﻿Morphology and phylogeny of four new species within Polycephalomycetaceae (Hypocreales) parasitising Ophiocordyceps species

﻿Abstract Species of the family Polycephalomycetaceae grow on insects or entomopathogenic fungi and are distributed from tropical to subtropical regions. This study proposed four new species of hyperparasitic fungi from China based on six molecular markers (ITS, SSU, LSU, TEF-1α, RPB1 and RPB2) phylogenetic analyses and morphological characteristics. The four new species, i.e. Pleurocordycepslitangensis, Polycephalomycesjinghongensis, Po.multiperitheciatae and Po.myrmecophilus, were described and illustrated. Pl.litangensis, exhibiting a hyperparasitic lifestyle on Ophiocordycepssinensis, differed from Pleurocordyceps other species in producing subulate β-phialides and ovoid or elliptic α-conidia. Po.jinghongensis was distinct from Polycephalomyces other species, being parasitic on Ophiocordyceps sp., as producing oval or long oval-shaped α-conidia and columns of β-conidia. Po.multiperitheciatae differed from Polycephalomyces other species as having synnemata with fertile head, linear β-conidia and parasitic on Ophiocordycepsmultiperitheciata. Po.myrmecophilus was distinct from Polycephalomyces other species, being parasitic on the fungus Ophiocordycepsacroasca, as producing round or ovoid α-conidia and elliptical β-conidia without synnemata from the colonies. These four species were clearly distinguished from other species in the family Polycephalomycetaceae by phylogenetic and morphological characteristics. The morphological features were discussed and compared to relevant species in the present paper.

South-western China is an area of high fungal biodiversity (Hyde et al. 2018).The rich biodiversity uncovered suggested that further collections could result in the discovery of numerous new taxa (Hyde et al. 2020a, b).In this study, the four novel species presented herein were collected from Yunnan Province and Sichuan Province in China.Morphological observations and phylogenetic analyses showed that these four species were novel and distinct from all other previously-described species in the family Polycephalomycetaceae.The four new species were discovered to be hyperparasites of Ophiocordyceps species.Pl. litangensis, Po. jinghongensis, Po. multiperitheciatae and Po.myrmecophilus were hyperparasitic on O. sinensis, Ophiocordyceps sp., O. multiperitheciata and O. acroasca, respectively.At present, relatively little is known about the mechanisms responsible for hyperparasitism in species of the family Polycephalomycetaceae and our findings provide ideal material for these studies.These findings have expanded the diversity of fungal species in the family Polycephalomycetaceae, providing taxonomic data to support species resource conservation and rational exploitation and utilisation of resources.

Specimens and isolates
Fungal specimens parasitising Ophiocordyceps sp. were collected from different regions of south-western China, including Sichuan Province (Litang County) and Yunnan Province (Jinghong City, Yuanyang County, Pu'er City).The specimens were found in moist soils.Geographic information (longitude, latitude and altitude) of collection were recorded in the field, then specimens were collected in sterilised plastic containers and transported to the laboratory.The micro-morphological characters (Synnemata) were examined using an Olympus SZ61 stereomicroscope (Olympus Corporation, Tokyo, Japan).To obtain axenic culture, the stromata was divided into 2-4 segments with sterilised blades.Each segment was immersed in hydrogen peroxide 30% (H 2 O 2 ) for 5 min and then rinsed five times in sterile water.After drying on sterilised filter paper, these segments were inoculated on Potato Dextrose Agar (PDA) plates.The conidial masses at the apex of the stipes were picked with an inoculating needle and immersed in 5 ml of sterilised water for blending.The homogenates were then spread on PDA plates containing 0.1 g/l streptomycin and 0.05 g/l tetracycline.The plates were maintained in a culture room at 25 °C.After purification, the cultures were stored at 4 °C (Wang et al. 2015a).Dry specimens were deposited in the Yunnan Herbal Herbarium (YHH) of Yunnan University.The cultures were stored in Yunnan Fungal Culture Collection (YFCC) of Yunnan University.

Morphological studies
Cultures on potato extract agar (PDA) were incubated for 21 days at 25 °C and photographed using a Canon 750 D camera (Canon Inc., Tokyo, Japan).For asexual morphological descriptions, microscope slide cultures were prepared by placing a small amount of mycelium on 5 mm diameter PDA medium blocks that were overlaid by a cover slip (Wang et al. 2015a;Tang et al. 2023b).The observations, measurements and photographs of the phialides and conidia were made using a light microscope (Olympus BX53).

Phylogenetic analysis
In order to construct a phylogeny of the major lineages in the family Polycephalomycetaceae, most of the DNA sequences used in this work were derived from previous phylogenetic studies (Xiao et al. 2023).Phylogenetic analyses were based on sequences of six molecular markers (ITS, SSU, LSU, TEF-1α, RPB1 and RPB2), all of which were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/).Then the nucleotide sequences were combined with those generated in our study (Table 1).Sequences were aligned using ClustalX v.2.0 (Larkin et al. 2007), adjusted manually and then concatenated in BioEdit v.7.1.1 (Hall 1999).Poorly-aligned regions were removed and adjusted manually using MEGA6 (v.6.0)(Tamura et al. 2013).ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the best-fitting likelihood model for the Maximum likelihood (ML) analyses and the Bayesian inference (BI) analyses were carried out for the fungi datasets.For ML analyses, tree searches were performed in IQ-tree (v.2.1.3)(Nguyen et al. 2015), based on the best-fit model GTR+F+I+I+R3 with 5000 ultrafast bootstraps (Hoang et al. 2017) in a single run.The BI search was according to the best-fit model GTR+F+I+G4, resorting to MrBayes (v.3.2.2) for BI analysis (Ronquist et al. 2012).The phylogenetic trees constructed using the ML and the BI analyses were largely congruent and strongly supported in most branches (Fig. 1).The final phylogenetic tree was visualised with its Maximum-Likelihood bootstrap proportions (ML-BS) and Bayesian posterior probability (BI-BPP) performed using FigTree v.1.4.2 and edited via Adobe Illustrator CS6.

Phylogenetic tree
Sequences of 113 samples were used for phylogenetic analysis.Tolypocladium ophioglossoides (NBRC 106330) and T. ophioglossoides (NBRC 100998) were designated as the outgroup taxa (Xiao et al. 2023).The total length of the concatenated dataset of six genes across the 113 samples was 6384 bp, including 859 bp for ITS, 1548 bp for SSU, 930 bp for LSU, 1037 bp for TEF-1α, 797 bp for   Terminal portion of a synnemata covered by a viscous mass, khaki.Colonies on PDA growing slowly, attaining a diameter of 1.4-1.6 cm in 3 weeks at 25 °C, filiform, dark yellow and reverse dry yellow.Phialides existing in two types: α-and β-phialides.Both types of phialides often reproduce new phialides at their own apices and yield catenulate β-conidia, collarettes not flared, periclinal thickening not visible.α-phialides acropleurogenous solitary on hyphae; spear point, tapering gradually from the base to the apex, 11.2-12.8μm long, 1.9-2.6 μm wide at the base and 0.7-0.9μm wide at the apex.β-phialides terminal on solitary on hyphae; subulate, tapering abruptly from the base to the apex, 9.9-27.8μm long, 1.6-2.5 μm wide at the base and 0.6-1.4μm wide at the apex.α-conidia ovoid or elliptic and occurring on the final portion of synnemata, 3.2-6.1 × 1.8-3.9μm; β-conidia fusiform, and produce on the surface mycelium of colony, multiple, usually in chains on a phialide, 3.5-6.1 × 1.4-2.5 μm.

Polycephalomyces myrmecophilus
Diagnosis.Polycephalomyces myrmecophilus are similar to that of Po. ramosus regarding the production of two types of conidia, but Po. myrmecophilus differ by α-conidia round or ovoid, β-conidia elliptical.

Discussion
Our taxonomic investigations revealed four new species of the family Polycephalomycetaceae, Pl. litangensis, Po. jinghongensis, Po. multiperitheciatae and Po.myrmecophilus.Morphological observations suggested that four species have sufficient morphological differences to justify their segregation into four species.A new species, Pl. litangensis, was described in the genus Pleurocordyceps.Pleurocordyceps litangensis was similar to Pl. agaricus, Pl. aurantiacus, Pl. lanceolatus, Pl. marginaliradians, Pl. sinensis, Pl. vitellina, Pl. yunnanensis, Pl. nutansis and Pl.heilongtanensis, by producing two types of conidia, while Pl.Parvicapitata and Pl.lianzhouensis had only one type of conidia.Pl. litangensis was distinct from other species of Pleurocordyceps, with having α-phialides spear point, β-phialides subulate, α-conidia ovoid or elliptic.Moreover, Pl. litangensis and Pl.sinensis both had the same host (O.sinensis) and β-Conidia, but their phialides, α-conidia size and shape were different (Table 2).Herein, we described three new species, namely, Po. jinghongensis, Po. multiperitheciatae and Po.myrmecophilus, enriching the species diversity in the genus Polycephalomyces.Six additional species are included in this genus (Table 1): Polycephalomyces baltica (Poinar and Vega 2020), Po. cylindrosporus (Matočec et al. 2014), Po. ditmarii (Van Vooren andAudibert, 2005), Po. paludosus (Mains 1948), Po. ramosus (Seifert 1985;Bischof et al. 2003) and Po.tomentosus (Seifert 1985).These species either lacked molecular data or their updated strain descriptions did not match those of the protologue (Wang et al. 2021).These three new species were similar to Po. ramosus, producing two types of conidia, while Po. baltica, Po. cylindrosporus, Po. ditmarii, Po. paludosus and Po. albiramus (Xiao et al. 2023) had only one type of conidia.Po. jinghongensis was distinct from Po. ramosus, being parasitic on Ophiocordyceps sp.producing longer α-conidia oval or long oval shape and β-conidia columns.Po. multiperitheciatae differed from Po. ramosus, being parasitic on O. multiperitheciata, having synnemata with fertile head and β-conidia linear.Po. myrmecophilus was distinguished from Po. ramosus, being parasitic on the fungus O. acroasca, producing synnemata, α-conidia round or ovoid, and β-conidia elliptical, without producing synnemata from the colonies, whereas Po. ramosus was parasitic on Lepidoptera larvae or Hirsutella guignardii, with α-conidia ovoid and β-conidia fusiform (Table 3).Some species of the family Polycephalomycetaceae have been reported from more than one host, indicating their non-host specific nature (Bischof et al. 2003;Wang et al. 2012Wang et al. , 2015a, b;, b;Matočec et al. 2014;Crous et al. 2017;Xiao et al. 2018).Pl. lianzhouensis (Wang et al. 2014) was found to parasitise insects along with the species of the genus Ophiocordyceps.The field investigation and studies showed that Pl. litangensis also parasitised O. sinensis, a phenomenon known as hyperparasitism.Most species of the genus Polycephalomyces parasitise insects in the orders Coleoptera and Hemiptera, and we have already discovered that Po. jinghongensis, Po. multiperitheciatae and Po.myrmecophilus are hyperparasitic on the species of Ophiocordyceps, expanding the diversity of hosts in Polycephalomyces.In subsequent studies, we should delve deeper into the ecological habits and hyperparasitic phenomena of the family Polycephalomycetaceae, explore the evolutionary relationship between hyperparasitic species and entomophytic fungi and promote their development and utilisation.Xiao et al. (2023) introduced Pl. nutansis as a new species under the genus Pleurocordyceps.However, Pl. sinensis and Pl.nutansis were found to be grouped together in the phylogenetic tree, which may be the reason and they are sister taxa to each other.Similarly, molecular phylogenetic analysis has shown that Pl. nipponica and Pl.kanzashianus are clustered together.Nevertheless, Wang et al. (2021) pointed out that they were distinct species, based on their sexual morphology characteristics.In addition, Wang et al. (2021) noted the description of the spore type of Pl. lianzhouensis was not clear and future research should strengthen the observation of its asexual morphology to determine its more accurate classification position.Cordyceps pleuricapitata has formed a monophyletic branch in the genus Polycephalomyces.Xiao et al. (2023) noted the paratype of C. pleuricapitata lacks molecular data and the two strains (NBRC 100745, NBRC 100746) named C. pleuricapitata for which there are molecular data lack morphological information.Hence, it was not possible to clarify the precise position of C. pleuricapitata and its classification at this time.These classifications issues require further research.Phylogeny based on our concatenated data also supported that our four new species belonged to the family Polycephalomycetaceae and were distinct from each other (Fig. 1).Four strains, namely, Pleurocordyceps sp.NBRC109990, Pleurocordyceps sp.NBRC109987, Pleurocordyceps sp.NBRC110224 and Pleurocordyceps sp.NBRC109988 and Pl.litangensis were aggregated into one branch.However, the four strains had only LSU sequences in the NCBI database and were classified as undefined species in Pleurocordyceps incertae sedis.Future research will require additional morphological and phylogenetic work to clarify their taxonomic status.

Figure 1 .
Figure1.Phylogenetic tree of Polycephalomycetaceae, based on the concatenation of ITS, SSU, LSU, TEF-1α, RPB1 and RPB2 sequence data.The tree was generated from an alignment of 6,384 sites and 113 taxa.The phylogeny was inferred using the IQ-tree.The Maximum likelihood bootstrap values greater than 75% (on the left) and the Bayesian posterior probabilities over 0.75 (on the right) were indicated above the nodes.The new species were indicated in back bold font.

Table 1 .
Sources of selected isolates and GenBank accession number for ITS and five genes of three genera within Polycephalomycetaceae were used in this study.