Four novel Pleurocordyceps (Polycephalomycetaceae) species from China

Entomopathogenic fungi comprise an ecologically important group of specialized pathogens infecting other fungi, invertebrates, and plants. These fungi are species-rich with high diversity and broad distribution worldwide. The majority of entomopathogenic fungi belong to clavicipitoids, which consist of the hypocrealean families, Clavicipitaceae, Cordycipitaceae, Ophiocordycipitaceae, and Polycephalomycetaceae. The latter is a newly established entomopathogenic family that recently separated from the family Ophiocordycipitaceae to accommodate the genera, Perennicordyceps, Pleurocordyceps, and Polycephalomyces. In recent years, Polycephalomycetaceae has been enriched with parasitic and hyperparasitic fungi. With 16 species spread across China, Ecuador, Japan, and Thailand, Pleurocordyceps is the most speciose genus in the family. In this study, we expand the number of taxa in the genus by introducing four new Pleurocordyceps species from China, namely, P. clavisynnema, P. multisynnema, P. neoagarica, and P. sanduensis. We provide detailed descriptions and illustrations and infer genus-level phylogenies based on a combined 6-loci gene sequence dataset comprising the internal transcribed spacer gene region (ITS), small subunit ribosomal RNA gene region (SSU), large subunit rRNA gene region (LSU), translation elongation factor 1-alpha gene region (TEF-1α), RNA polymerase II largest subunit gene region (RPB1), and RNA polymerase II second largest subunit (RPB2). This study contributes to knowledge with regard to the diversity of Pleurocordyceps specifically and entomopathogenic Hypocreales more broadly.


Sample collection, isolation, and morphological studies
Fresh specimens, comprising a total of eight, were collected from soil in Anhui and Guizhou provinces, China.The samples were transported in plastic boxes to the laboratory, and pertinent metadata (location, longitude, and latitude) were recorded.The fruiting bodies were examined using a stereomicroscope (SMZ 745 and SMZ 800N, Nikon, Tokyo, Japan) and free-hand sections were obtained for analysis.Micromorphological features such as synnemata, conidiophores, phialides, and conidia were captured using a Nikon DS-Ri2 digital camera connected to a Nikon ECLIPSE microscope (Tokyo, Japan).The strains were obtained from fresh tissue by removing a small piece of mycelium from the host, which was then transferred with a sterile needle onto PDA plates and incubated at 25 • C. The pure culture was stored in the Guizhou Culture Collection, China (GZCC).The specimens were deposited at the Guizhou Institute of Technology Herbarium (Herb.GZLG).The guidelines of the Facesoffungi database (https://www.indexfungorum.org)were followed to obtain Index Fungorum numbers, as outlined by Jayasiri et al. (2015).The morphological structures were measured using Tarosoft (R) v.0.9.7 Image Frame Work, and the photographic plates were processed using Adobe Photoshop CC 2022 (Adobe Systems, USA).
The ML phylogeny was inferred using IQ-TREE 2 with partitioned models and 1,000 exhaustive bootstrap replications (Minh et al., 2020).The model of evolution for each locus was chosen by the built-in ModelFinder tool (Kalyaanamoorthy et al., 2017).The BI analysis was conducted using MCMC sampling and MrBayes version 3.1.2(Ronquist et al., 2012).The sampling was performed with six simultaneous Markov chains for 1,850,000 generations based on the standard deviation of split frequencies being <0.01, with trees being sampled every 1,000 generations.The initial 25% of trees were considered as the burn-in phase and were discarded.The posterior probability (PP) was calculated using the remaining trees (Dissanayake et al., 2020).FigTree v.1.4.0 (http:// tree.bio.ed.ac.uk/software/figtree/) was used to visualize the ML tree.Based on the guidelines provided by Chethana et al. ( 2021
Notes: Pleurocordyceps sanduensis is the closest match to our new sample of P. clavisynnema.This is also confirmed by phylogenetic analyses, whereby the two are sister taxa with maximum statistical support (100% ML/1.00 PP; Figure 1).Base pair differences between P. clavisynnema and P. sanduensis are 23/824 in tef-1α, 8/1130 in SSU, 2/678 in rpb1, and 3/1050 in rpb2.Morphologically, P. clavisynnema differs from P. sanduensis by having longer synnemata, larger conidiophore, smaller phialides, and shorter conidia.Hence, this study introduces Pleurocordyceps clavisynnema as a new species based on morphological and phylogenetic analyses.
Colonies on PDA medium slow-growing, isolated from the tissue of synnemata, circular, attaining 3 cm in 35 days at 25 • C, dry yellow.Synnemata arising the margin of the colony after 30 days, without a fertile head, solitary or two-or three-branched, 2-6 × 0.9-1.8mm (x = 4 × 1.35 mm, n = 30), with several radiating ring-like distributions.Conidial masses pale yellow to yellow, covered the surface of the colony or generated from the middle part of the synnemata with hyaline to white yellow Frontiers in Microbiology frontiersin.org

FIGURE
Maximum likelihood phylogenetic tree of taxa and , sites combining LSU, SSU, ITS, tefα, rpb , and rpb sequence data.MLBS higher than % and PP > .are denoted near the nodes as MLBS/PP, and the newly generated sequences are in red bold font.The genus clade Pleurocordyceps is highlighted in green, while the outgroup is marked with a light orange background.
Frontiers in Microbiology frontiersin.orgNotes: Pleurocordyceps neoagarica (Host: Ophiocordyceps neogryllotalpae) differs from P. agarica (Host: Ophiocordyceps barnesii) morphologically due to its distinct host, longer synnemata and conidiophore, and shorter phialides (Wang et al., 2015b).P. neoagarica produces only one type of phialides and conidia, whereas P. agarica produces two.In the phylogenetic tree, the new collections (GZLG 23-103) shared a sister relationship with Pleurocordyceps agarica (Figure 1).The type of strain of P. neoagarica differs from P. agarica by 4 bp in ITS, 7 bp in SSU, 4 bp in rpb1, and 14 bp in rpb2 (Wang et al., 2015b).Given the significant morphological differences between these two taxa and their distinct phylogenetic placement, we conclude that they are separate species.
The discovery of the new species of Pleurocordyceps adds to the diversity of the genus and the associated family.Several Pleurocordyceps taxa have been found in China, indicating a high diversity of these organisms in the country.Pleurocordyceps species display variable host specialization (Wang et al., 2012;Xiao et al., 2023).A few are host-specific.Herein, Pleurocordyceps clavisynnema, P. neoagarica, and P. sanduensis were isolated from the same host, Ophiocordyceps neogryllotalpae.This is similar to the previous results, whereby P. nutansis and P. yunnanensis are parasitic on the same fungus, Ophiocordyceps nutans (Wang et al., 2015a;Xiao et al., 2023).Most Pleurocordyceps taxa are not hostspecific, and multiple species have been documented in the same host (Bischoff et al., 2003;Wang et al., 2012Wang et al., , 2015a,b;,b;Matočec et al., 2014;Crous et al., 2017;Xiao et al., 2018).Members of the genus parasitize insects and fungi, several species of which have broad geographic distributions possibly reflecting the diversity of Pleurocordyceps habitats.Future studies should focus on collecting additional Polycephalomycetaceae taxa to not only uncover the full extent of diversity of this family but also understand their distribution in relation to their hosts.
Colonies on PDA fast-growing, obtained from tissue, reaching 5 cm wide in 20 days at 25 • C, white, reverse yellow to brown, presenting multiple radiating ring-like distributions.Synnemata emerging after 25 days, solitary, unbranched, 0.1-0.5 mm long, distribution at the edge, with small or without a fertile head.Conidial masses covered the surface of the colony, pale yellow when young, later change to brown color, slime.China, Guizhou Province, Qiannan Buyi and Miao Autonomous Prefecture, Sandu Shui Autonomous County.Parasitic on Ophiocordyceps neogryllotalpae associated with the larva of Gryllotalpa species, in soil, collected on 10 April 2022, Xingcan Peng, SD16 (GZLG 23-104, holotype; ex-type living culture, GZCC 22-2044). characteristics: