<8) MycoKeys MycoKeys 99: 269-296 (2023) DOI: 10.3897/mycokeys.99.107565 Research Article Morphology, phylogeny and host specificity of two new Ophiocordyceps species belonging to the “zZombie-ant fungi" clade (Ophiocordycipitaceae, Hypocreales) Dexiang Tang'2®*, Jing Zhao'®*, Yingling Lu'®, Zhigin Wang'™®, Tao Sun'2®, Zuoheng Liu'®, Hong Yu'® 1 Yunnan Herbal Laboratory, College of Ecology and Environmental Sciences, Yunnan University, Kunming, 650504, China 2 School of Life Science, Yunnan University, Kunming, 650504, China Corresponding author: Hong Yu (hongyu@ynu.edu.cn; herbfish@163.com) OPEN Qrceess Academic editor: Marc Stadler Received: 6 June 2023 Accepted: 15 September 2023 Published: 16 October 2023 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 Copyright: © Dexiang Tang et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract Species of the genus Ophiocordyceps, which include species able to manipulate the be- haviour 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, TEF7a, 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 (CO/), which may be sharing the same host. Ophio- cordyceps tortuosa and O. ansiformis share the morphological character of producing lan- ceolate 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 routine- ly unmasked several cryptic species and have revealed this as a common phe- nomenon for the entomogenous fungi (Araujo 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 maturi- ty that do not disarticulate into part-spores. Then Ophiocordyceps was used as a * Those authors contributed equally to this work. 269 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi’ clade subgeneric classification of the genus Cordyceps by Kobayasi (1941). Ophiocordy- ceps 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 Ophiocordy- ceps 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; Araujo and Hughes 2016). Over forty species of Ophiocordyceps have been reported from adult ants (For- micidae, Hymenoptera) worldwide (Evans et al. 2011b; Kepler et al. 2011; Luang- sa-ard et al. 2011; Kobmoo et al. 2012, 2015; Araujo 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 uni- lateralis 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; Araujo 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 esti- mated 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, sin- gle 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 ana- morphs, whole and septate ascospores that do not disarticulate into part-spores that often exhibit secondary germination (capilliconidiophore) (Kobmoo et al. 2012; Araujo 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 (in- fected 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 | (CO/) gene as molecular marker was used for the ant’s phylo- genetic 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; Sid- diqui 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 MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 970 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi” clade previous studies, the CO/ 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 Araujo 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 sim- ilar 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 here- in 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. unilatertalis. 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 labo- ratory and examined within the same day if possible or stored at 4 °C. The speci- mens 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, To- kyo, 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 Araujo et al. (2018). MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 271 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi” clade 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 (Fore- gene Co., Ltd., Chengdu, China) according to the manufacturer's protocols. Poly- merase chain reaction (PCR) was used to amplify genetic markers using the fol- lowing primer pairs: NS1/NS4 for small subunit nuclear ribosomal DNA (SSU) (White et al. 1990), 2218R/983F for translation elongation factor 1-a (TEF7a) (Rehner and Buckley 2005), CRPB1/RPB1Cr_oph for partial RNA polymerase II largest subunit gene region (RPB7) (Castlebury et al. 2004; Araujo et al. 2018) and LCO1490/HC02198 for cytochrome oxidase subunit 1 (Hebert et al. 2003). Each 25 ul-PCR reaction contained 2.5 yl of PCR 10x Buffer (2 mmol/| Mg**) (Transgen Biotech, Beijing, China), 17.25 ul of sterile water, 2 ul of dNTP (2.5 mmol/l), 1 pl of each forward and reverse primer (10 umol/I), 0.25 pl of Taq DNA polymerase (Transgen Biotech, Beijing, China) and 1 ul of DNA template (500 ng/ul). 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 TEF7a, (1) 4 min at 95 °C, (2) 8 cycles of denaturation at 94 °C for 50 s, annealing at 52 °C for 50s 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 RPB7, (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, Bei- jing, 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 stud- ies (Sung et al. 2007; Quandt et al. 2014; Araujo 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.nim.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 MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 979 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi” clade Table 1. The taxa, GenBank accession numbers and host information in this study. information Hirsutella sp. NHJ 12525 EF469125 | EF469078 | EF469063 | EF469092 | EF469111 | _ Sungetal. (2007) OSC 128575 EF469126 | EF469079 | EF469064 | EF469093 | EF469110 | _ Sungetaall. (2007) Datigoneens ARSEF 5692 DQ522540 | DQ518754 | DQ522322 | DQ522368 | DQ522418 | Spatafora et al. (2007) OSC 128580 DQ522543 | DQ518757 | DQ522326 | DQ522371 | DQ522423 | Spatafora et al. (2007) Ophiocordyceps YFCC 9049 ON555837 | ON555918 | ON567757 | ON568677 | ON568130__‘ Tang et al. (2023b) ae ie YFCC 9019 ON555838 | ON555919 | ON567758 | ON568678 | ON568131| ‘Tang et al. (2023b) YFCC 9017 ON555839 | ON555920 | ON567759 | ON568679 | ON568132_—‘ Tang et al. (2023b) YFCC 9018 ON555840 | ON555921 | ON567760 | ON568680 | ON568133 |‘ Tang et al. (2023b) YFCC 9016 ON555841 | ON555922 | ON567761 | ON568681 | ON568134 ‘Tang et al. (2023b) YHH 20122 ONS55842| =| ON567762 | ONS68682} ==——|_—_—‘Tangetaal.. (2023b) Ophiocordyceps RC20 Camponotus sp. | KX713633 KX713670 Araujo et al. (2018) albacongiuae Ophiocordyceps CEM 303 KJ878915 | KJ878881 | KJ878962 | KJ878995 — | Quandt et al. (2014) annullata Ophiocordyceps aphodii | ARSEF 5498 DQ522541 | DQ518755 | DQ522323/ =| Q522419 | Spatafora et al. (2007) Ophiocordyceps australis | HUA 186097 | Pachycondyla sp. KC610786 | KC610765 | KC610735 KF658662, ==~—|_-Sanjuanetal. (2015) Ophiocordyceps YHH 20191 ON555828 | ON555910 | ON567748 | ON568672 | ON568121_—‘ Tang et al. (2023b) basiasca Ophiocordyceps bifertilis 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 MISSOUS5 Camponotus sp. | KX713641 | KX713610 | KX713688 | KX713716 Araujo et al. (2018 blakebarnesii MissouU4 | Camponotus sp. | KX713642 | KX713609 | KX713685 | KX713715 Aratijo et al. (2018) Ophiocordyceps OSC 128576 Coleoptera DQ522542 | DQ518756 | DQ522324 | DQ522369 | DQ522420 | Spatafora et al. (2007) brunneipunctata Ophiocordyceps buquetii_ | HMAS_199617 KJ878940 | KJ878905 | KJ878985 | KJ879020 Ophiocordyceps G143 Camponotus KX713658 | KX713595 | KX713690 | KX713705 camponoti-balzani balzani G104 Camponotus KX713660 | KX713593 | KX713689 | KX713703 balzani Ophiocordyceps OBIS5 Camponotus KX713636 | KX713616 | KX713693 | KX713721 camponoti-bispinosi bispinosus OBIS4 Camponotus KX713637 | KX713615 | KX713692 | KX713720 bispinosus Ophiocordyceps MF080 Camponotus MK874744 MK863824 camponoti-chartificis chartifex Ophiocordyceps FEMO2 Camponotus KX713663 | KX713590 | KX713678 | KX713702 camponoti-femorati femoratus Ophiocordyceps Flo4 Camponotus KX713662 | KX713591 camponoti-floridani femoratus FIx2 Camponotus KX713592 | KX713674 femoratus Ophiocordyceps HIPPOC Camponotus KX713655 | KX713597 | KX713673 | KX713707 camponoti-hippocrepidis hippocrepis Ophiocordyceps INDI2 Camponotus KX713654 | KX713598 camponoti-indiani indianus Ophiocordyceps C27 Camponotus JN819019 camponoti-leonardi leonardi C25 Camponotus JN819029 leonardi Ophiocordyceps NIDUL2 Camponotus KX713640 | KX713611 | KX713669 | KX713717 camponoti-nidulantis nidulans MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 273 SS ws ws | we |] we | Quandt et al. (2014) Araujo et al. (2018) Araujo et al. (2018) Araujo et al. (2018) Araujo et al. (2018) Araujo et al. (2018) Araujo et al. (2018) | Araujo et al. (2018) Araujo et al. (2018) Araujo et al. (2018) Araujo et al. (2018) Kobmoo et al. (2012) Kobmoo et al. (2012) Araujo et al. (2018) Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi’ clade Species name Ophiocordyceps camponoti- novogranadensis Ophiocordyceps camponoti-renggeri Ophiocordyceps camponoti-rufipedis Ophiocordyceps camponoti-saundersi Ophiocordyceps camponoti-saundersi Ophiocordyceps citrina Ophiocordyceps clavata Ophiocordyceps cochlidiicola Ophiocordyceps contiispora Ophiocordyceps curculionum Ophiocordyceps daceti Ophiocordyceps dipterigena Ophiocordyceps flabellata Ophiocordyceps formosana Ophiocordyceps formicarum Ophiocordyceps forquignonii Ophiocordyceps sp. Ophiocordyceps halabalaensis Ophiocordyceps halabalaensis Ophiocordyceps ansiformis Ophiocordyceps irangiensis Ophiocordyceps kimflemingiae Voucher information Mal63 Mal4 RENG2 ORENG G177 G108 C40 Co19 TNS F18537 CEM 1762 | HMAS_199612 YFCC 9025 YHH 20145 YFCC 9026 YFCC 9027 OSC 151910 MFO1 OSC 151911 OSC 151912 YFCC 8795 YFCC 8796 YHH 20038 YHH 20037 TNM F13893 TNS F18565 OSC 151902 OSC 151908 Gh41 MY1308 MY5151 YHH 2210007 OSC 128577 OSC 128578 OSC 128579 SC30 SC09B Host Camponotus novogranadensis Camponotus novogranadensis Camponotus renggeri Camponotus renggeri Camponotus rufipes Camponotus rufipes Camponotus saundersi Camponotus saundersi Hemiptera Coleoptera Lepidoptera Camponotus sp. Camponotus sp. Camponotus sp. Camponotus sp. Coleoptera Daceton armigerum Diptera Diptera Hymenoptera (Camponotus sp.) Hymenoptera (Camponotus sp.) Hymenoptera (Camponotus sp.) Camponotus sp. Coleoptera Hymenoptera Diptera Diptera Polyrhachis sp. Camponotus gigus Camponotus gigas Colobopsis sp. Hymenoptera Hymenoptera Hymenoptera Camponotus castaneus/ americanus Camponotus castaneus/ americanus SSU KX713648 KX713649 KX713632 KX713634 KX713657 | KX713659 KJ201519 KJ878916 KJ878917 ON555829 ON555830 ON555831 ON555832 KJ878918 | KJ878919 KJ878920 0L310721 OL310722 KJ878908 KJ878921 KJ878912 KJ878922 KX713656 | KM655825 KM655826 OR345230 DQ522546 DQ522556 EF469123 KX713629 KX713631 MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 LSU KX713603 KX713602 KX713617 KX713596 | KX713594 KJ878903 KJ878882 KJ878884 ON555911 ON555912 ON555913 KJ878885 KX713604 | | KJ878886 KJ878887 O0L310724 0L310723 KJ878888 KJ878876 KJ878889 DQ518760 DQ518770 EF469076 KX713622 KX713620 TEF1a KX713672 KX713671 KX713680 KX713679 JN819012 JN819018 KJ878983 KJ878963 KJ878965 ON567749 ON567750 ON567751 ON567752 KX713667 KJ878966 KJ878967 O0L322688 0L322692 O0L322694 0L322693 KJ878956 KJ878968 KX713668 GU797109 GU797110 ORO98435 DQ522329 DQ522345 EF469060 KX713699 KX713698 RPB1 KX713704 KJ878996 KJ878998 ON568139 ON568140 ON568141 ON568142 KJ878999 KJ879000 KJ879001 OL322687 OL322689 0L322691 0L322690 KJ878988 KJ879002 KJ878991 KJ879003 KX713706 OR351952 | DQ522374 DQ522391 EF469089 KX713727 KX713724 RPB2 KJ878954 ON568122 ON568123 ON568124 ON568125 0L322695 O0L322696 0L322697 KJ878943 KJ878946 KJ878945 KJ878947 DQ522427 DQ522445 EF469107 Reference Araujo et al. (2018) Araujo et al. (2018) Araujo et al. (2018) Araujo et al. (2018) Araujo et al. (2018) Araujo et al. (2018) Kobmoo et al. (2012) Kobmoo et al. (2012) Quandt et al. (2014) Quandt et al. (2014) Quandt et al. (2014) Tang et al. (2023b) Tang et al. (2023b) Tang et al. (2023b) Tang et al. (2023b) Quandt et al. (2014) Araujo et al. (2018) Quandt et al. (2014) Quandt et al. (2014) Tang et al. (2023a) Tang et al. (2023a) Tang et al. (2023a) Tang et al. (2023a) Quandt et al. (2014) Quandt et al. (2014) Quandt et al. (2014) Quandt et al. (2014) Araujo et al. (2018) Kobmoo et al. (2015) Kobmoo et al. (2015) This study Spatafora et al. (2007) Spatafora et al. (2007) Sung et al. (2007) Araujo et al. (2018) Araujo et al. (2018) 274 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi” clade Species name Ophiocordyceps kniphofioides Ophiocordyceps konnoana Ophiocordyceps lilacina Ophiocordyceps Iloydii Ophiocordyceps longissima Ophiocordyceps melolonthae Ophiocordyceps monacidis Ophiocordyceps myrmecophila Ophiocordyceps naomipierceae Ophiocordyceps neovolkiana Ophiocordyceps nigrella Ophiocordyceps nooreniae Ophiocordyceps nutans Ophiocordyceps nuozhaduensis Ophiocordyceps odonatae Ophiocordyceps oecophyllae Ophiocordyceps ootakii Ophiocordyceps ootakii Ophiocordyceps ponerinarum Ophiocordyceps polyrhachis-furcata Ophiocordyceps pulvinata Ophiocordyceps purpureostromata Ophiocordyceps rami Ophiocordyceps ravenelii Ophiocordyceps rhizoidea information HUA 186148 KC610790 | KF658679 | KC610739 | KF658667 | KC610717 roc TES | Comores H559 |_| ERE | eFasoor | FHS ‘er 20001 | Podrecis se Taz43|———~|OFPWEBS OFTHE ‘yr 220002 | Precis. | OPTez48| ——_—_—|OFPR867|OFPGHRD | "Tus eee | Hemiptera KiT825 | Ke7E82 |KeTEST | KITBI0S) as 15560/ Hemitee— KTOz6 | ———*|KTES72 | TBIOS | KEBI Reference Sanjuan et al. (2015) Sung et al. (2007) Sung et al. (2007) Tang et al. (2023a) Tang et al. (2023a) Quandt et al. (2014) Quandt et al. (2014) Quandt et al. (2014) OSC 110993 DQ522548 | DQ518762 | DQ522331 | DQ522376 | Spatafora et al. (2007) Ophgrc679 Coleoptera KC610768 | KC610744 | KF658666 a! MF74C Dolichoderus KX713646 | KX713606 bispinosus ME74 Dolichoderus | KX713647 | KX713605 KX713712 bispinosus CEM1710 KJ878928 | KJ878894 | KJ878974 | KJ879008 DAWKSANT | Polyrhachis cf. | KX713664 | KX713589 KX713701 robsonii OSC 151903 KJ878930 KJ878976 | KJ879010 EF468963 EF468758 | EF468866 | EF468920 NG065096 KX673812 KX673809 KJ878896 EFCC 9247 BRIP 55363 EF468818 NG059720 Chariomyrma cf. hookeri and Polyrhachis lydiae BRIP 64868 KX961142 KX961143 DQ518763 | DQ522333 ON555927 | ON567769 ON555928 | ON567770 KJ878877 KJ878878 ar 4 KX713682 | KX713709 KX713708 KF658668 Polyrhachis cf. hookeri and Polyrhachis lydiae DQ522549 ON555849 ON555850 Oecophyllas smaragdina J14 Polyrhachis moesta J13 Polyrhachis moesta HUA 186140 Paraponera KC610789 | KC610767 | KC610740 clavata P39 Polyrhachis KJ201504 JN819003 furcata P51 Polyrhachis furcata TNS-F-30044 Camponotus obscuripes KM655824 KJ878932 EF468969 EF468970 OSC 110994 YHH 20168 YHH 20169 TNS F18563 TNS F27117 OECO1 KX713635 KX713651 KX713652 | KX713600 | KX713681 KJ201505 JN819000 GU904208 GU904209 | GU904210 KJ878977 | KJ879011 KJ201532 TNS F18430 KJ878931 | KJ878897 MY6736 MY6738 OSC 151914 NHJ 12529 NHJ 12522 KM655823 KJ201534 KJ878978 | KJ879012 | KJ878950 EF468765 | EF468872 | EF468922 EF468764 | EF468873 | EF468923 EF468824 EF468825 MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 Sanjuan et al. (2015) Araujo et al. (2018) Araujo et al. (2018) Quandt et al. (2014) Araujo et al. (2018) Quandt et al. (2014) Sung et al. (2007) Crous et al. (2016) Crous et al. (2016) Spatafora et al. (2007) Tang et al. (2023b) Tang et al. (2023b) Quandt et al. (2014) Quandt et al. (2014) Aratijo et al. (2018) Araujo et al. (2018) Araujo et al. (2018) Araujo et al. (2018) Kobmoo et al. (2012) Kobmoo et al. (2012) Kepler et al. (2011) Araujo et al. (2018) Kobmoo et al. (2015) Kobmoo et al. (2015) Quandt et al. (2014) Sung et al. (2007) Sung et al. (2007) 275 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi” clade Species name Ophiocordyceps satoi Ophiocordyceps septa Ophiocordyceps sinensis Ophiocordyceps sobolifera Ophiocordyceps sphecocephala Ophiocordyceps stylophora Ophiocordyceps subtiliphialida Ophiocordyceps tianshanensis Ophiocordyceps tortuosa Ophiocordyceps tricentri Ophiocordyceps unilateralis Ophiocordyceps yakusimensis Paraisaria amazonica Paraisaria gracilis Paraisaria heteropoda Tolypocladium inflatum Tolypocladium ophioglossoides Voucher information J19 J7 YFCC 8807 YFCC 8809 YFCC 8810 Pur Pur2 C41 EFCC 7287 KEW 78842 OSC 110998 OSC 111000 OSC 110999 YFCC 8815 YFCC 8814 YFCC 8816 YFCC 8817 MFLU 19-1207 MFLU 19-1208 YHH 2210003 YHH 2210004 YHH 2210005 YHH 2210006 CEM 160 VIC 44303 VIC 44354 HMAS_199604 HUA 186113 EFCC 8572 EFCC 3101 OSC 106404 OSC 71235 CBS 100239 Host Polyrhachis lamellidens Polyrhachis lamellidens Polyrhachis sp. Polyrhachis sp. Polyrhachis sp. Camponotus sp. Camponotus sp. Camponotus sp. Lepidoptera Hemiptera Hymenoptera Coleoptera Coleoptera Camponotus sp. Camponotus sp. Camponotus sp. Camponotus sp. Camponotus japonicus Camponotus japonicus Colobopsis sp. Colobopsis sp. Colobopsis sp. Colobopsis sp. Hemiptera Camponotus sericeiventris Camponotus sericeiventris Hemiptera Orthoptera Lepidoptera Lepidoptera Hemiptera Coleoptera Elaphomyces sp. SSU KX713650 KX713653 OP782340 OP782341 OP782342 EF468971 EF468972 DQ522551 DQ522552 EF468982 ON555833 ON555834 ON555835 ON555836 MNO025409 MNO025410 OR067858 ORO67859 AB027330 KX713628 KX713627 KJ878938 KJ917566 EF468956 EF468955 AY489690 EF469124 KJ878910 LSU KX713601 KX713599 OP782345 OP782346 OP782347 EF468827 EF468828 DQ518765 DQ518766 EF468837 ON555914 ON555915 ON555916 ON555917 MNO25407 MNO025408 AB027376 KX713626 KJ878902 EF468811 EF468810 AY489722 EF469077 KJ878874 TEF1a KX713684 KX713683 OP796853 OP796854 OP796855 | KJ201528 KJ201529 JN819037 EF468767 DQ522336 DQ522337 EF468777 ON567753 ON567754 ON567755 ON567756 MK992784 MK992785 ORO98431 ORO98432 OROD98433 ORO98434 KX713675 KX713676 EF468751 EF468750 AY489617 EF469061 KJ878958 RPB1 KX713710 KX713711 OP796858 OP796859 OP796860 EF468874 EF468875 DQ522381 DQ522382 EF468882 ON568673 ON568674 ON568675 ON568676 ORO98436 OR098437 ORO98438 ORO98439 KX713730 KX713731 KJ879018 KP212903 EF468859 EF468858 AY489651 EF469090 KJ878990 RPB2 OP796863 OP796864 OP796865 EF468924 EF468925 DQ522432 DQ522433 EF468931 ON568126 ON568127 ON568128 ON568129 KJ878953 KM411980 EF468912 EF468913 EF469108 KJ878944 Reference Araujo et al. (2018) Araujo et al. (2018) Tang et al. (2023a) Tang et al. (2023a) Tang et al. (2023a) Araujo et al. (2018) Araujo et al. (2018) Kobmoo et al. (2015) Sung et al. (2007) Sung et al. (2007) Spatafora et al. (2007) Spatafora et al. (2007) Sung et al. (2007) Tang et al. (2023b) Tang et al. (2023b) Tang et al. (2023b) Tang et al. (2023b) Wei et al. (2020) Wei et al. (2020) This study This study This study This study Nikoh and Fukatsu (2000) Araujo et al. (2018) Araujo et al. (2018) Quandt et al. (2014) Sanjuan et al. (2015) Sung et al. (2007) Sung et al. (2007) Castlebury et al. (2004) Sung et al. (2007) Quandt et al. (2014) to determine the best fitting likelihood model for Maximum Likelihood (ML) and Bayesian Inference (BI) analyses according to the corrected Akaike Infor- mation 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+l+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% MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 276 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi” clade of samples were discarded as burn-in. The tree was visualised with its Maxi- mum-Likelihood bootstrap proportions (ML-BS) and Bayesian posterior prob- ability (BI-BPP) in Figtree v.1.4.3. Adobe Illustrator CS6 was used for editing. Phylogenetic analyses of host Phylogenetic analyses were based on CO/ 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 se- quences 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 ad- justed 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. Results Phylogenetic analysis of fungi Combining single gene trees (SSU, TEF7a, RPB7) in a concatenated tree, us- ing morphological features for comparison, enabled identification of two new species (0. tropiosa and O. ansiformis). We have inferred the phylogeny, based on each single gene (SSU, TEF1a, RPB1) and present the details below. Ophio- cordyceps 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 TEF7a or RPB1 (Suppl. materials 2, 3). For the concatenated tree (SSU, LSU, TEF7a, RPB7 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 RPB7 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 Likeli- hood 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 Araujo et al. (2018), phylogenetic analy- ses 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 support- ed (BS = 100%, BPP = 100%), 0. oecophyllae branched as its sister taxon with MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 977 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi’ clade Table 2. Specimen and GenBank accession numbers information for CO/ genes used in this study. Species name Camponotus americanus Camponotus castaneus Camponotus claripes Camponotus renggeri Colobopsis rufipes Camponotus simulans Camponotus sp. Colobopsis sp. Camponotus spanis Camponotus sericeiventris Camponotus sexguttatus Colobopsis badia Colobopsis explodens Colobopsis saundersi Colobopsis vitreus Camponotus wiederkehri Daceton armigerum Oecophylla smaragdina Polyrhachis abbreviata Polyrhachis anderseni Polyrhachis andromache Polyrhachis ammon Polyrhachis aurea Polyrhachis australis Polyrhachis arnoldiisolate Polyrhachis beccari Polyrhachis beccari Polyrhachis brevinoda Polyrhachis carbonaria Voucher information YNH-005 BIOUG03675-H07 BIOUG03675-H04 AECT Creng_1_B BIOUG24424-D11 AFR-CND-2010-47-F02 CASENT0441197-D01 GU710187 CASENT0043700-D01 YHH 20606 YHH 20607 YHH 20608 — YHH20168 — YHH20168 68 —NDAAO MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 1 R ° H i] | N 1 1 u A GenBank number MZ331828 KJ208900 KJ445248 JN134855 KP101600 OM314604 JN270684 KF200199 OP353541 OP353542 OP353543 OP353547 ee ee Reference Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Tang et al. (2023a) Tang et al. (2023b) Tang et al. (2023b) Tang et al. (2023b) Tang et al. (2023b) Tang et al. (2023b) Tang et al. (2023b) Tang et al. (2023b) Tang et al. (2023b) Tang et al. (2023b) Tang et al. (2023b) Tang et al. (2023b) This study This study Unpublished Unpublished Unpublished Unpublished Laciny et al. (2018) Unpublished Allio et al. (2020) Unpublished Unpublished Unpublished Unpublished Mezger and Moreau (2015 Mezger and Moreau (2015 Mezger and Moreau (2015 Mezger and Moreau (2015 Unpublished Mezger and Moreau (2015) Unpublished Unpublished Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) ) ) we me ma la Mezger and Moreau (2015 Mezger and Moreau (2015 278 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi’ clade Species name Polyrhachis cf. bismarckensis Polyrhachis cupreata Polyrhachis cyphonota Polyrhachis danum Polyrhachis delecta Polyrhachis flavibasis Polyrhachis furcata Polyrhachis gagates Polyrhachis hexacantha Polyrhachis hookeri Polyrhachis illaudata Polyrhachis jianghuaensis Polyrhachis latharis Polyrhachis lamellidens Polyrhachis lepida Polyrhachis lucidula Polyrhachis mackayi Polyrhachis monteithi Polyrhachis mucronata Polyrhachis nigropilosa Polyrhachis noesaensis Polyrhachis obesior Polyrhachis ornata Polyrhachis proxima Polyrhachis rastellata Polyrhachis robsoni Polyrhachis saevissima Polyrhachis schistacea Polyrhachis schoopae Polyrhachis sp. Voucher information FMNH-INS 2842022 CSM1015 CSM0682 FMNH-INS_2842221 CSM1841 CSM0965 RA0766 RA0763 YB-KHC51412 FMNH-INS_2842213 FMNH-INS_2842006 RA0747 FMNH-INS_2842112 FMNH-INS_2842222 GXJX0141 GXBLO0006 FMNH-INS_2842062 NSMK-IN-170100347 CSM1877 CSM1807 G160084 CSM0804 CSM0754 RA1154 RA1158 RA1164 CSM0696a FMNH-INS_2842045 FMNH-INS_2842106 FMNH-INS_2842054 CSM0797 CSM0842 FMNH-INS_2842042 FMNH-INS_2842129 FMNH-INS_2841999 CSM1050 FMNH-INS_2842115 FMNH-INS_2842058 FMNH-INS_2842071 FMNH-INS_2842067 CSM0626b FMNH-INS_2842139 FMNH-INS_2842198 FMNH-INS_2842195 FMNH-INS_2842179 FMNH-INS_2842190 FMNH-INS_2842193 FMNH-INS_2842194 FMNH-INS_2842074 FMNH-INS_2842082 FMNH-INS_2842039 MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 GenBank number KM348331 KY939064 KY939056 KM348234 KM348235 KY939013 KM348203 KY939081 MN618329 KM348270 KM348204 KM348215 KM348275 KM348271 JQ681065 JQ681069 KM348278 OL663445 KM348241 KM348239 OM420302 KM348242 KY939009 KM348338 KM348339 KM348340 KM348337 KM348284 KM348285 KM348286 KM348255 KY939061 KM348289 KM348288 KM348244 KY939017 KM348345 KM348297 KM348294 KM348293 KM348218 KM348305 KM348309 KM348308 KM348300 KM348304 KM348310 KM348307 KM348226 KM348306 KM348311 Reference Mezger and Moreau (2015) Unpublished Unpublished Mezger and Moreau (2015) Mezger and Moreau (2015) Unpublished Mezger and Moreau (2015) Unpublished Unpublished Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Unpublished Unpublished Mezger and Moreau (2015) Unpublished Mezger and Moreau (2015) Mezger and Moreau (2015) Unpublished Mezger and Moreau (2015) Unpublished Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Unpublished Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Unpublished Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) Mezger and Moreau (2015) 279 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi” clade Species name Polyrhachis sp. Polyrhachis tubifera Polyrhachis turneri Polyrhachis villipes Polyrhachis viscosa Voucher information GenBank number Reference CSM2738 KM348302 Mezger and Moreau (2015) FMNH-INS_2842043 KM348246 Mezger and Moreau (2015) RAO779 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 0P353533 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) CSM1108 KY939104 Unpublished CSM0827 KM348260 Mezger and Moreau (2015) FMNH-INS_28421186 KM348316 Mezger and Moreau (2015) FMNH-INS_2842064 KM348317 Mezger and Moreau (2015) 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 South- east 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 RPB7) tree (Suppl. materials 2 and 3). Ophiocordyceps ansiformis was recovered sis- ter 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 RPB7) 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 CO/ genetic marker matrix con- tained 660 bp. The matrix had 389 distinct patterns, 309 parsimony-informative, MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 980 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi” clade 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 CO/ 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 par- asitised 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” arrange- ment of ascospores in the asci. Diagnosis. The difference between Ophiocordyceps tortuosa and related species is that Ophiocordyceps tortuosa produces lanceolate and obvious sep- arate ascospores, while O. contiispora produces fusiform and no obvious sep- arate ascospores. Holotype. CHINA, Yunnan Province, Puer City, Simao District. Infected Colo- bopsis 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 — pre- served 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 api- cal part. Fertile region of lateral cushions, 1-3, commonly 2 per stroma, hemi- spherical, chocolate brown at maturity, 1-1.9 x 0.8-1.3 mm. Perithecia im- mersed to partially erumpent, flask-shaped, (211-) 218-298 (-305) x (94-) 99-142 (—158) um, with short, exposed neck or ostiole. Asci 8-spored, hyaline, cylindrical, (92—) 96-132 (-134) x 7-11 (-13) um. Ascus caps slightly promi- nent, hemispherical, 4—5 x (2—) 3-4 um. Ascospores hyaline, thin-walled, lance- olate, tortuous arrangement in the ascus, 47-64 x 5-7 um, 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 stro- mata. Hirsutella-C type produced from the leg and antennal joints. Phialides MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 281 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi’ clade lageniform, 54-99 um long, 4-6 um width at base, tapering to a long neck, 1-2 um in width. Conidia fusiform to limoniform, 6-8 x 3-5 um, slightly nar- rowing 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) x 1-2 um, bearing a single terminal capilliconidium, (5—) 6-9 x 3-4 (—5) um, 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 Colo- bopsis 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 pro- duced branched stromata, slightly narrowing conidia (Table 3). 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 — pre- served 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 x 0.7-1 mm. Perithecia immersed to partially erumpent, flask-shaped, (174—-) 189-290 x 99-126 (-128) ym, with short, exposed neck or ostiole. Asci 8-spored, hyaline, cylindrical, (88—) 92-108 (-112) x 7-10 (-11) um. As- cus caps prominent, hemispherical, 4—6 (-—7) x 2-3 (-4) um. Ascospores hya- line, thin-walled, lanceolate, having a handle-shape in the middle, 45-59 x 5-6 (-7) um, 6—9-septate, tapering at apex. MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 999 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi” clade 100/100 coe ceps camponoti-leonardi C27 arn, 99/100 Iph 97/100} i a s nuozhaduensis YHH 20169 s B/97- rire ile! ceps flabellata YFCC 8795 93/49] pphiocor ceps flabellata YHH 20038 ‘ 100/10D hiocordyceps flabellata YECC 8796 %, phiocordyceps flabellata YHH 20037 ‘, 68/61 100/100 aie: cheep Lett GS P39 \ 72/72 ae ceps polyrhachis-furcata P51 og/100 100/100 Deen eps halabalaensis MY 1308 \ vas lyceps halabalaensis MY5151 \ 100/100) Ophiocordyceps ootakii J\4 \ 140/100 Iphiocordyceps ootakii J13 100/100y Ophiocordyceps nooreniae BRIP 64868 } Iphiocordyceps nooreniae BRIP 55363 one? 100/100 ile eek ceps satoi J1 ry (84/65 hiocordyceps satoi J7 98/981. Ophiocordyceps satoi YFCC 8807 H sor eae Ophiocordyceps satoi YFCC 8809 - Ophiocordyceps satoi YFCC 8810 Ophiocordyceps naomipierceae DAWKSANT 99/100 100/98» Ophiocordyceps bifertilis Ophi bifertilis YHH 20162 oshOO] 1 ob2| Ophiocordyceps bifertilis YFCC 9013 1 oe Cphiocor Gia bifertilis YFCC 9012 iintioat Gphiocor cep bifertilis YFCC 904 - Iphiocordyceps bifertilis YAH 20163 Ophiocordyceps bifertilis YHH 20164 100/100) Ophiocordyceps camponoti-saundersi C40 hiocordyceps camponoti-saundersi Col9 99/77) Ophiocordyceps acroasca YFCC 9049 phiocordyceps acroasca YHH 20122 Ophiocordveeps acroasca YFCC 9018 poe ‘ceps acroasca YFCC 9016 hiocordyeeps acroasca YFCC 9019 93/79 hiocor i acroasca YFCC 9017 53/99 100/100 100/100 hiocordyceps septa Purl 100/100 Pea age ceps septa C4] hiocordyceps septa Pur2 71/6\¢ Ophiocordyceps subtiliphialida YFCC 8814 90/100 66/67 Ophiocordyceps subtiliphialida YFCC 8816 ART Fe} yl acena eas subtiliphialida YFCC 8817 phiocordyceps el ere YEFCC 8815 96/98 Ophiocordyceps basiasca YHH 20191 56/84 Cee ceps contiispora YFCC 9025 100/100 95/53 as de céps contiispora YFCC 9027 phiocordyceps contiispora YFCC 9026 Ophiocordyceps contiispora YHH 20145 100/100) | Ophiocordyceps tertuosa YHH 2210003 99/100 91/p9 phiocordyceps fortuosa YHH 2210005 Ophiocordyceps tortuosa ¥YHH 2210004 85/95 100/100! Ophiocordyceps tertuosa YAH 2210006 Ophiocordyceps ansiformis YHH 2210007 100/100 Opipocorayeens lilacina YHH 2210001 Iphiocordyceps lilacina YHH 2210002 96/100 Ophiocordyceps sp. Gh41 100/100) Ophiocordyceps rami MY 6736 hiocordyceps rami MY6738 100/100 Ophiocordyceps camponoti-rufipedis G177 i & dy pS camp ie 90/96. Ophiocordyceps unilateralis core clade 100/100 hiocordyceps camponoti-rufipedis G108 86/100 Ophiocordyceps camponoti-femorati FEMO2 ee Ophiocordyceps unilateralis VIC 44303 ‘Ba phiocordyceps unilateralis VIC 44354 100/100). Ophiocordyceps camponoti indiani INDI2 100/100 100/100) Ophiocordyceps camponoti-floridani Flo4 36/97 phiocordyceps camponoti-floridani F1x2 Pi | 100/100. Ophiocordyceps camponoti-renggeri RENG2 Af ‘fi aa 67/58 Ophiocordyceps camponoti-renggeri O) G by Ophiocordyceps albacongiuae RC20 97/97. Ophiocordyceps camponoti-nidulantis NIDUL2 Pale ORG ere camponoti-chartificis MF080 Ophiocordyceps camponoti- porte ae HIPPOC ! 100/100 ee ceps kimflemingiae $C30 96/97 ieee! kimflemingiae SCO9B 98/80 100/100 Ophiocordyceps Camponoti-novogranadensis Mal63 ‘ 1oor100) 483) | Ophiocordyceps camponoti-novogranadensis Mal4 é - 100/100 Ophiocordyceps camponoti-bispinosi OBISS a Ophiocordyceps camponoti-bispinosi OBIS4 Z 100/100) Ophiocordyceps camponoti-balzani G143 £ hiocordyceps camponoti-balzani G1 a 91199 100/100 op eatin blakebarnesii MISSOU5 - Ne eee eee eee ee eeeeEEeEeEey—eE—eE——eE——E———E—E—EE—EE EE 100/98 100/100 hiocordyceps blakebarnesii MISSOU3 - Cplunrane ceps tianshanensis MFLU 19-1207 - Iphiocordyceps tianshanensis MFLU 19-1208 nal | Ophiocordyceps pulvinata TRF s3 0044 — Ophiocordyceps ale ae ‘OLCOL 27/51 Op pont daceti Ol 100/100 phiocordyceps monacidis MF74C Ophiocordyceps monacidis MF74 88/71 100/100 Ophiaserineses kniphofioides HUA 186148 eg iocordyceps ponerinarum HUA 186140 100/100 Cpe ceps stylophora OSC 111000 100/100) Iphiocordyceps stylophora OSC 110999 70550 Ip focordvceps sinensis EFCC 7287 99/100] 100/10 S7/79 99/1001 - Puce citrina TNS F18537 100/100 ne iocordyceps acicularis OSC 128580 100/10 PRE cochlidiicola HMAS_199612 69/100 100/100¢ Hirsutella sp. NHJ 12525 2: Hirsutella sp. OSC 128575 9g/100 Ophiocordyceps acicularis ARSEF 5692 oo) Ophiocordyceps rhizoidea NHJ 12529 phiocordyceps rhizoidea NHJ 12522 99/100) Ophiocordyceps myrmecophila CEM 170 100/100 Ophiocordyeeps irangiensis OSC 128578 99/100 HAH 100/100 Opiuoceraycens irangiensis OSC 128577 : io8 Iphiocordyceps irangiensis OSC 128579 Ophiocordyceps tricentri CEM 160 _ SaT00 100/100 ¢ Ophiocordyceps odonatae TNS F18563 68/67 Ophiocardyceps adonatae TNS F27117 Ophiocor dveeny piecres hala OSC 110998 91/87 Ophiocordyceps Sears ‘ aie 18565 100/100¢ Ophiocordyceps forquignonii OSC | aa a a br Se OSC 151908 100/101 OP 3 Ne Ophiocordyceps lloydii OSC 151913 100/95, Ophea buquetii HMAS_ 199617 100/100 LOO oor Ophiocordyceps australis HUA 186097 : S283 Ophiocordyceps curculionum OSC 151910 Ophiocordyceps nutans OSC 110994 100/100¢ Ophiocordyceps dipterigena OSC 151911 Baio Ophinearaceas dipterigena OSC 151912 oa/100 = 00/100 Ophiocordyceps longissima TNS F18448 190/100 One longissima HMAS_ 199600 Vaated Ophiocordyceps yakusimensis HMAS_199604 00/98 Ophiocordyceps sobolifera KEW 78842— 00/98 jphiocordyceps aphodii ARSEF 5498 98 Ophiocordyceps annulata CEM 303 Ophiocordyeeps rare lancai OSC 128576 00/100) Ophiocordyceps konnoana EFCC 7295 99/99 Ophiocordyceps kKonnoana EFCC 7315 00/100 Ophiocordyceps nigrella EFCC 9247 Ophiocordyceps ravenelii OSC 151914 68/96 Ophiocordyceps purpureostromata TNS F18430 45/70 Ophiocordyceps clavata CEM 1762 68/90 Ophiocordyceps formosana TNM F13893 't loo/toor Paraisaria gracilis EFCC 3101 00/100 Paraisaria gracilis EFCC 8572 Paraisaria amazonica HUA 186113 92/97 1200100 Paraisaria heteropoda OSC 106404 | 00/100 0 Laat gracillisima Ohare 00/100 Iphiocordyceps melolonthae OSC 110993 Ophiocordyceps neovolkiana OSC 151903 00/100 Tolypocladium ophioglossoides CBS 100239 Tolypocladium inflatum OSC 71235 0.02 Figure 1. Phylogenetic tree of Ophiocordyceps and related genera, based on the concatenation of LSU, SSU, TEF1a, 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. MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 983 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi” clade 991100 Polyrhachis abbreviata CSMO776 Polyrhachis monteithi CSMO754 pl Polyrhachis australis RAQ757 Ree by) Polyrhachis delecta CSM0965 lyrhachis mackayi CSMO804 pithy Polyrhachis brevinoda CSM0773 Polyrha inoda CSM283 is Sp. 99 Polyrhachis lepida CSM1807 98/100 Polyrhachis danum CSM1841 00/100 Pobre colina Ta 2842221 is Sp. 0644 Polyrhachis furcata YB-KHC51412 Polyrhachis sp. FMNH-INS_2842103. Polyrhachis sp. YHH 20645 Polyrhachis sp. FMNH-INS_2842032 Polyrhachis sp. FMNH-INS_2842078 Polyrhachis saevissima FMNH-INS_2842115 Polyrhachis sp. FMNH-INS 2842044 Polyrhachis sp. YHH 20643 Polyrhachis sp. YHH 20646 6/9 Polyrhachis eget fae YHH 20162 Polyrhachis sp. YHH ‘hachi: Polyrhachis jianghuaensis GXBLO006 _ : 00/1007>— Polyrhachis cf. bismarckensis FMNH-INS_2 Polyrhachis mucronata CSM0696a Polyrhachis mucronata RA1158 0/100% Polyrhachis mucronata RA1164 Polyrhachis lucidula G160084 Polyrhachis sp. CSM2738 00¢ Polyrhachis sp. FMNH-INS_2842195 Polyrhachis sp. FMNH-INS_2842198 Polyrhachis latharis FMNH-INS_2842062 Louse Polyrhachis schistacea FMNH-INS_2842067 00/96 # Polyrhachis schistacea FMNH-INS_2842071 958 OWL Polyrhachis schistacea FMNH-INS_2842058 So 100 IL Polyrhachis sp. FMNH-INS_28421 See tek nee ce FMNH-INS_2842213 ‘hachis sp. FMNH-INS _ 2842194 Palyrhachis viscosa F| Palyrhachis sp. FMNH-INS_28421' Polyrhachis arnoldi NDA40 96/6 Polyrhachis noesaensis FMNH-INS_2842106 70/88 Polyrhachis villipes FMNH-INS_28421186 1 Polyrhachis nigropilosa FMNH-INS a Polyrhachis proxima FMNH-INS-284- Polyrhachis illaudata FMNH-INS_: 58491 12 Polyrhachis obesior FMNH-INS_2842054 as] 00/100¢ Polyrhachis illaudata FMNH-INS_2842222 Polyrhachis sp. FMNH-INS | 2842139 Polyrhachis sp. FMNH-INS_2842179 100/100¢° Polyrhachis sp. YHH 20601 SL 99/94 Polyrhachis sp. YHH 20636 00/1008. Polyrhachis illaudata GXIXO141 1eolioo] “Polyrhachis sp. YHH 20635 ia 00, Ba ‘hachis sp. YHH 20163 ‘hachis sp. YHH at pOrLNG Pela sp. YHH 2064 ‘hachis ee MN INS_2842129 ‘hachis sp. FMNH-INS_ 2842039 Polyrhachis pci FMNH-INS. 2842101 00 ¢ Polyrhachis beccari FMNH-INS | 284: Polyrhachis beceari FMNH-INS_2842169 Polyrhachis sp. YHH 20637 00 Polyrhachis andromache FMNH-INS_2842051 Polyrhachis sp. FMNH-INS_ 2842082 Polyrhachis lamellidens NSMK-IN-170100347 Polyrhachis hexacantha FMNH-INS_2842006 00/1004 Polyrhachis flavibasis RAO7T63 Polyrhachis flavibasis RAQT66 99/100p— Polyrhachis aurea RAO750 00 Polyrhachis sp. FMNH-INS_2842074 Polyrhachis hookeri RAQ747 Polyrhachis schoopae CSM0626b Polyrhachis ammon RAOQ751 Polyrhachis anderseni ANA42 Polyrhachis tubifera CSM1108 00/100) Polyrhachis cupreata CSM0682 q fl 00 Polyrhachis cupreata CSM1015 Polyrhachis turneri CSM0827 95/67 100/100) Polyrhachis ornata CSM0797 Polyrhachis ornata CSM0842 : sp. YHH 20608 6! Camponotus sp. YHH 20611 4 Camponotus sp. YHH 20607 eens 00/100) Colobopsis vitrea evel3410-1L Colobopsis vitrea evel = Camponotus sericeiventris BIOQUG24738-E05. Camponotus sp. CASENT0000633-D01 100/68) Camponotus castaneus BIOUG03675-H04 100/100% Camponotus castaneus BIOQUG03675-H07 "Camponotus americanus YNH 005 Camponotus spanis G191388 — Camponotus claripes AECT — Camponotus sp. CAMPO014 Camponotus simulans AFR-CND-2010-47-F02 Camponotus wiederkehri AEKB Camponotus sp. CASENT0043700-D01 53/55 Camponotus sp. CASENT0441197-D01 Oecophylla smaragdina CSM0633 Daceton armigerum USNM 0.05 Figure 2. Phylogenetic tree of some genera of the Formicinae based on CO/ 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. MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 284 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi” clade 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 I,m ascospore with capilliconidiophores n capilliconidium o-q phialides r conidia. Scale bars: 4000 um (a, b); 3000 um (c, d); 2000 um (e); 200 um (f); 100 um (g); 20 um (h, i); 10 um (j, k); 20 um (I, m); 10 um (n); 20 um (o-r). MycoKeys 99: 269-296 (2023), DOI: 10.3897/mycokeys.99.107565 985 Dexiang Tang et al.: Two new Ophiocordyceps species belonging to the “zombie-ant fungi” clade 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 asco- spores I-o ascospores with capilliconidiophores p capilliconidium q phialides. Scale bars: 4000 ym (a); 2000 um (b); 200 um (c); 100 um (d); 20 um (eh); 10 um (i, j); 20 um (k-o); 10 um (p, q); 5 um (r). 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Phialides lageni- form, 15-24 x 3-4 um, tapering to a short neck, 6-8 um 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) x 0.8-1.4 um, bearing a terminal capilliconidium, hyaline, smooth-walled, limoniform, 6-10 x 3-4 um, slightly narrowing apically. Host: Colobopsis sp. (Formicinae). Habitat. Subtropical monsoon evergreen broad-leaved forest. Infected Colo- bopsis 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 lin- eage 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 ansiform- is 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 pro- duced lanceolate ascospores and has a handle-shape in the middle (Table 3). Key to two novel taxa and related species within Ophiocordyceps unilateralis complex Jia Stroiiata TIEVeR DEANCNCO tec es cures