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

Dexiang Tang; Ou Huang; Weiqiu Zou; Yuanbing Wang; Yao Wang; Quanying Dong; Tao Sun; Gang Yang; Hong Yu

doi:10.1186/s43008-023-00114-9

. 2023 May 11;14:9. doi: 10.1186/s43008-023-00114-9

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

Dexiang Tang ^1,², Ou Huang ^1,², Weiqiu Zou ^1,², Yuanbing Wang ^1,³, Yao Wang ¹, Quanying Dong ^1,², Tao Sun ^1,², Gang Yang ⁴, Hong Yu ^1,^✉

PMCID: PMC10173673 PMID: 37170179

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.

Keywords: 6 new taxa, Camponotus, Living cultures, Morphology, Multi-gene phylogeny, Ophiocordyceps, Polyrhachis, Taxonomy

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

Species	Voucher information	SSU	LSU	TEF	RPB1	RPB2	Host	Location
Hirsutella sp.	NHJ 12525	EF469125	EF469078	EF469063	EF469092	EF469111	Hemiptera	–
Hirsutella sp.	OSC 128575	EF469126	EF469079	EF469064	EF469093	EF469110	Hemiptera	–
Ophiocordyceps acicularis	ARSEF 5692	DQ522540	DQ518754	DQ522322	DQ522368	DQ522418	Coleoptera	Korea
*Ophiocordyceps acroasca*	YFCC 9049	ON555837	ON555918	ON567757	ON568677	ON568130	*Camponotus* sp.	China
*Ophiocordyceps acroasca*	YFCC 9019	ON555838	ON555919	ON567758	ON568678	ON568131	*Camponotus* sp.	China
*Ophiocordyceps acroasca*	YFCC 9017	ON555839	ON555920	ON567759	ON568679	ON568132	*Camponotus* sp.	China
*Ophiocordyceps acroasca*	YFCC 9018	ON555840	ON555921	ON567760	ON568680	ON568133	*Camponotus* sp.	China
*Ophiocordyceps acroasca*	YFCC 9016^T	ON555841	ON555922	ON567761	ON568681	ON568134	*Camponotus* sp.	China
*Ophiocordyceps acroasca*	YHH 20122	ON555842	–	ON567762	ON568682	–	*Camponotus* sp.	China
Ophiocordyceps albacongiuae	RC20	KX713633	–	KX713670	–	–	Camponotus sp.	Colombia
Ophiocordyceps annullata	CEM 303	KJ878915	KJ878881	KJ878962	KJ878995	–	Coleoptera	Japan
Ophiocordyceps aphodii	ARSEF 5498	DQ522541	DQ518755	DQ522323	–	DQ522419	Coleoptera	–
Ophiocordyceps australis	HUA 186097	KC610786	KC610765	KC610735	KF658662	–	Hymenoptera	Colombia
*Ophiocordyceps basiasca*	YHH 20191	ON555828	ON555910	ON567748	ON568672	ON568121	*Camponotus* sp.	China
*Ophiocordyceps bifertilis*	YFCC 9012^T	ON555843	ON555923	ON567763	ON568143	ON568135	*Polyrhachis* sp.	China
*Ophiocordyceps bifertilis*	YHH 20162	ON555844	–	ON567764	ON568144	–	*Polyrhachis* sp.	China
*Ophiocordyceps bifertilis*	YHH 20163	ON555845	ON555924	ON567765	ON568145	ON568136	*Polyrhachis* sp.	China
*Ophiocordyceps bifertilis*	YHH 20164	ON555846	–	ON567766	ON568146	–	*Polyrhachis* sp.	China
*Ophiocordyceps bifertilis*	YFCC 9048	ON555847	ON555925	ON567767	ON568147	ON568137	*Polyrhachis* sp.	China
*Ophiocordyceps bifertilis*	YFCC 9013	ON555848	ON555926	ON567768	ON568148	ON568138	*Polyrhachis* sp.	China
Ophiocordyceps blakebarnesii	MISSOU5	KX713641	KX713610	KX713688	KX713716	–	Camponotus sp.	USA
Ophiocordyceps blakebarnesii	MISSOU4	KX713642	KX713609	KX713685	KX713715	–	Camponotus sp.	USA
Ophiocordyceps brunneipunctata	OSC 128576	DQ522542	DQ518756	DQ522324	DQ522369	DQ522420	Coleoptera	–
Ophiocordyceps buquetii	HMAS_199617	KJ878940	KJ878905	KJ878985	KJ879020	–	Hymenoptera	China
Ophiocordyceps camponoti-balzani	G143	KX713658	KX713595	KX713690	KX713705	–	Camponotus balzani	Brazil
Ophiocordyceps camponoti-balzani	G104	KX713660	KX713593	KX713689	KX713703	–	Camponotus balzani	Brazil
Ophiocordyceps camponoti-bispinosi	OBIS5	KX713636	KX713616	KX713693	KX713721	–	Camponotus bispinosus	Brazil
Ophiocordyceps camponoti-bispinosi	OBIS4	KX713637	KX713615	KX713692	KX713720	–	Camponotus bispinosus	Brazil
Ophiocordyceps camponoti-chartificis	MF080	MK874744	–	MK863824	–	–	Camponotus chartifex	Brazil
Ophiocordyceps camponoti-femorati	FEMO2	KX713663	KX713590	KX713678	KX713702	–	Camponotus femoratus	Brazil
Ophiocordyceps camponoti-floridani	Flo4	KX713662	KX713591	–	–	–	Camponotus femoratus	Brazil
Ophiocordyceps camponoti-floridani	Flx2	–	KX713592	KX713674	–	–	Camponotus femoratus	Brazil
Ophiocordyceps camponoti-hippocrepidis	HIPPOC	KX713655	KX713597	KX713673	KX713707	–	Camponotus hippocrepis	Brazil
Ophiocordyceps camponoti-indiani	INDI2	KX713654	KX713598	–	–	–	Camponotus indianus	Brazil
Ophiocordyceps camponoti-leonardi	C27	–	–	JN819019	–	–	Camponotus leonardi	Thailand
Ophiocordyceps camponoti-leonardi	C25	–	–	JN819029	–	–	Camponotus leonardi	Thailand
Ophiocordyceps camponoti-nidulantis	NIDUL2	KX713640	KX713611	KX713669	KX713717	–	Camponotus nidulans	Brazil
Ophiocordyceps camponoti-novogranadensis	Mal63	KX713648	KX713603	–	–	–	Camponotus novogranadensis	Brazil
Ophiocordyceps camponoti-novogranadensis	Mal4	KX713649	KX713602	–	–	–	Camponotus novogranadensis	Brazil
Ophiocordyceps camponoti-renggeri	RENG2	KX713632	–	KX713672	–	–	Camponotus renggeri	Brazil
Ophiocordyceps camponoti-renggeri	ORENG	KX713634	KX713617	KX713671	–	–	Camponotus renggeri	Brazil
Ophiocordyceps camponoti-rufipedis	G177	KX713657	KX713596	KX713680	–	–	Camponotus rufipes	Brazil
Ophiocordyceps camponoti-rufipedis	G108	KX713659	KX713594	KX713679	KX713704	–	Camponotus rufipes	Brazil
Ophiocordyceps camponoti-saundersi	C40	KJ201519	–	JN819012	–	–	Camponotus saundersi	Thailand
Ophiocordyceps camponoti-saundersi	Co19	–	–	JN819018	–	–	Camponotus saundersi	Thailand
Ophiocordyceps citrina	TNSF 18537	–	KJ878903	KJ878983	–	KJ878954	Hemiptera	Japan
Ophiocordyceps clavata	CEM 1762	KJ878916	KJ878882	KJ878963	KJ878996	–	Coleoptera	China
Ophiocordyceps cochlidiicola	HMAS_199612	KJ878917	KJ878884	KJ878965	KJ878998	–	Lepidoptera	China
*Ophiocordyceps contiispora*	YFCC 9025	ON555829	ON555911	ON567749	ON568139	ON568122	*Camponotus* sp.	China
*Ophiocordyceps contiispora*	YHH 20145	ON555830	-	ON567750	ON568140	ON568123	*Camponotus* sp.	China
*Ophiocordyceps contiispora*	YFCC 9026	ON555831	ON555912	ON567751	ON568141	ON568124	*Camponotus* sp.	China
*Ophiocordyceps contiispora*	YFCC 9027^T	ON555832	ON555913	ON567752	ON568142	ON568125	*Camponotus* sp.	China
Ophiocordyceps curculionum	OSC 151910	KJ878918	KJ878885	–	KJ878999	–	Coleoptera	Guyana
Ophiocordyceps daceti	MF01	–	KX713604	KX713667	–	–	Daceton armigerum	Brazil
Ophiocordyceps dipterigena	OSC 151911	KJ878919	KJ878886	KJ878966	KJ879000	–	Diptera	USA
Ophiocordyceps dipterigena	OSC 151912	KJ878920	KJ878887	KJ878967	KJ879001	–	Diptera	USA
Ophiocordyceps formicarum	TNSF 18565	KJ878921	KJ878888	KJ878968	KJ879002	KJ878946	Hymenoptera	Japan
Ophiocordyceps formosana	TNMF 13893	KJ878908	–	KJ878956	KJ878988	KJ878943	Coleoptera	Taiwan
Ophiocordyceps forquignonii	OSC 151902	KJ878912	KJ878876	–	KJ878991	KJ878945	Diptera	France
Ophiocordyceps forquignonii	OSC 151908	KJ878922	KJ878889	–	KJ879003	KJ878947	Diptera	France
Ophiocordyceps ghanensis	Gh41	KX713656	–	KX713668	KX713706	–	Polyrhachis sp.	Ghana
Ophiocordyceps halabalaensis	MY1308^T	KM655825	–	GU797109	–	–	Camponotus gigus	Thailand
Ophiocordyceps halabalaensis	MY5151	KM655826	–	GU797110	–	–	Camponotus gigas	Thailand
Ophiocordyceps irangiensis	OSC 128577	DQ522546	DQ518760	DQ522329	DQ522374	DQ522427	Hymenoptera	–
Ophiocordyceps irangiensis	OSC 128579	EF469123	EF469076	EF469060	EF469089	EF469107	Hymenoptera	–
Ophiocordyceps kimflemingiae	SC30	KX713629	KX713622	KX713699	KX713727	–	Camponotus castaneus/americanus	USA
Ophiocordyceps kimflemingiae	SC09B	KX713631	KX713620	KX713698	KX713724	–	Camponotus castaneus/americanus	USA
Ophiocordyceps kniphofioides	HUA 186148	KC610790	KF658679	KC610739	KF658667	KC610717	Hymenoptera	Colombia
Ophiocordyceps konnoana	EFCC 7295	EF468958	–	–	EF468862	EF468915	Coleoptera	Korea
Ophiocordyceps konnoana	EFCC 7315	EF468959	–	EF468753	EF468861	EF468916	Coleoptera	Korea
Ophiocordyceps lloydii	OSC 151913	KJ878924	KJ878891	KJ878970	KJ879004	KJ878948	Hymenoptera	Ecuador
Ophiocordyceps longissima	TNSF 18448	KJ878925	KJ878892	KJ878971	KJ879005	–	Hemiptera	Japan
Ophiocordyceps longissima	HMAS_199600	KJ878926	–	KJ878972	KJ879006	KJ878949	Hemiptera	China
Ophiocordyceps melolonthae	OSC 110993	DQ522548	DQ518762	DQ522331	DQ522376	–	Coleoptera	–
Ophiocordyceps melolonthae	Ophgrc 679	–	KC610768	KC610744	KF658666	–	Coleoptera	Colombia
Ophiocordyceps monacidis	MF74C	KX713646	KX713606	–	–	–	Dolichoderus bispinosus	Bazil
Ophiocordyceps monacidis	MF74	KX713647	KX713605	–	KX713712	–	Dolichoderus bispinosus	Brazil
Ophiocordyceps myrmecophila	CEM 1710	KJ878928	KJ878894	KJ878974	KJ879008	–	Hymenoptera	China
Ophiocordyceps naomipierceae	DAWKSANT	KX713664	KX713589	–	KX713701	–	Polyrhachis cf. robsonii	Australia
Ophiocordyceps neovolkiana	OSC 151903	KJ878930	KJ878896	KJ878976	KJ879010	–	Coleoptera	Japan
Ophiocordyceps nigrella	EFCC 9247	EF468963	EF468818	EF468758	EF468866	EF468920	–	Korea
Ophiocordyceps nooreniae	BRIP 55363^T	NG065096	NG059720	KX673812	–	KX673809	Chariomyrma cf. hookeri and Polyrhachis lydiae	Australia
Ophiocordyceps nooreniae	BRIP 64868	KX961142	–	KX961143	–	–	Polyrhachis cf. hookeri and Polyrhachis lydiae	Australia
Ophiocordyceps nutans	OSC 110994	DQ522549	DQ518763	DQ522333	DQ522378	–	Hemiptera	–
*Ophiocordyceps nuozhaduensis*	YHH 20168	ON555849	ON555927	ON567769	ON568683	–	*Camponotus* sp.	China
*Ophiocordyceps nuozhaduensis*	YHH 20169	ON555850	ON555928	ON567770	ON568684	–	*Camponotus* sp.	China
Ophiocordyceps odonatae	TNSF 18563	–	KJ878877	–	KJ878992	–	Odonata	Japan
Ophiocordyceps odonatae	TNS 27117	–	KJ878878	–	–	–	Odonata	Japan
Ophiocordyceps oecophyllae	OECO1	KX713635	–	–	–	–	Oecophyllas maragdina	Australia
Ophiocordyceps ootakii	J14	KX713651	–	KX713682	KX713709	–	Polyrhachis moesta	Japan
Ophiocordyceps ootakii	J13	KX713652	KX713600	KX713681	KX713708	–	Polyrhachis moesta	Japan
Ophiocordyceps ponerinarum	HUA 186140^T	KC610789	KC610767	KC610740	KF658668	–	Paraponera clavata	Brazil
Ophiocordyceps pulvinata	TNS-F 30044^T	GU904208	–	GU904209	GU904210	–	Camponotus obscuripes	Japan
Ophiocordyceps purpureostromata	TNSF 18430	KJ878931	KJ878897	KJ878977	KJ879011	–	Coleoptera	Japan
Ophiocordyceps polyrhachis-furcata	P39	KJ201504	–	JN819003	–	–	Polyrhachis furcata	Thailand
Ophiocordyceps polyrhachis-furcata	P51	KJ201505	–	JN819000	–	–	Polyrhachis furcata	Thailand
Ophiocordyceps ravenelii	OSC 151914	KJ878932	–	KJ878978	KJ879012	KJ878950	Coleoptera	USA
Ophiocordyceps rhizoidea	NHJ 12529	EF468969	EF468824	EF468765	EF468872	EF468922	Coleoptera	–
Ophiocordyceps rhizoidea	NHJ 12522	EF468970	EF468825	EF468764	EF468873	EF468923	Coleoptera	–
Ophiocordyceps rami	MY6736^T	KM655823	–	KJ201532	–	–	Camponotus sp.	Thailand
Ophiocordyceps rami	MY6738	KM655824	–	KJ201534	–	–	Camponotus sp.	Thailand
Ophiocordyceps satoi	J19	KX713650	KX713601	KX713684	KX713710	–	Polyrhachis lamellidens	Japan
Ophiocordyceps satoi	J7	KX713653	KX713599	KX713683	KX713711	–	Polyrhachis lamellidens	Japan
Ophiocordyceps septa	Pur1	–	–	KJ201528	–	–	Camponotus sp.	Thailand
Ophiocordyceps septa	Pur2	–	–	KJ201529	–	–	Camponotus sp.	Thailand
Ophiocordyceps septa	C41	–	–	JN819037	–	–	Camponotus sp.	Thailand
Ophiocordyceps sinensis	EFCC 7287	EF468971	EF468827	EF468767	EF468874	EF468924	Lepidoptera	–
Ophiocordyceps sobolifera	KEW 78842	EF468972	EF468828	–	EF468875	EF468925	Hemiptera	–
Ophiocordyceps sphecocephala	OSC 110998	DQ522551	DQ518765	DQ522336	DQ522381	DQ522432	Hymenoptera	–
Ophiocordyceps stylophora	OSC 111000	DQ522552	DQ518766	DQ522337	DQ522382	DQ522433	Coleoptera	–
Ophiocordyceps stylophora	OSC 110999	EF468982	EF468837	EF468777	EF468882	EF468931	Coleoptera	–
*Ophiocordyceps subtiliphialida*	YFCC 8815^T	ON555833	ON555914	ON567753	ON568673	ON568126	*Camponotus* sp.	China
*Ophiocordyceps subtiliphialida*	YFCC 8814	ON555834	ON555915	ON567754	ON568674	ON568127	*Camponotus* sp.	China
*Ophiocordyceps subtiliphialida*	YFCC 8816	ON555835	ON555916	ON567755	ON568675	ON568128	*Camponotus* sp.	China
*Ophiocordyceps subtiliphialida*	YFCC 8817	ON555836	ON555917	ON567756	ON568676	ON568129	*Camponotus* sp.	China
Ophiocordyceps tricentri	CEM 160	AB027330	AB027376	–	–	–	Hemiptera	–
Ophiocordyceps tianshanensis	MFLU 19-1207^T	MN025409	MN025407	MK992784	–	–	Camponotus japonicus	China
Ophiocordyceps tianshanensis	MFLU 19-1208	MN025410	MN025408	MK992785	–	–	Camponotus japonicus	China
Ophiocordyceps unilateralis	VIC 44303	KX713628	KX713626	KX713675	KX713730	–	Camponotus sericeiventris	Brazil
Ophiocordyceps unilateralis	VIC 44354	KX713627	–	KX713676	KX713731	–	Camponotus sericeiventris	Brazil
Ophiocordyceps yakusimensis	HMAS_199604	KJ878938	KJ878902	–	KJ879018	KJ878953	Hemiptera	China
Paraisaria amazonica	HUA 186113	KJ917566	–	–	KP212903	KM411980	Orthoptera	Colombia
Paraisaria gracilis	EFCC 8572	EF468956	EF468811	EF468751	EF468859	EF468912	Lepidoptera	–
Paraisaria gracilis	EFCC 3101	EF468955	EF468810	EF468750	EF468858	EF468913	Lepidoptera	–
Paraisaria heteropoda	OSC 106404	AY489690	AY489722	AY489617	AY489651	–	Hemiptera	Australia
Tolypocladium inflatum	OSC 71235	EF469124	EF469077	EF469061	EF469090	EF469108	Coleoptera	–
Tolypocladium ophioglossoides	CBS 100239	KJ878910	KJ878874	KJ878958	KJ878990	KJ878944	Elaphomyces sp.	–

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^TType material. New species were shown in bold

Table 3.

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

Gene name	ML	BI
SSU	TNe + I + G4	K2P + I + G4
LSU	GTR + F + I + G4	GTR + F + I + G4
TEF	GTR + F + I + G4	GTR + F + I + G4
RPB1	GTR + F + I + G4	GTR + F + I + G4
RPB2	TIM + F + I + G4	GTR + F + I + G4
COI	GTR + F + I + G4	GTR + F + I + G4

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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

Species name	Voucher information	GenBank number
Camponotus americanus	YNH-005	MZ331828
Camponotus americanus	BKH-019	MW802204
Camponotus badia	TUCIM:6601	MF993268
Camponotus badia	TUCIM:6461	MF993266
Camponotus castaneus	BIOUG03675-H07	KJ208900
Camponotus castaneus	BIOUG03675-H04	KJ445248
Camponotus claripes	AECT	JN134855
Camponotus cylindricus	–	EF634204
Camponotus explodens	TUCIM:5080	MF993254
Camponotus novogranadensis	–	MT904506
Camponotus renggeri	Creng_1_B	KP101600
Camponotus rufipes	BIOUG24424-D11	OM314604
Camponotus saundersi	–	BK012313
Camponotus saundersi	–	MT904541
Camponotus simulans	AFR-CND-2010-47-F02	JN270684
Camponotus sp.	CASENT0441197-D01	GU710187
Camponotus sp.	CASENT0043700-D01	KF200199
Camponotus sp.	CAMPO014	MH290634
Camponotus sp.	CASENT0000633-D01	HM373060
*Camponotus* sp.	YHH 20122	OP353539
*Camponotus* sp.	YHH 20605	OP353540
*Camponotus* sp.	YHH 20606	OP353541
*Camponotus* sp.	YHH 20607	OP353542
*Camponotus* sp.	YHH 20608	OP353543
*Camponotus* sp.	YHH 20609	OP353544
*Camponotus* sp.	YHH 20610	OP353545
*Camponotus* sp.	YHH 20611	OP353546
*Camponotus* sp.	YHH 20612	OP353547
*Camponotus* sp.	YHH 20168	OP353548
*Camponotus* sp.	YHH 20191	OP353549
Camponotus spanis	G191388	OM420293
Camponotus sericeiventris	BIOUG13980-G06	OM558348
Camponotus sericeiventris	BIOUG24738-E05	OM556713
Camponotus sexguttatus	CASENT0612243	JF863527
Camponotus vitreus	gvc13410-1L	HM914891
Camponotus vitreus	gvc13412-1L	HM914893
Camponotus wiederkehri	AEKB	JN134865
Dolichoderus bispinosus	–	KU187256
Dolichoderus quadridenticulatus	–	KU187255
Dolichoderus bispinosus	MACN-bar-ins-07510	MN625067
Daceton armigerum	USNM:ENT:01566820	MW983875
Oecophylla smaragdina	CSM0633	KM348012
Oecophylla smaragdina	EM898	MN619431
Polyrhachis anderseni	ANA42	KM348248
Polyrhachis ammon	RA0751	KY939110
Polyrhachis aurea	RA0750	KM348211
Polyrhachis arnoldi isolate	NDA40	MK591916
Polyrhachis beccari	FMNH-INS_2842133	KM348266
Polyrhachis carbonaria	FMNH-INS_2842101	KM348267
Polyrhachis cf. bismarckensis	FMNH-INS_2842022	KM348331
Paraponera clavata	YB-BCI150685	MK769309
Polyrhachis cupreata	CSM1015	KY939064
Polyrhachis cupreata	CSM0682	KY939056
Polyrhachis flavibasis	RA0766	KM348203
Polyrhachis flavibasis	RA0763	KY939081
Polyrhachis furcata	YB-KHC51412	MN618329
Polyrhachis gagates	FMNH-INS_2842213	KM348270
Polyrhachis hookeri	RA0747	KM348215
Polyrhachis illaudata	FMNH-INS_2842112	KM348275
Polyrhachis illaudata	FMNH-INS_2842222	KM348271
Polyrhachis jianghuaensis	GXBL0006	JQ681069
Polyrhachis latharis	FMNH-INS_2842062	KM348278
Polyrhachis lamellidens	NSMK-IN-170100347	OL663445
Polyrhachis lucidula	G160084	OM420302
Polyrhachis mucronata	RA1154	KM348338
Polyrhachis mucronata	RA1158	KM348339
Polyrhachis mucronata	RA1164	KM348340
Polyrhachis mucronata	CSM0696a	KM348337
Polyrhachis nigropilosa	FMNH-INS_2842045	KM348284
Polyrhachis noesaensis	FMNH-INS_2842106	KM348285
Polyrhachis obesior	FMNH-INS_2842054	KM348286
Polyrhachis ornata	CSM0797	KM348255
Polyrhachis ornata	CSM0842	KY939061
Polyrhachis proxima	G191229	OM420306
Polyrhachis proxima	FMNH-INS_2842042	KM348289
Polyrhachis proxima	FMNH-INS_2842129	KM348288
Polyrhachis schistacea	FMNH-INS_2842059	KM348296
Polyrhachis schistacea	FMNH-INS_2842058	KM348297
Polyrhachis schistacea	FMNH-INS_2842065	KM348295
Polyrhachis schistacea	FMNH-INS_2842071	KM348294
Polyrhachis schistacea	FMNH-INS_2842072	KM348292
Polyrhachis schlueteri	CASENT	KM348298
Polyrhachis sp.	RA0784	KM348355
Polyrhachis sp.	FMNH-INS_2842139	KM348305
Polyrhachis sp.	FMNH-INS_2842198	KM348309
Polyrhachis sp.	FMNH-INS_2842195	KM348308
Polyrhachis sp.	FMNH-INS_2842179	KM348300
Polyrhachis sp.	FMNH-INS_2842190	KM348304
Polyrhachis sp.	FMNH-INS_2842193	KM348310
Polyrhachis sp.	FMNH-INS_2842194	KM348307
Polyrhachis sp.	FMNH-INS_2842074	KM348226
Polyrhachis sp.	RA736b	KM348229
*Polyrhachis* sp.	YHH 20162	OP353532
*Polyrhachis* sp.	YHH 20163	OP353533
*Polyrhachis* sp.	YHH 20164	OP353534
*Polyrhachis* sp.	YHH 20601	OP353535
*Polyrhachis* sp.	YHH 20602	OP353536
*Polyrhachis* sp.	YHH 20603	OP353537
*Polyrhachis* sp.	YHH 20604	OP353538
Polyrhachis turneri	CSM0722	KY939058
Polyrhachis villipes	FMNH-INS_28421186	KM348316

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Boldface: data generated in this study

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).

Table 4.

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

Species	Host	Death position	Stromata	Ascomata	Perithecia (μm)	Asci (μm)	Prominent caps	Ascospores (μm)	Septa	Hirsutella asexual morph (μm)	Conidia (μm)	Country	References
*Ophiocordyceps acroasca*	*Camponotus* sp.	Biting leaf	Single	Hemispherical, 3 × 2–3 mm	Ovoid, 247–296 × 176–225	Cylindrical, 8-spored, 131–172 × 5–8	Prominent, 3–5 × 4–6	Vermiform, 83–108 × 2–3	4–5	*Hirsutella* A-type and *Hirsutella* C-type, 17–30 × 1–4	Limoniform, 2–3 × 1–2	China	This study
Ophiocordyceps albacongiuae	Camponotus sp.	Biting epiphites	One or two	Disc-shaped	Flask-shaped, 240–290 × 105–135	Cylindrical to clavate, 8-spored, 130–160 × 8–11	Hemispherical, 3–5 × 4–5	Cylindrical, 80–100 × 5	5–6	–	–	Colombia	Araújo et al. (2018)
*Ophiocordyceps basiasca*	*Camponotus* sp.	Biting leaf	Single	Spherical, 3 × 2 mm	Flask-shaped or ovoid, 202–242 × 102–149	Cylindrical, 8-spored, 96–188 × 4–9	Hemispherical, 3–5 × 4–5	Vermiform, 89–119 × 2–3	4–5	*Hirsutella* A-type, 10–23 × 1–5	Oviform, 1–4 × 1–2	China	This study
*Ophiocordyceps bifertilis*	*Polyrhachis* sp.1 and *Polyrhachis* sp.2	Biting leaf	Multiple	Disc-shaped or hemispherical, 3 × 2–3 mm	Flask-shaped, 156–211 × 102–129	Cylindrical, 8-spored, 130–198 × 6–10	Prominent, 3–5 × 5–6	Fusiform, 70–94 × 2–4	4–5	*Hirsutella* A-type, 9–24 × 2–4	–	China	This study
Ophiocordyceps blakebarnesii	Camponotus sp.	Biting inside log	Single	Discshaped to irregular, 1.5 × 1 mm	Flask-shaped, 300–320 × 105–120	Cylindrical to clavate, 8-spored, 220–250 × 12–14	–	Cylindrical, 140–160 × 4	6–7	Hirsutella A-type, 75 × 3–4	Limoniform, 8–9 × 3	USA	Araújo et al. (2018)
*Ophiocordyceps contiispora*	*Camponotus* sp.	Biting leaf	Single	Disc-shaped, 0.7–1 mm	Flask-shaped, 158–212 × 69–122	Cylindrical, 8-spored, 89–130 × 4–9	Hemispherical or square, 1–3 × 3–5	Fusiform, 38–48 × 2–4	No obvious separation	*Hirsutella* C-type, 57–92 × 1–4	Olivary or flask-shaped, 4–6 × 1–2	China	This study
Ophiocordyceps camponoti-leonardi	Camponotus leonardi	Biting leaf	Single	–	Fusoid-ellipsoid, 400–430 × 200–230	Cylindrical, 8-spored, 130–175 × 7–8	–	Lanceolate, 110–125 × 2–3	Multiseptate	Hirsutella, 22.5 × 2.0–3.5	Fusoid 2–4 × 1–2	Thailand	Kobmoo et al. (2012)
Ophiocordyceps camponoti-saundersi	Camponotus saundersi	Biting leaf	Single	–	Fusoid-ellipsoid, 280–320 × 160–180	Cylindrical, 8-spored, 80–160 × 6–7	–	Lanceolate, 75–85 × 2–3	Multiseptate	Hirsutella, 25 × 2–3	Fusoid, 2–3 × 1–2	Thailand	Kobmoo et al. (2012)
Ophiocordyceps halabalaensis	Camponotus gigas	Biting leaf	Three	–	Fusoid-ellipsoid, 350–420 × 180–210	Cylindrical, 8-spored, 150–200 × 7–10	–	Cylindrical, 60–75 × 3–5	Multiseptate	–	–	Thailand	Luangsa-ard et al. (2011)
Ophiocordyceps nooreniae	Polyrhachis cf. hookeri	Biting leaf	–	–	–	–	–	–		Hirsutella A-type, 30–55; Hirsutella C-type, 35–50 × 1.5–8	Ovoid, 5–6 × 2–3	Australia	Crous et al. (2016)
*Ophiocordyceps nuozhaduensis*	*Camponotus* sp.	Biting leaf	Single	Spherical, 2.4 × 1.6 mm	Flask-shaped, 222–274 × 153–159	–		Vermiform, 91–126 × 2–5	7–13	*Hirsutella* A-type, 6–22 × 2–4	Ellipsoidal or oviform, 2–5 × 2–3	China	This study
Ophiocordyceps naomipierceae	Polyrhachis cf. robsonii	Biting leaf	–	Hemispherical to irregular, 0.75 × 0.5–0.65 mm	Flask-shaped, 260–320 × 150–200	Vermiform, cylindrical, 8-spored, 150–180 × 7	Prominent	Vermiform, 75–105 × 5–6	4–6	Paraisaria-like, 15–35 × 3	Conidium, 5–7 × 3	Australia	Araújo et al. (2018)
Ophiocordyceps ootakii	Polyrhachis sp.	Biting leaf	Single or branched	Fisc-shaped, 1.1 × 0.8 mm	Flask-shaped, 230–260 × 120–150	Cylindrical to clavate, 8-spored, 130–180 × 8–9	Prominent	Vermiform, 85–100 × 3	5	Hirsutella A-type, 6–8 × 3–4	5 × 3	Japan	Araújo et al. (2018)
Ophiocordyceps polyrhachis-furca	Polyrhachis furca	Biting leaf	Single	–	Fusoid-ellipsoid, 380–400 × 160–180	Cylindrical, 8-spored, 140–190 × 7–8 µm	–	Lanceolate, 90–100 × 2–3	Multi-septate	Hirsutella, 30 × 2–3	Fusoid, 3–5 × 2–3	Thailand	Kobmoo et al. (2012)
Ophiocordyceps rami	Camponotus sp.	Biting leaf	Single	Hemispherical, 2 mm	Fusoid-ellipsoid, 325–500 × 275–300	Cylindrical, 8-spored, 200–340 × 7–10	–	Filiform, 200–215 × 2–3	7–8	Hisutella A-type, 9–10 × 3–4; Hisutella C-type, 30 × 3–5	Cylindrical to narrow fusiform, 3.5–6.5 × 1–2; fusiform to narrowly lemoniform, 9 × 5	Thailand	Kobmoo et al. (2015)
Ophiocordyceps satoi	Polyrhachis lamellidens	Biting twing	Three	1 × 0.8 mm	Flask-shaped, 230–270 × 120–160	Cylindrical to clavate, 8-spored, 120–160 × 8–10	Prominent	Cylindrical, 85–100 × 4	5	Hirsutella A-type, 12 × 7	–	Japan	Araújo et al. (2018)
Ophiocordyceps septa	Camponotus sp.	Biting leaf	Single	Hemispherical, 2 mm	Fusoid-ellipsoid, 280–300 × 100–150	Cylindrical, 8-spored, 125–165 × 12.5–15	–	Lanceolate, 45–50 × 6–8	7–8	Hisutella A-type, 25 × 2–3; Hisutella C-type, 50 × 5.5	Fusiform, 5–6 × 1–2; fusiform to narrowly lemoniform, 9 × 5	Thailand	Kobmoo et al. (2015)
*Ophiocordyceps subtiliphialida*	*Camponotus* sp.	Biting leaf	Single	Disc-shaped, 2 × 1.2–1.9 mm	Flask-shaped, 195–296 × 87–161	Cylindrical, 8-spored, 89–119 × 5–9	Hemispherical, 2–4 × 5–7	Lanceolate, 52–72 × 5–8	6–7	*Hirsutella* C-type, 70–116 × 1–3	Olivary, 6–10 × 3–6	China	This study
Ophiocordyceps camponoti-atricipis	Camponotus atriceps	Biting leaf	Single	Hemispherical, 1.5 × 0.5–0.8 mm	Flask-shaped, 240–280 × 100–150	Cylindrical to clavate, 8-spored, hyaline, 110–140 × 6–6.5	5 × 5.5	Vermiform 80–85 × 3	5	Hirsutella A-type, 5–7 × 2–3	–	Brazil	Araújo et al. (2015)
Ophiocordyceps camponoti-balzani	Camponotus balzani	Biting leaf	Single	1.5 × 1.0 mm	Flask-shaped, 400–450 × 100–150	Cylindrical, 8-spored, 200–240 × 12–16	Prominent, 8–10 × 6–8	Cylindrical, 135–175 × 4.0–5.0	14–22	Hirsutella A-type, Hirsutella C-type 20–25 × 3–4	Cylindric to fusiform, 12–14 × 2–3	Brazil	Evans et al. (2011b)
Ophiocordyceps camponoti-bispinosi	Camponotus bispinosus	Biting spines	Single	0.8 × 0.4–0.7 mm	Globose to flask-shaped, 250–290 × 150–170	Cylindrical to clavate, 8-spored, hyaline, 110–130 × 8–8.5	3.5 × 4.5	Cylindrical, 70–75 × 4.5–5	4–5	Hirsutella A-type, 6 × 2.5–3	Narrow limoniform, 6–7 × 2	Brazil	Araújo et al. (2015)
Ophiocordyceps camponoti-chartificis	Camponotus chartifex	Biting leaf	Single	Hemispherical, 1.5 × 1 mm	Globose to hemispherical shaped, 200–235 × 135–175	Cylindrical to clavate, 8-spored, 100–125 × 6	6–7 × 3–4	Vermiform 75–85 × 5	9–13	Hirsutella A-type, 5–6 × 3	Fusiform to limoniform, 7 × 2.6	Brazil	Araújo et al. (2018)
Ophiocordyceps camponoti-femorati	Camponotus femoratus	Biting leaf/spines	Single	Disc-shaped to hemispherical, 1.2–2.2 × 0.8–1.4 mm	Flask-shaped, 200–230 × 135–165	Cylindrical to clavate, 8-spored, 110–130 × 8–9	6 × 3	75–90 × 3	5	Hirsutella A-type, 7–10 × 3–4	Limoniform, 7–9 × 3	Brazil	Araújo et al. (2018)
Ophiocordyceps camponoti-floridani	Camponotus floridanus	Biting leaf	Single	Disc-shaped	Flask-shaped, 265 × 100	Cylindrical to clavate, 8-spored, 145 × 9–10	–	Cylindrical, 75–90 × 4–5	5	Hirsutella A-type, 8–9 × 3–4	Limoniform, 8–9 × 3	USA	Araújo et al. (2018)
Ophiocordyceps camponoti-hippocrepidis	Camponotus hippocrepis	Biting spines	Single	2–2.5 × 0.25–0.45 mm	Flask-shaped, 225–250 × 135–165	Cylindrical to clavate, 8-spored, 115–135 × 7–10	Prominent, 6–7 × 4	Cylindrical, 75–85 × 4–5	5	Hirsutella A-type, 8–9 × 4	Limoniform, 5 × 2	Brazil	Araújo et al. (2018)
Ophiocordyceps camponoti-indiani	Camponotus indianus	Biting leaf	Multiple	Hemispherical	Ovoid to flask-shaped, 230–310 × 120–175	Cylindrical, 8-spored, 170 × 8.5	Prominent, 4.5 × 5	Cylindrical, 75 × 4.5	5	Hirsutella A-type, 7.5 × 3.5; Hirsutella C-type	–	Brazil	Araújo et al. (2015)
Ophiocordyceps camponoti-melanotici	Camponotus melanoticus	Biting leaf	Single	1.3 × 0.8 mm	Flask-shaped, 400–450 × 100–150	8-spored, 200–275 × 12–16	8–10 × 6–8	Cylindrical, 170–210 × 4–5	27–35	Hirsutella A-type	–	Brazil	Evans et al. (2011b)
Ophiocordyceps camponoti-nidulantis	Camponotus niduland	Biting saplings	Single	Disc-shaped to hemispherical, 1.5 × 1 mm	Flask-shaped, 200–240 × 100–150	Vermiform to clavate, 8-spored, 110–145 × 6–8	4 × 6	Vermiform, 90–105 × 3–4	5	Hirsutella A-type; Hirsutella C-type, 70–120 × 4–6	Limoniform, 8 × 3	Brazil	Araújo et al. (2015)
Ophiocordyceps camponoti-novogranadensis	Camponotus novogranadensis	Biting epiphites	Single	0.8–1.0 × 0.5–0.6 µm	225–250 × 125–155	Cylindrical, 8-spored, 95–120 × 9–10	Prominent, 5–6 × 3–4	Filiform, 75–95 × 2.5–3.5	5–10	Hirsutella A-type; Hirsutella B-type, 80–100 × 35–40	Narrowly clavate to obclavate, 10–12 × 1.5–2.0	Brazil	Evans et al. (2011b)
Ophiocordyceps camponoti-renggeri	Camponotus renggeri	Biting leaf/moss	Single	Hemispherical to globose, 1–1.5 × 0.8–1 mm	Flask-shaped, 220–250 × 100–165	Cylindrical, 8-spored, 130–145 × 8–10	Prominent, 7–8 × 3	Vermiform 90–120 × 4	5–8	Hirsutella C-type, 40–60 × 3–5	–	Brazil	Araújo et al. (2018)
Ophiocordyceps camponoti-rufipedis	Camponotus rufipes	Biting leaf	Single	Discshaped to hemisphaerical, 1 × 0.5 mm	Flask-shaped, 175–260 × 100–130	Cylindrical to clavate, 8-spored, 120–160 × 8–10	Prominent, 4.0–5.5 × 3.0–4.5	Vermiform, 80–95 × 2–3	4–7	Hirsutella A-type, 10 × 2	Fusiform to narrowly limoniform, 5 × 1.5	Brazil	Evans et al. (2011b)
Ophiocordyceps camponoti-sexguttati	Camponotus sexguttatus	Biting leaf	Single	Disc-shaped, 1 × 1 mm	Flask-shaped, 225–230 × 135	Cylindrical, 8-spored, 150–160 × 8–9	Prominent, 6 × 3	Cylindrical, 120–140 × 3	7	Hirsutella A-type, 5–8 × 3–4	Limoniform, 5 × 2	Brazil	Araújo et al. (2018)
Ophiocordyceps kimflemingiae	Camponotus castaneus	Biting twig	Single	Disc-shaped, 1.5–2 × 1.3 mm	Flask-shaped, 250–275 × 120–160	Cylindrical to clavate, 8-spored, 120–150 × 10–11	Prominent	Cylindrical, 80–90 × 5	5–6	Hirsutella A-type; Hirsutella C-type	–	USA	Araújo et al. (2018)
Ophiocordyceps oecophyllae	Oecophylla smaragdina	Biting leaf	–	–	–	–	–	–	–	30–50 × 3–4	Ovoid to cylindrical, 5.5–10 × 1.5–3	Australia	Araújo et al. (2018)
Ophiocordyceps monacidis	Dolichoderus bispinosus	Base of trunk	Single	–	–	–	–	–	–	–	–	Brazil	Araújo et al. (2018)
Ophiocordyceps daceti	Daceton armigerum	Leaf (not biting)	Single	–	–	–	–	–	–	Hirsutella, 16–18 × 4	Cylindrical, 7–10 × 3	Brazil	Araújo et al. (2018)
Ophiocordyceps kniphofioides	Cephalotes atratus	Base of trunk	Single	5–6 × 0.7–1 mm	Ovoid to lageniformia, 170–250 × 110–140	Narrow cylindrical, 140–200 × 6–12	–	Filiform, 110–150 × 1.5–3	3–5	Hirsutella A-type, 10–16 × 0.6–4; Hirsutella B-type	Narrowly clavate, 7–9 × 1.5–2.5; ovoid to cylindrical, 8–12 × 4–5	Brazil	Evans and Samson (1982)
Ophiocordyceps ponerinarum	Paraponera clavata	Base of trunk	–	8–14 × 0.8–1 mm	Flask-shaped, 210–320 × 140–190	–	–	–	–	Hirsutella A-type, 10–14 × 1.8–2.5	Clavate, 7–9 × 1.8–3	Brazil	Evans and Samson (1982)
Ophiocordyceps pulvinata	Camponotus obscuripes	Clinging to twigs	Single	–	400–600 × 150–250	Clavate, 8-spored, 220–300 × 9–19	4–5.4 × 6–9	Filiform, 160–220 × 3–5	–	–	–	Japan	Kepler et al. (2011)
Ophiocordyceps tianshanensis	Camponotus japonicus	The bark of a dilapidated (not biting)	–	Disc-shaped, 1.1–1.6 × 0.5–1.1 mm	Flask-shaped, 220–260 × 100–140	–	–	–	–	Hirsutella A-type, 8–9 × 2.5–3.5	Fusiform to obpyriform, 6–9.2 × 2.2–3	China	Wei et al. (2020)
Ophiocordyceps unilateralis	Camponotus sericeiventris	Biting leaf	Single	–	Flask-shaped, 200–250 × 140–160	Cylindrical, 8-spored, 95–125 × 6–8	5–6 × 4–5	Filiform, 75–85 × 2–2.5	4–5	Hirsutella A-type, 10–12 × 3–3.5; Hirsutella B-type, 14–16 × 2.5–3	Limoniform, 6.5–8 × 2–2.5; cylindrical-fusoid, 8–11 × 2.5–3	Brazil	Evans et al. (2018)

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New species are shown in bold

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).

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.

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

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.

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.

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.

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

Acknowledgements

We thank Jing Zhao for providing important support to the phylogenetic analysis in this work. We thank Tahir Khan for providing English editing.

Abbreviations

BI: Bayesian inference
BP: Bayesian posterior probability
bp: Base pair
CTAB: Cetyl-trimethyl-ammonium bromide
DNA: Deoxyribonucleic acid
ML: Maximum likelihood
SSU: The nuclear ribosomal small subunit
LSU: The nuclear ribosomal large subunit
PCR: Polymerase chain reaction
PP: Posterior probabilities
PDA: Potato dextrose agar
RPB1: The largest subunits of RNA polymerase II
RPB2: The second largest subunits of RNA polymerase II
TEF: The translation elongation factor 1α

Author contributions

D-XT, W-QZ, GY, and HY collected samples. D-XT and W-QZ isolated cultures and performed DNA isolation and PCR amplification. OH, Y-BW, YW, Q-YD, and TS analyzed data. D-XT wrote the original draft. HY reviewed and edited the draft. All authors read and approved the final manuscript.

Funding

This work was funded by the National Natural Science Foundation of China (31870017, 32060007).

Availability of data and materials

All sequence data generated for this work can be accessed via GenBank: https://www.ncbi.nlm.nih.gov/genbank/. All alignments for phylogenetic analyses were deposited in TreeBASE (http://www.treebase.org; the following links were available: http://purl.org/phylo/treebase/phylows/study/TB2:S29994?x-access-code=8e258e97fca38d4f834975a2fefb47a1&format=html.)

Declarations

Ethics approval and consent to participate

Not applicable.

Adherence to national and international regulations

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

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Data Availability Statement

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PERMALINK

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

Dexiang Tang

Ou Huang

Weiqiu Zou

Yuanbing Wang

Yao Wang

Quanying Dong

Tao Sun

Gang Yang

Hong Yu

Abstract

INTRODUCTION

MATERIALS AND METHODS

Sampling and isolation

Morphological observations

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

Phylogenetic analyses

Phylogenetic analyses of fungi

Table 1.

Table 3.

Phylogenetic analyses of ants

Table 2.

RESULTS

Phylogenetic analysis of the genus Ophiocordyceps

Fig. 1.

Phylogenetic analysis of host ants

Fig. 2.

TAXONOMY

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

Fig. 3.

Table 4.

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

Fig. 4.

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

Fig. 5.

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

Fig. 6.

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

Fig. 7.

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

Fig. 8.

Discussion

Conclusions

Key to Ophiocordyceps unilateralis complex species worldwide

Acknowledgements

Abbreviations

Author contributions

Funding

Availability of data and materials

Declarations

Ethics approval and consent to participate

Adherence to national and international regulations

Consent for publication

Competing interests

Footnotes

References

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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