There is gold in the graveyard: a new lineage of zombie-ant fungi in the genus Ophiocordyceps (Ophiocordycipitaceae: Hypocreales) from Minas Gerais, Brazil

Abstract

Ophiocordyceps serves as a key model for studying cryptic fungal diversity and behavioural manipulation of hymenopterous insects. Here, we describe Ophiocordyceps acanthoponerae, a newly discovered species infecting Acanthoponera mucronata (Heteroponerini: Formicidae) in a Brazilian Atlantic rainforest-Cerrado ecotone. Morphological analyses revealed mixed traits characteristic of Ophiocordyceps lineages associated with ants and wasps, including leaf biting behaviour manipulation, dark brown ascostromata covering 360º of the stalk, ascospores producing capilliconidia and hirsutelloid asexual morphs. Phylogenetic analyses based on four genomic regions (SSU, LSU, TEF and RPB1) placed this species outside the traditional myrmecophilous hirsutelloid clades O. unilateralis and O. kniphofioides, and within a novel clade closely related to the wasp pathogen O. humbertii. This discovery represents the first record of Ophiocordyceps infecting Heteroponerini and highlights an unexplored lineage of manipulative fungi. Our findings expand the known host range for myrmecophilous Ophiocordyceps and underscore the importance of studying fungal diversity in under-sampled ecological niches.

Citation: Lima-Santos SJ, Araújo JPM, Feitosa RM, Mendes-Pereira T, Elliot SL, Evans HC (2025). There is gold in the graveyard: a new lineage of zombie-ant fungi in the genus Ophiocordyceps (Ophiocordycipitaceae: Hypocreales) from Minas Gerais, Brazil. Fungal Systematics and Evolution 16: 243–264. doi: 10.3114/fuse.2025.16.14

INTRODUCTION

The order Hypocreales (Ascomycota) comprises most of the fungal species that are known as insect pathogens, or entomopathogenic fungi distributed predominantly across three families: Clavicipitaceae, Cordycipitaceae and Ophiocordycipitaceae (Sung et al. 2007, 2008). The ancestral trait of the order is an association with plants (decomposers, phytopathogens, epiphytes and endophytes), with the insect-associated lineages arising around 160 million years ago (Sung et al. 2008). The evolution of hypocrealean entomopathogens probably occurred via the process of inter-kingdom host-jumping (or host-switching), in which closely-related fungi are able to exploit nutritional resources from even distantly related host taxa (e.g. animals, plants and other fungi) that share the same habitat or niche (Spatafora et al. 2007, Sung et al. 2008, Yu et al. 2021, Wei et al. 2022). This phenomenon is considered to be a major driver of diversity, lifestyle differentiation and speciation for the entomopathogenic fungi (Thines 2019, Wei et al. 2022).

Ophiocordyceps (Ophiocordycipitaceae: Hypocreales) is a flagship genus for the study of cryptic fungal diversity, particularly in tropical forests (Evans et al. 2011a). Current hypotheses suggest that Ophiocordyceps arose nearly 100 million years ago (Sung et al. 2008) and is almost exclusively comprised of pathogens of insects (nine insect orders) with approximately 350 legitimate species described (Sung et al. 2007, Quandt et al. 2014, Araújo & Hughes 2019, MycoBank 2025). The diversification process of Ophiocordyceps might have been favoured by successive and independent host-jumps from the ancestral host-association with Coleoptera larvae to other orders of insects and also between taxa within these orders (Araújo & Hughes 2019). A characteristic of the genus is firm, mostly darkly pigmented stromata, in contrast to the fleshy and brightly pigmented stromata of Cordyceps (Sung et al. 2007). The most widely known species is the lepidopteran pathogen O. sinensis, which has been studied extensively because of its pharmaceutical applications, especially in traditional Chinese medicine (Shrestha & Bawa 2013, Baral 2017). Within the genus, O. unilateralis s. l. — the so-called “zombie-ant fungus” — has also been a subject of interest to scientists and the general public alike because of its behavioural manipulation of the ant host. This has inspired successful video games and television series, as well as number of books (Andersen et al. 2009, Araújo & Hughes 2016, Hughes et al. 2016, Araújo et al. 2018).

Following infection by O. unilateralis and related taxa, the ant climbs to an elevated position in the forest understorey where it bites into the plant stem or leaf (the so-called death-grip; Hughes et al. 2011); dying attached to the vegetation and locked into it by its mandibles (Evans 1982, Evans & Samson 1982, Andersen et al. 2009). This manipulation strategy allows the fungus to optimise spore release from an elevated position onto the forest floor where foraging hosts are found (Andersen et al. 2009, Hughes et al. 2016). Dead ants are commonly found aggregated at high densities — especially, in tropical forests — forming “graveyards” or “cemeteries”, which can persist in the same location for months or even years (Evans & Samson 1982, Pontoppidan et al. 2009, Loreto et al. 2014, Neto et al. 2019, Will et al. 2023). These aggregations are constantly resupplied with new infected hosts, revealing the chronic or enzootic nature of Ophiocordyceps-ant interactions at the colony level, rather than epizootic events (Andersen et al. 2012, Loreto et al. 2014).

Evans et al. (2011b) determined that the fungus previously identified as O. unilateralis was, in fact, a complex of species strictly associated with ants of the tribe Camponotini (Formicinae: Formicidae). Evans et al. (2011a, b) also hypothesized that species within the complex were each restricted to one species of host and, therefore, that on a global scale, tens or hundreds of species would eventually be delimited within this complex. If the one-species-one-fungus hypothesis holds, then the cosmopolitan nature of the Camponitini across the tropics — with almost 2,500 described species with a wide range of habitats (Feitosa & Dias 2024, Bolton 2025) — seems to make this conjecture feasible. Since 2011, new fungal species strictly associated with a particular Camponotini host are constantly being discovered and described worldwide, increasing the support for this hypothesis (Kepler et al. 2011, Luangsa-ard et al. 2011, Kobmoo et al. 2012, 2015, Sanjuan et al. 2015, Araújo et al. 2015, 2018). A recent study, however, has shown that this interaction might not be as strict or host-specific as previously thought (Tang et al. 2023) and more research is needed to determine the level or levels of host specificity.

These new myrmecophilous species associated with Camponotini are currently inserted in what is now recognized as the O. unilateralis clade (Kepler et al. 2011, Luangsa-ard et al. 2011, Kobmoo et al. 2012, 2015, Sanjuan et al. 2015, Araújo et al. 2015, 2018, Tang et al. 2023). This considers their phylogenetic proximity and shared morphological traits, especially ascospore morphology and their Hirsutella-like (or hirsutelloid) asexual morphs, but also host specificity (Araújo & Hughes 2017, Araújo et al. 2018). Among all Ophiocordyceps lineages that infect ants, the O. unilateralis core clade and the O. kniphofioides sub-clade are, to date, the only hirsutelloid lineages that induce ‘death-grip’ manipulation in their hosts (Araújo & Hughes 2017, Araújo et al. 2018, 2020). The O. unilateralis core clade exclusively infects Camponotini and can be recognized by the brown or black fertile region (or ascoma) that forms laterally on the stalk (or stroma) (Evans & Samson 1984, Araújo & Hughes 2017, Araújo et al. 2018). The O. kniphofioides sub-clade comprises five known species and infects other ant tribes (Cephalotini, Dacetini, Dolichoderini and Paraponerini) and produces a single ascoma that grows laterally from the host’s thorax, with the fertile portion covering all sides of the stalk (Evans & Samson 1982, Araújo & Hughes 2017, Araújo et al. 2018).

The myrmecophilous and manipulative Ophiocordyceps (O. unilateralis core clade and O. kniphofioides sub-clade) are closely related to another hirsutelloid lineage, composed of O. humbertii, which infects and induces behavioural manipulation in social wasps similar to that seen in O. unilateralis infections (Petch 1935, Araújo & Hughes 2019, Somavilla et al. 2020, Sobczak et al. 2020, Barbosa & Somavilla 2022). It is considered that behavioural manipulation might have occurred first in social wasps, rather than in ants, during the evolution of manipulative Ophiocordyceps (Araújo & Hughes 2019). All hymenopterans descend from a common ancestor, so it is possible that the morpho-physiological similarities between them follows their co-existence in the same habitat (host-habitat hypothesis) which could have facilitated host-jumps during the evolution of Ophiocordyceps (Branstetter et al. 2017, Huber et al. 2017, Araújo & Hughes 2019). It is also possible that the evolution of manipulative Ophiocordyceps was favoured by the social behaviour exhibited by the hosts, given that behavioural manipulation is considered to be a parasite strategy that helps to cope with social immunity inside colonies (Loreto et al. 2014, Araújo & Hughes 2019). To address these questions, it is important to expand our knowledge of Ophiocordyceps-infecting social hymenopterans and, in particular, those where there are features in common or in closely-related lineages with a scarcity of taxon sampling.

Here, we describe a new species of Ophiocordyceps infecting Acanthoponera mucronata (Heteroponerini: Ectatomminae: Formicidae) in Brazil: an ant species for which life-history and ecological data are scarce. This species has morphological traits similar to known myrmecophilous Ophiocordyceps species but also to the wasp-parasitic species O. humbertii. Our phylogenetic analyses have revealed that this new taxon also represents a new origin of myrmecophily and of host-manipulative Ophiocordyceps species, closely-related to the wasp pathogen, O. humbertii. This is also the first record of ants belonging to the Heteroponerini tribe being infected by Ophiocordyceps, expanding the host range for the genus.

MATERIALS AND METHODS

In this study, we combined morphological, ecological and molecular data for the description of the new species. Our methodologies for collection, field observations, morphological characterisation and molecular analyses are based on previous studies with other myrmecophilous Ophiocordyceps species (Araújo et al. 2015, 2018, 2020, Evans et al. 2011b, 2018).

Field collections

Collection of specimens was concentrated at the Estação Ecológica do Tripuí (EET), Ouro Preto municipality, Minas Gerais, Brazil (BR 356, km 90, R. Nossa Senhora, da Conceição, 375, 20°23’5”S, 43°32’34.8”W) (Fig. 1). The forest reserve covers approximately 392 ha, with altitudes varying between 1,180 and 1,300 m.a.s.l., and is located in the transition (or ecotone) between Atlantic Forest and Cerrado biomes, with a mixed vegetation belonging to both; including semi-deciduous mesophytic forests, Cerrado and permanent swamps (FEAM 1995, Pedralli et al. 1997, Werneck et al. 2001). The forest reserve is also hyperdiverse in arthropods, especially Coleoptera (Gatti & Carneiro 2019). Surveys were conducted along the “Trilha da Guarita” trail (Fig. 1) in July and October 2023, March and November 2024 and February 2025. Collections were concentrated at the same spot (20°22’48”S, 43°32’24”W) (Fig. 1) where the infected ants formed a major graveyard. These collections consisted of careful examination of the vegetation (mainly understory shrubs), from ground level up to ca 2 m in height. Infected ants attached to the substratum (leaves and twigs) were then transferred to sterile containers and taken to the Laboratory of Insect-Microbe Interactions at the Universidade Federal de Viçosa (LIIM-UFV) for morphological and molecular analyses. Permits for collection and transport were provided by SISBIO (No. 86685–4) and the Instituto Estadual de Florestas (IEF; No. 037/2023). Images of the specimens were taken in situ and their positions on the vegetation were noted.

Fig. 1.

Sampling site in Ouro Preto, Minas Gerais, Brazil. A. Map with an overview of the forest reserve, Estação Ecológica do Tripuí. B, D. Sampling sites at the higher and drier elevations showing the relatively open canopy and the understorey dominated by tree ferns (Cyathea spp.), where ant graveyards predominantly occurred. C. Lower, wetter parts of the trail being invaded by exotic wild ginger (Hedychium coronarium).

The ant hosts were identified by RMF, specialist in the taxonomy of Heteroponerini. Voucher specimens of O. acanthoponerae (holotype and paratypes) were deposited at the VIC Fungarium, located at UFV, at Royal Botanic Gardens Kew Fungarium (K-M: IMI) and at the Natural History Museum of Denmark, University of Copenhagen (NHMD). The remaining specimens are conserved at LIIM-UFV and at the Entomological Collection Padre Jesus Santiago Moure at the Universidade Federal do Paraná (DZUP-UFPR).

DNA extraction, amplification and sequencing

For molecular identification, DNA was extracted from ascostromata (ascoma + stroma) and synnemata which had been excised from the host and macerated in microtubes and then placed in liquid nitrogen. The DNA extraction was performed using the Wizard™ Genomic DNA Purification Kit (Promega™, Madison, USA), following the manufacturer’s recommendations. The final concentrations of the DNA extractions were determined by NanoDrop™ spectrophotometry (Thermo Scientific™, Waltham, USA).

In total, four genomic regions were amplified using polymerase chain reaction (PCR): i) the nuclear ribosomal small subunit (SSU), ii) the nuclear ribosomal large subunit (LSU), iii) the translation elongation factor 1-α (TEF), and iv) RNA polymerase II largest subunit (RPB1). Primers used for each region were: (SSU) NS1 (5′-GTAGTCATATGCTTGTCTC-3′) and NS4 (5′-CTTCCGTCAATTCCTTTAAG-3′) (White et al. 1990), (LSU) LR0R (5′-ACCCGCTGAAC-TTAAGC-3′) and LR5 (5′-TCCTGAGGGAAACTTCG-3′) (Vilgalys & Sun 1994), (TEF) 983F (5′-GCYCCYGGHCAYCGTGAYTTYAT-3′) and 2218R (5′-ATGACACCRACRGCRACRGTYTG-3′) (Rehner & Buckley 2005), (RPB1) RPB1-Ac: (5′-GARTGYCCDGGDCAYTTYGG-3′) and RPB1-Cr (5′-CCNGCDATNTCRTTRTCCATRTA-3′) (Murata et al. 2014).

Each 25 μL PCR amplification reaction contained 12.5 μL KAPA Taq ReadyMix (KAPA Biosystems™), 1 μL of each forward and reverse primers (10 pmol/μL), 5 μL of DNA template, and 5.5 μL of DNAse/RNAse Free Water (ZymoBIOMICS™). The amplification reactions were placed in an Eppendorf™ thermocycler under the following conditions: for SSU and LSU, 2 min at 95 °C, 4 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 2 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 50.5 °C for 1 min, and extension at 72 °C for 2 min and final extension at 5 min at 72 °C. For TEF, 2 min at 94 °C, 10 cycles of denaturation at 94 °C for 30 s, annealing at 64 °C for 1 min, and extension at 72 °C for 1 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 1 min, extension at 72 °C for 1 min and final extension at 3 min at 72 °C. For RPB1, 2 min at 95 °C, 10 cycles of denaturation at 95 °C for 30 s, annealing at 66 °C for 1 min, and extension at 72 °C for 1 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 56.6 °C for 1 min, extension at 72 °C for 1 min and final extension at 3 min at 72 °C. After amplification, PCR products were purified and sequenced by Macrogen™ (Seoul, South Korea).

Phylogenetic analyses

The obtained contigs were compared to sequences of related species deposited in the NCBI (https://www.ncbi.nlm.nih.gov/) nucleotide database using BLASTn at Geneious Prime™ 2025.0.3 (Kearse et al. 2012) to confirm that they belonged to the genus Ophiocordyceps. We aligned the O. acanthoponerae to a database with 530 OTUs from species (Table S1) that broadly represent the order Hypocreales. The final database consisted of 323 SSU, 479 LSU, 360 TEF and 444 RBP1 sequences.

We generated, improved (excluding gaps and removing ambiguously aligned regions) and combined individual gene alignments with Geneious Prime v. 2025.0.3. The final alignment length was 3972 bp with 1242 bp for SSU, 999 bp for LSU, 1006 bp for TEF and 725 bp for RBP1. We used the ModelTest-NG v. 0.1.7 (Darriba et al. 2020) on CIPRES Science Gateway v. 3.3 (Miller et al. 2012) for selecting the nucleotide substitution models, considering the lowest Akaike Information Criterion (AIC). The final dataset consisted of eight data partitions (one for SSU, one for LSU, three for TEF and three for RBP1).

Concatenated phylogenetic trees for all four genes were reconstructed performing Maximum Likelihood analysis using IQ-TREE v. 2.2.2.7, based on the fit-model for each region (GTR+I+G4) and generated 1000 bootstrap replicates, (Nguyen et al. 2015, Chernomor et al. 2016, Kalyaanamoorthy et al. 2017, Hoang et al. 2018, Minh et al. 2020) on the CIPRES webserver. The species Verticillium dahliae (ATCC 16535) was used as the outgroup. The resulting consensus phylogenetic tree was visualized in FigTree v. 1.4.4 (Rambaut 2017) and edited in Adobe Illustrator™.

Morphological characterisation

The samples were examined and photographed under a stereomicroscope Zeiss Discovery V20™ with an attached camera (AxioCam MRc). The photos were processed with the AxionVisionSE64™ software. Characteristics such as the host’s position and attachment to the plant substrate, shape, colour and size of the sexual and asexual structures were considered for macro-morphological characterisation.

For micro-morphological characterisation, ascostromata and synnemata were removed from hosts and were hand-sectioned using a single-sided razor blade and placed on slides with lacto-glycerol (20 mL lactic acid in 40 mL glycerol and 20 mL distilled water) or lacto-fuchsin 1 % (0.1 g acid fuchsin in 100 mL lactic acid). Specimens were examined under an Olympus™ BX53 light photomicroscope with an attached digital camera (Olympus Q-Color 3). The microphotographs were processed with the cellSens Dimensions™ software.

To obtain mature ascospores and to follow the germination process, specimens bearing ascostromata were taped to the lids of 9-cm-diam. Petri dish and suspended with Vaseline above the dish containing a thin layer of distilled water agar (DWA) and kept in an incubator in the dark at 20 °C, to await the release of ascospores (Evans et al. 2011b). Specimens were examined daily under a dissecting microscope to check for spore discharge and germination. A minimum of 50 discharged (mature) ascospores were examined and measured for comparative morphology.

RESULTS

Phylogenetic analyses

Sequences of the genomic regions SSU (= 2), LSU (= 2), TEF (= 2) and RPB1 (= 1) (4) from infected Acanthoponera samples clustered together with strong bootstrap support value (BS = 100 %) (Fig. 2). Single-locus phylogenies for TEF and RPB1 also supported the new species (Figs S1, S2). Ophiocordyceps acanthoponerae was placed in a strongly supported clade (BS = 100 %) alongside the wasp pathogen O. humbertii and the lepidopteran pathogen O. elongata (Fig. 2). This clade clustered as a sister group of the clade O. unilateralis (BS = 90 %). Building on the previous work by Araújo et al. (2018), O. acanthoponerae was positioned outside the two traditional clades of hirsutelloid Ophiocordyceps associated with ants, the O. unilateralis core clade, O. kniphofioides sub-clade and O. oecophyllae. The O. unilateralis core clade included 22 combined sequences of different species and was strongly supported (BS = 100 %) (Fig. 2; Table 1). Ophiocordyceps oecophyllae clustered as a sister-group of O. unilateralis core clade (BS = 91 %). The sub-clade O. kniphofioides was placed as a sister group of the O. unilateralis core clade together with O. oecophyllae, but this relationship had no support.

Fig. 2.

Maximum likelihood tree of Ophiocordyceps obtained with a concatenated dataset of SSU, LSU, TEF and RPB1. Numbers on nodes represent bootstrap values > 70 %. Ophiocordyceps acanthoponerae sp. nov. is indicated in bold font and highlighted in orange. The wasp-parasitic species, O. humbertii (yellow), and the myrmecophilous clades, O. unilateralis core clade (pink) and O. kniphofioides sub-clade (green), are also highlighted in the tree. The whole analysis tree of the order Hypocreales is depicted in the top-left corner, with the position of Ophiocordyceps highlighted in orange.

Table 1.

List of Ophiocordyceps species, vouchers, GenBank accession numbers and host associations. Ophiocordyceps acanthoponerae sp. nov. is highlighted in bold font.

Species	Voucher	nSSU rDNA	nLSU rDNA	TEF	RPB1	Host	Reference
Ophiocordyceps acanthoponerae	VIMI_2023_0001	PV283217	PV283219	PV356076	PV356074	Acanthoponera mucronata	This study
	VIMI_2023_0002	PV283216	PV283218	PV356075	—	Acanthoponera mucronata	This study
Ophiocordyceps acicularis	OSC 128580	DQ522543	DQ518757	DQ522326	DQ522371	Coleoptera	Quandt et al. (2014)
Ophiocordyceps agriotidis	ARSEF 5692	DQ522540	DQ518754	DQ522322	DQ522368	Coleoptera	Quandt et al. (2014)
Ophiocordyceps albacongiuae	RC20	KX713633	—	KX713670	—	Hymenoptera	Araújo et al. (2019)
Ophiocordyceps amazonica	HUA 186113	KJ917566	KJ917571	—	KP212903	Orthoptera	Sanjuan et al. (2015)
Ophiocordyceps annulata	CEM303	KJ878915	KJ878881	KJ878962	KJ878995	Coleoptera	Quandt et al. (2014)
Ophiocordyceps aphodii	ARSEF 5498	DQ522541	DQ518755	DQ522323	—	Coleoptera	Quandt et al. (2014)
Ophiocordyceps australis	HUA 186097	KC610786	KC610765	KC610735	KF658662	Hymenoptera	Sanjuan et al. (2015)
	HUA 186147	KC610784	KC610764	KC610734	KF658678	Hymenoptera	Quandt et al. (2014)
Ophiocordyceps blakebarnesii	MISSOU5	KX713641	KX713610	KX713688	KX713716	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps brunneipunctata	OSC 128576	DQ522542	DQ518756	DQ522324	DQ522369	Coleoptera	Quandt et al. (2014)
Ophiocordyceps buquetii	HMAS_199613	KJ878939	KJ878904	KJ878984	KJ879019	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps camponoti-atricipis	A25	KX713666	—	KX713677	—	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps camponoti-balzani	G104	KX713660	KX713593	KX713689	KX713703	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps camponoti-bispinosi	OBIS5	KX713636	KX713616	KX713693	KX713721	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps camponoti-femorati	FEMO2	KX713663	KX713590	KX713678	KX713702	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps camponoti-floridani	Flx1	KX713661	—	—	—	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps camponoti-hippocrepidis	HIPPOC	KX713655	KX713597	KX713673	KX713707	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps camponoti-leonardi	C36	KJ201512	—	JN819013	—	Hymenoptera	Kobmoo et al. (2018)
Ophiocordyceps camponoti-nidulantis	NIDUL2	KX713640	KX713611	KX713669	KX713717	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps camponoti-renggeri	RENG2	KX713632	—	KX713672	—	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps camponoti-rufipedis	G108	KX713659	KX713594	KX713679	KX713704	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps camponoti-saundersi	C40	KJ201519	—	JN819012	—	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps citrina	TNS F18537	—	KJ878903	KJ878983	—	Hemiptera	Quandt et al. (2014)
Ophiocordyceps clavata	CEM1762	KJ878916	KJ878882	KJ878963	KJ878996	Coleoptera	Quandt et al. (2014)
Ophiocordyceps cochlidiicola	HMAS_199612	KJ878917	KJ878884	KJ878965	KJ878998	Lepidoptera	Quandt et al. (2014)
Ophiocordyceps communis	NHJ 12581	EF468973	EF468831	EF468775	—	Coleoptera	Quandt et al. (2014)
Ophiocordyceps curculionum	OSC 151910	KJ878918	KJ878885	—	KJ878999	Coleoptera	Quandt et al. (2014)
Ophiocordyceps daceti	MF01	—	KX713604	KX713667	—	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps diabolica	BDS 32	MK393830	MK393322	—	—	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps dipterigena	OSC 151912	KJ878920	KJ878887	KJ878967	KJ879001	Diptera	Quandt et al. (2014)
Ophiocordyceps elongata	OSC 110989	—	EF468808	EF468748	EF468856	Lepidoptera	Quandt et al. (2014)
Ophiocordyceps entomorrhiza	16250	KJ878942	—	KJ878987	KJ879021	Coleoptera	Quandt et al. (2014)
Ophiocordyceps evansii	HUA 186159	KC610796	KC610770	KC610736	KP212916	Hymenoptera	Sanjuan et al. (2015)
Ophiocordyceps formicarum	TNS F18565	KJ878921	KJ878888	KJ878968	KJ879002	Hymenoptera	Quandt et al. (2014)
Ophiocordyceps formosana	TNM F13893	KJ878908	—	KJ878956	KJ878988	Coleoptera	Quandt et al. (2014)
Ophiocordyceps forquignonii	OSC 151902	KJ878912	KJ878876	—	KJ878991	Diptera	Quandt et al. (2014)
	OSC 151908	KJ878922	KJ878889	—	KJ879003	Diptera	Quandt et al. (2014)
Ophiocordyceps fulgoromorphila	HUA 186139	KC610794	KC610760	KC610729	KF658676	Hemiptera	Sanjuan et al. (2015)
	HUA 186142	KC610795	KC610761	KC610730	KF658677	Hemiptera	Sanjuan et al. (2015)
Ophiocordyceps gracilis	EFCC 8572	EF468956	EF468811	EF468751	EF468859	Lepidoptera	Quandt et al. (2014)
Ophiocordyceps gracillisima	HUA 186132	—	KC610768	KC610744	KF658666	Coleoptera	Sanjuan et al. (2015)
Ophiocordyceps halabalaensis	MY1308	KM655825	—	GU797109	—	Hymenoptera	Luangsa-ard et al. (2011)
Ophiocordyceps heteropoda	EFCC 10125	EF468957	EF468812	EF468752	EF468860	Hemiptera	Quandt et al. (2014)
Ophiocordyceps humbertii	MF116A	MK874747	MK875537	—	MK863828	Hymenoptera	Araújo et al. (2019)
	MF116B	MK874748	MK875536	—	MK863829	Hymenoptera	Araújo et al. (2019)
Ophiocordyceps irangiensis	128578	DQ522556	DQ518770	DQ522345	DQ522391	Hymenoptera	Quandt et al. (2014)
	OSC 128577	DQ522546	DQ518760	DQ522329	DQ522374	Hymenoptera	Sanjuan et al. (2015)
Ophiocordyceps kimflemingiae	SC30	KX713629	KX713622	KX713699	KX713727	Hymenoptera	Araújo et al. (2019)
Ophiocordyceps kniphofioides	HUA 186148	KC610790	KF658679	KC610739	KF658667	Hymenoptera	Sanjuan et al. (2015)
Ophiocordyceps konnoana	EFCC 7315	EF468959	—	EF468753	EF468861	Coleoptera	Quandt et al. (2014)
Ophiocordyceps lloydii	OSC 151913	KJ878924	KJ878891	KJ878970	KJ879004	Hymenoptera	Quandt et al. (2014)
Ophiocordyceps longissima	HMAS_199600	KJ878926	—	KJ878972	KJ879006	Hemiptera	Quandt et al. (2014)
Ophiocordyceps melolonthae	OSC 110993	DQ522548	DQ518762	DQ522331	DQ522376	Coleoptera	Quandt et al. (2014)
Ophiocordyceps monacidis	MF74	KX713646	KX713606	—	—	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps myrmecophila	HMAS_199620	KJ878927	KJ878893	KJ878973	KJ879007	Hymenoptera	Quandt et al. (2014)
Ophiocordyceops naomipierceae	DAWKSANT	KX713664	KX713589	—	KX713701	Hymenoptera	Araújo et al (2018)
Ophiocordyceps neovolkiana	OSC 151903	KJ878930	KJ878896	KJ878976	KJ879010	Coleoptera	Quandt et al. (2014)
Ophiocordyceps nigrella	EFCC 9247	EF468963	EF468818	EF468758	EF468866	Coleoptera	Quandt et al. (2014)
Ophiocordyceps nutans	OSC 110994	DQ522549	DQ518763	DQ522333	DQ522378	Hemiptera	Quandt et al. (2014)
Ophiocordyceps oecophyllae	OECO1	KX713635	—	—	—	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps ootakii	J13	KX713652	KX713600	KX713681	KX713708	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps palthothyreum	Palt1	MK393848	MK393345	—	—	Hymenoptera	Araújo et al. (2019)
Ophiocordyceps polyrhachis-furcata	P39	KJ201504	—	JN819003	—	Hymenoptera	Kobmoo et al. (2015)
Ophiocordyceps ponerinarum	HUA 186140	KC610789	KC610767	KC610740	KF658668	Hymenoptera	Sanjuan et al. (2015)
Ophiocordyceps pruinosa	NHJ 12994	EU369106	EU369041	EU369024	EU369063	Lepidoptera	Quandt et al. (2014)
Ophiocordyceps pulvinata	TNS-F 30044	GU904208	—	GU904209	GU904210	Hymenoptera	Kepler et al. (2011)
Ophiocordyceps purpureostromata	TNS F1843	KJ878931	KJ878897	KJ878977	KJ879011	Coleoptera	Quandt et al. (2014)
Ophiocordyceps rami	MY6736	KM655823	—	KJ201532	—	Hymenoptera	Kobmoo et al. (2015)
Ophiocordyceps ravenelii	OSC 110995	DQ522550	DQ518764	DQ522334	DQ522379	Coleoptera	Quandt et al. (2014)
Ophiocordyceps rhizoidea	NHJ 12522	EF468970	EF468825	EF468764	EF468873	Coleoptera	Quandt et al. (2014)
Ophiocordyceps satoi	J7	KX713653	KX713599	KX713683	KX713711	Hymenoptera	Araújo et al. (2018)
Ophiocordyceps sinensis	EFCC 7287	EF468971	EF468827	EF468767	EF468874	Lepidoptera	Quandt et al. (2014)
Ophiocordyceps sobolifera	KEW 78842	EF468972	EF468828	—	EF468875	Hemiptera	Quandt et al. (2014)
Ophiocordyceps sp.	TNS F18495	KJ878937	KJ878901	—	KJ879017	Coleoptera	Quandt et al. (2014)
	OSC 151904	KJ878934	KJ878899	KJ878980	KJ879014	Hemiptera	Quandt et al. (2014)
	OSC 151905	KJ878935	—	KJ878981	KJ879015	Hemiptera	Quandt et al. (2014)
	CatKNZ01_OP	LC370816	LC370818	LC370821	LC370819	Hemiptera (Symbiont)	Matsuura et al. (2018)
	CfaKSD05_OP	LC370788	LC370790	LC370801	LC370791	Hemiptera (Symbiont)	Matsuura et al. (2018)
	EUTCHI_OP	LC370888	LC370890	LC370893	LC370891	Hemiptera (Symbiont)	Matsuura et al. (2018)
	EokKNG01_OP	LC370894	LC370896	LC370899	LC370897	Hemiptera (Symbiont)	Matsuura et al. (2018)
	GbiNNJ01_OP	LC370850	LC370852	LC370855	LC370853	Hemiptera (Symbiont)	Matsuura et al. (2018)
	GniKSD01_OP	LC370844	LC370846	LC370849	LC370847	Hemiptera (Symbiont)	Matsuura et al. (2018)
	HmaTKB05_OP	LC370983	LC370985	LC370988	LC370986	Hemiptera (Symbiont)	Matsuura et al. (2018)
	MiwITN01_OP	LC370967	LC370969	LC370972	LC370970	Hemiptera (Symbiont)	Matsuura et al. (2018)
	MkuYGJ01_OP	LC370960	LC370962	LC370965	LC370963	Hemiptera (Symbiont)	Matsuura et al. (2018)
	MopTKB06_OP	LC370921	LC370923	LC370926	LC370924	Hemiptera (Symbiont)	Matsuura et al. (2018)
	MosKNG01	LC370940	LC370942	LC370945	LC370943	Hemiptera (Symbiont)	Matsuura et al. (2018)
	MmiNNJ01_OP	LC370999	LC371001	LC371004	LC371002	Hemiptera (Symbiont)	Matsuura et al. (2018)
	TERVAC_OP	LC370866	LC370868	LC370871	LC370869	Hemiptera (Symbiont)	Matsuura et al. (2018)
	TjaTKB04_OP	LC370902	LC370904	LC370907	LC370905	Hemiptera (Symbiont)	Matsuura et al. (2018)
	MopTKB01_01	LC371006	LC371008	LC371011	LC371009	Hemiptera (Symbiont)	Matsuura et al. (2018)
	TniTKB04_OP	LC370872	LC370874	LC370877	LC370875	Hemiptera (Symbiont)	Matsuura et al. (2018)
	Gh41	KX713656	—	KX713668	KX713706	Hymenoptera	Araújo et al. (2019)
	OSC 151909	KJ878936	KJ878900	KJ878982	KJ879016	Hymenoptera	Quandt et al. (2014)
Ophiocordyceps sphecocephala	OSC 110998	DQ522551	DQ518765	DQ522336	DQ522381	Hymenoptera	Quandt et al. (2014)
Ophiocordyceps stylophora	OSC 111000	DQ522552	DQ518766	DQ522337	DQ522382	Coleoptera	Quandt et al. (2014)
Ophiocordyceps tiputini	QCNE 186287	KC610792	KC610773	KC610745	KF658671	Megaloptera	Sanjuan et al. (2015)
Ophiocordyceps variabilis	OSC 111003	EF468985	EF468839	EF468779	EF468885	Coleoptera	Quandt et al. (2014)
Ophiocordyceps yakusimensis	HMAS_199604	KJ878938	KJ878902	—	KJ879018	Hemiptera	Quandt et al. (2014)

Taxonomy

Based on morphology, and supported by the molecular data, a new species in the genus Ophiocordyceps is proposed:

Ophiocordyceps acanthoponerae

S.J. Lima-Santos, H.C. Evans & J.P.M. Araújo, sp. nov. MB 859808. Figs 3–7.

Fig. 3.

Ophiocordyceps acanthoponerae. A. Holomorph with mature sexual and asexual morphs. B, C. Ascostromata produced from the dorsal neck region, dark brown, non-fleshy. D. Transversal section of the fertile part showing the buried perithecia. E. Perithecium with asci. F. Ascus, 8–spored with an apical cap. G, H. 7-septate ascospores. I. Germinated ascospore with three capilliconidiophores. J. Germinated ascospore bearing a capilliconidium on the right corner. Images A–C by Frederico Salles. Scale bars: A = 1 mm; B = 0.5 mm; C = 50 µm; D = 30 µm; E = 0.2 µm; F = 10 µm; G–J = 10 µm.

Etymology: Named after the host ant genus Acanthoponera (Heteroponerini: Formicidae).

Typus: Brazil, Minas Gerais state, Ouro Preto municipality, Estação Ecológica do Tripuí (20°22′48”S, 43°32′24”W), on Acanthoponera mucronata, in forest understorey, 23 Nov. 2024, S.J. Lima-Santos, B.L. Clemente, N.L.S. Lima & T. Mendes-Pereira (holotype VIC 49609). Paratypes: All from Brazil, Minas Gerais state, Ouro Preto municipality, Estação Ecológica do Tripuí (20°22′48”S, 43°32′24”W), on Acanthoponera mucronata, in forest understorey, 12 Oct. 2023, S.J. Lima-Santos, G.S. de Paula, M.C. Castro, M.M. Moreira & R.C. Silva, VIC 49613; ibid., 11 Mar. 2024, S.J. Lima-Santos, L.C. dos Reis, S.L. Elliot & H.C. Evans, VIC 49614; ibid., 23 Nov. 2024, B.L. Clemente, N.L.S. Lima & T. Mendes-Pereira, VIC 49610, VIC 49611, VIC 49612 VIC 49615, NHMD001788818, NHMD001788819, NHMD001788820; ibid., 19 Feb. 2025, S.J. Lima-Santos, B.L. Clemente, G.S. de Paula & H.C. Evans, IMI 507615.

Additional specimens examined: In total 75 infected Acanthoponera mucronata ants were collected at different stages of infection. All of them were used to conduct fungus diagnosis. Specimens that are not deposited can be located at LIIM-UFV and in the Entomological Collection Padre Jesus Santiago Moure at the Universidade Federal do Paraná (DZUP-UFPR).

Description: External mycelium white in early stages, becoming light brown with age, emerging from all host orifices and sutures. Sexual morph (Fig. 3): One or up to three stromata produced from dorsal pronotum, simple or branched, clavate to cylindrical, 2.8–4 mm in length, 0.2–0.3 mm thick, pale brown, clavate; fertile region consisting of a dark brown ascoma, covering 360° and mostly not reaching the tip of the stalk, averaging 1.6 × 0.5 mm (Fig. 3A–C). Perithecia immersed to partially erumpent, flask-shaped, (209–)247(–289) × (103–)134(–179) µm (Fig. 3D–E). Asci with 8 intertwined spores, cylindrical, hyaline, (131–)150(–175) × (6.0–)7.4(–9.0) µm; with a prominent apical cap with a narrow pore, (2.0–)4.9(–6.0) × (4.5–)5.2(–6.0) µm (Fig. 3F). Ascospores vermiform, hyaline, (94–)126(–152) × (2.0–)2.6(–3.0) µm, 7–septate, slightly curved, tapering towards the apices (Fig. 3G–H). Asexual morph (Fig. 4): Hirsutella-like phialides produced along the stromatal stalk and on synnemata emerging from sutures and joints. Synnemata pale brown or tan-coloured, single or branched, usually with secondary smaller and delicate branches produced particularly near the base; main stalks often subcapitate, produced from the dorsal pronotum, the leg joints, thorax and abdominal sutures; averaging 2.5 mm in length (Fig. 4A, B). Phialides lageniform, guttulate, hyaline, (10.0–)13.7(–16.5) × (3.0–)4.5(–6.5) µm, tapering to a short neck, (4.5–)8.3(–10.5) × (0.5–)1.2(–2.0) µm (Fig. 4C, D); occasionally with much longer, needle-like neck, especially on the ascostromata, up to 60 µm in length; conidia aseptate, tear-shaped, smooth, guttulate, hyaline, (7.5–)8.6(–10.0) × (2.0–)2.2(–2.5) µm (Fig. 4E). Hyphal bodies ovoid to pyriform, swollen, aseptate to septate, guttulate, hyaline, (16.0–)29.8(–58) × (8.0–)11.5(–14) µm, excised from abdomen, glistening white en masse (Fig. 4F).

Fig. 4.

Ophiocordyceps acanthoponerae asexual morph. A, B. Clavate and cylindrical branched synnemata, pale or tan-coloured arising from sutures on the body and appendages. C, D. Phialides along the synnemata. E. Tear-shaped conidia. F. Chains of hyphal bodies located internally in the host’s abdomen. Image A by Frederico Salles. Scale bars: A = 1 mm; B = 0.2 mm; C–F = 10 µm; F = 20 µm.

Germination process: Ascospores, ejected onto DWA, germinated within 24–48 h, often accompanied by spore swelling, to produce up to four straight capilliconidiophores, averaging 17.3 × 1.3 µm (Fig. 3I); each bearing a single, terminal, fusiform capilliconidium (Fig. 3J), averaging 8.4 × 1.6 µm. Germ-tubes were not produced and no further growth was observed.

Habitat: Semi-deciduous, mesophytic forest in a transition area between Atlantic rainforest and Cerrado in the municipality of Ouro Preto, Minas Gerais, Brazil. Ouro Preto — so-named because of the dark layer of iron oxide covering the gold — was the centre of a gold rush in the 18^th century; becoming famous for its many Baroque-style churches, often adorned with gold leaf: hence, the gold reference in the title of this article. Also refers to the golden colour of the workers of A. mucronata.

Notes: Infected ants were found biting mostly on the edges of leaves — but also on the abaxial side, typically on the midrib — as well as clinging and biting onto twigs and side branches, at heights ranging from 0.5 up to 2 m above ground level. All the infected ants were found in the same location, forming one major graveyard at a specific spot along the Trilha da Guarita trail (20°22′48”S, 43°32′24”W). All specimens (n = 75) of A. mucronata infected by O. acanthoponerae were found consistently attached to the vegetation with their mandibles embedded in the substratum, often with evidence of scarring; a characteristic of the so-called zombie ants, as typified by species of the O. unilateralis complex attacking formicine ants of the tribe Camponotini.

DISCUSSION

The unusual morphologies of both the asexual and sexual morphs of O. acanthoponerae were the first indication that this could represent a new hirsutelloid species within Ophiocordyceps (Table 2). Myrmecophilous hirsutelloid Ophiocordyceps can be divided into two clades: O. unilateralis core clade and O. kniphofioides sub-clade (Fig. 2). The O. unilateralis core clade usually produces brown or black cushion-like fertile regions (single or multiple) that emerge unilaterally on the stalk, hence the name O. unilateralis, with ascospores that germinate via capilliconidiophores (Evans & Samson 1984, Evans et al. 2011b, Araújo et al. 2015, 2018). In comparison, the O. kniphofioides sub-clade typically produces a single ascostroma, covering 360° of the stalk, with ascospores that do not germinate (at least in vitro) via capilliconidiophores (Evans & Samson 1982, Sanjuan et al. 2015, Araújo et al. 2018). We have shown that O. acanthoponerae shares mixed traits with both clades, such as the dark brown ascostromata covering 360° of the stalk and ascospores producing capilliconidiophores and capilliconidia (Fig. 3I, J). Whilst in the asexual morph, the pale-coloured, multi-branched synnemata producing tear-shaped conidia (Fig. 4E) readily distinguish this species from other myrmecophilous hirsutelloid species described thus far.

Table 2.

Comparison of host, death position (first part of table), ascospore morphology and geographic allocation (second part of table) of hirsutelloid species of Ophiocordyceps. Ophiocordyceps acanthoponerae sp. nov. is highlighted in bold font.

Species Complex and Species	Host	Death position	Stromata	Fertile region	Source
Unnamed lineages
Ophiocordyceps acanthoponerae sp. nov.	Acanthoponera mucronata	biting the edges of leaves, abaxial faces of leaves and twigs	1–3 stromata, simple or branched, produced from dorsal pronotum	Dark brown, covering 360° and mostly not reaching the tip of the stalk	This study
O. elongata	Apatela americana (Lepidoptera) larva and pupa	—	1–3 stromata, arising from each host	Pale yellow, cylindric, terete, equal	Petch (1937)
O. humbertii	Hymenoptera	biting leaf	Two stromata produced from points of insertion of the wings on each side	Dark brown, covering 360° and not reaching the tip of the stalk	Petch (1935)
O. tiputini	Megaloptera	leaf litter	Single, simple or branched, capitate, slender	Dark orange to deep orange, having a central core and sterile acuminate tip	Sanjuan et al. (2015)
O. unilateralis s. l.
O. unilateralis (Epitype)	Camponotus sericeiventris	biting leaf	Solitary, arising from the dorsal pronotum	Dark brown, hemisphaerical produced unilaterally	Evans et al. (2018)
O. camponoti-atricipis	Camponotus atriceps	biting leaf	Single, produced from dorsal pronotum	Chocolate brown, 1–2 lateral cushions, hemispherical	Araújo et al. (2015)
O. camponoti-balzani	Camponotus balzani	biting leaf	Single, produced from dorsal pronotum	Brown to black, lateral cushions, disc-shaped to hemisphaerical	Evans et al. (2011b)
O. camponoti-bispinosi	Camponotus bispinosus	biting spines	Single, produced anterior to pronotum	Dark brown becoming black with age, lateral globose cushion	Araújo et al. (2015)
O. camponoti-chartificis	Camponotus chartifex	biting leaf	Single, produced from dorsal pronotum	Chocolate brown, 1–2 lateral cushions	Araújo et al. (2018)
O. camponoti-femorati	Camponotus femoratus	biting leaf/spines	Single, produced from dorsal pronotum	Chestnut-brown, terminal of 1–3 lateral cushions, disc-shaped to hemispherical	Araújo et al. (2018)
O. camponoti-hippocrepidis	Camponotus hippocrepidis	biting spines	Single, produced from dorsal pronotum	Chestnut-brown, circular, produced at the middle part of stroma, laterally attached	Araújo et al. (2018)
O. camponoti-indiani	Camponotus indianus	biting leaf	Multiple, arising from right and left sides of pronotum, and leg joints	Chocolate to dark brown with age, 1–4 lateral cushions, hemispherical	Araújo et al. (2015)
O. camponoti-melanotici	Camponotus melanoticus	biting leaf	Single, produced from dorsal pronotum	Dark brown to black, 1–2 lateral cushions, hemispherical	Evans et al. (2011b)
O. camponoti-nidulantis	Camponotus nidulans	biting saplings	Single, produced from dorsal pronotum	Light brown, 1–2 lateral cushions, disc-shaped to hemispherical	Araújo et al. (2018)
O. camponoti-novogranadensis	Camponotus novogranadensis	biting epiphytes	Single, produced from dorsal pronotum	Brown, 1–2 lateral cushions, disc-shaped to hemisphaerical	Evans et al. (2011b)
O. camponoti-renggeri	Camponotus renggeri	biting leaf/moss	Single, rarely branched, produced from dorsal pronotum	Dark-brown to black, 1–2 lateral cushions, hemispherical to globose	Araújo et al. (2018)
O. camponoti-rufipedis	Camponotus rufipes	biting leaf	Single, produced from dorsal pronotum	Pale to chestnut-brown, 1–3 lateral cushions, disc-shaped to hemisphaerical	Evans et al. (2011b)
O. camponoti-sexguttati	Camponotus sexguttatus	biting leaf	Single, arising from the dorsal pronotum, never branching	Chestnut-brown, single lateral cushion, disc-shaped	Araújo et al. (2018)
O. kniphofioides s. l.
O. monacidis	Dolichoderus bispinosus	base of trunk (moss)	Single, emerges laterally from the pronotum, rarely from the gaster	Dark orange, terminal	Evans et al. (1982); Araújo et al. (2018)
O. daceti	Daceton armigerum	leaf (not biting)	Arising from the dorsal pronotum	—	Araújo et al. (2018)
O. kniphofioides	Cephalotes atratus	base of trunk	Simple or branched, arising laterally from neck or upper thorax (pronotum), occasionally dorsally	Light to deep orange, terminal, clavate to conical	Evans et al. (1982)
O. ponerinarum	Paraponera clavata	base of trunk	Simple or branched, emerging laterally from the pronotum	Acuminate	Evans et al. (1982)

The Maximum Likelihood phylogenetic analysis confirmed this novelty. Surprisingly, O. acanthoponerae clustered in a strongly supported clade (BS = 100 %) outside of the traditional hirsutelloid myrmecophilous clade (O. unilateralis core clade + O. oecophyllae + O. kniphofioides sub-clade) (Fig. 2). This unexpected phylogenetic position corroborates the existence of a novel lineage of myrmecophilous and hirsutelloid Ophiocordyceps.

It is important to compare the current host association, death position and mechanisms of infection of O. acanthoponerae, with the two traditional clades of manipulative Ophiocordyceps; firstly, with the O. unilateralis core clade, whose species exclusively infect ants of the tribe Camponotini (Evans et al. 2011b). Their hosts are commonly found biting on leaves — the so-called death grip (Hughes et al. 2011) — usually at the edges or tips, but also can occur attached to palm spines, epiphytes and mosses (in tropical forests), and twigs (in temperate forests) (Table 2), with heights varying up to 2 m (Evans et al. 2011b, Hughes et al. 2016, Araújo et al. 2018, Loreto et al. 2018, van Roosmalen & de Bekker 2024). It is posited that ants are infected on or near their foraging trails when coming into contact with sticky capilliconidia, a secondary and infective spore produced on hirsutelloid conidiogenous cells (capilliconidiophores) arising from germinating ascospores that have been ejected from ascostromata on infected ants in the forest understorey (Evans et al. 2011a, b). This infection process has been likened, somewhat fancifully, to “a delayed-action landmine” (Evans & Boddy 2010), and the germination of the ascospores, essential for the infection process, can be simulated in vitro, following the protocol described previously in the Materials and Methods (Evans et al. 2011b). The morphology of the ascus, with its thickened apical cap and a narrow pore — diagnostic of the genus (Petch 1931) — is crucial for the explosive release of the ascospores and the purported infection cycle with ascospores raining on and ‘contaminating’ the ant trails. As graphically shown in Figs 5 & 6, the death positions and the biting strategy of Acanthoponera ants infected with O. acanthoponerae replicate the behaviours of Camponotini ants infected with O. unilateralis sensu lato. This evidence of fungal manipulation, with the ants positioned in the lower understorey above the nests and the trails emanating from them on the forest floor, justifies the classification of O. acanthoponerae within the zombie-ant fungi.

Fig. 5.

Sequence of events following infection of Acanthoponera mucronata by Ophiocordyceps acanthoponerae: development of asexual morph. A. Recently dead ant biting into leaf midrib with no external evidence of infection. B–D. White silky hyphae emerging from sutures on body and appendages, including the antennae, on ants clinging to leaf margins. E, F. Developing synnemata, pale-coloured emerging from body and appendages. Scale bars: A–E = 1.5 mm; F = 2.5 mm.

Fig. 6.

Sequence of events following infection of Acanthoponera mucronata by Ophiocordyceps acanthoponerae: development of sexual morph. A–D. Single immature, pale-coloured ascostroma emerging from dorsal pronotum of each ant, dying in various positions biting into leaf margins, midribs and small branches. E, F. Mature, dark brown ascostromata (short arrows); atypically, in E, it is emerging from the lateral thorax. Note the leaf scars in A, C and E (long arrows), caused by repetitive biting prior to death. Scale bars = 2.5 mm.

Species of the O. kniphofioides sub-clade infect ant hosts belonging to the distantly related genera Cephalotes, Daceton, Dolichoderus and Paraponera. The most studied interaction involves O. kniphofioides sensu stricto and Cephalotes atratus — an arboreal myrmicine ant highly adapted to living in the forest canopy where it nests and forages on pollen and homopteran exudates with the aid of adhesive tarsal pads (Stark & Yanoviak 2018)— where infected ants aggregate on tree trunks (Table 2) (Evans & Samson 1982, Sanjuan et al. 2015, Araújo et al. 2018, Imirzian et al. 2020). Healthy workers scavenge on the trees attempting to remove infected cadavers and it is most probable that these become infected when encountering the mucilaginous conidia produced on hirsutelloid, rhizoid-like synnemata that radiate out on the tree bark from the ant host (Evans & Samson 1982, Araújo et al. 2018).

A closely-related species within the O. kniphofioides complex is O. monacidis on the myrmicine ant Dolichoderus (Monacis) bispinosus (Araújo et al. 2018): a strictly arboreal ant with similar foraging habits to C. atratus (Mackay 1993). As with diseased C. atratus ants, infected D. bispinosus ants descend from their arboreal habitats and die amongst clumps of moss on the buttress roots of upperstorey trees, where the dark-orange, poker-like ascostromata of O. monacidis mimick the moss sporophores (Araújo et al. 2018). If, as the evidence suggests (Mackay 1993), D. bispinosus is an arboreal ant — foraging and nesting in the canopy — with only occasional sorties to the forest floor, it is difficult to determine how the ascospores reach their target hosts and function in the infection cycle. Rather than being manipulated by the fungus, infected ants would appear to be moving away from their arboreal habitats and actively hiding within the moss substrate, which could be interpreted to be altruistic by avoiding infection of the nests and trails in the canopy. Such an interpretation has been posited regarding both C. atratus and D. bispinosus infected by another Ophiocordyceps species complex, O. cucumispora sensu lato, where both hosts when infected descend from the forest upperstorey and die hidden within the litter layer (Evans 1982, 1988, Evans & Samson 1982). The ascospores readily break into highly-distinct, cucumber-like, longitudinally-ridged and echinulate part-spores. The form and function of these unusual spores is unknown, as is how they reach their arboreal targets, although the striation and echinulation indicates the likely involvement of phoresy on a vector.

In the phylogenetic analyses, O. acanthoponerae clustered in a clade (BS = 100 %) with sequences obtained for the wasp pathogen O. humbertii and the lepidopteran pathogen O. elongata (Fig. 2). We were able to identify shared traits between the taxa associated with hymenopteran insects. Both taxa, O. acanthoponerae and O. humbertii, have conspicuous hirsutelloid asexual morphs, dark brown ascostromata covering 360° of the stalk and vermiform (or narrow-fusoid) ascospores (Table 2) (Petch 1935, Somavilla et al. 2020, Sobczak et al. 2020, Barbosa & Somavilla, 2022). Wasps are also manipulated by O. humbertii and are mostly found biting on the border of leaves (Somavilla et al. 2020, Sobczak et al. 2020, Barbosa & Somavilla 2022), in a similar way to O. acanthoponerae. An analysis by Araújo & Hughes (2019) placed O. humbertii as a sister group of O. unilateralis clade (BS = 100 %) and it was hypothesized that an intermediate step might have occurred via social wasps, in the sense that an ancient Ophiocordyceps lineage jumped from wasps to ants, during the evolution of the myrmecophilous clade. Our new findings could represent such a missing link between manipulative ant- and wasp-associated Ophiocordyceps. Currently, studies are under way to provide a more robust phylogenetic framework for wasp-associated species, in the O. humbertii clade or complex, with the objective of elucidating the origins and evolution of manipulative Ophiocordyceps lineages.

This study is also the first record of ants belonging to the genus Acanthoponera (Heteroponerini: Ectatomminae: Formicidae) being infected by Ophiocordyceps. The genus Acanthoponera is restricted to the Neotropics and these relatively-large, golden-coloured ponerines are characterized by their prominent spines, the well-developed eyes and their long palps (Feitosa 2015, Camacho et al. 2022). They represent a lineage of nocturnal ants which forage exclusively on arboreal substrates, mostly in humid forests (Feitosa 2015, Feitosa & Prada-Achiardi 2019, Camacho et al. 2022). Workers are usually found foraging alone on the vegetation searching for small arthropods (Feitosa 2015, Feitosa & Prada-Achiardi 2019). It is thought that their colonies may harbour dozens to a few hundred individuals but data on nest structure and colony size are lacking (Feitosa 2015, and pers. obs.). The species A. mucronata has the largest distribution of the genus and is found in all Brazilian regions (Feitosa 2015, Feitosa & Prada-Achiardi 2019). This raises the questions of whether O. acanthoponerae is to be found across its host’s range and whether other Ophiocordyceps species exist that parasitize the rarer members of the genus. Acanthoponera mucronata shares arboreal nesting and foraging habits with other genera of ants commonly infected by manipulative Ophiocordyceps (i.e., Camponotus, Cephalotes, Daceton, Paraponera and Polyrhachis) (Feitosa & Dias 2024). This life-history trait might have favoured infection and ultimately host-switching by Ophiocordyceps between hosts sharing the same ecological niche or habitat (Sanjuan et al. 2015, Araújo & Hughes 2019). The fact that workers forage alone rather than in collective trails is likely to influence how O. acanthoponerae infects and manipulates them. Their colony size, with fewer individuals, might also indicate host-parasite dynamics different from that described for O. unilateralis sensu lato on Camponotini (Andersen et al. 2012, Loreto et al. 2014).

In their recent review of behavioural manipulation, van Roosmalen & de Bekker (2024) stressed that more studies are needed to fully elucidate the infection biology of Ophiocordyceps. There is accumulating evidence which indicates “that the secretion of parasite proteins and light-driven biological rhythms are likely involved in the infection and manipulation biology” (van Roosmalen & de Bekker 2024). In particular, it is posited that Ophiocordyceps turn their ant hosts into light seekers (Andriolli et al. 2019). How this would relate to a nocturnal ant species, such as A. mucronata, would be worthy of further investigation.

CONCLUSIONS

The morphological and phylogenetic analyses confirm O. acanthoponerae as a new species within the genus Ophiocordyceps. The fungus presents mixed morphological traits related to the myrmecophilous and manipulative clade O. unilateralis (O. unilateralis core clade and O. kniphofioides sub-clade) and shows similarities with the wasp-parasitic fungus, O. humbertii. Significantly, the phylogenetic reconstruction showed that O. acanthoponerae falls outside of these ‘traditional’ clades and indicates the existence of a new lineage of myrmecophilous and manipulative fungi within the genus Ophiocordyceps. This is also the first record of Heteroponerini ants being infected by Ophiocordyceps, which expands our knowledge of the host range for these fungi. More work is needed, especially on the little-researched ant-host species, to further elucidate the biological and ecological factors that may influence this host-parasite interaction.

Table 2.

(Part 2).

Species Complex and Species	Ascospores					Distribution
		Shape	Size (µm)	Septation	Capilliconidiophore		Capilliconidia
Unnamed lineages
Ophiocordyceps acanthoponerae sp. nov.	vermiform	(94–)126(–152) × (1.9–)2.6(–3.2)	7	straight, averaging 17.3 × 1.3 µm	fusiform, averaging 8.4 × 1.6 µm	Brazilian Atlantic Forest-Cerrado ecotone
O. elongata	cylindrical	—	—	—	—	USA
O. humbertii	narrow-fusoid	75 × 2.5	at intervals of 6 to 16 µm	—	—	Sarawak, Malaysia
O. tiputini	elongate-fusiform	120–180 × 1.5–1.5	—	—	—	Amazon region, Ecuador and Bolivia
O. unilateralis s. l.
O. unilateralis (Epitype)	filiform	(70–)75–85 × 2–2.5	4–5	—	—	Brazilian Atlantic Forest
O. camponoti-atricipis	vermiform	(75–)80–85(–100) × (2–)3(–3.5)	5	straight, thread-like structures, 55 μm in length	allantoid, 10–11 × 2–2.5 μm	Brazilian Central Amazon
O. camponoti-balzani	cylindrical	(120–)135–175 × 4–5	14–22(–27)	—	—	Brazilian Atlantic Forest
O. camponoti-bispinosi	cylindrical	(60–)70–75(–80) × 4.5–5(–6)	4–5	straight, (50–)65(–80) μm	slightly truncate at the base, 10–11 × 3–4 μm	Brazilian Central Amazon
O. camponoti-chartificis	vermiform	75–85 × 5	9–13	long and extremely narrow hair-like, (65–)75–90(–95) μm	fusoid	Brazilian Central Amazon
O. camponoti-femorati	sinuous to curved	75–90 × 3	5	narrow, 35–40 μm long	clavate, 9 × 3 μm	Brazilian Central Amazon
O. camponoti-hippocrepidis	cylindrical	75–85 × 4–5	5	straight, 45–50 μm long	truncate at base, narrowing apically, 10–11 × 4 μm	Brazilian Central Amazon
O. camponoti-indiani	cylindrical	(60–)75(–80) × (3.5–)4.5(–5)	5	hair-like, 120–130 μm in length	fusoid, 13–14 μm	Brazilian Central Amazon
O. camponoti-melanotici	cylindrical	170–210 × 4–5	27–35	—	—	Brazilian Atlantic Forest
O. camponoti-nidulantis	vermiform	90–105(–115) × 3–4	5	straight, extremely narrow hair-like, 50–60 μm	clavate, 9 × 2 μm	Brazilian Central Amazon
O. camponoti-novogranadensis	filiform	75–95 × 2.5–3.5	5–10	20–25 × 0.2–0.4 μm	reniform, (5–)7–8 × 2.5–3.0 μm	Brazilian Atlantic Forest
O. camponoti-renggeri	vermiform	90–120 × 4	5–8	—	—	Brazilian Central Amazon
O. camponoti-rufipedis	vermiform	(75–)80–95(–115) × 2–3	4–7	extremely narrow hair-like structures, (45–)60–70(–80) μm	clavate, (7–)9–11 × 1.5–2.5 μm	Brazilian Atlantic Forest
O. camponoti-sexguttati	cylindrical	120–140 × 3	7	straight, 25–30 μm	narrowing apically, 5–9 × 2 μm	Brazilian Central Amazon
O. kniphofioides s. l.
O. monacidis	—	(80–)95–110(–120)	3–4	—	—	Brazilian Amazon
O. daceti	—	—	—	—	—	Brazilian Central Amazon
O. kniphofioides	filiform	110–150 × 1.5–3.0	3–5	—	—	Brazilian Amazon, Colombia
O. ponerinarum	—	—	—	—	—	Brazilian Amazon, Colombia

DATA AVAILABILITY

The sequences generated in this study are deposited with NCBI GenBank (Table 1) and the alignment and phylogenetic trees in Zenodo (doi: 10.5281/zenodo.16687083).

Conflict of Interest:

The authors declare that they have no conflicts of interest.

Supplementary Material: http://fuse-journal.org/

Fig. S1

Single-locus Maximum likelihood tree of Ophiocordyceps for the translation elongation factor 1-α (TEF) genomic region. Numbers on nodes represent bootstrap values > 70 %. Ophiocordyceps acanthoponerae sp. nov. is indicated in bold font. The whole analysis tree of the order Hypocreales is depicted in the top-left corner, with the position of the clade containing O. acanthoponerae highlighted in orange.

Fig. S2

Single-locus Maximum likelihood tree of Ophiocordyceps for the RNA polymerase II largest subunit (RPB1) genomic region. Numbers on nodes represent bootstrap values > 70 %. Ophiocordyceps acanthoponerae sp. nov. is indicated in bold font. The whole analysis tree of the order Hypocreales is depicted in the top-left corner, with the position of the clade containing O. acanthoponerae highlighted in orange.

Table S1

List of species from the order Hypocreales, vouchers, GenBank accession numbers and host associations, with exception of Ophiocordyceps which is in Table 1.