (Kobayasi 1941;Sung et al. 2007).
Ophiocordyceps Petch and Polycephalomyces Kobayasi are two morphologically, phylogenetically and ecologically closely-related genera placed in Ophiocordycipitaceae. They produce rigid, pliant or wiry stipes that are usually darkly coloured; their asexual morphs are mainly Hirsutella-like, but phialides of Polycephalomyces lack the swollen base and are concentrated at the tips of synnemata; and they are typically found on hosts buried in soil or in rotting wood, especially wireworms Kepler et al. 2013). Ophiocordyceps is the largest genus of Cordyceps s.l., with O. blattae (Petch) Petch as the type species, linking with Didymobotryopsis-, Hirsutella-, Hymenostilbe-, Sorosporella-, Synnematium-and Troglobiomyces-like asexual states (Quandt et al. 2014) and currently comprising approximately 200 species (Wei et al. 2020). Polycephalomyces, with P. formosus Kobayasi as its type and linking with Acremonium-, Hirsutella-and Polycephalomyces-like asexual states, includes 19 known species thus far, some of which are found on stromata of Ophiocordyceps spp. (Kepler et al. 2013;Wang 2016;Index Fungorum 2021).
In nature, Cordyceps s.l. species develop mainly on insects, spiders, other Cordyceps s.l. species and hypogeous fungi of the genus Elaphomyces. These ascomycetes can reproduce via ascospores, conidia and mycelia that generally inhabit soil, plants, invertebrates, nematodes, mushrooms and other organisms . The ecology and habits of different host groups are generally different and this often determines the species specificity of Cordyceps s.l. on them. As a result, in practice, Cordyceps s.l. species have commonly been classified according to their host groups. With respect to the taxonomy of Cordyceps s.l. on insects, early systematic work mainly came from Petch (e.g. 1934), Kobayasi (e.g. 1941) and Shimizu (1997) who all classified Cordyceps s.l. species according to their host orders. Later, Shrestha et al. (2016Shrestha et al. ( , 2017 reviewed Cordyceps s.l. species on their Coleoptera, Lepidoptera, Hymenoptera and Hemiptera hosts. Recently, Zha et al. (2020) systematically studied the Orthoptera hosts and investigated the relationships with their pathogens.
A diverse range of Cordyceps s.l. species have been reported as pathogens of wireworms. Due to the difficulities in identifying wireworms, hosts of these fungal species have generally been recorded as Elateridae larvae, Tenebrionidae larvae or Coleoptera larvae (e.g. Petch 1933Petch , 1937Kobayasi 1941;Shimizu 1982b, 1983). Shimizu (1997) provided beautiful drawings for many Cordyceps s.l. species, which included more than 30 species on wireworms and wireworm-like insects. A recent report for wireworm-infecting Cordyceps s.l. involved only 20 species (Shrestha et al. 2016), which is fewer than the number recorded by Shimizu (1997). It should be noticed that these fungi affect the populations of wireworms and have the potential to control these agricultural pests (Barsics et al. 2013;Rogge et al. 2017). Therefore, we need a deeper knowledge of species diversity, taxonomy, distribution and lifestyle of these wireworminfecting Cordyceps s.l.
In this study, the species diversity of wireworm-infecting Cordyceps s.l. (Elateroidea and Tenebrionoidea) is reviewed. We discuss their hosts and geographic distribution and provide taxonomic notes for species. In addition, we describe two new members of this group, Ophiocordyceps borealis sp. nov. and O. spicatus sp. nov. Polycephalomyces formosus Kobayasi is also described because it represents the first report of this species on wireworms (Elateroidea). We reconstructed a multilocus (SSU, LSU and TEF1-α) phylogeny to support morphological results.

Sample collections and morphological studies
Wireworm-infecting species of Cordyceps s.l. were collected from south-western China and the Russian Far East. Specimens were placed in plastic boxes and carried to the laboratory for further study. The macro-characteristics and ecology were photographed using a Nikon Coolpix P520 camera in the field. Specimens were examined and photographed using an Optec SZ660 stereo dissecting microscope and a Nikon Eclipse 80i compound microscope connected with a Canon EOS 600D camera. Microscopic measurements were made using Tarosoft (R) Image Framework software. Images were processed using Adobe Photoshop CS v. 8.0.1 (Adobe Systems Incorporated, San Jose, California, USA). Voucher specimens are deposited in the Fungarium of the Centre of Excellence in Fungal Research, Mae Fah Luang University (MFLU), Chiang Rai, Thailand and the Herbarium of Guizhou University (GACP), Guiyang, China.

DNA extraction, sequencing, sequence assembly and alignment
Total DNA was extracted from dried specimens using E.Z.N.A.TM Fungal DNA MiniKit (Omega Biotech, CA, USA). The ribosomal internal transcribed spacers (ITS), small and large subunits (SSU and LSU) and translation elongation factor 1α (TEF1-α) genes were amplified and sequenced using the PCR programmes and primer pairs listed in Table 1. PCR amplification reactions were performed in an ABI 2720 thermal cycler (Applied Biosystems, Foster City, CA, USA). PCR products were purified using Bioteke's Purification Kit (Bioteke Corporation, Beijing, China) and were sequenced using an ABI 3730 DNA analyser and an ABI BigDye 3.1 terminator cycle sequencing kit (Sangon Co., Shanghai, China). Sequences were aligned and assembled visually and manually using Clustalx1.81, Chromas230, ContigExpress and MEGA6 software.

Construction of molecular phylogenetic trees
BLAST searches were performed to reveal the closest matches in the GenBank database that would allow the selection of appropriate taxa for phylogenetic analyses. Each gene region was independently aligned and improved manually, then the SSU, LSU and TEF1-α gene sequences were combined to form a concatenated dataset. The ITS region was not included in our multilocus analyses because of: 1) insufficient ITS sequence data (Table 2) which may lead to inaccurate phylogenetic results; 2) distinct different rate of evolution from SSU, LSU and TEF genes and with many irregular insertions and deletions of bases. Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian Inference (BI) analyses were performed using the concatenated sequence dataset. Sequence information of the three described species and their allies is listed in Table 2.
Maximum Likelihood (ML) analysis was done via the CIPRES Science Gateway platform (Miller et al. 2010) using RAxML-HPC2 on XSEDE (8.2.10) with the GTRGAMMA nucleotide substitution model and 1000 bootstrap iterations (Jeewon et al. 2003;Hongsanan et al. 2017). An MP tree was constructed with PAUP* 4.0b10 (Swofford 2002) using the heuristic search option with TBR branch swapping and bootstrapping with 1,000 replicates (Cai et al. 2006;Tang et al. 2007). BI analysis was conducted using MrBayes v. 3.1.2 with Markov Chain Monte Carlo sampling to Table 1. Primers and PCR programmes used in this study (White et al. 1990, Spatafora et al. 2006, Ban et al. 2015.

Molecular phylogeny of the three described species
The combined concatenated dataset included 36 samples including 32 species of Ophiocordycipitaceae (Ophiocordyceps, Paraisaria and Polycephalomyces) as ingroups and Cordyceps militaris (L.) Fr. (strain OSC 93623, Kepler et al. 2012) as the outgroup.
Sexual morph. Parasitising Elateroidea larvae (Coleoptera) living in fallen wood. The larvae are cylindrical, 11 mm long and 1.1-1.3 mm thick, yellowish-brown; their body cavity stuffed with milky yellow mycelia and their intersegmental membranes covered with many milky yellow and flocculent funiculi. Stromata arising from any part of larval body, single or paired, unbranched. Stipe grey, slender and cylindrical, fibrous and flexible, curved more or less, 10-13 mm long and 0.25-0.6 mm thick, sur- face relatively smooth but with many longitudinal wrinkles, apex pointed. Fertile part irregularly attached on one side of the surface of distal part of stipe, which resembles a mass of insect eggs that are clustered together or separated into several lumps; substrate layer milky white, surface milky yellow accompanied by lavender and dotted with numerous black ostioles. Perithecia immersed, densely arranged, obliquely or at right angles to the surface of stipe, pyriform, neck unconspicuous, 220-290 × 120-150 µm and their tops obtuse; walls dark brown and 25-32 µm thick; ostioles slightly thickened and slightly protruding over the surface of fertile part. Asci cylindrical, 6-8 µm in diameter; caps hemispherical, 5-6 (x -= 5.5, n = 30) µm wide and 3.5-5 (x -= 4.2, n = 30) µm high. Ascospores filiform and elongate, multi-septate (far more than 3), not easy to break into part-spores; part-spores cylindrical, truncated at both ends, 10-15 (x -= 12.2, n = 30) × 2 µm. Asexual morph. Unknown.
Hosts. Growing on Elateroidea larvae (Coleoptera) living in fallen wood in a deciduous forest.
Nucleotide sequences of O. borealis are most similar to those of O. purpureostromata (specimen TNS F18430, Quandt et al. 2014), but there is 2.3% bp difference across the 804 bp in TEF1-α, 0.5% bp difference across the 845 bp in LSU and 0.1% bp difference across 1,061 bp in SSU. ITS of O. borealis is > 14.1% different to all ITS available in GenBank (ITS are not available for O. purpureostromata). On the phylogenetic tree, the new species is also nearest (100% ML/100% MP/1.00 PP) to O. purpureostromata, but they form into two distinct branches which support them being two separate species (Fig. 1). Etymology. Referring to the spicate fertile head.
Host. Growing on a Tenebrionoidea larva (Coleoptera) living in humid and decayed wood in a broad-leaved forest.
Notes. Ophiocordyceps spicatus is morphologically somewhat similar to O. formosana (Kobayasi and Shimizu 1981;Li et al. 2016), but it has a much smaller stroma (stipes 6-10 (or 19-37) mm long and 1. formosana, but they form into two distinct branches which also support them being two separate species (Fig. 1).  (Kobayasi 1941;Samson and Evans 1985;Wang 2016). We collected a P. formosus-like specimen on the stroma of Ophiocordyceps sp. on an Elateroidea larva from Guizhou, China. Morphological and phylogenetic data showed that it is P. formosus. This is the first report of P. formosus on wireworms.
Host and ecology. On the stroma of Ophiocordyceps sp. on an Elateroidea larva on the ground in a humid bamboo (Chimonobambusa quadrangularis (Franceschi) Makino) forest in Guizhou karst regions.
The larva might live in soil or decayed wood at first, but was then infected by Ophiocordyceps sp. and produced a sexual stroma. Following heavy rainfall, the host, together with the stroma of Ophiocordyceps sp., was washed away and exposed on the ground and at last, was parasitised by Polycephalomyces formosus. The fertile head of the stroma might have been lost during the floods. Hosts. Spiders, insects from various orders, including Coleoptera (e.g. Tenebrionidae: Alphitobius diaperinus); inhabiting phytopathogenic fungi and plant-parasitic nematodes (Humber and Hansen 2005;Shinya et al. 2008).
Distribution. Widely distributed in tropical and temperate regions, for example: Dominican Republic, Jamaica, Indonesia, Peru, Sri Lanka, the West Indies, Turkey and USA (Zare and Gams 2001).
Notes. The species was originally and frequently reported on scale insects (Hemiptera: Coccidae (syn. Lecaniidae)) (Zare and Gams 2001). Humber and Hansen (2005) listed its hosts involving spiders, many insect orders and found on the mushroom Puccinia striiformis (Pucciniaceae). The species was also found on phytopathogenic fungi and plant-parasitic nematodes (Shinya et al. 2008). Zare and Gams (2001)  Distribution. Widely distributed. Note. Beauveria bassiana sensu lato includes a large complex of cryptic species with wide host ranges, including many Coleoptera families (Rehner et al. 2011;Imoulan et al. 2017).
Known distribution. China (Keissler and Lohwag 1937). Note. Taxonomically uncertain species which was described from the previous Cordyceps Fr. (differs from the current Cordyceps Fr. emend. G.H. Sung et al., same as below).
Note. Hosts of the species were recorded as Coleoptera larvae (Tasanathai et al. 2016). According to the picture provided, the hosts are wireworms.
Note. Under laboratory conditions and injection of hyphal bodies into the haemocoel of insects, C. militaris can infect many insect orders (Shrestha et al. 2012), including pupae of Tenebrio molitor (Tenebrionidae) (De Bary 1867; Sato and Shimazu 2002). Therefore, the conclusion that wireworms (e.g. Tenebrio molitor) are the natural hosts of C. militaris is probably untenable and we temporarily reject it.
Known distribution. Japan (Kobayasi and Shimizu 1983). Notes. Taxonomically uncertain species from the previous Cordyceps. Its host was originally recorded as a Coleoptera larva (Kobayasi and Shimizu 1983) and then Shimizu (1997) identified it as a Tenebrionidae larva.
Known distribution. South America (Petch 1933). Notes. Taxonomically uncertain species from the previous Cordyceps. Hosts of the species were recorded as beetle larvae in rotten wood (Petch 1933). Petch (1933) considered the species as a synonym of Nigelia martiale (≡ C. martialis). According to the information given by Petch (1933), hosts of the species are wireworms.
Note. Hosts of the species were originally recorded as Coleoptera larvae (Kobayasi and Shimizu 1978) and then Shimizu (1997) identified them as Tenebrionidae larvae.
Note. Hosts of the species were originally recorded as Coleoptera larvae (Mongkolsamrit et al. 2020). According to the illustration and the information provided, the hosts are wireworms. Tasan
Note. Hosts of the species were generally identified as wireworms or Coleoptera larvae Shimizu 1980a, Liang 2007). Shimizu (1997) identified the hosts of the species from Japan and Taiwan as Elateridae larvae.
Notes. The specific epithet of this species was adopted from the generic name of its host insect 'Agriotes' (Kobayasi and Shimizu 1980a). The epithet 'agriotidis', used in Index Fungorum (2021) and related literature (e.g. Sung et al. 2007), is incorrect. Yang (2004) and Liang (2007) also recorded its hosts as Elateridae larvae.
Note. Host of the species was originally recorded as a Coleoptera larva (Kobayasi and Shimizu 1982a). According to the illustration by Shimizu (1997), the host is a wireworm.
Known distribution. China (Taiwan), Japan (Shimizu 1997 Hosts. Larvae and adults of many Coleoptera families, for example, Tenebrionidae larva (Shrestha et al. 2016) and Lampyridae larvae. Distribution. Widely distributed. Note. According to the illustrations by Shimizu (1997), we identify the hosts of the species from Japan as Lampyridae larvae (Elateroidea).
Note. Host of the species was originally recorded as a Coleoptera larva (Kobayasi and Shimizu 1980a). According to the illustration by Shimizu (1997), the host is a wireworm.
Note. Liang (2007) recorded the hosts of the species as Lepidoptera larvae, but his provided picture (a specimen collected from Jilin, China) appears to be a wireworm host. ( Hosts. Elateridae larvae (Shimizu 1997).

Ophiocordyceps subflavida
Known distribution. Japan (Shimizu 1997), Venezuela (Mains 1959). Note. The species was originally reported from Venezuela and its host was recorded as an insect larva (Mains 1959). Shimizu (1997) identified the host of a specimen from Japan as an Elateridae larva.
Notes. In early literature, O. variabilis was recorded on Coleoptera (e.g. Elateridae) and Diptera larvae in rotten wood (Petch 1937;Mains 1958;Liang 2007). Hodge et al. (1998) checked many samples and confirmed the hosts to be Xylophagidae larvae (Diptera). More than 40 samples of O. variabilis were collected in Russia (Far East, Western Siberia) and all of them developed on Xylophagidae larvae (Yaroslavtseva et al. 2019;Kryukov et al., unpublished). Ecological habits and morphology of Xylophagidae larvae and wireworms are closely similar, but their last abdominal segments are distinctly different. As with O. ferruginosa listed above, we conclude that O. variabilis is not a pathogen of wireworms. Hosts. Elateridae larvae (Shimizu 1997;Yahagi 2008).
Note. Host of the species was originally recorded as a Coleoptera larva (Kobayasi and Shimizu 1983). According to the illustration by Shimizu (1997), the host is a Tenebrionoidea larva.
Note. Hosts of the species were recorded as Coleoptera larvae (Crous et al. 2017). According to the picture provided, the hosts are wireworms.

Discussion
The superfamilies Elateroidea and Tenebrionoidea are two very large groups of beetles and comprise more than 50 families of Coleoptera (Catalogue of Life 2021). These include Lampyridae (fireflies), Elateridae (click beetles), Phengodidae (glowworm beetles), Cantharidae (soldier beetles) and their relatives in Elateroidea; and Meloidae (blister beetles), Anthicidae (ant-like flower beetles), Mordellidae (tumbling flower beetles), Tenebrionidae (darkling beetle), Ciidae (the minute tree-fungus beetles), Zopheridae (ironclad beetles) and their relatives in Tenebrionoidea. Most of Elateroidea and Tenebrionoidea larvae (wireworms) are closely similar and morphology alone could hardly distinguish them. In practice, hosts of many wireworm-infecting Cordyceps s.l. species are commonly identified as Elateridae (mainly) or Tenebrionidae larvae. Considering the difficulties in identifying wireworms, we suggest to use the superfamily names (Elateroidea or Tenebrionoidea) to record the hosts of the fungi, unless we can definitely know the species identity (e.g. by barcoding techniques).
In present paper, we summarised the data of wireworm-infecting species of Cordyceps s.l. To date, a total of 63 species have been reported, including 17 species (Akanthomyces, Beauveria and Cordyceps) in Cordycipitaceae, 11 species (Metarhizium and Nigelia) in Clavicipitaceae and 35 species (Ophiocordyceps, Paraisaria, Perennicordyceps, Polycephalomyces and Tolypocladium) in Ophiocordycipitaceae. Amongst these, C. militaris, O. ferruginosa and O. variabilis are rejected; the remaining 60 species are accepted as natural pathogens of wireworms. It is likely that a significant portion of fungi, associated with wireworms, is represented by specialised forms. Thirteen of the reported species (20%) have broad host ranges, that is, they can infect different arthropod taxa and may also parasitise fungi and nematodes. The other 47 species (80%) have, thus far, been registered on wireworms only. Generalist fungi are mostly widespread, whereas specialised fungi are generally reported from warm and humid environments of Southeast Asia (Japan, south-western China and Thailand), the Amazon of South America and the Russian Far East. It should be noted that many animal-associated fungi are awaiting description, especially in groups, such as Hypocreales (Antonelli et al. 2020;Cheek et al. 2020) and many taxonomically-uncertain Cordyceps s.l. species infecting Elateroidea and Tenebrionoidea remain to be studied. Apart from the description of novel taxa, further studies should focus on revisions of these uncertain species and further information of wireworm hosts. Limited by lack of information and taxonomic knowledge of larvae, species diversity of wireworm-infecting Cordyceps s.l. may not have been completely accounted for and many wireworm hosts cannot be or are incorrectly assigned to their families. This is the first study summarising species diversity of wireworm-infecting Cordyceps s.l. A checklist of 60 species is provided and two novel species are described. Our work provides basic information for future research on species diversity of Cordyceps s.l. associated with wireworms, management and biocontrol of wireworm populations, as well as on edible and medicinal insects and fungi.