Collections
We collected 26 samples in two locations in Okinawa, 27 in Yakushima isl., Kagoshima, and 5 in Miyazaki. Four specimens from Yonahadake and Kunigami (Okinawa) and one from Kagoshima were used for DNA extraction and sequencing for each prefecture (see Fig. 4, Table 3). Almost all fungal specimens were collected from adults of S. esakii and S. taiwanensis between April – June from 2004 to 2019.
Fig. 4. Southwestern part of Japan showing collection sites of O. salganeicola specimens used in this study. Specimens details are indicated in Table 3. The map is retrieved and edited from Geospatial Information Authority of Japan.
Taxonomy
OphiocordycepssalganeicolaAraújo, Moriguchi & Matsuura, sp. nov. – Mycobank MB836091; Fig. 5–6.
Etymology: Named after the host genus Salganea.
Specimen examined: Japan. Okinawa, Kunigami-son, Yonahadake, 26°43'45.0"N 128°12'48.2"E on Salganea taiwanensis (Blattodea, Blaberidae), 21 June 2017, M.G. Moriguchi, Holotype: XXX (to be provided by National Museum of Nature and Science - Japan).
External mycelium sparse, light to dark brown, arising from the host’s sutures. One of two stromata, 1–7 cm long, 1.3–5 mm thick, cream to light or dark brown, clavate to cylindrical in shape. Perithecia immersed, usually covering the apical part descending until about the middle of the stromata, immersed to semi-immersed, ovoid to flask-shaped, (325–) 365 (–408) ‘ 100–140 µm. Asci hyaline, 8-spored, elongated clavate, 150–200 ‘ 7–11 µm with prominent cap. Ascospores hyaline, 70–100 x 3 µm, 7-septa, not disarticulating into partspores. Hirsutella-like phialides occurring sparsely on the surface of stromata where perithecia are absent, 7–16 ‘ 6–8 µm with neck measuring 18–30 ‘ 5–7.5. Conidia ovoid, 7 ‘ 5 µm, hyaline to pale brown.
Habitat. Forests of Miyazaki Prefecture, Yakushima and Okinawa islands of Japan. Host invariably dead inside rotten logs with only the fungus sprouting out.
Distribution. Only known for Japan.
Host association. Salganea taiwanesnsis and S. esakii.
Fig. 5. Ophiocordyceps salganeicola on Salganea taiwanensis (dried specimen) from Kunigami-son, Okinawa (Holotype – JPMA124). a. Two ascomata arising from S. taiwanensis. b. Close-up of dried ascoma; c) Cross section of ascoma showing the perithecial arrangement; d. Ascus with twisted ascospores; e. Perithecial ostiole; f–g. 8-celled ascospore.
Fig. 6. Ophiocordyceps salganeicola on Salganea esakii (fresh specimen) from Yakushima, Kagoshima (JPMA106). a.Salganea esakii with a single robust ascoma; b. Close-up showing early stage ascoma arising from ventral pronotum; c–d. Close-up of ascoma; e. Cross-section of ascoma; f–g. Perithecia; h. Ascospores within Ascus; i–j. 8-celled ascospores.
Molecular phylogeny and Evolutionary origins of cockroach-associated Ophiocordyceps
We obtained 20 new sequences from 5 specimens of O. salganeicola (Fig. 4, Table 3). Our phylogenetic analysis is in accordance with previously published Ophiocordyceps topology (Quandt et al. 2014, Sanjuán et al. 2015, Araújo et al. 2018, Tasanathai et al. 2019). All O.salganeicola specimens we collected, from different parts of Japan and infecting two species of Salganea, clustered together as a single species with considerably high degree of genetic similarity with a long branch (Fig. 7). It formed a monophyletic group with another cockroach-associated species, O. blattae, which is the type species for Ophiocordyceps. This is the first time O. blattae is included in a phylogenetic study.
Our results indicate that Ophiocordyceps originated from a beetle-associated ancestral (72% ACSR), corroborating with previous studies (Araújo & Hughes 2019). For the cockroach parasites, we found at least two independent origins within Ophiocordyceps, one within Paraisaria clade, i.e. Paraisaria blattarioides (Fig. 7 node A), and the other within the hirsutelloid species, i.e. O. salganeicola sp nov and O. blattae (Fig. 7 node B). The ancestral host association for the cockroach-associated Paraisaria lineage was ambiguously recovered, while for the hirsutelloid cockroach-associated species our data show it has originated likely from a termite-associated ancestor, although not strongly supported (44% ACSR). We also found that the association with termites is older than cockroaches, evolving independently at least twice (Fig. 7 nodes C and D). The oldest, would have arisen from beetles to termites (65% ACSR, Fig. 7 node C). However, the origins of O. brunneirubra remains uncertain as part of the ancient termite-associated lineage (Fig. 7 node C) or if it jumped more recently from Hymenoptera to termites (Fig. 7 Node C).
Paraisaria clade is an ecologically heterogeneous group composed by species parasitic on Coleoptera, Orthoptera, Lepidoptera and Hemiptera (Mongkolsamrit et al. 2019). Our ACSR analysis provided weak resolution for the origins of O. amazonica/O. blattarioides/O. gracilis clade with 50.1% for Orthoptera, 25.9% for Blattodea (cockroaches) and 10.9% for Lepidoptera (Fig. 7 Node A). Our data also did not provide strong support for the ancestral of O. blattarioides with 51.1% for Blattodea (cockroaches), 21% for Orthoptera and 20.6% for Lepidoptera (Fig. 7 Node A). Nevertheless, the whole Paraisaria lineage was strongly supported as evolved from a beetle parasite (Fig. 7 Node F, ACSR = 81.1%).
On the other hand, for the novel clade composed by O. salganeicola sp. nov. and O. blattae, our results suggest (BS=87; ACSR=72.4%) that it evolved from an ancestral parasite on termites (Fig. 7 – Node E). Ophiocordyceps salganeicola/blattae was retrieved as a sister group to a clade composed mostly by termite (Blattodea, Termitidae) parasites with also species associated with hemipterans (Pseudococcidae) and mites (Acari, Eriophyidae). According to our results, all host switches in this clade occurred from termites (i.e. termites to Coleoptera, termites to Hemiptera, termites to Acari and termites to cockroaches). Unexpectedly, our analyses also suggest (ACSR=61.7%) the clade composed by parasites of Coleoptera, Lepidoptera and Hemiptera, including the economically and culturally important O. sinensis, could have been originated from an ancestor infecting termite, instead of beetle larvae as previously proposed (Araújo & Hughes 2019).
Fig. 7. Maximum likelihood tree of Ophiocordycipitaceae obtained from RAxML analyses based on a concatenated set of 5 genes (SSU, LSU, TEF, RPB1 and RPB2). Colored branches reflect Ancestral Character State Reconstruction (ACSR) analyses based on host associations (See legend at the bottom-left) and pie-charts represent the probability for the association with host orders. Host pictures by Alex Wild and Shizuma Yanagisawa.