Six new species of zombie-ant fungi from Yunnan in China

Abstract

Some Ophiocordyceps species infecting ants are able to manipulate the host behavior. The hosts are manipulated in order to move to location that are advantageous for fungal spore transmission. Ophiocordyceps species that are able to manipulate the ant's behavior are called "zombie-ant fungi". They are widespread within tropical forests worldwide, with relatively few reports from subtropical monsoon evergreen broad-leaf forest. Zombie-ant fungi have been described and reported in different countries worldwide. However, there were a few reports from China. This study proposed six new species of zombie-ant fungi from China based on multi-gene (SSU, LSU, TEF, RPB1 and RPB2) phylogenetic analyses and morphological characteristics. Six novel species of Ophiocordyceps from China were identified as the Ophiocordyceps unilateralis core clade, forming a separate lineage with other species. Six novel species of Ophiocordyceps with hirsutella-like asexual morphs exclusively infecting ants were presented herein, namely, Ophiocordyceps acroasca, Ophiocordyceps bifertilis, Ophiocordyceps subtiliphialida, Ophiocordyceps basiasca, Ophiocordyceps nuozhaduensis and Ophiocordyceps contiispora. Descriptions and illustrations for six taxon were provided. Five of these species were collected from the subtropical monsoon evergreen broad-leaf forest, and one was collected from the rainforest and subtropical monsoon evergreen broad-leaf forest. This work proposes that the same host of Camponotus can be infected by multiple ant pathogenic fungi, while multiple ants of Polyrhachis can be infected by the same pathogenic fungi at the same time. This study contributes towards a better understanding of the evolutionary relationship between hosts and fungi, and provides novel insights into the morphology, distribution, parasitism, and ecology of Ophiocordyceps unilateralis sensu lato. We have provided a method for obtaining living cultures of Ophiocordyceps unilateralis complex species and their asexual morphs based on the living cultures, which is of significant value for further studies of Ophiocordyceps unilateralis complex species in the future.

INTRODUCTION

Evolutionary relationships between fungi and insects, from parasitism to mutualism, have been widely studied (Suh et al. 2005; Cheek et al. 2020; Haelewaters et al. 2020). Insects are diverse, with more than a million described species (Foottit and Adler 2009), in 29 orders (Misof et al. 2014). The fungal pathogens are able to colonize 19 of 29 orders, resulting in the evolution of extensive diversity of strategies and morphologies, by using the insect body for infection and onward transmission (Araújo and Hughes 2016). Among these insects and fungi strategies, one of the most impressive and sophisticated involved ants and species of fungi within the genus Ophiocordyceps (Andersen et al. 2009). The species of Ophiocordyceps had colonized 13 orders of insects (Crous et al. 2004; Araújo and Hughes 2016), comprised of more than 300 species of entomopathogens (Kepler et al. 2011; Sanjuan et al. 2015; Crous et al. 2016; Araújo et al. 2018; Khonsanit et al. 2018; Araújo and Hughes 2019; Wei et al. 2020; Tang et al. 2022; Xu et al. 2022). The insect hosts orders infected by these fungi included Coleoptera, Diptera, Hemiptera, Hymenoptera, Isoptera, Lepidoptera, Neuroptera, Dragonflies, and Orthoptera (Araújo et al. 2015; Araújo and Hughes 2019). Ants (Hymenoptera) were widely distributed in the arctic to tropical, occupying a wide range of habitats from high canopy to leaf litter; their colonies ranged from a few dozen (Jahyny et al. 2002) to millions of individuals (Currie et al. 2003). In tropical forests, they contributed as much as 50% of animal biomass (Hölldobler et al. 2009). Among the hosts of many entomopathogenic fungi, ants were also the most common host of species within Ophiocordyceps (Evans and Samson 1982; Evans et al. 2011b; Kepler et al. 2011; Luangsa-ard et al. 2011; Kobmoo et al. 2012, 2015; Araújo et al. 2015, 2018, 2020; Sanjuan et al. 2015; Spatafora et al. 2015; Crous et al. 2016; Tasanathai et al. 2019; Wei et al. 2020; Tang et al. 2022; Xu et al. 2022).

Ophiocordyceps was erected by Petch (1931) to accommodate the species of Cordyceps that produce non-disarticulating ascospores. The term as a subgeneric classification was used by Kobayasi, based solely on ascospores morphology, and essentially adopted the diagnosis of Petch (Kobayasi 1941; Petch 1931). Then the subgenera Ophiocordyceps was transferred as subgenus of Cordyceps sensu lato (Mains 1958). The three new families were well-supported in Sung et al. (2007) study, hence their proposition to split them into 3 families (Ophiocordycipitaceae, Clavicipitaceae and Cordycipitaceae). Ophiocordyceps was proposed as a genus of Ophiocordycipitaceae. The classification system of Cordyceps sensu lato was widely accepted (Sung et al. 2007). Ophiocordyceps unilateralis sensu stricto was originally published as Torrubia unilateralis (Tulasne and Tulasne 1865). Torrubia unilateralis was transferred to Ophiocordyceps (Petch 1931). Evans et al. (2018) moved to epitypify O. unilateralis sensu stricto and to clarify its description, providing an interpretive type that was more effective in a biological sense than the illustrations by Tulasne; it was proposed to distinguish O. unilateralis sensu stricto and O. unilateralis sensu lato. Asexual morphs associated with Ophiocordyceps included Hirsutella, Syngliocladium, Stilbella, Paraisaria, Hymenostilbe and Sorosporella (Quandt et al. 2014). Hirsutella, Stilbella, Paraisaria and Hymenostilbe were recorded to be associated with ants. Asexual morphs Hymenostilbe and Hirsutella were commonly found associated with ants (Evans and Samson 1982, 1984; Araújo et al. 2015; Araújo and Hughes 2017).

Members of the O. unilateralis complex were ordinary among the pathogenic fungi on ants (Evans et al. 2011a, 2011b). These fungi could change ant behavior controlling it to leave the nest to die, usually in an exposed position in which they were attached or biting leaves or branches in a "death grip" (Hughes et al. 2011). The manipulative behavior caused by species within O. unilateralis complex has attracted extensive attention (Moore 1995, Thomas et al. 2010, Poulin and Maure 2015, de Bekker et al. 2018, Hafer-Hahmann 2019, Will et al. 2020). However, the mechanism of manipulating host behavior remained unknown (Herbison 2017; Will et al. 2020). Many studies have often used the term O. unilateralis sensu lato for the zombie-ant fungus, including the evolutionary relationship between fungi and hosts, the mechanism of manipulating host behavior, and genomes (Andersen et al. 2009; Hughes et al. 2009; Pontoppidan et al. 2009; Evans et al. 2011a). Regarding the evolutionary relationship between fungi and hosts, Evans et al. (2011b) found that different fungi parasitized different ants; their appearances were very similar but differed in morphological characters. A total of thirty-six species of the O. unilateralis sensu lato have been described. Although this group was estimated to be tens or even hundreds of species worldwide (Evans et al. 2011a), or 580 species discussed by Araújo et al. (Araújo et al. 2018, Araújo and Hughes, unpublished data). There are many species of O. unilateralis sensu lato need further global collections to provide more new taxa to support for exploring the evolutionary relationship between the fungus and its host.

Previous some taxonomic works supported the “one ant-one Ophiocordyceps species” hypothesis (Evans et al. 2011b; Kobmoo et al. 2012; Araújo et al. 2018). They pointed out that host-specific fungal species seemed to be associated to each ant species, leading to the "one ant-one fungus", and the host identity was used as a proxy for fungal identification, such as O. camponoti-atricipis, O. camponoti-balzani, O. camponoti-bispinosi, O. camponoti-chartificis, O. camponoti-femorati, O. camponoti-floridani, O. camponoti-hippocrepidis, O. camponoti-indiani, O. camponoti-leonardi, O. camponoti-melanotici, O. camponoti-nidulantis, O. camponoti-novogranadensis, O. camponoti-renggeri, O. camponoti-rufipedis, O. camponoti-saundersi, O. camponoti-sexguttati, and O. polyrhachis-furca (Evans et al. 2011b; Kobmoo et al. 2012; Araújo et al. 2015, 2018). However, with the deepening of research, different views have emerged, two hosts of the genus Polyrhachis were infected by the ant pathogenic fungus "O. nooreniae" (Crous et al. 2016). Lin et al. (2020) showed that a single species of O. unilateralis sensu lato can infect eight ant species. In addition, Kobmoo et al. (2019) indicated that the ant pathogenic fungus may parasitize the same host based on population genomics study, and constitute further cryptic species, challenging the one ant-one fungus paradigm. The relationship between O. unilateralis sensu lato complex and Formicine ants is still uncertain. Host identification was an important feature to describe and report new taxa. However, in our research, observing hundreds of specimens, we identified that some vital characteristics of the host (such as mouthparts, antennae, legs and abdomens) have been destroyed by pathogenic fungi. Therefore, constructing a host phylogenetic tree using molecular data (COI genes) is of great significance to explore the evolutionary relationship between host and species of O. unilateralis sensu lato.

Ophiocordyceps unilateralis sensu lato has been described and reported in the past two decades. Eighteen species were described from Brazil (Evans and Samson 1982; Evans et al. 2011b; Araújo et al. 2015, 2018), one from Colombia (Araújo et al. 2018), three from the USA (Araújo et al. 2018), one from Ghana (Spatafora et al. 2015), three from Australia (Crous et al. 2016; Araújo et al. 2018), three from Japan (Kepler et al. 2011; Araújo et al. 2018), six from Thailand (Luangsa-ard et al. 2011; Kobmoo et al. 2012, 2015), one from China (Wei et al. 2020). In the past three years, we have also found the species of O. unilateralis sensu lato in Laos and Vietnam (unpublished data). Although multiple taxa of O. unilateralis sensu lato have been described, many questions remain open within the group, such as the evolutionary relationship between host and O. unilateralis sensu lato species, the origins of the group, and the mechanisms that manipulate host behavior. The description and record of the new taxa of O. unilateralis sensu lato is of great importance for the solution of the above problems.

Most species of O. unilateralis sensu lato have been collected from tropical rainforests. There are few or no record of O. unilateralis sensu lato species in the subtropical monsoon evergreen broad-leaf forest. Few species of O. unilateralis sensu lato were reported in China (Wei et al. 2020). The unique geographical location of southwest China is an important area for the diversity of Cordyceps sensu lato. Many species of Ophiocordyceps have been reported from Yunnan province, for example, O. laojunshanensis (Chen et al. 2011), O. lanpingensis (Chen et al. 2013), O. alboperitheciata (Fan et al. 2021), O. pingbianensis (Chen et al. 2021). Our team has spent the past more than two decades investigating and collecting entomopathogenic fungi to describe more new species and to solve taxonomic problems. The six novel species presented herein were collected from Yunnan province in China. Based on morphological and phylogenetic analyses, all species were identified as part of the core clade of O. unilateralis. This study aims to provide additional new taxa that support understanding of the evolutionary relationships between fungi and their hosts, providing novel insights into their living cultures, morphology, ecology, parasitism, and distribution.

MATERIALS AND METHODS

Sampling and isolation

All specimens were collected from Yunnan Province in China in this work. Most specimens were collected from Sun River National Park; some were from Nuozhadu Nature Reserve and Mohan Town, Mengla County. Specimens were noted (e.g., vegetation type, death position, altitude above ground) and photographed in the field, then placed in a sterilized boxes, returned to the laboratory, and stored at 4 °C. Before obtaining axenic cultures, the specimens' fertile region (ascomata) was examined using an Olympus SZ61 stereomicroscope (Olympus Corporation, Tokyo, Japan). Stromata was removed from the head of the ant for morphological observation (sexual and asexual morph). The sclerotium (body of the ant) was immersed in 30% H₂O₂ for 5–8 min, immersed in 75% ethanol for 1 min, and rinsed five times in sterilized water (the specimens must be complete). After drying on sterilized filter paper, the sclerotium was divided into four segments (the head and abdomen were divided into the same two-part, respectively) and inoculated onto solid medium plates (potato 200 g/L, dextrose 20 g/L, agar 20 g/L, yeast powder 10 g/L and peptone 5 g/L), cultured at 25–28 °C (normal temperature was the best condition). Pure cultures were transplanted to a PDA slant, and stored at 4 °C. The specimens were deposited in the Yunnan Herbal Herbarium (YHH) of Yunnan University. The cultures were stored in Yunnan the Fungal Culture Collection (YFCC) of Yunnan University.

Morphological observations

For sexual morph observation, ascomata were photographed and measured by using an Olympus SZ61 stereomicroscope (Olympus Corporation, Tokyo, Japan). Free-hand or frozen sections of the fruiting structures were mounted in lactophenol cotton blue solution for microscopic study and photomicrography. The frozen sections were used by Freezing Microtome HM525NX (Thermo Fisher Scientific, Massachusetts, America). Micro-morphological characteristics (perithecia, asci, apical caps and ascospores) of fungi were examined using Olympus CX40 and BX53 microscopes. Two methods were used for asexual morphological observations. One was directly observed from stromata, sutures, legs and joints of specimens, and another was observed from the pure culture on solid medium plates. Cultures on solid medium plates were incubated for 30–40 days at 25 °C and photographed using a Canon 750 D camera (Canon Inc., Tokyo, Japan). The solid medium was made 0.5–1 mm thick, then divided into 5 mm long and 5 mm wide. Finally, the medium was placed on the glass slide in the sterile culture dish (there was a glass rod to cushion that it could not be submerged in sterile water). The colony was placed on a solid medium, gently covered the cover slide, added sterile water 3 ml, and placed at 25 °C for 30–40 days. The BX53 microscope and Olympus CX40 were used to examine the asexual characteristics such as conidiophores, conidiogenous cells and conidia. Unfortunately, we were not able to study the germination process in most species because the samples had been previously dried.

DNA extraction, polymerase chain reaction (PCR), and sequencing

Specimens and axenic living cultures were prepared for DNA extraction, and the specimens were treated in the same way as the axenic cultures prior to DNA extraction. Total DNA was extracted using the CTAB method, following the described by Liu et al. (2001). Five genes (SSU, LSU, TEF, RPB1, RPB2) and COI genes were amplified and sequenced. The primer pair NS1 and NS4 were used to amplify a fraction of the nuclear ribosomal small subunit (SSU) (White et al. 1990). The primer pair LR0R (Hopple 1994) and LR5 (Vilgalys and Hester 1990) were used to amplify the nuclear ribosomal large subunit (LSU). The primer pair 2218R and 983F were used to amplify the translation elongation factor 1α (TEF) (Rehner and Buckley 2005). The primer pairs RPB1 and RPB1Cr_oph, fRPB2-7cR and fRPB2-5F, were used to amplify the largest and second largest subunits of RNA polymerase II (RPB1 and RPB2), respectively (Liu et al. 1999; Castlebury et al. 2004; Araújo et al. 2018). The primer pair, LCO1490 and HCO2198 (Hebert et al. 2003) was used to amplify the COI gene. The polymerase chain reaction (PCR) matrix was performed in a final volume of 25 µl, composed of 17.25 µl of sterile water, 2.5 µl of PCR 10 × Buffer (2 mmol/l Mg2+) (Transgen Biotech, Beijing, China), 2 µl of dNTP (2.5 mmol/l), 1 µL of forwarding primers (10 µmol/), 1 µl of reverse primers (10 µmol/l), 0.25 µl of Taq DNA polymerase (Transgen Biotech, Beijing, China), 1 µl of DNA template (500 ng/µl). Amplification reactions were performed in a BIO-RAD T100TM thermal cycler (BIO-RAD Laboratories, Hercules, CA, United States). The PCR program of five genes was conducted as described by Wang et al. (2020), and the COI gene was conducted as described by Hebert et al. (2003). The Beijing Genomics Institute (Chongqing, China) performed the target gene amplification and sequencing.

Phylogenetic analyses

Phylogenetic analyses of fungi

Phylogenetic analyses were based on sequences of five genes (SSU, LSU, TEF, RPB1 and RPB2). Sequences of multiple genes from various species (see Table 1) were retrieved from GenBank and the nucleotide sequences were combined with those generated in our study. Information on specimens and GenBank accession numbers were listed in Table 1. Sequences were aligned using Clustal X (v.2.0) (Larkin et al. 2007), poorly-aligned regions were removed and adjusted manually using MEGA6 (v.6.0) (Tamura et al. 2013). We generated one fungi dataset (SSU, LSU, TEF, RPB1 and RPB2). Modelfinder (Kalyaanamoorthy et al. 2017) was used to select the best-fitting likelihood model for maximum likelihood (ML) analyses, and Bayesian inference (BI) analyses were carried out for the fungi datasets. The Corrected Akaike Information Criterion (AIC) was used to select the model for each gene, and the best-fitting models were provided in Table 3. For ML analyses, tree searches were performed in IQ-tree (v.2.1.3) (Nguyen et al. 2015) based on the best-fit model with 5000 ultrafast bootstraps (Hoang et al. 2017) in a single run. BI analyses were conducted using MrBayes (v.3.2.2) (Ronquist et al. 2012). Four Markov Chain Monte Carlo chains were run, each beginning with a random tree and sampling, one tree every 100 generations of 2000,000 generations, and the first 25% of samples were discarded as burn-in. Each tree was visualized with its maximum-likelihood bootstrap support values (ML-BS) and Bayesian inference posterior probability (BI-PP) in Figtree (v.1.4.3). Adobe Illustrator CS6 was used for editing.

Table 1 Voucher information, GenBank accession numbers, host and location of the taxa used in this study

Phylogenetic analyses of ants

Phylogenetic analyses were based on COI gene sequences. Sequences of COI gene from various species (see Table 2) were retrieved from GenBank and the nucleotide sequences were combined with those generated in our study. Information on specimens and GenBank accession numbers were listed in Table 2. Sequences were aligned using Clustal X (v.2.0) (Larkin et al. 2007), poorly-aligned regions were removed and adjusted manually using MEGA6 (v.6.0) (Tamura et al. 2013). One host dataset (COI) was generated. Modelfinder (Kalyaanamoorthy et al. 2017) was used to select the best-fitting likelihood model for maximum likelihood (ML) analyses, and Bayesian inference (BI) analyses were carried out for the host datasets. The Corrected Akaike Information Criterion (AIC) was used to select the model for each gene, and the best-fitting models were provided in Table 3. The latter method was consistent with the phylogenetic analyses of fungi.

Table 2 The COI genes and GenBank accession numbers of the taxa were used in this study

Table 3 Results of the best-fitting likelihood model for maximum likelihood (ML) and Bayesian inference (BI) for the two datasets

RESULTS

Phylogenetic analysis of the genus Ophiocordyceps

Sequences of 129 samples were used for phylogenetic analysis. Tolypocladium inflatum OSC 71235 and Tolypocladium ophioglossoides CBS 100239 were designated as outgroups. The total length of the concatenated dataset of five genes across the 129 samples was 4785 bp, including 1057 bp for SSU, 952 bp for LSU, 965 bp for TEF, 738 bp for RPB1, and 1073 bp for RPB2. The phylogenetic relationships showed four clades in Ophiocordyceps, including the Hirsutella clade, O. sphecocephala clade, O. sobolifera clade and O. ravenelii clade. Ophiocordyceps unilateralis clade (34 species; BP = 100%, PP = 99%), O. kniphofioides sub-clade (3 species; BP = 94%, PP = 96%) and O. oecophyllae clade (1 species; BP = 99%, PP = 100%) were strongly supported by BI and ML analyses (Fig. 1). All the species collected and described in this work were clustered in the O. unilateralis core clade and clustered into a clade with O. unilateralis sensu lato species reported in Asian African (Ghana, Japan, Thailand) and Oceania (Australia) countries.

Fig. 1

The phylogenetic tree of Ophiocordyceps and its related genera was inferred from five-gene dataset (SSU, LSU, TEF, RPB1, RPB2) based on Bayesian inference and maximum likelihood analyses. The illustration indicated to characteristics of new species. Tolypocladium inflatum OSC 71235 and Tolypocladium ophioglossoides CBS 100239 were designated as outgroups

Phylogenetic analysis of host ants

Sequences of 97 specimens were used for phylogenetic analysis. Dolichoderus bispinosus was designated as the outgroup. Phylogenetic relationships have demonstrated that the phylogenetic trees consist of Camponotus, Polyrhachis, Paraponera, Oecophylla and Dolichoderus. Phylogenetic tree showed that O. bifertilis had two ant hosts (Fig. 2), namely, Polyrhachis sp.1 (Polyrhachis sp. YHH 20163, Polyrhachis sp. YHH 20164, Polyrhachis sp. 20601) and Polyrhachis sp.2 (Polyrhachis sp. YHH 20603, Polyrhachis sp. YHH 20604, Polyrhachis sp. YHH 20602, Polyrhachis sp. YHH 20162), with being a higher bootstrap value and posterior probability. Camponotus leonardi was sister to Camponotus sp. based on the host phylogenetic relationships. Their pathogenic fungi, such as O. nuozhaduensis and O. camponoti-leonardi, were also sister species. Notably, the phylogenetic relationships also showed that these ant pathogenic fungi, i.e., O. basiasca, O. contiispora, O. acroasca, O. subtiliphialida, parasitized on the same host Camponotus sp. (Fig. 2).

Fig. 2

The phylogenetic tree of Polyrhachis and Camponotus including 97 taxa reconstructed using Bayesian inference and maximum likelihood. Each value at a node indicates a Bayesian posterior probability and bootstrap proportions. The Latin name refered to the pathogenic fungus that infected the host ant

TAXONOMY

Ophiocordyceps acroasca Hong Yu bis & D.X. Tang, sp. nov.

Mycobank: MB 844350 (Fig. 3)

Etymology: The epithet refered to ascomata of lateral cushions produced from the top of stromata.

Diagnosis: Similar to O. septa in immersed and ostiole perithecia, but O. acroasca differs by ascomata arising from the top of stromata.

Type: China: Yunnan, Puer City, Sun River Natioal Park. Camponotus sp. was infected and bited into a leaf of tree seedling, 22°35′38″ N, 101°6′36″ E, alt. 1452 m, 18 Aug. 2020, Hong Yu bis (YHH 20121 – holotype preserved in the Yunnan Herbal Herbarium; living culture YFCC 9016 – ex-holotype stored in Yunnan Fungal Culture Collection).

Description: Sexual morph: External mycelia produced from the legs and body of the host. Stromata single and curved at the top, produced from dorsal pronotum of the ant, cylindrical, clavate, dark brown at maturity, the top was lighter than other parts of stromata. Fertile regions (ascomata) of lateral cushions produced from the top of stromata, one to two ascomata were found, hemispherical, brown, averaging 3 × 2–3 mm. Perithecia ovoid, immersed to partially erumpent, with short, exposed neck or rounded ostiole, 247–296 × (170–) 176–225 (–238) μm. Asci cylindrical, hyaline, curved, thick, 8-spored, (126–) 131–172 (–180) × 5–8 μm. Ascus caps hemispherical, prominent and small, 3–5 µm high and 4–6 µm wide. Ascospores vermiform, thin-walled, hyaline, 4–5-septate, slightly curved to sinuous, round to slightly tapered at the apex, (76–) 83–108 (–113) × 2–3 µm. Asexual morph: Colonies on PDA slow-growing, 26–27 mm diameter in 60 days at 25 °C, milky white to light brown, hard, with protuberant mycelial at the surface, the pigment produced around colonies, dark brown, reverse light brown to dark brown. Hyphae branched, septate, smooth-walled, hyaline. Hirsutella type-A and Hirsutella type-C produced from colonies, Hirsutella not examined from sutures and joints because the specimens were used to isolated strains. Conidiogenous cells monophialidic, produced from hyphae, smooth, swollen base, cylindrical to lageniform, tapering gradually or abruptly a long neck, slight bending, 17–30 × 1–4 µm. Conidia limoniform, solitary, hyaline, smooth-walled, 2–3 × 1–2 µm.

Germination process: No germination observed because the specimens were dried.

Host: Camponotus sp. (Formicinae)

Habitat: Subtropical monsoon evergreen broad-leaf forest. Infected Camponotus sp. was found biting into a leaf of tree seedling; from 0.5 to 2 m above the ground.

Distribution: China, Yunnan Province, Puer City

Material examined: China: Yunnan, Puer City, Sun River National Park. Infected ants were found biting into a leaf of tree seedling, 22°38′2″ N, 101°6′7″ E, alt. 1468 m, 19 Aug. 2020, Hong Yu bis (living culture YFCC 9017, YFCC 9018, YFCC 9019, YFCC 9049) and 22°34′34″ N, 101°6′24″ E, alt. 1095 m, 23 Aug. 2021, D.X. Tang (YHH 20122).

Notes: Phylogenetic analyses showed that O. acroasca formed a sister lineage with O. septa, and was clustered in the O. unilateralis core clade of Hirsutella, with strong statistical supported by bootstrap proportions (BP = 90%) (Fig. 1). Ophiocordyceps acroasca was similar to O. septa in the behavior of the host biting a leaf, cylindrical or clavate stromata, immersed and ostiole perithecia. However, it differed from O. septa by ascomata of lateral cushion arising from the top of stromata, vermiform ascospores, producing Hirsutella type-A and Hirsutella type-C, cylindrical to lageniform conidiogenous cells, limoniform conidia. In addition, the sizes of perithecia, ascomata, asci, ascospores, phialides, and conidia also differed from O. septa (Table 4).

Fig. 3

Ophiocordyceps acroasca. A: Infected Camponotus sp. was biting into a leaf of tree seedling. B: The ascoma was produced from the stroma. C, D: Cross-section of the ascoma showing the perithecial arrangement. E, F: Asci. G, H: Ascospores. I, J: Colonies on PDA medium. K, L: Conidiogenous cells and conidia. M: Conidia. Scale bars: A = 3000 µm; B = 2000 µm; C = 200 µm; D = 100 µm; E–G = 50 µm; H = 20 µm; I, J = 2 µm; K, L = 10 µm; M = 2 µm

Table 4 Comparison of morphological characters and host of Ophiocordyceps unilateralis sensu lato in this study

Ophiocordyceps bifertilis Hong Yu bis & D.X. Tang, sp. nov.

Mycobank: MB 844351 (Fig. 4)

Etymology: The epithet refered to two fertile regions produced from stromata.

Diagnosis: Ophiocordyceps bifertilis similar to O. satoi regarding the production of multiple stalks, but O. bifertilis differed by stromata branching, with only two ascomata.

Type: China: Yunnan, Puer City, Sun River National Park. An adult Polyrhachis sp. was hanging upside down on the underside of the leaves, 2°20′24″ N, 101°6′43″ E, alt. 1487 m, 18 August 2020, Hong Yu bis (YHH 20160 – holotype preserved in the Yunnan Herbal Herbarium; living culture YFCC 9012 – ex-holotype stored in Yunnan Fungal Culture Collection).

Description: Sexual morph: External mycelia scarce, produced from sutures and joints. One to multiple stromata at the head of the ant, few branching, curved, cylindrical, clavate, dark brown. Ascomata of lateral cushions produced from stromata, two ascomata were observed, disc-shaped or hemispherical, brown, averaging 3 × 2–3 mm. Perithecia flask-shaped, immersed to partially erumpent, with short, exposed neck or rounded ostiole, (149–) 156–211 (–236) × (91–) 102–129 (–134) μm. Asci cylindrical, hyaline, 8-spored, (123–) 130–198 (–211) × 6–10 μm. Ascus caps were hemispherical, prominent, 3–5 µm high, and 5–6 µm wide. Ascospores fusiform, hyaline, 4–5-septate, round to tapered at the apex, 70–94 (–96) × 2–4 µm. Asexual morph: Colonies on PDA grows slowly, 19–20 mm diameter in 120 days at 25 °C, light purple to light brown, hard, with protuberant mycelia at the edge, reverse light brown to dark brown, pigment light brown to dark brown. Hirsutella type-A was present along stromata; Hirsutella was not observed from the sutures and joints. Phialides lageniform, smooth, swollen base, tapering abruptly a neck, short, 9–24 (–29) × 2–4 µm. Conidia were not observed.

Germination process: No germination observed because the specimens were dried.

Host: Polyrhachis sp.1 and Polyrhachis sp.2 (Formicinae)

Habitat: Subtropical monsoon evergreen broad-leaf forest. Infected Polyrhachis sp.1 was found biting into a leaf of Pteridophyta, and Polyrhachis sp.2 biting into a leaf of Gramineae, always at lower heights, ranging from 0.5 to 1.5 m.

Distribution: China, Yunnan Province, Puer City

Material examined: China: Yunnan, Puer City, Sun River National Park. Adult Polyrhachis sp.1 and Polyrhachis sp.2 were hanging upside down on the underside of the leaves of Pteridophyta and Gramineae, 22°35′50″ N, 101°6′39″ E, alt. 1529 m, 19 Aug. 2020, Hong Yu bis (living culture YFCC 9013, YFCC 9048) and 22°35′51″ N, 101°6′40″ E, alt. 1532 m, 23 Aug. 2021, D.X. Tang (YHH 20162, YHH 20163, YHH 20164).

Notes: Phylogenetic analyses revealed that O. bifertilis formed a sister lineage with O. satoi and O. naomipierceae, was clustered in the O. unilateralis core clade of Hirsutella, with statistical support from BI posterior probabilities (PP = 95%) and ML bootstrap proportions (BP = 89%) (Fig. 1). Ophiocordyceps bifertilis was similar to O. satoi and O. naomipierceae in the behavior of the host Polyrhachis infected and biting a leaf. In addition, it was also similar to O. satoi in clavate stromata, flask-shaped perithecia, Hirsutella type-A, lageniform phialides. However, it differed from O. satoi by branching stromata, fusiform ascospores. Moreover, the sizes of phialides also differed from O. satoi and O. naomipierceae (Table 4).

Fig. 4

Ophiocordyceps bifertilis. A: Infected Polyrhachis sp.1was biting a leaf of Pteridophyta. B: Two ascomata plates attached to stromata. C, D: Cross-section of the ascoma showing the perithecial arrangement. E, F: Asci. G, H: Ascospores. I, J: Colonies on PDA medium. K, L: Phialides. Scale bars: A = 4000 µm; B = 1000 µm; C = 200 µm; D = 100 µm; E, F = 50 µm; G, H = 20 µm; I, J = 2 cm; K–M = 10 µm

Ophiocordyceps subtiliphialida Hong Yu bis & D.X. Tang, sp. nov.

Mycobank: MB 844352 (Fig. 5)

Etymology: The epithet refered to the phialides slender than related species.

Diagnosis: Similar to O. contiispora in phialides monophialidic or rarely polyphialidic, but phialides of O. subtiliphialida (70–116 × 1–3 µm) was slender than O. contiispora (57–92 × 1–4 µm).

Type: China: Yunnan, Puer City, Sun River National Park. Camponotus sp. was infected and bited into a leaf of tree seedling, 22°34′34″ N, 101°6′24″ E, alt. 1420 m, 18 Aug. 2020, Hong Yu bis (YHH 20139 – holotype preserved in the Yunnan Herbal Herbarium; living culture YFCC 8815 – ex-holotype stored in Yunnan Fungal Culture Collection).

Description: Sexual morph: External mycelia produced from the sutures and joints of the ant. Stromata single, produced from dorsal pronotum of the ant, cylindrical, clavate, brown at maturity. Fertile part of lateral cushions produced from stromata, 1–2, disc-shaped, brown, averaging 2 × 1.2–1.9 mm. Perithecia flask-shaped, immersed to partially erumpent, with short, exposed ostiole, (195–) 199–296 (–303) × (87–) 97–161 (–168) μm. Asci cylindrical, hyaline, short and wide, 8-spored, 89–119 × 5–9 μm. Ascus caps hemispherical, 2–4 µm high, 5–7 µm wide. Ascospores lanceolate, hyaline, 6–7-septate, slightly curved, round to tapered at the apex, 52–72 × 5–7 (–8) µm. Asexual morph: Colonies grows slowly on PDA medium, 19–20 mm diameter in 60 days at 25 °C, milky white to light brown, raising cottony-shaped mycelia density at the edge, protuberant mycelia light brow at the centrum, reverse light brown to dark brown. Hyphae immersed in the medium, milky white, branched, septate, smooth-walled, hyaline. Hirsutella type-C only. Conidiophores rare, cylindrical, produced from the hyphae, septate, short and wide. Phialides monophialidic or rarely polyphialidic, forming on side hyphae or the conidiophores, smooth, slight swollen base, lageniform, septate, tapering gradually a slender neck, slight bending, 70–116 (–124) × 1–3 µm. Conidia olivary, solitary, hyaline, smooth-walled, 6–10 × 3–6 µm.

Germination process: Ascospores germinating in 72 h to produce 1–4, long and narrow capilliconidiophore, (44–) 58–79 μm long, 0.8–1.9 μm wide, bearing a single capilliconidium, averaging (6–) 7–9 × 2–3 μm.

Host: Camponotus sp. (Formicinae).

Habitat: Subtropical monsoon evergreen broad-leaf forest. Infected Camponotus sp. was found biting into a leaf of a sapling. Died in the lower position, collected from 0.5 to 1 m.

Distribution: China, Yunnan Province, Puer City.

Material examined: China: Yunnan, Puer City, Sun River National Park. ​Infected Camponotus sp. was found biting into a leaf of a sapling, 22°35′51″ N, 101°6′40″ E, alt. 1430 m, 19 Aug. 2020, Hong Yu bis (living culture YFCC 8814, YFCC 8816, YFCC 8817).

Notes: Phylogenetic analyses showed that the four samples of the O. subtiliphialida group together with high statistical support (PP = 60%; BP = 100%), were clustered within the O. unilateralis core clade of Southeast Asian countries (Fig. 1). It was similar to O. septa, O. acroasca and O. basiasca in swollen and lageniform base. However, it differed from O. septa, O. acroasca and O. basiasca by lanceolate ascospores, rare conidiophores, monophialidic or rarely polyphialidic phialides, tapering a narrow and slender neck, olivary conidia.

Fig. 5

Ophiocordyceps subtiliphialida. A, C: Camponotus sp. was infected and bited into a leaf of a sapling. D: Fertile structure produced from the stroma; E–F: Cross-section of the ascoma showing the perithecial arrangement; G: Asci; H, I: Ascospores. J, K: Ascospore with long capilliconidia. L, M: Colonies on PDA medium. N–V: Conidiogenous cells and conidia. Scale bars: A, B = 0.4 cm; C = 0.2 cm; D = 0.1 cm; E = 200 µm; F = 100 µm; G = 50 µm; H–J = 20 µm; K = 50 µm; L, M = 2 cm; N, O = 20 µm; P–T = 50 µm; U, V = 5 µm

Ophiocordyceps basiasca Hong Yu bis & D.X. Tang, sp. nov.

Mycobank: MB 844353 (Fig. 6)

Etymology: The epithet refered to ascomata of lateral cushions produced from the basal of stromata.

Diagnosis: Similar to O. contiispora in conidia olivary, however, ascospores vermiform of O. basiasca was differed to O. contiispora (fusiform).

Type: China: Yunnan, Puer City, Sun River National Park. Camponotus sp. was infected and bited the middle vein of a leaf of tree seedling, 22°38′2″ N, 101°6′7″ E, alt. 1468 m, 19 Aug. 2020, Hong Yu bis (YHH 20190 – holotype preserved in the Yunnan Herbal Herbarium).

Description: Sexual morph: External mycelia produced from the sutures and joints, one stroma at the head of the ant, curved at the top, cylindrical, clavate, the base of stromata were dark brown, pale white at the top. Ascomata of lateral cushions produced from the basal of stromata, one ascoma was observed, spherical, brown, averaging 3 × 2 mm. Perithecia flask-shaped or ovoid, immersed to partially erumpent, with short, exposed neck or rounded ostiole, (195–) 202–242 (–248) × (92–) 102–149 μm. Asci cylindrical, hyaline, 8-spored, 96–188 (–212) × 4–9 (–10) μm. Ascus caps hemispherical, 3–5 µm high, 4–5 µm wide. Ascospores vermiform, hyaline, 4–5-septate, round to slightly tapered at the apex, 89–119 (–122) × 2–3 µm. Asexual morph: Hirsutella type-A only. Phialides lageniform, smooth, swollen base, tapering abruptly a neck, short, (8–) 10–23 (–26) × 1–5 µm. Conidia oviform, hyaline, smooth-walled, 1–4 × 1–2 µm.

Germination process: No ascospores examined from dried specimens.

Host: Camponotus sp. (Formicinae)

Habitat: Subtropical monsoon evergreen broad-leaf forest. Camponotus sp. was infected and bited into a leaf of tree seedling. It was collected from 1.5 m above the ground.

Distribution: China, Yunnan Province, Puer City

Material examined: China: Yunnan, Puer City, Sun River National Park. Infected ants were found biting into a leaf of tree seedling, 22°38′2″ N, 101°6′7″ E, alt. 1468 m, 19 August 2020, Hong Yu bis (YHH 20191).

Notes: Phylogenetic analyses showed that O. basiasca formed a separate clade in the O. unilateralis core clade; it was closed to O. subtiliphialida and O. contiispora, with statistical supported from BI posterior probabilities (PP = 100%) and ML bootstrap proportions (BP = 97%) (Fig. 1). Ophiocordyceps basiasca was similar to O. subtiliphialida and O. contiispora in lageniform phialides, olivary conidia. However, it differed from O. subtiliphialida and O. contiispora by vermiform ascospores, Hirsutella type-A.

Fig. 6

Ophiocordyceps basiasca. A: Infected Camponotus sp. was biting into a leaf of tree sapling. B, C: Cross-section of the ascoma showing the perithecial arrangement. D, E: Asci. F–H: Ascospores. I–L: Phialides and conidia. Scale bars: A = 2000 µm; B = 200 µm; C = 100 µm; D, E = 50 µm; F–H = 20 µm; I–L = 5 µm

Ophiocordyceps nuozhaduensis Hong Yu bis & D.X. Tang, sp. nov.

Mycobank: MB 844354 (Fig. 7)

Etymology: The epithet refered to the locality (Nuozhadu) where the holotype was collected.

Diagnosis: Similar to O. camponoti-leonardi in perithecia rounded ostiole, but O. nuozhaduensis differs by ellipsoidal or oviform conidia, smaller flask-shaped perithecia (215–285 × 128–172 μm).

Type: China: Yunnan, Puer City, Nuozhadu Nature Reserve. Camponotus sp. was infected and bited into a leaf of tree sapling, 22°38′27″ N, 100°29′53″ E, alt. 1107 m, 24 Aug. 2021, Hong Yu bis (YHH 20167 – holotype preserved in the Yunnan Herbal Herbarium).

Description: Sexual morph: External mycelia produced from sutures and joints of the ant. One stroma at the head of the ant, curved at the top, cylindrical, clavate, and dark brown at maturity. Fertile regions of lateral cushions produced from the middle of stromata, one ascoma was observed, spherical, brown, averaging 2.4 × 1.6 mm. Perithecia flask-shaped, immersed to partially erumpent, with short, exposed neck or rounded ostiole, (215–) 222–274 (–285) × (128–) 153–159 (–172) μm. Asci were not observed. Ascospores vermiform, hyaline, 7–13-septate, round to slightly tapered at the apex, 91–126 (–132) × 2–5 µm. Asexual morph: Hirsutella type-A present on the stroma and the legs. Phialides cylindrical or lageniform, smooth, swollen base, tapering abruptly a neck, short, 6–22 (–22) × 2–4 µm. Conidia ellipsoidal or oviform, 2–5 × 2–3 µm.

Germination process: No germination examined because the specimens were dried.

Host: Camponotus sp. (Formicinae)

Habitat: Subtropical monsoon evergreen broad-leaf forest. Camponotus sp. was infected and bited into a leaf of tree sapling. Always at lower heights, collected from 25 to 50 cm above the ground.

Distribution: China, Yunnan Province, Puer City.

Material examined: China: Yunnan, Puer City, Nuozhadu Nature Reserve. Infected ants were found biting a leaf of tree seedling, 22°38′27″ N, 100°29′53″ E, alt. 1107 m, 24 Aug. 2021, Hong Yu bis (YHH 20168, YHH 20169).

Notes: Phylogenetically, this species was closed to O. camponoti-leonardi, was clustered in the O. unilateralis core clade, with high statistical supported by BI (PP = 98%) and ML (BP = 100%) (Fig. 1). It was similar to sister O. camponoti-leonardi in rounded ostiole perithecia. However, it differed from O. camponoti-leonardi in vermiform ascospores, ellipsoidal or oviform conidia.

Fig. 7

Ophiocordyceps nuozhaduensis. A: Camponotus sp. was infected and bited into a leaf of tree sapling. B: The ascoma was produced from the stroma. C: Cross-section of the ascoma showing the perithecial arrangement. D–G: Ascospores. H: Conidiogenous cells and conidia. I–K: Phialides. L, M: Conidia. Scale bars: A = 3000 µm; B = 1000 µm; C = 100 µm; D–G = 20 µm; H = 5 µm; I–K = 10 µm; L, M = 2 µm

Ophiocordyceps contiispora Hong Yu bis & D.X. Tang, sp. nov.

Mycobank: MB 844355 (Fig. 8)

Etymology: The epithet refered to the top of conidia having a protuberance like a spear.

Diagnosis: Similar to O. subtiliphialida in the top of conidia has a protuberance, but the protuberance of O. contiispora was more prominent and the width of conidia was smaller (4–6 × 1–2 μm) than O. subtiliphialida (6–10 × 3–6 μm).

Type: China: Yunnan, Mengla County, Mohan Town, Xinming Village. Camponotus sp. was infected and bited into a leaf of epiphytes, 21°9′35″ N, 101°45′49″ E, alt. 1173 m, 2 Oct. 2019, Hong Yu bis (YHH 20144 – holotype preserved in the Yunnan Herbal Herbarium; living culture YFCC 9027 – ex-holotype stored in Yunnan Fungal Culture Collection).

Description: Sexual morph: External mycelia produced dense from the joints, covering the host body, sparsely when touching the substrate. Stromata single, produced from dorsal pronotum of the ant, cylindrical, clavate, brown at maturity. Fertile part of lateral cushions produced from stromata, one ascoma was observed, disc-shaped, brown, averaging 1.3–1.8 × 1–1.5 mm. Perithecia flask-shaped, immersed to partially erumpent, with short, exposed ostiole, (146–) 158–212 (–224) × 69–122 μm. Asci cylindrical, hyaline, curved, 8-spored, (74–) 89–130 (–134) × 4–9 μm. Ascus caps hemispherical or square, small, 1–3 µm high, 3–5 µm wide. Ascospores fusiform, hyaline, no obvious separation, occasionally curved, round to slightly tapered at the apex, (29–) 38–48 (–62) × 2–4 µm. Asexual morph: Colonies on PDA medium slow-growing, 28–30 mm diameter in 30 days at 25 °C, milky white to light brown, raising cottony-shaped mycelia density, protuberant mycelia at the centrum, reverse light brown to dark brown. Hyphae immersed in the medium, milky white, branched, septate, smooth-walled, hyaline. Hirsutella type-C only. Conidiophores rare, cylindrical, produced from the hyphae, septate, short, 11–12 × 3–4 µm. Conidiogenous cells monophialidic or rarely polyphialidic, forming on side hyphae or conidiophores, smooth, swollen base, lageniform, tapering gradually a long neck, straight, (42–) 57–92 (–97) × 1–4 µm. Conidia olivary or flask-shaped, hyaline, the top of conidia has a protuberance like a spear, smooth-walled, 4–6 × 1–2 µm.

Germination process: No germination observed from dried specimens.

Host: Camponotus sp. (Formicinae)

Habitat: Rainforest and subtropical monsoon evergreen broad-leaf forest. Camponotus sp. was infected and bited into a leaf of epiphytes. Dying in an elevated position, collected from 1 to 2 m above the ground.

Distribution: China, Yunnan Province, Puer City and Jinghong City.

Material examined: China: Yunnan, Mengla County, Mohan Town, Xinming Village. Camponotus sp. was infected and bited into a leaf of epiphytes, 22°21′20″ N, 101°69′01″ E, alt. 865 m, 3 Oct. 2019, D.X. Tang (YHH 20145; living culture YFCC 9026). Other specimens were collected from China, Yunnan Province, Puer City, Sun River National Park. Infected ants were found biting into a leaf of tree sapling, 22°38′2″ N, 101°6′7″ E, alt. 1468 m, 19 Aug. 2020, Hong Yu bis (living culture YFCC 9025).

Notes: Ophiocordyceps contiispora was phylogenetically sister to O. basiasca with high statistical supported by BP = 100% and PP = 100%. It was similar to O. basiasca in flask-shaped perithecia, cylindrical asci, lageniform phialides. However, it differed from O. basiasca by fusiform ascospores, producing Hirsutella type-C.

Fig. 8

Ophiocordyceps contiispora. A: Infected Camponotus sp. was biting into a leaf of epiphytes. B: Close-up of the ascoma. C, D: Cross-section of the ascoma showing the perithecial arrangement. E, F: Asci. G, H: Ascospores. I, J: Colonies on PDA medium. K: Conidiophores and phialides. L–N: Conidiogenous cells and conidia. O, P: Conidia. Scale bars: A = 1000 µm; B = 500 µm; C = 200 µm; D = 100 µm; E, F = 20 µm; G, H = 10 µm; I, J = 2 cm; K–N = 20 µm; O, P = 2 µm

Discussion

Many phylogenetic classifications have been undertaken for O. unilateralis sensu lato (Evans et al. 2011a; kobmoo et al. 2012, 2015; Araújo et al. 2018; Wei et al. 2020), these groups have been continuously supplemented and improved based on morphology, molecular phylogeny and ecology. This study focused on the phylogenetic investigation of O. unilateralis sensu lato  species collected from Yunnan Province, China. The phylogenetic tree showed that six new species were clustered in the O. unilateralis core clade of Hirsutella (Fig. 1). Four species (O. contiispora, O. basiasca, O. subtiliphialida, and O. acroasca) were formed a sister lineage with O. septa. In addition, O. bifertilis formed a sister lineage with O. satoi and O. naomipierceae, and O. nuozhaduensis also formed a sister lineage with O. camponoti-leonardi. The phylogenetic framework was consistent with previous studies (Kobmoo et al. 2012; Crous et al. 2016; Araújo et al. 2018; Wei et al. 2020). However, some species were lower support for topologies, including O. camponoti-leonardi, O. polyrhachis-furcata, O. nuozhaduensis, O. ootakii and O. nooreniae. The reason might be that a few genes were used for O. camponoti-leonardi and O. polyrhachis-furcata. Both phylogenetic analysis and morphological characters supported that the six fungi were distinctive in the core clade of O. unilateralis. Six new species were proposed to be located in the O. unilateralis core clade of the Hirsutella clade within Ophiocordyceps.

The O. unilateralis complex was composed of O. unilateralis core clade, O. oecophyllae clade, O. kniphofioides sub-clades (Araújo et al. 2018). Many species were described in the O. unilateralis core clade, with most of the hosts being Camponotus and Polyrhachis. Species within the O. unilateralis core clade shared many macro-morphological characteristics that made them easily recognized in this study, such as stromata, ascomata, type of host, and location of host attachment. Ophiocordyceps unilateralis complex species commonly bitten and attached leaves, spines, epiphites, saplings, moss, twing in a "death grip" (Evans et al. 2011b, 2018; Hughes et al. 2011; Kepler et al. 2011; Luangsa-ard et al. 2011; Kobmoo et al. 2012, 2015; Araújo et al. 2015, 2018; Crous et al. 2016), with dying in an elevated position, from 0.25 to 2 m or higher above the ground. Fewer O. unilateralis complex species did not have biting and grasping behavior, such as O. tianshanensis (Wei et al. 2020). These species, i.e., O. acroasca, O. basiasca, O. bifertilis, O. contiispora, O. nuozhaduensis, and O. subtiliphialida, died by biting onto the middle vein of a sapling, Pteridophyta, Gramineae, and epiphytes in death position, and at an elevated position, from 0.25 to 2 m above the ground, with results being consistent with previous work (Andersen et al. 2009; Araújo et al. 2018). Species of the O. unilateralis complex have been investigated at the same site in this survey for two years. It was found that the height at which the host died on vegetation from the ground appeared to be affected by climate, such as rainfall, humidity, temperature, etc.. The death position of species in the O. unilateralis complex was 0.5 to 1 m or higher above the ground in the first year, while these species died 0.25 m or less above the ground in the second year. This adaption might occupy a niche and provide for effective spores dispersal (Andersen et al. 2009; Hughes et al. 2011).

The ant manipulation behavior of the O. unilateralis complex occurred only in host-specific species, especially those entomopathogenic fungi that were parasitic on the ants of Camponotus (Evans et al. 2011b; de Bekker et al. 2014; Araújo et al. 2018; Sakolrak et al. 2018). Crous et al. (2016) reported that O. nooreniae infected two host species of Polyrhachis (Polyrhachis cf. hookeri and Polyrhachis lydiae). Our survey also found that a fungus infected multiple hosts of Polyrhachis, and behavior manipulation of the ant almost tended to be consistent, such as the host Polyrhachis sp.1 and Polyrhachis sp.2 were infected by O. bifertilis. The majority of ant pathogenic fungi parasitic on the host of the genus Polyrhachis were reported from Southeast Asia, and some species found in Australia (Crous et al. 2016; Araújo et al. 2018). Pathogenic fungi infecting Polyrhachis ants, such as O. bifertilis, often induced the host to bite onto the main vein of a Pteridophyta leaf. It was similar to O. naomipierceae, O. ootakii, O. polyrhachis-furca, O. nooreniae and O. satoi in phylogeny, habitat, biting and attachment behaviour. The phylogenetic tree of the host ants also indicated that they were closely related species (Fig. 2). This evidence showed that the pathogenic fungi and the host ants were closely related in genetic evolution, and that the diversity of the host might affect the diversity of the pathogenic fungus. Polyrhachis was the second most species-rich genus in Formicinae, currently comprising 706 valid species (http://antcat.org/2022). Polyrhachis originated in Southeast Asia, and dispersed out of Southeast Asia to Australia (Mezger and Moreau 2015). They were widely distributed, ranging from tropical regions in Africa and Asia to Australia and a few Pacific islands. The highest species richness and diversity were in China and Australia. Currently, at least five pathogenic fungi of the host Polyrhachis had been reported in Australia and Southeast Asia. There might exist many pathogenic fungi hosted by Polyrhachis ants to be discovered worldwide, especially in China, Southeast Asia, and Austrilia.

Parasite manipulation of host behavior was an active research topics in various fields (Evans et al. 2011b, 2018; Hughes et al. 2011; Kepler et al. 2011; Luangsa-ard et al. 2011; Kobmoo et al. 2012, 2015; Araújo et al. 2015, 2018; Crous et al. 2016; de Bekker et al. 2018; Will et al. 2020). Multiple reports indicated that manipulation of ant behavior was host-specific (de Bekker et al. 2014, 2018). Host-specific fungal species seemed to be associated with each ant species, leading to the "one ant, one fungus", and the host identity used as criteria for fungal species identification (Evans et al. 2011b; Kobmoo et al. 2012; Araújo et al. 2015, 2018). Population genomics also supported the host-specificity in ant pathogenic fungi by Kobmoo et al. (2019). In this work, the result suggested that multiple ant pathogenic fungi, including O. acroasca, O. basiasca, O. contiispora, O. subtiliphialida (Fig. 2), infecting the same host Camponotus sp.. Interestingly, Kobmoo et al. (2019) revealed that genetic clusters in ant pathogenic fungi sharing the same host. This study supports previous studies that the same host of Camponotus can be infected by different ant pathogenic fungi, while the ant pathogenic fungi of Polyrhachis can infect multiple hosts at the same time. It is not known that an ant fungus infects multiple hosts of the genus Camponotus at the same time. Ophiocordyceps unilateralis complex species were composed of distinct evolutionary species leads to a global diversity of the ant pathogenic fungi (Kobmoo et al. 2012, 2015; Araújo et al. 2015, 2018; Crous et al. 2016). Camponotus was the most species-rich genus in Formicinae, currently comprising 1087 valid species (http://antcat.org/2022). Camponotus ants were distributed in the terrestrial environment worldwide. However, up to now, less than 30 pathogenic fungi have been reported to parasitize Camponotus ants, and some ant pathogenic fungi tend to between sharing the same microhabitat and niche overlap, which might lead to a diversity of the ant pathogenic fungi.

In recent decades, a large amount of research has been conducted to discuss how many fungi exist in the world (Weir and Hammond 1997; Hawksworth 2001; Hawksworth and Lücking 2017). Through the continuous efforts of scientists, the original estimate of 1.5 million (Hawksworth 2001) fungi has changed to 2.2 to 3.8 million fungi (Hawksworth and Lücking 2017). However, relatively few studies have discussed the number of entomopathogenic fungi worldwide. One significant study on host-specificity by Weir and Hammond (1997) in relation to insects was that on the Laboulbeniales on beetles. These studies suggest a beetle (Coleoptera): fungus ratio of 1.68–2: 1. Araújo and Hughes (2019) research shows that zombie-ant fungal lineage likely arose from an ancestor that infected beetle (Coleoptera) larvae. At present, it has been reported that seven genera in the family Formicidae were infected by the O. unilateralis complex species, including Camponotus, Cephalotes, Daceton, Dolichoderus, Oecophylla, Paraponera and Polyrhachis (Evans and Sampson 1982, Kepler et al. 2011, Evans et al. 2011b, Kobmoo et al. 2012, Luangsa ard et al. 2011, Araújo et al. 2015, Kobmoo et al. 2015, Crous et al. 2016, Araújo et al. 2018, Evans et al. al. 2018, Wei et al. 2020). There were 2046 valid species in seven genera of Formicidae (excluding valid subspecies) (https://antcat.org/2022). If all entomopathogenic fungi accord with the beetle (Coleoptera): fungus ratio of 1.68–2: 1 by Weir and Hammond (1997), then there may be 1217–1023 species of entomopathogenic fungi in the world that can infect ants of seven genera in Formicidae, including the O. unilateralis complex.

Morphological characters were diverse for O. unilateralis sensu lato species. Most of the morphological features of O. unilateralis sensu lato species included cylindrical and clavate stromata that arose from the dorsal pronotum of the host, at least one ascoma that grew from lateral cushions of stromata. Some species produced multiple stromata, such as O. camponoti-indiani, O. halabalaensis, O. satoi (Araújo et al. 2015, 2018). Similar results were obtained in this study, the species, O. bifertilis, two stromata produced from the head of Polyrhachis sp.1 and Polyrhachis sp.2, resulting in two ascomata from stromata. Ascomata of O. unilateralis sensu lato were usually characterized by hemispherical, disc-shaped, spherical, one to multiple. All species in this group produced ascospores that were not disarticulate into part spores, and the shape includes vermiform, cylindrical, lanceolate, and fusiform. These shapes might to better dispersal for their spores.

Most species formed an asexual morph characterized by Hirsutella type-A phialides, tapering to a long neck and bearing a single conidium at their apices. There were also two types of asexual morphs, i.e., Hirsutella type-B and Hirsutella type-C. Most species produced phialides along stromata, legs and joints. The phialides of these species, such as O. basiasca (Hirsutella type-A), O. bifertilis (Hirsutella type-A), O. nuozhaduensis (Hirsutella type-A), were also observed from stromata, legs and joints. However, their phialides were shorter than O. acroasca (Hirsutella type-A and Hirsutella type-C), O. subtiliphialida (Hirsutella type-C) and O. contiispora (Hirsutella type-C) (Table 4). The phialides of O. acroasca, O. subtiliphialida, O. contiispora were produced from pure culture. This structure, rarely polyphialidic and conidiophores, were observed in the species of O. subtiliphialida, O. contiispora. Ophiocordyceps subtiliphialida and O. contiispora were only observed in Hirsutella type-C, and Hirsutella A-type was not been observed. The same result was also reported in Araújo et al. (2018). Unfortunately, the phialides produced from pure cultures and specimens were not compared, as specimens were used to isolate strains, or were dried, or made into permanent specimens. Conidia were diverse in O. acroasca (limoniform), O. basiasca (oviform), O. nuozhaduensis (ellipsoidal or oviform), O. subtiliphialida (olivary) and O. contiispora (olivary or flask-shaped) (Table 4). In addition, characteristics of the living cultures were introduced in the present work more than in previous studies (Kobmoo et al. 2012; Crous et al. 2016; Araújo et al. 2018; Wei et al. 2020). They were slow-growing, hard, light brown to dark brown in color, and produced pigment. This work has provided a method (see materials and methods for details) for obtaining living cultures of O. unilateralis complex species and asexual morph based on pure culture, which is of real value for further studies of O. unilateralis complex species in the future.

Conclusions

Six zombie-ant fungi were described from Yunnan Province, China. These novel species of Ophiocordyceps with hirsutella-like asexual morphs exclusively infecting ants were well supported based on molecular phylogenetic data and morphological evidence. This work proposes that the same host of Camponotus can be infected by multiple ant pathogenic fungi, while multiple species of Polyrhachis can be infected by the same pathogenic fungi at the same time. This study provides six new taxa support to explore the evolutionary relationship between the host and the fungus, and provides novel insights into the morphology, parasitism, distribution and ecology of O. unilateralis sensu lato within Ophiocordyceps. It has provided a method to obtain living cultures of the O. unilateralis complex and asexual morphs based on pure culture, which is of great value for further future studies of zombie-ant fungi.

Key to Ophiocordyceps unilateralis complex species worldwide

1a. On host Camponotus…………………………………………………………………………………………………………...2

1b. On host Cephalotes…………………………………………………………………………………….Ophiocordyceps kniphofioides

1c. On host Daceton………………… …………………………………………………………………..Ophiocordyceps daceti

1d. On host Dolichoderus………………………………………………………………………………..Ophiocordyceps monacidis

1e. On host Oecophylla…………………………………………………………………………………..Ophiocordyceps oecophyllae

1f. On host Paraponera…………………………………………………………………………………..Ophiocordyceps ponerinarum

1 g. On host Polyrhachis………………………………………...............................................................................................14

2a. The host without a biting behavior…………………………………………………………………...Ophiocordyceps tianshanensis

2b. The host with biting behavior………………………………………...................................................................................3

3a. Death position of the host was biting leaf………………………………….................................................................4

3b. Death position of the host was biting twing………………………………..............................................................12

4a. Ascospores not obvious separation………………............................................................................Ophiocordyceps contiispora

4b. Ascospores obvious separation………………………………………..................................................................................5

5a. The widest ascospore was not more than 3 μm……………………………………..........................................................6

5b. The widest ascospore was more than 3 μm…………………………………................................................................7

6a. Ascospores 83–108 × 2–3 μm, perithecia 247–296 × 176–225 μm, asci 131–172 × 5–8 μm………...Ophiocordyceps acroasca

6b. Ascospores 89–119 × 2–3 μm, perithecia 202–242 × 102–149 μm, asci 96–188 × 4–9 μm………….Ophiocordyceps basiasca

6c. Ascospores 110–125 × 2–3 μm, perithecia 400–430 × 200–230 μm, asci 130–175 × 7–8 μm……….Ophiocordyceps camponoti-leonardi

6d. Ascospores 75–85 × 2–3 μm, perithecia 280–320 × 160–180 μm, asci 80–160 × 6–7 μm…………...Ophiocordyceps camponoti-saundersi

6e. Ascospores 200–215 × 2–3 μm, perithecia 325–500 × 275–300 μm, asci 200–340 × 7–10 μm……...Ophiocordyceps rami

6f. Ascospores 80–85 × 3 μm, perithecia 240–280 × 100–150 μm, asci 110–140 × 6–6.5 μm…………...Ophiocordyceps camponoti-atricipis

6g. Ascospores 75–90 × 3 μm, perithecia 200–230 × 135–165 μm, asci 110–130 × 8–9 μm…………….Ophiocordyceps camponoti-femorati

6h. Ascospores 80–95 × 2–3 μm, perithecia 175–260 × 100–130 μm, asci 120–160 × 8–10 μm………..Ophiocordyceps camponoti-rufipedis

6i. Ascospores 120–140 × 3 μm, perithecia 225–230 × 135 μm, asci 150–160 × 8–9 μm……………….Ophiocordyceps camponoti-sexguttati

6j. Ascospores 75–85 × 2–2.5 μm, perithecia 200–250 × 140–160 μm, asci 95–125 × 6–8 μm………....Ophiocordyceps unilateralis

7a. Ascospores vermiform………………………………………...........................................................................................8

7b. Ascospores cylindrical…………………………………..............................................................................................10

7c. Ascospores filiform………………...................................................................................................Ophiocordyceps camponoti-novogranadensis

7d. Ascospores lanceolate…………………………………………...............................................................................................11

8a. Ascospores the longest was not more than 85 μm………………......................................................Ophiocordyceps camponoti-chartificis

8b. Ascospores the longest was more than 85 μm………………………………............................................................9

9a. Ascospores 91–126 × 2–5 μm, perithecia 222–274 × 153–159 μm……………….............................Ophiocordyceps nuozhaduensis

9b. Ascospores 90–105 × 3–4 μm, perithecia 200–240 × 100–150 μm……………….............................Ophiocordyceps camponoti-nidulantis

9c. Ascospores 90–120 × 4 μm, perithecia 220–250 × 100–165 μm………………..................................Ophiocordyceps camponoti-renggeri

10a. Ascospores 80–100 × 5 μm, distributed in Colombia………………................................................Ophiocordyceps albacongiuae

10b. Ascospores 60–75 × 3–5 μm, distributed in Thailand………………................................................Ophiocordyceps halabalaensis

10c. Ascospores 135–175 × 4–5 μm, distributed in Brazil……………….................................................Ophiocordyceps camponoti-balzani

10d. Ascospores 70–75 × 4.5–5 μm, distributed in Brazil………………..................................................Ophiocordyceps camponoti-bispinosi

10e. Ascospores 75–90 × 4–5 μm, distributed in USA……………….......................................................Ophiocordyceps camponoti-floridani

10f. Ascospores 75–85 × 4–5 μm, distributed in Brazi…………………...................................................Ophiocordyceps camponoti-hippocrepidis

10g. Ascospores 75 × 4.5 μm, distributed in Brazil………………............................................................Ophiocordyceps camponoti-indiani

10h. Ascospores 170–210 × 4–5 μm, distributed in Brazil…………………..............................................Ophiocordyceps camponoti-melanotici

11a. Ascospores 45–50 × 6–8 μm, distributed in Thailand…………………..............................................Ophiocordyceps septa

11b. Ascospores 52–72 × 5–8 μm, distributed in China………………......................................................Ophiocordyceps subtiliphialida

12a. Ascospores cylindrical………………………………………………................................................................................................13

12b. Ascospores filiform………………....................................................................................................Ophiocordyceps pulvinata

13a. Two types of Hirsutella asexual morph………………......................................................................Ophiocordyceps kimflemingiae

13b. One types of Hirsutella asexual morph………………......................................................................Ophiocordyceps blakebarnesii

14a. Biting leaf……………………………………..................................................................................................................15

14b. Biting twing………………...............................................................................................................Ophiocordyceps satoi

15a. Paraisaria-like phialides………………............................................................................................Ophiocordyceps naomipierceae

15b. Hirsutella-like phialides………………………………….............................................................................................16

16a. Two types of Hirsutella asexual morph……………….......................................................................Ophiocordyceps nooreniae

16b. One types of Hirsutella asexual morph…………………………….......................................................................17

17a. Phialides 9–24 × 2–4 μm, distributed in China………………............................................................Ophiocordyceps bifertilis

17b. Phialides 6–8 × 3–4 μm, distributed in Japan………………..............................................................Ophiocordyceps ootakii

17c. Phialides 30 × 2–3 μm, distributed in Thailand………………...........................................................Ophiocordyceps polyrhachis-furca