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Research Article
Morphology, phylogeny and host specificity of two new Ophiocordyceps species belonging to the “zombie-ant fungi” clade (Ophiocordycipitaceae, Hypocreales)
expand article infoDexiang Tang‡, Jing Zhao‡, Yingling Lu‡, Zhiqin Wang‡, Tao Sun‡, Zuoheng Liu‡, Hong Yu‡
‡ Yunnan University, Kunming, China

Corresponding author: Hong Yu ( hongyu@ynu.edu.cn )

Academic editor: Marc Stadler
© 2023 Dexiang Tang, Jing Zhao, Yingling Lu, Zhiqin Wang, Tao Sun, Zuoheng Liu, Hong Yu.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Tang D, Zhao J, Lu Y, Wang Z, Sun T, Liu Z, Yu H (2023) Morphology, phylogeny and host specificity of two new Ophiocordyceps species belonging to the “zombie-ant fungi” clade (Ophiocordycipitaceae, Hypocreales). MycoKeys 99: 269-296. https://doi.org/10.3897/mycokeys.99.107565
Open Access

Abstract

Species of the genus Ophiocordyceps, which include species able to manipulate the behaviour of ants, are known as the “zombie-ant fungi” and have attracted much attention over the last decade. They are widespread within tropical, subtropical and even temperate forests worldwide, with relatively few reports from subtropical monsoon evergreen broad-leaved forest. Fungal specimens have been collected from China, occurring on ants and producing hirsutella-like anamorphs. Based on a combination of morphological characters, phylogenetic analyses (LSU, SSU, TEF1a, RPB1 and RPB2) and ecological data, two new species, Ophiocordyceps tortuosa and O. ansiformis, are identified and proposed herein. Ophiocordyceps tortuosa and O. ansiformis are recorded on the same species of Colobopsis ant, based on phylogenetic analyses (COI), which may be sharing the same host. Ophiocordyceps tortuosa and O. ansiformis share the morphological character of producing lanceolate ascospores. They have typical characteristics distinguished from other species. The ascospore of O. tortuosa are tortuously arranged in the ascus and the ascospore of O. ansiformis have a structure like a handle-shape in the middle. Our molecular data also indicate that O. tortuosa and O. ansiformis are clearly distinct from other species.

Key words

Colobopsis, Entomopathogenic fungi, Ophiocordyceps, Taxonomy

Introduction

Fungi associated with insects, morphologically similar, but genetically distinct cryptic closely-related species, have given rise to spectacular diversity across a wide range of taxa in the kingdom of fungi. Molecular studies have routinely unmasked several cryptic species and have revealed this as a common phenomenon for the entomogenous fungi (Araújo et al. 2018; Tasanathai et al. 2019; Tasanathai et al. 2022; Tang et al. 2023a, b). Ophiocordyceps Petch is a large genus in the Ophiocordycipitaceae, with approximately 330 accepted species names (Indexfungorum.org. 2023). It was established originally by Petch (Petch 1924, 1931) to accommodate the species of Cordyceps Fr. producing asci with conspicuous apical caps and whole ascospores with distinct septation at maturity that do not disarticulate into part-spores. Then Ophiocordyceps was used as a subgeneric classification of the genus Cordyceps by Kobayasi (1941). Ophiocordyceps was restored to the rank of genus to include those Cordyceps species in the Ophiocordycipitaceae by Sung et al. (2007). The type of the genus Ophiocordyceps was O. blattae Petch to be found on cockroach (Blattoidea). Hirsutella Pat., Hymenostilbe Petch and Paraisaria Samson & B.L. Brady are commonly asexual morphs within Ophiocordyceps. Species of Hirsutella typically produced one to several conidia in a limited number of mucus droplets borne on basally subulate phialides that tapered into slender necks (Gams and Zare 2003). Typically, most of the Ophiocordyceps species parasitic to ants and associated with Hirsutella included the O. unilateralis complex. Entomogenous fungi within Ophiocordyceps have a wide range of insect hosts, ranging from solitary beetle larvae to social insects. They are able to colonise insects across 13 orders, including Blattoidea, Coleoptera, Dermaptera, Diptera, Hemiptera, Hymenoptera, Isoptera, Lepidoptera, Mantodea and Megaloptera etc. (Crous et al. 2004; Araújo and Hughes 2016).

Over forty species of Ophiocordyceps have been reported from adult ants (Formicidae, Hymenoptera) worldwide (Evans et al. 2011b; Kepler et al. 2011; Luangsa-ard et al. 2011; Kobmoo et al. 2012, 2015; Araújo et al. 2015, 2018; Spatafora et al. 2015; Crous et al. 2016; Wei et al. 2020; Tang et al. 2023a, b). These ant pathogenic fungi with biting behaviour belong to the O. unilateralis complex in the Hirsutella clade. The Ophiocordyceps unilateralis complex species are able to manipulate the ant behaviour by controlling it to leave the nest to die attached on to an ideal location for the fungus to develop, to produce the fruiting body and to begin spore transmission (Hughes et al. 2011). The Ophiocordyceps unilateralis complex is widely distributed around the world, for example, Australia, Brazil, China, Colombia, Ghana, Japan, Thailand and USA (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; Spatafora et al. 2015; Crous et al. 2016; Wei et al. 2020; Tang et al. 2023a, b). Although many taxa of the O. unilateralis complex have been reported and described in previous studies, there are estimated to be tens or even hundreds of undescribed species worldwide (Evans et al. 2011a). Many cryptic species in the O. unilateralis complex need to be further collected globally to explore the diversity of this species complex.

The diagnostic for the O. unilateralis complex is the ant biting behaviour, single or (sometimes multiple) stalk(s) arising from the dorsal pronotum of dead ants, with one or multiple lateral cushions from the base to the top along the stroma attached unilaterally (hence the epithet), exhibiting hirsutella-like anamorphs, whole and septate ascospores that do not disarticulate into part-spores that often exhibit secondary germination (capilliconidiophore) (Kobmoo et al. 2012; Araújo et al. 2018; Tang et al. 2023b). In addition, the host species is also a very useful characteristic for identification amongst species. Although the ant identification depends upon morphological features, for the zombie-ants (infected from fungi), some vital characteristics may have been obscured by the pathogenic fungi, therefore posing a challenge to identify the infected ants. With the further application of molecular technology, the mitochondrial cytochrome c oxidase subunit I (COI) gene as molecular marker was used for the ant’s phylogenetic studies, for exploring the diversity of the ants and distinguishing species and subspecies in the ant complex (Hebert et al. 2003; Narain et al. 2013; Siddiqui et al. 2019). There are few studies from the zombie-ant fungi elucidating the molecular details or DNA barcodes of these hosts (Tang et al. 2023a, b). In previous studies, the COI gene was used to construct a phylogenetic tree of the host ants by Tang et al. (2023b) and for species identification of the hosts. These studies showed that four species of the O. unilateralis complex were recorded on the same ant Camponotus sp. (Tang et al. 2023b). Evans et al. (2011b) and Araújo et al. (2015, 2018) have suggested that each fungal species seemed to be specifically associated with a given ant species and the host identity used as a proxy for fungal identification. Therefore, reconstructing the host phylogeny is important to understand the evolutionary event between fungi and the ants.

In China, nine species occurring on Formicinae (Formicidae) exhibiting similar behavioural manipulation have been reported in previous studies (Wei et al. 2020; Tang et al. 2023a, b), including O. acroasca Hong Yu bis & D.X. Tang, O. bifertilis Hong Yu bis & D.X. Tang, O. subtiliphialida Hong Yu bis & D.X. Tang, O. basiasca Hong Yu bis & D.X. Tang, O. nuozhaduensis Hong Yu bis & D.X. Tang, O. contiispora Hong Yu bis & D.X. Tang, O. flabellata Hong Yu bis & D.X. Tang, O. lilacina Hong Yu bis & D.X. Tang and O. tianshanensis L. S. Zha, D. P. Wei & K. D. Hyde. Most species were found in subtropical monsoon evergreen broad-leaved forest in southwest China. The two novel species presented herein have been collected from Yunnan Province in China. Based on morphological and phylogenetic characteristics, they were identified as belonging to the core clade of O. unilateralis. This study aims to present two novel species of the “zombie-ant fungi” belonging to the O. unilateralis core clade, O. tortuosa and O. ansiformis, from China and to investigate their phylogenetic relationships.

Materials and methods

Specimen collection

The specimens were collected from south-western China. Collections took place in subtropical monsoon evergreen broad-leaved forest. The ant’s death location from above the ground and the ants attached (biting) to substrate types (e.g. leaf, spine, trunk, moss and base of trunk) were measured and recorded in the field, then all specimens were collected in sterilised plastic containers, transported to the laboratory and examined within the same day if possible or stored at 4 °C. The specimens were deposited in the Yunnan Herbal Herbarium (YHH) of Yunnan University.

Morphological studies

For ecological characteristics, the quantity of stromata and ascomata per specimen and their colour, size and position were recorded, photographed and examined using a stereomicroscope Olympus SZ61 (Olympus Corporation, Tokyo, Japan). The stromata and the legs from the same ant host were moved for morphological studies. A cryosectioning of the ascoma was performed using a Freezing Microtome HM525NX (Thermo Fisher Scientific, Massachusetts, America). Samples were mounted on a slide with sterile water or lactophenol cotton blue solution for light microscopy examination using an Olympus BX53 (Tokyo, Japan). Micro-morphological characteristics (perithecia, asci, apical caps and ascospores) were examined. The naturally released ascospores and germination events were examined using an Olympus BX53 and the detailed method was based on the research of Araújo et al. (2018).

DNA extraction, PCR amplification and sequencing

DNA templates (contains the host and fungus from the same specimen) were obtained directly from fresh specimens using the Plant DNA Isolation Kit (Foregene Co., Ltd., Chengdu, China) according to the manufacturer’s protocols. Polymerase chain reaction (PCR) was used to amplify genetic markers using the following primer pairs: NS1/NS4 for small subunit nuclear ribosomal DNA (SSU) (White et al. 1990), 2218R/983F for translation elongation factor 1-α (TEF1a) (Rehner and Buckley 2005), CRPB1/RPB1Cr_oph for partial RNA polymerase II largest subunit gene region (RPB1) (Castlebury et al. 2004; Araújo et al. 2018) and LCO1490/HCO2198 for cytochrome oxidase subunit 1 (Hebert et al. 2003).

Each 25 µl-PCR reaction contained 2.5 µl of PCR 10× Buffer (2 mmol/l Mg2+) (Transgen Biotech, Beijing, China), 17.25 µl of sterile water, 2 µl of dNTP (2.5 mmol/l), 1 µl of each forward and reverse primer (10 µmol/l), 0.25 µl of Taq DNA polymerase (Transgen Biotech, Beijing, China) and 1 µl of DNA template (500 ng/µl). The PCR reactions were placed in a Bio-Rad T100 thermocycler (Bio-Rad Laboratories Co., Ltd, Shanghai, China) under the following conditions: For SSU, (1) 4 min at 95 °C, (2) 22 cycles of denaturation at 94 °C for 1 min, annealing at 53 °C for 1 min, and extension at 72 °C for 1.3 min, followed by (3) 12 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, and extension at 72 °C for 1.35 min and (4) 8 min at 72 °C (Wang et al. 2015). For TEF1a, (1) 4 min at 95 °C, (2) 8 cycles of denaturation at 94 °C for 50 s, annealing at 52 °C for 50 s and extension at 72 °C for 1 min, followed by (3) 30 cycles of denaturation at 94 °C for 50 s, annealing at 51 °C for 50 s and extension at 72 °C for 1 min and (4) 10 min at 72 °C (Wang et al. 2015). For RPB1, (1) 4 min at 95 °C, (2) 30 cycles of denaturation at 94 °C for 50 s, annealing at 52 °C for 50 s and extension at 72 °C for 1 min, followed by (3) 8 cycles of denaturation at 94 °C for 50 s, annealing at 51 °C for 50 s and extension at 72 °C for 1 min and (4) 10 min at 72 °C (Wang et al. 2015). For COI, (1) 1 min at 95 °C, (2) 5 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 1.5 min and extension at 72 °C for 1.5 min, followed by (3) 35 cycles of denaturation at 94 °C for 1 min, annealing at 54 °C for 1.5 min and extension at 72 °C for 1 min and (4) 5 min at 72 °C (Hebert et al. 2003). PCR products were purified using the Gel Band Purifcation Kit (Bio Teke Co., Ltd, Beijing, China) and sequenced by Beijing Genomics Institute (Chongqing, China). All LSU and RPB2 sequences were downloaded from GenBank.

Phylogenetic analyses

Phylogenetic analyses of fungi

To construct a phylogeny of major lineages in Ophiocordyceps, most of the DNA sequences used in this work were based on previous phylogenetic studies (Sung et al. 2007; Quandt et al. 2014; Araújo et al. 2018). Phylogenetic analyses were based on sequences of five molecular markers: SSU, LSU, TEF1a, RPB1 and RPB2, all of which were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/). Then the nucleotide sequences were combined with those generated in our study (Table 1). Sequences were aligned using ClustalX v.2.0 (Larkin et al. 2007), adjusted manually and then concatenated in BioEdit v.7.1.1 (Hall 1999). ModelFinder (Kalyaanamoorthy et al. 2017) was employed to determine the best fitting likelihood model for Maximum Likelihood (ML) and Bayesian Inference (BI) analyses according to the corrected Akaike Information Criterion (AIC). For ML analyses, tree searches were performed in IQ-tree v.2.1.3 (Nguyen et al. 2015), based on the best-fit model (TIM2+F+I+G4) with 5000 ultrafast bootstraps (Hoang et al. 2017) in a single run. BI analyses were performed in MrBayes v.3.2.7 (Ronquist et al. 2012). The BI search was based on the GTR+F+I+G4 model. Four Markov Chain Monte Carlo chains (one cold, three heated) were run, each beginning with a random tree and sampling one tree every 100 generations of 2,000,000 generations and the first 25% of samples were discarded as burn-in. The tree was visualised with its Maximum-Likelihood bootstrap proportions (ML-BS) and Bayesian posterior probability (BI-BPP) in Figtree v.1.4.3. Adobe Illustrator CS6 was used for editing.

Table 1.
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The taxa, GenBank accession numbers and host information in this study.

Species name Voucher information Host SSU LSU TEF1α RPB1 RPB2 Reference
Hirsutella sp. NHJ 12525 Hemiptera EF469125 EF469078 EF469063 EF469092 EF469111 Sung et al. (2007)
OSC 128575 Hemiptera EF469126 EF469079 EF469064 EF469093 EF469110 Sung et al. (2007)
Ophiocordyceps acicularis ARSEF 5692 Coleoptera DQ522540 DQ518754 DQ522322 DQ522368 DQ522418 Spatafora et al. (2007)
OSC 128580 Coleoptera DQ522543 DQ518757 DQ522326 DQ522371 DQ522423 Spatafora et al. (2007)
Ophiocordyceps acroasca YFCC 9049 Camponotus sp. ON555837 ON555918 ON567757 ON568677 ON568130 Tang et al. (2023b)
YFCC 9019 Camponotus sp. ON555838 ON555919 ON567758 ON568678 ON568131 Tang et al. (2023b)
YFCC 9017 Camponotus sp. ON555839 ON555920 ON567759 ON568679 ON568132 Tang et al. (2023b)
YFCC 9018 Camponotus sp. ON555840 ON555921 ON567760 ON568680 ON568133 Tang et al. (2023b)
YFCC 9016 Camponotus sp. ON555841 ON555922 ON567761 ON568681 ON568134 Tang et al. (2023b)
YHH 20122 Camponotus sp. ON555842 ON567762 ON568682 Tang et al. (2023b)
Ophiocordyceps albacongiuae RC20 Camponotus sp. KX713633 KX713670 Araújo et al. (2018)
Ophiocordyceps annullata CEM 303 Coleoptera KJ878915 KJ878881 KJ878962 KJ878995 Quandt et al. (2014)
Ophiocordyceps aphodii ARSEF 5498 Coleoptera DQ522541 DQ518755 DQ522323 DQ522419 Spatafora et al. (2007)
Ophiocordyceps australis HUA 186097 Pachycondyla sp. KC610786 KC610765 KC610735 KF658662 Sanjuan et al. (2015)
Ophiocordyceps basiasca YHH 20191 Camponotus sp. ON555828 ON555910 ON567748 ON568672 ON568121 Tang et al. (2023b)
Ophiocordyceps bifertilis YFCC 9012 Polyrhachis sp. ON555843 ON555923 ON567763 ON568143 ON568135 Tang et al. (2023b)
YHH 20162 Polyrhachis sp. ON555844 ON567764 ON568144 Tang et al. (2023b)
YHH 20163 Polyrhachis sp. ON555845 ON555924 ON567765 ON568145 ON568136 Tang et al. (2023b)
YHH 20164 Polyrhachis sp. ON555846 ON567766 ON568146 Tang et al. (2023b)
YFCC 9048 Polyrhachis sp. ON555847 ON555925 ON567767 ON568147 ON568137 Tang et al. (2023b)
YFCC 9013 Polyrhachis sp. ON555848 ON555926 ON567768 ON568148 ON568138 Tang et al. (2023b)
Ophiocordyceps blakebarnesii MISSOU5 Camponotus sp. KX713641 KX713610 KX713688 KX713716 Araújo et al. (2018)
MISSOU4 Camponotus sp. KX713642 KX713609 KX713685 KX713715 Araújo et al. (2018)
Ophiocordyceps brunneipunctata OSC 128576 Coleoptera DQ522542 DQ518756 DQ522324 DQ522369 DQ522420 Spatafora et al. (2007)
Ophiocordyceps buquetii HMAS_199617 Hymenoptera KJ878940 KJ878905 KJ878985 KJ879020 Quandt et al. (2014)
Ophiocordyceps camponoti-balzani G143 Camponotus balzani KX713658 KX713595 KX713690 KX713705 Araújo et al. (2018)
G104 Camponotus balzani KX713660 KX713593 KX713689 KX713703 Araújo et al. (2018)
Ophiocordyceps camponoti-bispinosi OBIS5 Camponotus bispinosus KX713636 KX713616 KX713693 KX713721 Araújo et al. (2018)
OBIS4 Camponotus bispinosus KX713637 KX713615 KX713692 KX713720 Araújo et al. (2018)
Ophiocordyceps camponoti-chartificis MF080 Camponotus chartifex MK874744 MK863824 Araújo et al. (2018)
Ophiocordyceps camponoti-femorati FEMO2 Camponotus femoratus KX713663 KX713590 KX713678 KX713702 Araújo et al. (2018)
Ophiocordyceps camponoti-floridani Flo4 Camponotus femoratus KX713662 KX713591 Araújo et al. (2018)
Flx2 Camponotus femoratus KX713592 KX713674 Araújo et al. (2018)
Ophiocordyceps camponoti-hippocrepidis HIPPOC Camponotus hippocrepis KX713655 KX713597 KX713673 KX713707 Araújo et al. (2018)
Ophiocordyceps camponoti-indiani INDI2 Camponotus indianus KX713654 KX713598 Araújo et al. (2018)
Ophiocordyceps camponoti-leonardi C27 Camponotus leonardi JN819019 Kobmoo et al. (2012)
C25 Camponotus leonardi JN819029 Kobmoo et al. (2012)
Ophiocordyceps camponoti-nidulantis NIDUL2 Camponotus nidulans KX713640 KX713611 KX713669 KX713717 Araújo et al. (2018)
Ophiocordyceps camponoti-novogranadensis Mal63 Camponotus novogranadensis KX713648 KX713603 Araújo et al. (2018)
Mal4 Camponotus novogranadensis KX713649 KX713602 Araújo et al. (2018)
Ophiocordyceps camponoti-renggeri RENG2 Camponotus renggeri KX713632 KX713672 Araújo et al. (2018)
ORENG Camponotus renggeri KX713634 KX713617 KX713671 Araújo et al. (2018)
Ophiocordyceps camponoti-rufipedis G177 Camponotus rufipes KX713657 KX713596 KX713680 Araújo et al. (2018)
G108 Camponotus rufipes KX713659 KX713594 KX713679 KX713704 Araújo et al. (2018)
Ophiocordyceps camponoti-saundersi C40 Camponotus saundersi KJ201519 JN819012 Kobmoo et al. (2012)
Ophiocordyceps camponoti-saundersi Co19 Camponotus saundersi JN819018 Kobmoo et al. (2012)
Ophiocordyceps citrina TNS F18537 Hemiptera KJ878903 KJ878983 KJ878954 Quandt et al. (2014)
Ophiocordyceps clavata CEM 1762 Coleoptera KJ878916 KJ878882 KJ878963 KJ878996 Quandt et al. (2014)
Ophiocordyceps cochlidiicola HMAS_199612 Lepidoptera KJ878917 KJ878884 KJ878965 KJ878998 Quandt et al. (2014)
Ophiocordyceps contiispora YFCC 9025 Camponotus sp. ON555829 ON555911 ON567749 ON568139 ON568122 Tang et al. (2023b)
YHH 20145 Camponotus sp. ON555830 ON567750 ON568140 ON568123 Tang et al. (2023b)
YFCC 9026 Camponotus sp. ON555831 ON555912 ON567751 ON568141 ON568124 Tang et al. (2023b)
YFCC 9027 Camponotus sp. ON555832 ON555913 ON567752 ON568142 ON568125 Tang et al. (2023b)
Ophiocordyceps curculionum OSC 151910 Coleoptera KJ878918 KJ878885 KJ878999 Quandt et al. (2014)
Ophiocordyceps daceti MF01 Daceton armigerum KX713604 KX713667 Araújo et al. (2018)
Ophiocordyceps dipterigena OSC 151911 Diptera KJ878919 KJ878886 KJ878966 KJ879000 Quandt et al. (2014)
OSC 151912 Diptera KJ878920 KJ878887 KJ878967 KJ879001 Quandt et al. (2014)
Ophiocordyceps flabellata YFCC 8795 Hymenoptera (Camponotus sp.) OL310721 OL310724 OL322688 OL322687 OL322695 Tang et al. (2023a)
YFCC 8796 Hymenoptera (Camponotus sp.) OL310722 OL310723 OL322692 OL322689 OL322696 Tang et al. (2023a)
YHH 20038 Hymenoptera (Camponotus sp.) OL322694 OL322691 Tang et al. (2023a)
YHH 20037 Camponotus sp. OL322693 OL322690 OL322697 Tang et al. (2023a)
Ophiocordyceps formosana TNM F13893 Coleoptera KJ878908 KJ878956 KJ878988 KJ878943 Quandt et al. (2014)
Ophiocordyceps formicarum TNS F18565 Hymenoptera KJ878921 KJ878888 KJ878968 KJ879002 KJ878946 Quandt et al. (2014)
Ophiocordyceps forquignonii OSC 151902 Diptera KJ878912 KJ878876 KJ878991 KJ878945 Quandt et al. (2014)
OSC 151908 Diptera KJ878922 KJ878889 KJ879003 KJ878947 Quandt et al. (2014)
Ophiocordyceps sp. Gh41 Polyrhachis sp. KX713656 KX713668 KX713706 Araújo et al. (2018)
Ophiocordyceps halabalaensis MY1308 Camponotus gigus KM655825 GU797109 Kobmoo et al. (2015)
Ophiocordyceps halabalaensis MY5151 Camponotus gigas KM655826 GU797110 Kobmoo et al. (2015)
Ophiocordyceps ansiformis YHH 2210007 Colobopsis sp. OR345230 OR098435 OR351952 This study
Ophiocordyceps irangiensis OSC 128577 Hymenoptera DQ522546 DQ518760 DQ522329 DQ522374 DQ522427 Spatafora et al. (2007)
OSC 128578 Hymenoptera DQ522556 DQ518770 DQ522345 DQ522391 DQ522445 Spatafora et al. (2007)
OSC 128579 Hymenoptera EF469123 EF469076 EF469060 EF469089 EF469107 Sung et al. (2007)
Ophiocordyceps kimflemingiae SC30 Camponotus castaneus/americanus KX713629 KX713622 KX713699 KX713727 Araújo et al. (2018)
SC09B Camponotus castaneus/americanus KX713631 KX713620 KX713698 KX713724 Araújo et al. (2018)
Ophiocordyceps kniphofioides HUA 186148 Hymenoptera KC610790 KF658679 KC610739 KF658667 KC610717 Sanjuan et al. (2015)
Ophiocordyceps konnoana EFCC 7295 Coleoptera EF468958 EF468862 EF468915 Sung et al. (2007)
EFCC 7315 Coleoptera EF468959 EF468753 EF468861 EF468916 Sung et al. (2007)
Ophiocordyceps lilacina YHH 2210001 Polyrhachis sp. OP782343 OP796856 OP796861 Tang et al. (2023a)
YHH 2210002 Polyrhachis sp. OP782344 OP796857 OP796862 Tang et al. (2023a)
Ophiocordyceps lloydii OSC 151913 Hymenoptera KJ878924 KJ878891 KJ878970 KJ879004 KJ878948 Quandt et al. (2014)
Ophiocordyceps longissima TNS F18448 Hemiptera KJ878925 KJ878892 KJ878971 KJ879005 Quandt et al. (2014)
HMAS_199600 Hemiptera KJ878926 KJ878972 KJ879006 KJ878949 Quandt et al. (2014)
Ophiocordyceps melolonthae OSC 110993 Coleoptera DQ522548 DQ518762 DQ522331 DQ522376 Spatafora et al. (2007)
Ophgrc679 Coleoptera KC610768 KC610744 KF658666 Sanjuan et al. (2015)
Ophiocordyceps monacidis MF74C Dolichoderus bispinosus KX713646 KX713606 Araújo et al. (2018)
MF74 Dolichoderus bispinosus KX713647 KX713605 KX713712 Araújo et al. (2018)
Ophiocordyceps myrmecophila CEM1710 Hymenoptera KJ878928 KJ878894 KJ878974 KJ879008 Quandt et al. (2014)
Ophiocordyceps naomipierceae DAWKSANT Polyrhachis cf. robsonii KX713664 KX713589 KX713701 Araújo et al. (2018)
Ophiocordyceps neovolkiana OSC 151903 Coleoptera KJ878930 KJ878896 KJ878976 KJ879010 Quandt et al. (2014)
Ophiocordyceps nigrella EFCC 9247 EF468963 EF468818 EF468758 EF468866 EF468920 Sung et al. (2007)
Ophiocordyceps nooreniae BRIP 55363 Chariomyrma cf. hookeri and Polyrhachis lydiae NG065096 NG059720 KX673812 KX673809 Crous et al. (2016)
BRIP 64868 Polyrhachis cf. hookeri and Polyrhachis lydiae KX961142 KX961143 Crous et al. (2016)
Ophiocordyceps nutans OSC 110994 Hemiptera DQ522549 DQ518763 DQ522333 DQ522378 Spatafora et al. (2007)
Ophiocordyceps nuozhaduensis YHH 20168 Camponotus sp. ON555849 ON555927 ON567769 ON568683 Tang et al. (2023b)
YHH 20169 Camponotus sp. ON555850 ON555928 ON567770 ON568684 Tang et al. (2023b)
Ophiocordyceps odonatae TNS F18563 Odonata KJ878877 KJ878992 Quandt et al. (2014)
TNS F27117 Odonata KJ878878 Quandt et al. (2014)
Ophiocordyceps oecophyllae OECO1 Oecophyllas smaragdina KX713635 Araújo et al. (2018)
Ophiocordyceps ootakii J14 Polyrhachis moesta KX713651 KX713682 KX713709 Araújo et al. (2018)
Ophiocordyceps ootakii J13 Polyrhachis moesta KX713652 KX713600 KX713681 KX713708 Araújo et al. (2018)
Ophiocordyceps ponerinarum HUA 186140 Paraponera clavata KC610789 KC610767 KC610740 KF658668 Araújo et al. (2018)
Ophiocordyceps polyrhachis-furcata P39 Polyrhachis furcata KJ201504 JN819003 Kobmoo et al. (2012)
P51 Polyrhachis furcata KJ201505 JN819000 Kobmoo et al. (2012)
Ophiocordyceps pulvinata TNS-F-30044 Camponotus obscuripes GU904208 GU904209 GU904210 Kepler et al. (2011)
Ophiocordyceps purpureostromata TNS F18430 Coleoptera KJ878931 KJ878897 KJ878977 KJ879011 Araújo et al. (2018)
Ophiocordyceps rami MY6736 Camponotus sp. KM655823 KJ201532 Kobmoo et al. (2015)
MY6738 Camponotus sp. KM655824 KJ201534 Kobmoo et al. (2015)
Ophiocordyceps ravenelii OSC 151914 Coleoptera KJ878932 KJ878978 KJ879012 KJ878950 Quandt et al. (2014)
Ophiocordyceps rhizoidea NHJ 12529 Coleoptera EF468969 EF468824 EF468765 EF468872 EF468922 Sung et al. (2007)
NHJ 12522 Coleoptera EF468970 EF468825 EF468764 EF468873 EF468923 Sung et al. (2007)
Ophiocordyceps satoi J19 Polyrhachis lamellidens KX713650 KX713601 KX713684 KX713710 Araújo et al. (2018)
J7 Polyrhachis lamellidens KX713653 KX713599 KX713683 KX713711 Araújo et al. (2018)
YFCC 8807 Polyrhachis sp. OP782340 OP782345 OP796853 OP796858 OP796863 Tang et al. (2023a)
YFCC 8809 Polyrhachis sp. OP782341 OP782346 OP796854 OP796859 OP796864 Tang et al. (2023a)
YFCC 8810 Polyrhachis sp. OP782342 OP782347 OP796855 OP796860 OP796865 Tang et al. (2023a)
Ophiocordyceps septa Pur1 Camponotus sp. KJ201528 Araújo et al. (2018)
Pur2 Camponotus sp. KJ201529 Araújo et al. (2018)
C41 Camponotus sp. JN819037 Kobmoo et al. (2015)
Ophiocordyceps sinensis EFCC 7287 Lepidoptera EF468971 EF468827 EF468767 EF468874 EF468924 Sung et al. (2007)
Ophiocordyceps sobolifera KEW 78842 Hemiptera EF468972 EF468828 EF468875 EF468925 Sung et al. (2007)
Ophiocordyceps sphecocephala OSC 110998 Hymenoptera DQ522551 DQ518765 DQ522336 DQ522381 DQ522432 Spatafora et al. (2007)
Ophiocordyceps stylophora OSC 111000 Coleoptera DQ522552 DQ518766 DQ522337 DQ522382 DQ522433 Spatafora et al. (2007)
OSC 110999 Coleoptera EF468982 EF468837 EF468777 EF468882 EF468931 Sung et al. (2007)
Ophiocordyceps subtiliphialida YFCC 8815 Camponotus sp. ON555833 ON555914 ON567753 ON568673 ON568126 Tang et al. (2023b)
YFCC 8814 Camponotus sp. ON555834 ON555915 ON567754 ON568674 ON568127 Tang et al. (2023b)
YFCC 8816 Camponotus sp. ON555835 ON555916 ON567755 ON568675 ON568128 Tang et al. (2023b)
YFCC 8817 Camponotus sp. ON555836 ON555917 ON567756 ON568676 ON568129 Tang et al. (2023b)
Ophiocordyceps tianshanensis MFLU 19-1207 Camponotus japonicus MN025409 MN025407 MK992784 Wei et al. (2020)
MFLU 19-1208 Camponotus japonicus MN025410 MN025408 MK992785 Wei et al. (2020)
Ophiocordyceps tortuosa YHH 2210003 Colobopsis sp. OR098431 OR098436 This study
YHH 2210004 Colobopsis sp. OR067858 OR098432 OR098437 This study
YHH 2210005 Colobopsis sp. OR067859 OR098433 OR098438 This study
YHH 2210006 Colobopsis sp. OR098434 OR098439 This study
Ophiocordyceps tricentri CEM 160 Hemiptera AB027330 AB027376 Nikoh and Fukatsu (2000)
Ophiocordyceps unilateralis VIC 44303 Camponotus sericeiventris KX713628 KX713626 KX713675 KX713730 Araújo et al. (2018)
VIC 44354 Camponotus sericeiventris KX713627 KX713676 KX713731 Araújo et al. (2018)
Ophiocordyceps yakusimensis HMAS_199604 Hemiptera KJ878938 KJ878902 KJ879018 KJ878953 Quandt et al. (2014)
Paraisaria amazonica HUA 186113 Orthoptera KJ917566 KP212903 KM411980 Sanjuan et al. (2015)
Paraisaria gracilis EFCC 8572 Lepidoptera EF468956 EF468811 EF468751 EF468859 EF468912 Sung et al. (2007)
EFCC 3101 Lepidoptera EF468955 EF468810 EF468750 EF468858 EF468913 Sung et al. (2007)
Paraisaria heteropoda OSC 106404 Hemiptera AY489690 AY489722 AY489617 AY489651 Castlebury et al. (2004)
Tolypocladium inflatum OSC 71235 Coleoptera EF469124 EF469077 EF469061 EF469090 EF469108 Sung et al. (2007)
Tolypocladium ophioglossoides CBS 100239 Elaphomyces sp. KJ878910 KJ878874 KJ878958 KJ878990 KJ878944 Quandt et al. (2014)

Phylogenetic analyses of host

Phylogenetic analyses were based on COI gene sequences. Most of the DNA sequences used in this work were based on previous phylogenetic studies (Mezger and Moreau 2015; Tang et al. 2023a, 2023b) and partial sequences were retrieved using the BLASTn searches in GenBank. The nucleotide sequences downloaded from NCBI were then combined with those generated in our study. Information on specimens and GenBank accession numbers are listed in Table 2. Sequences were initially aligned using ClustalX, manually adjusted and then concatenated in BioEdit. ModelFinder was used to select the best-fitting likelihood model (GTR+F+I+G4) for ML analyses and BI analyses according to the AIC. The host dataset used the same tree search setting as for the fungi phylogenetic inference.

Table 2.
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Specimen and GenBank accession numbers information for COI genes used in this study.

Species name Voucher information GenBank number Reference
Camponotus americanus YNH-005 MZ331828 Unpublished
Camponotus castaneus BIOUG03675-H07 KJ208900 Unpublished
BIOUG03675-H04 KJ445248 Unpublished
Camponotus claripes AECT JN134855 Unpublished
Camponotus renggeri Creng_1_B KP101600 Unpublished
Colobopsis rufipes BIOUG24424-D11 OM314604 Unpublished
Camponotus simulans AFR-CND-2010-47-F02 JN270684 Unpublished
Camponotus sp. CASENT0441197-D01 GU710187 Unpublished
CASENT0043700-D01 KF200199 Unpublished
CAMPO014 MH290634 Unpublished
CASENT0000633-D01 HM373060 Unpublished
YHH20648 OP783989 Tang et al. (2023a)
YHH 20605 OP353540 Tang et al. (2023b)
YHH 20606 OP353541 Tang et al. (2023b)
YHH 20607 OP353542 Tang et al. (2023b)
YHH 20608 OP353543 Tang et al. (2023b)
YHH 20609 OP353544 Tang et al. (2023b)
YHH 20610 OP353545 Tang et al. (2023b)
YHH 20611 OP353546 Tang et al. (2023b)
YHH 20612 OP353547 Tang et al. (2023b)
YHH 20168 OP353548 Tang et al. (2023b)
YHH 20191 OP353549 Tang et al. (2023b)
YHH 20122 OP353539 Tang et al. (2023b)
Colobopsis sp. YHH 2210006 OR068149 This study
YHH 2210007 OR068150 This study
Camponotus spanis G191388 OM420293 Unpublished
Camponotus sericeiventris BIOUG13980-G06 OM558348 Unpublished
BIOUG24738-E05 OM556713 Unpublished
Camponotus sexguttatus CASENT0612243 JF863527 Unpublished
Colobopsis badia TUCIM:6601 MF993268 Laciny et al. (2018)
Colobopsis explodens TUCIM:5080 MF993254 Unpublished
Colobopsis saundersi BK012313 Allio et al. (2020)
Colobopsis vitreus gvc13410-1L HM914891 Unpublished
gvc13412-1L HM914893 Unpublished
Camponotus wiederkehri AEKB JN134865 Unpublished
Daceton armigerum USNM:ENT:01566820 MW983875 Unpublished
Oecophylla smaragdina CSM0633 KM348201 Mezger and Moreau (2015)
Polyrhachis abbreviata CSM0776 KM348230 Mezger and Moreau (2015)
Polyrhachis anderseni ANA42 KM348248 Mezger and Moreau (2015)
Polyrhachis andromache FMNH-INS_2842051 KM348264 Mezger and Moreau (2015)
Polyrhachis ammon RA0751 KY939110 Unpublished
Polyrhachis aurea RA0750 KM348211 Mezger and Moreau (2015)
Polyrhachis australis RA0757 KM348231 Unpublished
Polyrhachis arnoldiisolate NDA40 MK591916 Unpublished
Polyrhachis beccari FMNH-INS_2842133 KM348266 Mezger and Moreau (2015)
Polyrhachis beccari FMNH-INS_2842169 KM348265 Mezger and Moreau (2015)
Polyrhachis brevinoda CSM2831 KY939023 Mezger and Moreau (2015)
CSM0773 KM348232 Mezger and Moreau (2015)
Polyrhachis carbonaria FMNH-INS_2842101 KM348267 Mezger and Moreau (2015)
Polyrhachis cf. bismarckensis FMNH-INS 2842022 KM348331 Mezger and Moreau (2015)
Polyrhachis cupreata CSM1015 KY939064 Unpublished
CSM0682 KY939056 Unpublished
Polyrhachis cyphonota FMNH-INS_2842221 KM348234 Mezger and Moreau (2015)
Polyrhachis danum CSM1841 KM348235 Mezger and Moreau (2015)
Polyrhachis delecta CSM0965 KY939013 Unpublished
Polyrhachis flavibasis RA0766 KM348203 Mezger and Moreau (2015)
RA0763 KY939081 Unpublished
Polyrhachis furcata YB-KHC51412 MN618329 Unpublished
Polyrhachis gagates FMNH-INS_2842213 KM348270 Mezger and Moreau (2015)
Polyrhachis hexacantha FMNH-INS_2842006 KM348204 Mezger and Moreau (2015)
Polyrhachis hookeri RA0747 KM348215 Mezger and Moreau (2015)
Polyrhachis illaudata FMNH-INS_2842112 KM348275 Mezger and Moreau (2015)
FMNH-INS_2842222 KM348271 Mezger and Moreau (2015)
GXJX0141 JQ681065 Unpublished
Polyrhachis jianghuaensis GXBL0006 JQ681069 Unpublished
Polyrhachis latharis FMNH-INS_2842062 KM348278 Mezger and Moreau (2015)
Polyrhachis lamellidens NSMK-IN-170100347 OL663445 Unpublished
Polyrhachis lepida CSM1877 KM348241 Mezger and Moreau (2015)
CSM1807 KM348239 Mezger and Moreau (2015)
Polyrhachis lucidula G160084 OM420302 Unpublished
Polyrhachis mackayi CSM0804 KM348242 Mezger and Moreau (2015)
Polyrhachis monteithi CSM0754 KY939009 Unpublished
Polyrhachis mucronata RA1154 KM348338 Mezger and Moreau (2015)
RA1158 KM348339 Mezger and Moreau (2015)
RA1164 KM348340 Mezger and Moreau (2015)
CSM0696a KM348337 Mezger and Moreau (2015)
Polyrhachis nigropilosa FMNH-INS_2842045 KM348284 Mezger and Moreau (2015)
Polyrhachis noesaensis FMNH-INS_2842106 KM348285 Mezger and Moreau (2015)
Polyrhachis obesior FMNH-INS_2842054 KM348286 Mezger and Moreau (2015)
Polyrhachis ornata CSM0797 KM348255 Mezger and Moreau (2015)
CSM0842 KY939061 Unpublished
Polyrhachis proxima FMNH-INS_2842042 KM348289 Mezger and Moreau (2015)
FMNH-INS_2842129 KM348288 Mezger and Moreau (2015)
Polyrhachis rastellata FMNH-INS_2841999 KM348244 Mezger and Moreau (2015)
Polyrhachis robsoni CSM1050 KY939017 Unpublished
Polyrhachis saevissima FMNH-INS_2842115 KM348345 Mezger and Moreau (2015)
Polyrhachis schistacea FMNH-INS_2842058 KM348297 Mezger and Moreau (2015)
FMNH-INS_2842071 KM348294 Mezger and Moreau (2015)
FMNH-INS_2842067 KM348293 Mezger and Moreau (2015)
Polyrhachis schoopae CSM0626b KM348218 Mezger and Moreau (2015)
Polyrhachis sp. FMNH-INS_2842139 KM348305 Mezger and Moreau (2015)
FMNH-INS_2842198 KM348309 Mezger and Moreau (2015)
FMNH-INS_2842195 KM348308 Mezger and Moreau (2015)
FMNH-INS_2842179 KM348300 Mezger and Moreau (2015)
FMNH-INS_2842190 KM348304 Mezger and Moreau (2015)
FMNH-INS_2842193 KM348310 Mezger and Moreau (2015)
FMNH-INS_2842194 KM348307 Mezger and Moreau (2015)
FMNH-INS_2842074 KM348226 Mezger and Moreau (2015)
FMNH-INS_2842082 KM348306 Mezger and Moreau (2015)
FMNH-INS_2842039 KM348311 Mezger and Moreau (2015)
Polyrhachis sp. CSM2738 KM348302 Mezger and Moreau (2015)
FMNH-INS_2842043 KM348246 Mezger and Moreau (2015)
RA0779 KY939027 Unpublished
FMNH-INS_2842044 KM348350 Mezger and Moreau (2015)
FMNH-INS_2842078 KM348314 Mezger and Moreau (2015)
FMNH-INS_2842032 KM348313 Mezger and Moreau (2015)
FMNH-INS_2842103 KM348315 Mezger and Moreau (2015)
YHH 20635 OP783990 Tang et al. (2023a)
YHH 20636 OP783991 Tang et al. (2023a)
YHH 20637 OP783992 Tang et al. (2023a)
YHH 20638 OP783993 Tang et al. (2023a)
YHH 20639 OP783994 Tang et al. (2023a)
YHH 20640 OP783995 Tang et al. (2023a)
YHH 20641 OP783996 Tang et al. (2023a)
YHH 20642 OP783997 Tang et al. (2023a)
YHH 20643 OP783998 Tang et al. (2023a)
YHH 20644 OP783999 Tang et al. (2023a)
YHH 20645 OP784000 Tang et al. (2023a)
YHH 20646 OP784001 Tang et al. (2023a)
YHH 20647 OP784002 Tang et al. (2023a)
YHH 20162 OP353532 Tang et al. (2023b)
YHH 20163 OP353533 Tang et al. (2023b)
YHH 20164 OP353534 Tang et al. (2023b)
YHH 20601 OP353535 Tang et al. (2023b)
YHH 20602 OP353536 Tang et al. (2023b)
YHH 20603 OP353537 Tang et al. (2023b)
YHH 20604 OP353538 Tang et al. (2023b)
Polyrhachis tubifera CSM1108 KY939104 Unpublished
Polyrhachis turneri CSM0827 KM348260 Mezger and Moreau (2015)
Polyrhachis villipes FMNH-INS_28421186 KM348316 Mezger and Moreau (2015)
Polyrhachis viscosa FMNH-INS_2842064 KM348317 Mezger and Moreau (2015)

Results

Phylogenetic analysis of fungi

Combining single gene trees (SSU, TEF1a, RPB1) in a concatenated tree, using morphological features for comparison, enabled identification of two new species (O. tropiosa and O. ansiformis). We have inferred the phylogeny, based on each single gene (SSU, TEF1a, RPB1) and present the details below. Ophiocordyceps tortuosa was recovered as sister to O. lilacina (BS = 88%) (Suppl. material 1) and the relationship between O. tortuosa and O. contiispora was recovered with strong support (BS = 100%), based on TEF1a or RPB1 (Suppl. materials 2, 3). Sequences of Ophiocordyceps ansiformis, O. subtiliphialida, O. contiispora and O. basiasca were clustered together into a clade with weak bootstrap support (BS = 49%), based on SSU (Suppl. material 1), O. ansiformis was recovered sister to O. tortuosa + O. contiispora with weak to strong support (BS = 63–91%), based on TEF1a or RPB1 (Suppl. materials 2, 3).

For the concatenated tree (SSU, LSU, TEF1a, RPB1 and RPB2), the alignment comprised 143 taxa (Table 1). Tolypocladium ophioglossoides CBS 100239 and T. inflatum OSC 71235 were used as the outgroup taxa. The final trimmed five genetic marker matrix contained 4,827 bp, including 1,059 bp for SSU, 966 bp for LSU, 967 bp for TEF1a, 762 bp for RPB1 and 1,073 bp for RPB2. This matrix has 2,688 distinct patterns, 1,699 parsimony-informative, 410 singleton sites and 2,718 constant sites. The likelihood of the best scoring IQ tree was −54,690.799. The best-fit model TIM2+F+I+G4 was used for Maximum Likelihood analysis and the GTR+F+I+G4 model was used for the Bayesian analysis. The generic level relationships of ML and BI trees were topologically similar. In agreement with the previous study by Araújo et al. (2018), phylogenetic analyses showed that the Hirsutella ant pathogen consisted of three major groups, i.e. O. unilateralis core clade, O. oecophyllae and O. kniphofioides sub-clade. The O. unilateralis core clade included 38 species and was strongly supported (BS = 100%, BPP = 100%), O. oecophyllae branched as its sister taxon with BS = 98%, BPP = 80%. The subclade O. kniphofioides was sister to the core clade O. unilateralis + O. oecophyllae clade with strong support (BS = 100%, BPP = 98%). The phylogenetic analysis indicated that the two species in this study were clustered together in the O. unilateralis core clade within the Southeast Asian clade and that the two new taxa formed distinct lineages from the other species, respectively. The sister relationships between O. tortuosa and O. contiispora were recovered with strong support (BS = 100%, BPP = 100%) and obtained the same topological structure as the single gene (TEF1a and RPB1) tree (Suppl. materials 2 and 3). Ophiocordyceps ansiformis was recovered sister to O. tortuosa + O. contiispora with strong support (BS = 85%, BPP = 95%) and also obtained the same topological structure as the single gene (TEF1a and RPB1) tree (Suppl. materials 2, 3).

Phylogenetic analysis of host ants

The alignment consisted of 131 taxa (Table 2). Daceton armigerum USNM was used as the outgroup taxa. The final trimmed COI genetic marker matrix contained 660 bp. The matrix had 389 distinct patterns, 309 parsimony-informative, 42 singleton sites and 309 constant sites. The likelihood of the best scoring IQ tree was −16,415.047. The best-fit model GTR+F+I+G4 was used for Maximum Likelihood analysis and Bayesian analysis. The generic level relationships of ML and BI trees were topologically similar.

Phylogenetic analyses showed that the genera Colonopsis (BS = 98%, BPP = 100%) and Polyrhachis (BS = 91%, BPP = 99%) within Formicinae formed each a monophyletic clade with strong supports and statistical topology. The phylogenetic analysis indicated that the hosts Colonopsis sp. (YHH 2210006 and YHH 2210007) formed a clade and were infected by both O. tortuosa and O. ansiformis (Fig. 2). Ants infected with the two new fungi were identified by morphological features and COI phylogenetic analysis as belonging to the same host, Colonopsis sp. The ant species could not be further identified because the ant characteristics were not obvious. Interestingly, these ant pathogenic fungi, including O. basiasca, O. contiispora, O. acroasca and O. subtiliphialida also parasitised on the same host (Camponotus sp. YHH 20606, 20609, 20608, 20611, 20607, 20191, 20610, 20122, 20605 and 20612) in Tang et al. studies (2023b). The results in this work suggest that the fungal pathogen infects the same host as Colonopsis sp. and that the two species may share the same niche.

Taxonomy

Ophiocordyceps tortuosa Hong Yu bis, D.X. Tang & J. Zhao, sp. nov.

MycoBank No: 849060
Fig. 3

Etymology

Tortuosa = tortuous, the epithet referred to the “tortuous” arrangement of ascospores in the asci.

Diagnosis

The difference between Ophiocordyceps tortuosa and related species is that Ophiocordyceps tortuosa produces lanceolate and obvious separate ascospores, while O. contiispora produces fusiform and no obvious separate ascospores.

Holotype

China, Yunnan Province, Puer City, Simao District. Infected Colobopsis sp. (Formicinae) biting into a leaf of Lauraceae Juss., 22°42'40"N, 100°57'28"E, alt. 1345 m, 03 October 2022, Hong Yu bis (YHH 2210035 – preserved in the Yunnan Herbal Herbarium).

Description

Sexual morph : External mycelia produced from all orifices and sutures, often covering the host body, initially white turning brown. Stromata single to multiple, produced from dorsal pronotum, part branched, 16–24 mm in length, cylindrical, pale white to light brown, becoming pinkish at the apical part. Fertile region of lateral cushions, 1–3, commonly 2 per stroma, hemispherical, chocolate brown at maturity, 1–1.9 × 0.8–1.3 mm. Perithecia immersed to partially erumpent, flask-shaped, (211–) 218–298 (–305) × (94–) 99–142 (–158) μm, with short, exposed neck or ostiole. Asci 8-spored, hyaline, cylindrical, (92–) 96–132 (–134) × 7–11 (–13) μm. Ascus caps slightly prominent, hemispherical, 4–5 × (2–) 3–4 μm. Ascospores hyaline, thin-walled, lanceolate, tortuous arrangement in the ascus, 47–64 × 5–7 μm, 6–7-septate, gently curved at round apex, tapered end shorter than round apex.

Asexual morph : Hirsutella-A type associated with the apical part of stromata. Hirsutella-C type produced from the leg and antennal joints. Phialides lageniform, 54–99 μm long, 4–6 μm width at base, tapering to a long neck, 1–2 μm in width. Conidia fusiform to limoniform, 6–8 × 3–5 μm, slightly narrowing at the top.

Germination process : The released ascospores germinated within 48 h to produce 1–2 long and extremely narrow hair-like capilliconidiophores, (27–) 44–65 (–69) × 1–2 μm, bearing a single terminal capilliconidium, (5–) 6–9 × 3–4 (–5) μm, hyaline, smooth-walled, limoniform to fusiform, slightly narrowing and curved at the top.

Host : Colobopsis sp. (Formicinae).

Habitat

Subtropical monsoon evergreen broad-leaved forest. Infected Colobopsis sp. biting into a leaf of Lauraceae Juss., from 1.2 to 2.4 m above the ground.

Distribution

China, Yunnan Province, Puer City.

Material examined

China, Yunnan, Puer City, Simao District. Infected Colobopsis sp. biting into a leaf of Lauraceae Juss., 22°42'40"N, 100°57'28"E, alt. 1,345 m, 03 October 2022, D.X. Tang (YHH 2210003, YHH 2210004, YHH 2210005, YHH 2210006).

Notes

In the phylogenetic tree, the new species O. tortuosa was sister to O. contiispora (Fig. 1: BS = 100%, BPP = 100%) within O. unilateralis core clade (Fig. 1: BS = 100%, BPP = 100%). Ophiocordyceps tortuosa was distinct from other species of the O. unilateralis core clade in that it produced lanceolate, obvious separate and tortuous arrangement ascospores in the ascus and produced branched stromata, slightly narrowing conidia (Table 3).

Figure 1. 

Phylogenetic tree of Ophiocordyceps and related genera, based on the concatenation of LSU, SSU, TEF1α, RPB1 and RPB2 sequence data. The tree was generated from an alignment of 4,827 sites and 143 taxa (38 within O. unilateralis). The phylogeny was inferred using the IQ-tree. Values at the nodes represent IQ-tree bootstrap proportions (on the left) and posterior probabilities (on the right). All values were shown at the nodes. The scale bar 0.02 indicates the number of expected mutations per site. The two new species were indicated in blue and red font within O. unilateralis core clade. Two species (T. inflatum OSC 71235 and T. ophioglossoides CBS 100239) in Tolypocladium were used as the outgroup taxa.

Figure 2. 

Phylogenetic tree of some genera of the Formicinae based on COI sequence data. The tree was generated from an alignment of 660 sites and 131 taxa. The phylogeny was inferred using the IQ-tree. Values at the nodes represent IQ-tree bootstrap proportions (on the left) and posterior probabilities (on the right). All values were shown at the nodes. The scale bar 0.05 indicates the number of expected mutations per site. The species (Colonopsis sp. YHH 2210006 and Colonopsis sp. YHH 2210007) are indicated in black and bold font in this work. The Latin name on the right of the tree refers to the pathogenic fungi infecting the host ants and the illustration refers to the fungi infecting ants in the wild. Daceton armigerum USNM was used as the outgroup taxa.

Figure 3. 

Ophiocordyceps tortuosa a–d infected Colobopsis sp. biting into a leaf of Lauraceae Juss e the three ascomata produced from the stroma f, g cross-section of ascomata showing the perithecial arrangement h, i asci j, k ascospores l, m ascospore with capilliconidiophores n capilliconidium o–q phialides r conidia. Scale bars: 4000 µm (a, b); 3000 µm (c, d); 2000 µm (e); 200 µm (f); 100 µm (g); 20 µm (h, i); 10 µm (j, k); 20 µm (l, m); 10 µm (n); 20 µm (o–r).

Table 3.
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Morphological comparison of two novel taxa and related species within Ophiocordyceps unilateralis complex.

Species Host Death position Stromata Ascomata Perithecia (μm) Asci (μm) Prominent cap Ascospores (μm) Septation Hirsutella asexual morph (μm) Conidia (μm) 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-Ctype, 17–30 × 1–4 limoniform, 2–3 × 1–2 Tang et al. (2023b)
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 Tang et al. (2023b)
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 Tang et al. (2023b)
Ophiocordyceps ansiformis Colobopsis sp. biting leaf single hemispherical, 1–1.3 × 0.7–1 mm flask-shaped, 174–290 × 99–128 cylindrical, 88–112 × 7–11 hemispherical, 4–7 × 2–4 lanceolate, 45–59 × 5–7 6–9 Hirsutella-A type, 15–24 × 3–4 This study
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 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 Tang et al. (2023b)
Ophiocordyceps tortuosa Colobopsis sp. biting leaf single to multiple hemispherical, 1–1.9 × 0.8–1.3 mm flask-shaped, 211–305 × 94–158 cylindrical, 92–134 × 7–13 hemispherical, 4–5 × 2–4 lanceolate, 47–64 × 5–7 6–7 Hirsutella-A type, 54–99 × 1–6 fusiform to limoniform, 6–8 × 3–5 This study

Ophiocordyceps ansiformis Hong Yu bis, D.X. Tang & J. Zhao, sp. nov.

MycoBank No: 849061
Fig. 4

Etymology

Ansi- = handle, formis = forms, the epithet refers to ascospores having a handle-shape.

Diagnosis

Ophiocordyceps ansiformis differs from closely-related species by producing lanceolate ascospores with a structure resembling a handle-shape in the middle, while O. contiispora produces fusiform ascospores that do not exhibit a similar structure in the middle.

Holotype

China, Yunnan Province, Jinghong City, Puwen Town. Infected Colobopsis sp. (Formicinae) biting into a leaf of Rubiaceae Juss., 22°31'24"N, 100°58'57"E, alt. 1,029 m, 02 October 2022, Hong Yu bis (YHH 2210036 – preserved in the Yunnan Herbal Herbarium).

Description

Sexual morph : External mycelia produced from all orifices and sutures, brown at maturity. Stroma single, produced from dorsal pronotum, never branched, 25–28 mm in length, cylindrical, dark brown at maturity, light brown at the apical part. Fertile region of lateral cushions, 1–3, hemispherical, 1–1.3 × 0.7–1 mm. Perithecia immersed to partially erumpent, flask-shaped, (174–) 189–290 × 99–126 (–128) μm, with short, exposed neck or ostiole. Asci 8-spored, hyaline, cylindrical, (88–) 92–108 (–112) × 7–10 (–11) μm. Ascus caps prominent, hemispherical, 4–6 (–7) × 2–3 (–4) μm. Ascospores hyaline, thin-walled, lanceolate, having a handle-shape in the middle, 45–59 × 5–6 (–7) μm, 6–9-septate, tapering at apex.

Figure 4. 

Ophiocordyceps ansiformis a infected Colobopsis sp. biting into a leaf of Rubiaceae Juss b the three ascomata produced from the stroma c, d cross-section of ascomata showing the perithecial arrangement e–h ascus i–k ascospores l–o ascospores with capilliconidiophores p capilliconidium q phialides. Scale bars: 4000 µm (a); 2000 µm (b); 200 µm (c); 100 µm (d); 20 µm (e–h); 10 µm (i, j); 20 µm (k–o); 10 µm (p, q); 5 µm (r).

Asexual morph : Hirsutella-A type present along stromata. Phialides lageniform, 15–24 × 3–4 μm, tapering to a short neck, 6–8 μm in length. Conidia were not observed.

Germination process : Ascospores released on agar germinated after 48 h to produce 1–2 capilliconidiophores, (54–) 60–79 (–84) × 0.8–1.4 μm, bearing a terminal capilliconidium, hyaline, smooth-walled, limoniform, 6–10 × 3–4 μm, slightly narrowing apically.

Host : Colobopsis sp. (Formicinae).

Habitat

Subtropical monsoon evergreen broad-leaved forest. Infected Colobopsis sp. biting into a leaf of Rubiaceae Juss., from 0.8 to 1 m above the ground.

Distribution

China, Yunnan Province, Jinghong City.

Material examined

China, Yunnan Province, Jinghong City, Puwen Town. Infected Colobopsis sp. biting into a leaf of Rubiaceae Juss., 22°31'24"N, 100°58'57"E, alt. 1,029 m, 02 October 2022, D.X. Tang (YHH 2210007).

Notes

Phylogenetic analyses showed that O. ansiformis formed a sister lineage with O. tortuosa and O. contiispora, was clustered in the O. unilateralis core clade, with statistical support from bootstrap proportions (BS = 85%) and Bayesian posterior probabilities (BPP = 95%) (Fig. 1). Ophiocordyceps ansiformis was similar to O. tortuosa and O. contiispora in the same host Colobopsis sp. (Fig. 2). However, it differed from O. tortuosa and O. contiispora in that it produced lanceolate ascospores and has a handle-shape in the middle (Table 3).

1a Stromata never branched 2
1b Stromata part branched Ophiocordyceps tortuosa
2a Ascomata hemispherical 3
2b Ascomata disc-shaped 4
2c Ascomata spherical Ophiocordyceps basiasca
3a Perithecia ovoid, ascospores vermiform, 83–108 × 2–3 µm Ophiocordyceps acroasca
3b Perithecia flask-shaped, ascospores lanceolate, 45–59 × 5–7 µm Ophiocordyceps ansiformis
3c Perithecia fusoid-ellipsoid, ascospores lanceolate, 45–50 × 6–8 Ophiocordyceps septa
4a Ascospores fusiform, 38–48 × 2–4 µm Ophiocordyceps contiispora
4b Ascospores lanceolate, 52–72 × 5–8 µm Ophiocordyceps subtiliphialida

Discussion

Many closely-related species parasitising Hymenoptera are considered cryptic species within the genus Ophiocordyceps. These species are distinguished by morphological features and molecular phylogenetic studies. Known examples of these fungi were found occurring on adult ants in the O. myrmecophila species complex, for example, O. megacuculla and O. granospora (Khonsanit et al. 2018). Over ten species were parasitic on termites, for example, O. asiatica, O. khokpasiensis, O. mosingtoensis, O. pseudorhizoidea and O. termiticola (Tasanathai et al. 2019), O. radiciformis, O. isopterorum, O. globosa, O. fusiformis (Tasanathai et al. 2022), O. puluongensis (Xu et al. 2022) and O. ovatospora (Tang et al. 2022). Other groups such as Ophiocordyceps nutans species complex attack stink bugs (Friedrich et al. 2018; Khao-ngam et al. 2021) and Ophiocordyceps pseudoacicularis species complex were found occurring on Lepidoptera larvae (Tasanathai et al. 2020). These complexes have been proposed as distinct species, based on molecular phylogenetic studies and morphological characteristics.

In this study, two new species, namely O. tortuosa and O. ansiformis, were established within Ophiocordyceps, based on a combination of morphological features, phylogenetic analyses (LSU, SSU, TEF1a, RPB1 and RPB2) and ecological data. The O. unilateralis complex species was sister to O. oecophyllae and both are sister to the O. kniphofioides sub-clade. The species within O. unilateralis clade infects exclusively Camponotini ants (e.g. Camponotus, Polyrhachis, Colobopsis, Dinomyrmex) (Evans et al. 2018). Entomopathogenic fungi in the O. unilateralis complex occurring on the host ants Camponotus show host specificity (Araújo et al. 2018; Kobmoo et al. 2019; Lin et al. 2020; Tang et al. 2023b). However, pathogenic fungi infecting Polyrhachis ants do not exhibit species specificity, for example, more than nine species of Polyrhachis ants infected by O. satoi (Tang et al. 2023a) and two species of Polyrhachis ants (Polyrhachis cf. hookeri and Polyrhachis lydiae) infected by O. nooreniae (Crous et al. 2016). The same ant host, Camponotus sp. (YHH 20606, 20609, 20608, 20611, 20607, 20191, 20610, 20122, 20605 and 20612), was infected by ant pathogenic fungi, including O. basiasca, O. contiispora, O. acroasca, O. subtiliphialida, O. tortuosa and O. ansiformis (Tang et al. 2023b). The ants, Camponotus sp. and Colonopsis sp. (Fig. 2), may be the same ant, separated only by a small genetic distance, based on COI molecular phylogenetic studies. The Tang et al. (2023b) result showed that the ant pathogenic fungi parasitising the genus Camponotus have host specificity and Kobmoo et al. (2019) indicated that more than one ant pathogenic species might parasitise the same host species. Based on a population genomics study, our results in this work fully prove and support the basis of the above research.

The two novel species within the O. unilateralis core clade showed slightly micro-morphological characteristics (the shape of ascospore, secondary germination) that made them recognised from other species. Ophiocordycep ansiformis differed from O. contiispora by producing lanceolate ascospores with a handle-shape in the middle, while O. tortuosa differed from O. contiispora by producing lanceolate ascospore with obvious separation and tortuous arrangement in the ascus. In addition, O. tortuosa and O. ansiformis differed in the size of perithecia (211–305 × 94–158 µm vs. 174–290 × 99–128 µm), asci (92–134 × 7–13 µm vs. 88–112 × 7–11 µm) and ascospores (47–64 × 5–7 µm, 6–7-separate vs. 45–59 × 5–7 µm, 6–9-separate), this work supporting the idea of cryptic species (Evans et al. 2011b; Khonsanit et al. 2018; Tasanathai et al. 2019). The species in the O. unilateralis complex commonly bite and attach themselves on to spines, leaves, saplings, epiphytes, moss and twigs in a “death grip”, dying in an elevated position, from 0.25 m to 2 m (Evans et al. 2011b; Hughes et al. 2011; Kepler et al. 2011; Luangsa-ard et al. 2011; Kobmoo et al. 2012; Araújo et al. 2015; Crous et al. 2016; Araújo et al. 2018; Evans et al. 2018; Tang et al. 2023a, 2023b). The two new species O. ansiformis were biting and attached in a leaf of Rubiaceae Juss., while O. tortuosa was biting in a leaf of Lauraceae Juss., dying in an elevated position (O. ansiformis 0.8 to 1 m vs. O. tortuosa 1.2 to 2.4 m). We conducted a two-year tracking survey on one of the O. unilateralis complex species at the same location and season in subtropical monsoon evergreen broad-leaved forest. It was found that the death location of the host ant on the underside of leaves above the ground seems to be influenced by environmental factors such as rainfall, humidity, temperature etc. We found that there were differences in the location of death from the ground in the O. unilateralis complex at different years in the same species. In addition, Loreto et al. (2018) have shown that environmental conditions affect the biting type of host ants.

Therefore, future studies are recommended to examine the impact of changes in environmental conditions on the height at which host ants die.

We had inferred the phylogeny, based on each single gene and also used a concatenated dataset in this study. Ophiocordyceps tortuosa was recovered sister to O. contiispora with strong support and consistent topology, based on the concatenated and single gene (TEF1a and RPB1) tree. The species O. ansiformis was also recovered sister to O. tortuosa + O. contiispora with weak to strong support and consistent topology, based on the concatenated and single gene (TEF1a and RPB1) tree (Suppl. materials 2, 3). The sister relationship between O. tortuosa and O. lilacina was recovered, based on single gene SSU tree. Sequences of Ophiocordyceps ansiformis, O. subtiliphialida, O. contiispora and O. basiasca clustered together into a clade, based on the SSU tree. Single gene SSU trees were different from the topological structure of other trees (concatenated, TEF1a and RPB1 tree) for the two species proposed in this work. Other species (O. camponoti-femorati, O. camponoti-rufipedis and O. unilateralis), based on SSU, TEF1a and RPB1 trees have similar topological structures in this work. SSU phylogenies indicate their utility as a well marker to infer phylogenetic relationships at the subclass level (Tang et al. 2006), but it may be difficult to distinguish cryptic species only using the single gene SSU. To sum up, the two new species, O. tortuosa and O. ansiformis, proposed in this study, were fully supported by morphological features, phylogenetic analyses (concatenated, TEF1a and RPB1 tree) and ecological data.

Acknowledgements

We special thank Dr. Hao Ran who provided important support during identification of host ants in this work. We thank all those who have provided assistance for this work. We thank the National Natural Science Foundation of China (31870017) for the financial support.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by National Natural Science Foundation of China (31870017).

Author contributions

Funding acquisition: HY. Investigation: ZW, TS, ZL, YL. Methodology: JZ. Writing - original draft: DT.

Author ORCIDs

Dexiang Tang https://orcid.org/0000-0002-7662-224X

Jing Zhao https://orcid.org/0000-0001-7871-2209

Yingling Lu https://orcid.org/0009-0008-8119-1975

Zhiqin Wang https://orcid.org/0000-0001-9022-3635

Tao Sun https://orcid.org/0000-0001-7837-2101

Zuoheng Liu https://orcid.org/0000-0003-4118-3694

Hong Yu https://orcid.org/0000-0002-2149-5714

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

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Supplementary materials

Supplementary material 1 

Phylogenetic tree of Ophiocordyceps and related genera, based on single gene SSU sequence

Dexiang Tang, Jing Zhao, Yingling Lu, Zhiqin Wang, Tao Sun, Zuoheng Liu, Hong Yu

Data type: tif

Explanation note: The phylogeny was inferred using the IQ-tree. Values at the nodes represent IQ-tree bootstrap proportions. The scale bar 0.05 indicates the number of expected mutations per site. The two new species were indicated in bold. Two species (T. inflatum OSC 71235 and T. ophioglossoides CBS 100239) in Tolypocladium were used as the outgroup taxa.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (3.69 MB)
Supplementary material 2 

Phylogenetic tree of Ophiocordyceps and related genera, based on single gene TEF1a sequence

Dexiang Tang, Jing Zhao, Yingling Lu, Zhiqin Wang, Tao Sun, Zuoheng Liu, Hong Yu

Data type: tif

Explanation note: The phylogeny was inferred using IQ-tree. Values at the nodes represent IQ-tree bootstrap proportions. The scale bar 0.05 indicates the number of expected mutations per site. The two new species were indicated in bold. Two species (T. inflatum OSC 71235 and T. ophioglossoides CBS 100239) in Tolypocladium were used as the outgroup taxa.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (3.63 MB)
Supplementary material 3 

Phylogenetic tree of Ophiocordyceps and related genera, based on single gene RPB1 sequence

Dexiang Tang, Jing Zhao, Yingling Lu, Zhiqin Wang, Tao Sun, Zuoheng Liu, Hong Yu

Data type: tif

Explanation note: The phylogeny was inferred using the IQ-tree. Values at the nodes represent IQ-tree bootstrap proportions. The scale bar 0.05 indicates the number of expected mutations per site. The two new species were indicated in bold. Two species (T. inflatum OSC 71235 and T. ophioglossoides CBS 100239) in Tolypocladium were used as the outgroup taxa.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (3.63 MB)
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