Phylogenetic and morphological classification of Ophiocordyceps species on termites from Thailand

Abstract Seven new species occurring on termites are added to Ophiocordyceps – O. asiatica, O. brunneirubra, O. khokpasiensis, O. mosingtoensis, O. pseudocommunis, O. pseudorhizoidea and O. termiticola, based on morphological and molecular phylogenetic evidence. O. brunneirubra possesses orange to reddish-brown immersed perithecia on cylindrical to clavate stromata. O. khokpasiensis, O. mosingtoensis and O. termiticola have pseudo-immersed perithecia while O. asiatica, O. pseudocommunis and O. pseudorhizoidea all possess superficial perithecia, reminiscent of O. communis and O. rhizoidea. Phylogenetic analyses based on a combined dataset comprising the internal transcribed spacer regions (ITS) and the largest subunit (LSU) of the ribosomal DNA, partial regions of the elongation factor 1-α (TEF) and the largest and second largest subunits for the RNA polymerase genes (RPB1, RPB2) strongly support the placement of these seven new species in Ophiocordyceps.


Introduction
The entomopathogenic genus Ophiocordyceps was established by Petch in 1931. His description was based on four specimens including O. blattae Petch, the type species, occurring on a cockroach collected from Sri Lanka, O. unilateralis (Tul. & C. Tul.) Petch on ants, O. peltata (Wakef.) Petch on Coleoptera larva (Cryptorhynchus sp.) and O. rhizoidea (Höhn.) Petch on Coleoptera larva. The distinction of the genus from Cordyceps Fr. was made due the presence of clavate asci that gradually narrowed to a thickened apex, as opposed to the cylindrical asci in many Cordyceps species. The ascospores in Ophiocordyceps sensu Petch are elongated fusoid, multi-septate that remain whole after discharge. Sung et al. (2007) & Spatafora]. The ascospores are usually cylindrical, multi-septate that could either dissociate into part-spores (O. sphecocephala, O. nutans) or remain whole ascospores (O. unilateralis). To date, Ophiocordyceps is the most speciose genus in Ophiocordycipitaceae with 235 names of accepted species (Spatafora et al. 2015;Khonsanit et al. 2018;Luangsa-ard et al. 2018). Most Asian species of Ophiocordyceps have fibrous, hard and pliant to wiry, dark coloured stromata with superficial to immersed perithecia (Kobayasi 1941;Kobmoo et al. 2012Kobmoo et al. , 2015Luangsa-ard et al. 2018).
Termites (Isoptera) are one of the eusocial and soil insects that have successfully evolved since the Cretaceous Period and are classified into 7 families, 14 subfamilies, 280 genera and 2,500 species (Pearce 1999). They occur throughout tropic and subtropic regions and can also be found in many temperate areas and semi-arid environments of the world (Eggleton et al. 2000). Termites are abundant in Thailand and are found in natural forests as well as urban areas, mostly considered as serious pests of wooden constructions. Current records of termite species from Thailand have been 199 species, 39 genera, 10 subfamilies and 4 families (Sornnuwat et al. 2004). Relationships between termites and fungi are classified into two categories. Firstly, termites cultivate fungi (Termitomyces spp.) in their fungus gardens within the subterranean nest or mound of fungus-growing termites (subfamily Macrotermitinae). Secondly, a parasitic interaction, in which fungi infect and consume termites as food for its nutrient value (Abe et al. 2000). Some species of fungi are known as pathogens of termites and they can be used as potential agents of biological control for each of the host's (i.e. termites) specificities (Rath 2000).
In surveys of entomopathogenic fungi in national parks and community forests collections of termite pathogens, most with superficial perithecia and rarely with immersed perithecia were found. The phenotypic characters of the collections in having wiry and pliant, darkly pigmented stromata identifies them primarily to be members of the Ophiocordycipitaceae, mostly as Ophiocordyceps communis. The aims of this study are (1) to clarify the relationships of these collections to known members of the Ophiocordycipitaceae, (2) to uncover hidden species in O. communis species complex and (3) to describe new taxa to accommodate species diversity in Ophiocordyceps.

Collection and isolation
Species occurring on termites (Isoptera) were found in the ground. The specimens were excavated carefully so as not to lose the host, which could be buried as deep as 15 cm under the ground and were placed in small plastics boxes before returning to the laboratory for isolation. The materials were examined under a stereomicroscope (OLYMPUS SZ61, Olympus Corporation, Japan). The fertile heads of the specimens containing mature perithecia were carefully placed over the Potato Dextrose Agar plate (PDA; fresh diced potato 200 g, dextrose 20 g, agar 15 g, in 1 litre distilled water). These were placed in a plastic box with moist tissue paper overnight to create a humid chamber. The following morning plates were examined with a stereomicroscope to check the discharged ascospores. Discharged ascospores were examined daily for germination and also for fungal contaminants.

Morphological study
The newly collected specimens were noted and photographed in the field using a digital Nikon D5100 camera and were taken to the laboratory and photographed using an Olympus SZX12 before they were placed in a moist chamber to facilitate ascospore discharge. The colour of the freshly collected specimens and cultures were characterised with the colour standard of the Online Auction Colour Chart. One to two perithecia were removed from the stroma and mounted on a glass slide using lactophenol cotton blue to measure their sizes and shapes, as well as the sizes and shapes of the asci and ascospores. Cultures on PDA, Potato Sucrose Agar plate (PSA: potato 200 g/l, sucrose 20 g/l, calcium carbonate 5g/l, agar 20g/l) and quarter strength Sabouraud Dextrose Yeast Agar (SDYA/4; Difco) were observed using light microscopy (Olympus SZ60, CX 30) daily to check for germination and contamination for 2-3 wks. Colony growth rates and characteristics (colour, texture, pigmentation) under dark/light condition (L:D = 14:10) were recorded and photos were taken using the Nikon D5100 camera.
For micro-morphological description, microscope slide cultures were prepared from a block of media (PDA, PSA and SDYA/4, ca. 5 × 5 mm 2 ) inoculated with the fungus and overlaid by a glass coverslip. The cultures were incubated at 25 °C. Observations, measurements of the conidiogenous cells and conidia of the asexual morphs and photographs were taken with an Olympus DP11 microscope.

Host identification
Dead termite hosts were identified, based on morphological characteristics, such as mandibulate mouthparts, antennae, shape of head and thoraxes. The identification of dead insects was conducted after pure cultures were acquired. Termites were identified by using the extant families of Isoptera after Sornnuwat et al. (2004) and Krishna et al. (2013).

DNA extraction, PCR amplification and sequencing
Cultivation of fungi for molecular work. -Pure cultures were grown on PDA. After approximately 2 wks, the plates were checked for contaminants and small agar blocks were inoculated into sterile Erlenmeyer flasks containing 50 ml Sabouraud Dextrose Broth (Difco) and incubated for 1-2 wks at 25 °C without shaking. Mycelium was then harvested by filtration and washed several times with sterile distilled water. Filtered mycelium was lyophilised. The material was extracted from mycelium by a modified CTAB method as previously described (Luangsa-ard et al. 2004(Luangsa-ard et al. , 2005. PCR amplification. -Five nuclear loci including the nuc rDNA region encompassing the internal transcribed spacers 1 and 2, along with the 5.8S rDNA (ITS), nuc 28S rDNA (LSU), the translation elongation factor 1-α gene (TEF) and the genes for RNA polymerase II largest (RPB1) and second largest (RPB2) subunits were sequenced. PCR primers used to amplify the gene regions for this study were: ITS5, ITS4 for ITS, LROR and LR7 for LSU (White et al. 1990), 983F and 2218R for TEF, CRPB1 and RPB1Cr for RPB1, RPB2-5F2 and RPB2-7Cr for RPB2 (Castlebury et al. 2004). The PCR reaction mixture consisted of 1× PCR buffer, 200 μM of each of the four dNTPs, 2.5 mM MgCl 2 , 0.4 M Betaine, 1 U Taq DNA Polymerase, recombinant (Thermo Scientific, US) and 0.2 μM of each primer in a total volume of 50 μl. PCR cycle conditions were as previously described in Sung et al. (2007). PCR amplicons were visualised by ethidium bromide staining after gel electrophoresis of 4 μl of the product in 0.8% agarose gel. Quantification of the PCR products was performed using a standard DNA marker of known size and weight. PCR products were purified using Qiagen columns (QIAquick PCR Purification Kit). Purified PCR products were sequenced with the PCR amplification primers.

Sequencing alignment and phylogenetic analyses
The DNA sequences, generated in this study, were examined for ambiguous bases using BioEdit 7.2.5 (Hall 2004) and then submitted to GenBank (Table 1). The dataset of taxa in Cordycipitaceae was assembled from previously published studies (Sung et al. 2007;Kepler et al. 2017) and were downloaded from GenBank for the construction of the phylogenetic tree (Table 1). Alignments were performed using MUSCLE 3.6 software with default settings (Edgar 2004). Sequences of Cordyceps kyusyuensis and Cordyceps militaris in the Cordycipitaceae were used as the outgroup.
Maximum Likelihood (ML) analyses was performed with RAxML-HPC2 on XSEDE v8.2.10 (Stamatakis 2014) with the use of GAMMA Model parameters. The reliability of ML internal branches was assessed using a non-parametric bootstrap method with 1000 replicates. Bayesian (BI) phylogenetic inference was performed with MrBayes on XSEDE v3.2.6 (Ronquist and Huelsenbeck 2003) using the GTR+I+G model as selected by MrModeltest v2.2 (Nylander 2004). The chain length of the Bayesian analyses was 5,000,000 generations, sampled every 1000 generations and a burn-in of 10% of the total run. Maximum parsimony analysis was conducted on the combined dataset using PAUP 4.0b10 (Swofford 2002).
Distribution. Thailand, only known from Khao Yai National Park. Ecology. Parasitic on a pair of termites from a reproductive caste (Order Isoptera: Family Termitidae, Subfamily Macrotermitinae) and these specimens were buried in the soil. The fungus emerged from the segment between the prothorax and mesothorax of one of the termite pairs.
Distribution. Only reported from Khao Yai National Park. Ecology. Parasitic on a pair of termites from a reproductive caste (Order Isoptera: Family Termitidae, Subfamily Macrotermitinae) and these specimens were buried in the soil. The fungus emerged from the segment between the prothorax and mesothorax of one of the termite pairs. Additional specimens examined. THAILAND. Nakhon Ratchasima Province, Khao Yai National Park; 14°711'N, 101°421'E; on termite; 22 July 2003; R. Nasit, N.L. Hywel-Jones, J.W. Spatafora (NHJ12581, NHJ12582). Tasanathai Etymology. 'pseudorhizoidea' referring to close affinity to what was called Ophiocordyceps rhizoidea on termites by NHJ.
Notes. Both O. termiticola and O. khokpasiensis produce pseudo-immersed reddish perithecia on a stroma. In O. termiticola, the perithecia are tightly packed, while in O. khokpasiensis, they are loosely aggregated and the length of the anamorphic layer at the end of the fertile part is longer in the latter.

Discussion
Out of the 230+ species of Ophiocordyceps worldwide, less than 10 species occur on termites. The majority of these species produce cylindrical, wiry to pliant, mostly simple, seldom multiple, stromata. Species found in Africa and Mexico, O. bispora (Cordycepioideus bisporus) and O. octospora (Cordycepioideus octosporus), produce thick-walled, multiseptate ascospores, suggesting an adaptation to the harsh environmental conditions in these countries (Ochiel et al. 1997;Gilbertson 1981, 1984). All termite pathogenic species in Thailand including O. asiatica, O. brunneirubra, O. communis, O. khokpasiensis, O. mosingtoensis, O. pseudocommunis, O. pseudorhizoidea and O. termiticola produce filiform, multiseptate, whole ascospores on predominantly superficial and pseudo-immersed perithecia. The dark to pallidly coloured stroma of these species are cylindrical, wiry and pliant and the anamorph is produced at the terminal part of the stroma, after the fertile part.
Interestingly, our results clearly present Ophiocordyceps species occurring on reproductive castes of termites, especially subterranean termite species in the Family Termitidae, Subfamily Macrotermitinae. All species of subterranean termites construct their nests below ground or build mounds above ground and excavate their foraging tunnel in several ways (Eggleton 2010;Ahmad et al. 2018). Usually, the reproductive caste of termites, i.e. flying termites, includes male and female swarms during mating season at the start of the rainy season. The winged queen emerges from the colony for her nuptial flight or the mating flight, releasing pheromones to attract the males to mate. When the male finds the queen, they do a tandem run that lasts for as long as the pair finds a suitable place to start a new colony, during which they shed their wings. In termites, both male and female are the same size (Howard and Thorne 2010;Ahmad et al. 2018). Specimens of termites might have been infected by Ophiocordyceps species after their nuptial flight, when they bury themselves in the ground to establish a nesting area for starting a new colony.
Fungi represent a silent threat to the termite community. Termites have many predators, such as other amphibians (toads), birds, reptiles (lizards, gecko, snakes), small mammals, rodents and even humans. The percentage of the infection to these reproductive castes may be low in comparison to the individuals in a termite swarm, however, only few survive or evade the imminent threat of arthropods and other animals. Eventually, the number of infections caused by Ophiocordyceps becomes significant when only a few can actually survive to start a new colony.
The number of available morphological characters needed to delimit species in fungi are so limited and this may be an important reason why cryptic species are abundant in Kingdom Fungi, i.e. morphologically indistinguishable biological/phylogenetic units present within taxonomic species (Balasundaram et al. 2015) or, as Bickford et al. (2007) put it: 'two or more distinct species that are erroneously classified (and hidden) under one species name'. Many species of entomopathogenic fungi in Ophiocordycipitceae belong to species complexes or are cryptic species. Zombie ant pathogens in Ophiocordyceps have all been classified as Ophiocordyceps unilateralis sensu lato until morphological and molecular studies, including host identification, were completed (Araujo et al. 2015(Araujo et al. , 2018Luangsa-ard et al. 2010;Kobmoo et al. 2012Kobmoo et al. , 2015. The use of DNA-based molecular analyses has subsequently uncovered several new species in the genus Luangsa-ard et al. 2018). In culture, the conidiogenous cells of these termite pathogens produce phialides that are either monophialidic or have several lateral necks. The anamorphs of these species do not always form Hirsutella asexual states but more of an intermediate between Hirsutella and Hymenostilbe. This could either be a transition into a different genus or forming a diverging lineage in Ophiocordyceps -in the process of a speciation event or that the production of these anamorphs are so plastic that they cannot be used in taxonomy.
The knowledge that Ophiocordyceps species infect reproductive castes of termites can be used as basic information to study the biological control of subterranean termite pests and to better implement them. All specimens of termites collected are subterranean termites and produce relatively fast growing synnemata with numerous infectious propagules (ascopores) which can be developed further for biological control strategies.