Ophiocordyceps salganeicola, a parasite of social cockroaches in Japan and insights into the evolution of other closely-related Blattodea-associated lineages

The entomopathogenic genus Ophiocordyceps includes a highly diverse group of fungal species, predominantly parasitizing insects in the orders Coleoptera, Hemiptera, Hymenoptera and Lepidoptera. However, other insect orders are also parasitized by these fungi, for example the Blattodea (termites and cockroaches). Despite their ubiquity in nearly all environments insects occur, blattodeans are rarely found infected by filamentous fungi and thus, their ecology and evolutionary history remain obscure. In this study, we propose a new species of Ophiocordyceps infecting the social cockroaches Salganea esakii and S. taiwanensis, based on 16 years of collections and field observations in Japan, especially in the Ryukyu Archipelago. We found a high degree of genetic similarity between specimens from different islands, infecting these two Salganea species and that this relationship is ancient, likely not originating from a recent host jump. Furthermore, we found that Ophiocordyceps lineages infecting cockroaches evolved around the same time, at least twice, one from beetles and the other from termites. We have also investigated the evolutionary relationships between Ophiocordyceps and termites and present the phylogenetic placement of O. cf. blattae. Our analyses also show that O. sinensis could have originated from an ancestor infecting termite, instead of beetle larvae as previously proposed.


INTRODUCTION
The genus Ophiocordyceps (Hypocreales, Ophiocordycipitaceae) comprises species typically pathogenic to insect hosts. Recently, however, there are reports of beneficial, endosymbiotic species of sap-sucking hemipterans hosts (Quandt et al. 2014;Gomez-Polo et al. 2017;Matsuura et al. 2018). The genus was erected by Petch (1931) to accommodate species of Cordyceps exhibiting clavate asci containing spores that do not disarticulate into partspores, contrasting with "the majority of the species of Cordyceps which have been described" at that time, exhibiting cylindrical asci and spores that readily disarticulate into numerous short partspores upon maturity. The diversity of Ophiocordyceps has been increasingly unraveled in the last decade, especially with discoveries of species associated with Hymenoptera, Lepidoptera and Hemiptera (Araújo et al. 2016(Araújo et al. , 2018Luangsa-ard et al. 2018). However, our knowledge about species associated with blattodean insects (cockroaches and termites) is still restricted, especially regarding cockroach parasites. Currently, we know of only 11 species infecting termites, i.e. O. bispora (Stifler 1941); O. octospora (Blackwell and Gilbertson 1981); O. communis (Sung et al. 2007 (Tasanathai et al. 2019). Furthermore, there are only three species described infecting cockroaches, i.e. O. blattae from Sri Lanka (formerly Ceylon) - (Petch 1931); O. blattarioides from Colombia - (Sanjuan et al. 2015) and O. salganeicola sp. nov. from Japanthis study).
The cockroaches (Blaberidae, Blattodea) are ubiquitous organisms occupying almost all habitats where insects occur (Schall et al. 1984). They play a key ecological role as decomposers, with many examples of species living within and feeding on rotting wood, including a lineage of social species that evolved into the termites (Bell et al. 2007;Maekawa et al. 2008;Bourguignon et al. 2018;Evangelista et al. 2019). Parasitism by microsporidians and protozoans on these insects is relatively common and known for more than a century (Crawley 1905;Woolever 1966;Purrini et al. 1988). However, the cases of infection by filamentous fungi on cockroaches have been rarely reported.
Although examples of Ophiocordyceps parasitizing Blattodea are scarce, the type of the genus is O. blattae infecting a cockroach identified as Blatta germanica (Fig. 1, adapted from Petch 1924). Only two specimens were originally collected in Sri Lanka (See Fig. 1a-d) with recent few records from Thailand (Luangsa-ard et al. 2018). Another example of an Ophiocordyceps infecting a cockroach is O. blattarioides (syn. Paraisaria blattarioides; Mongkolsamrit et al. 2019). This species was described from Colombia with records in Belize and tropical lowlands in the eastern (Amazonian) Ecuador (Sanjuan et al. 2015). Both O. blattae and O. blattarioides exhibit striking morphological and ecological differences. For example, O. blattae forms a cylindric ascoma producing elongated-fusoid ascospores, which do not disarticulate into part-spores, measuring 50-80 × 3-4 μm, while O. blattarioides has a stalk bearing a globoid fertile part at the tip, producing spores that disarticulate into part-spores of 6-12 × 1.5 μm. The host death location is also distinct with O. blattae occuring on the underside of leaves while O. blattarioides is found buried in the leaf litter. Besides these studies, there is no detailed information on the evolution and ecology of cockroach-associated entomoparasitic fungi.
In this study, we propose a new species of Ophiocordyceps that parasitizes two social wood-feeding cockroach species distributed in the southwestern part and Nansei Islands of Japan, both living inside decaying logs. We provide morphological, molecular and ecological data to support the new species proposal with insights into the evolutionary origins of the closely related parasitic fungi of cockroaches and  (Petch 1924 termites. We also present, for the first time, the phylogenetic position of O. cf. blattae.

Sampling
Surveys were undertaken in the Japanese warm temperate and subtropical evergreen forests mainly consisting of trees belonging to Fagaceae, Lauraceae and Theaceae in Kunigami-son, Okinawa, Yakushima, Kagoshima and Nobeoka, Miyazaki. The parasitized cockroach samples of two host species, namely Salganea esakii and S. taiwanensis, were mainly collected in the small humid valley or riparian forests where Castanopsis sieboldii, Distylium racemosum and Schefflera heptaphylla grow, but also in the secondary forest harboring Alnus japonica after the deforestation of Castanopsis in Okinawa.
The specimens used in this study were always found hidden inside soft rotten logs or large fallen branches of those trees, with only the fungus emerging (Fig. 2). The infected cockroaches, and the substrata they were attached to, were collected in plastic containers and transported to the laboratory. Some specimens were investigated immediately after the collection, while others were dried and preserved for many years before being analyzed. The specimens were photographed individually, using a Canon 7D camera, equipped with an EF-100 mm macro lens or a MP-E 65 mm (5X) lens with a MT-24EX Canon macro lite flash attached.

Collections
We collected 26 samples in two locations in Okinawa, 27 in Yakushima island, Kagoshima, and five in Miyazaki. Four specimens from Yonahadake and Kunigami (Okinawa) and two from Kagoshima were used for DNA extraction and sequencing for each prefecture (see Fig. 3, Table 1). Almost all fungal specimens were collected from adults of S. esakii and S. taiwanensis between April -June from 2004 to 2019.

Morphological studies
For macro-morphological characterization, specimens were examined using a stereoscopic microscope (Leica S8 APO) and sorted for further micro-morphological investigation. The characters investigated were ascomatal size, color, position, presence/absence and characterization of asexual morphs and perithecial insertion (e.g. immersed, semi-immersed, erumpent, superficial). For micro-morphological characterization, either free-hand or cryo-sectioning of the ascoma was performed using a Leica CM1850 Cryostat. Samples were mounted on a slide with plain lactic acid or lacto-fuchsin (0.1 g of acid fuchsin in 100 mL of lactic acid) for light microscopy examination using a Nikon Eclipse Ni-U. A minimum of 50 ascospores were measured for morphological comparison. The illustrations of fungal specimens were drawn based on the observation of photographs using drawing pens 0.13 mm and 0.2 mm (Rotring, Hamburg, Germany), painted by watercolors (HOLBEIN Art Materials Inc., Osaka, Japan) and scanned for imaging (Fig. 4). We also present the morphological comparison between Ophiocordyceps species infecting cockroaches and termites (Table 2).

Phylogenetic analyses
The raw sequence reads (.ab1 files) were edited manually using Geneious 11.1.5 (https://www. geneious.com). Individual gene alignments were generated by MAFFT (Katoh and Standley 2013). The alignment of every gene was improved manually, annotated and concatenated into a single combined dataset using Geneious 11.1.5. Ambiguously aligned   Table 3.

Ancestral character state reconstruction (ACSR)
To understand the evolutionary pathways of host association of Ophiocordyceps and blattodean insects, we conducted ancestral character reconstruction in Mesquite (Maddison and Maddison 2018) of the whole genus, using the best-scoring ML tree produced in RAxML (Stamatakis 2014 Diagnosis: Ophiocordyceps salganeicola can be easily differentiated from other closely-related species by its unique host association, ascomatal morphology and its strict distribution across the Southern Islands of Japan. Other closely related species are associated with termites, mites and hemipterans and exhibit completely different macro morphology, being easily distinguished still in the field. Type: 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 (TNS-F-60532holotype).
Host: Salganea taiwanesnsis and S. esakii. Habitat: Forests of Miyazaki Prefecture, Yakushima and Okinawa islands of Japan. On remains of the hosts inside rotten logs, with only the fungus sporophores emerging.
Distribution: Only currently known from Japan.

Molecular phylogeny and evolutionary origins of cockroach-associated Ophiocordyceps
We obtained 20 new sequences from five specimens of O. salganeicola (Fig. 3, Table 3). Our phylogenetic analysis is in accordance with previously published Ophiocordyceps topologies (Quandt et al. 2014;Sanjuan 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 a high degree of genetic similarity with a long branch (Fig. 5). 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. cf. blattae is included in a phylogenetic study.
Our results indicate that Ophiocordyceps originated from a beetle-associated ancestor (72% ACSR), corroborating previous studies (Araújo and Hughes 2019). For the cockroach parasites, we found at least two independent origins within Ophiocordyceps, one within the Paraisaria clade, i.e. Paraisaria blattarioides (Fig. 5 node A), and the other within the hirsutelloid species, i.e. O. salganeicola and O. blattae (Fig. 5 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 this is not strongly supported (44% ACSR). We also found that the association with termites is older than cockroaches, evolving independently at least twice ( Fig. 5 nodes C and D). The oldest, would have arisen from beetles to termites (65% ACSR, Fig. 5 node C). However, the origins of O. brunneirubra remains uncertain as part of the ancient termite-associated lineage (Fig. 5 node C) or if it jumped more recently from Hymenoptera to termites (Fig. 5 Node D).
The Paraisaria clade is an ecologically heterogeneous group composed of species parasitic on Coleoptera,  Orthoptera, Lepidoptera, and Hemiptera (Mongkolsamrit et al. 2019). Our ACSR analysis provided weak resolution for the origins of P. amazonica/P. blattarioides/P. gracilis clade with 50.1% for Orthoptera, 25.9% for Blattodea (cockroaches) and 10.9% for Lepidoptera (Fig. 8 Node A). Our data also did not provide strong support for the ancestor of P. blattarioides with 51.1% for Blattodea (cockroaches), 21% for Orthoptera and 20.6% for Lepidoptera ( Fig. 8 Node A). Nevertheless, the whole Paraisaria lineage was strongly supported as having evolved from a beetle parasite ( Fig. 8 Node F, ACSR = 81.1%). Conversely, for the novel clade composed of O. salganeicola and O. blattae, our results suggest (BS=87; ACSR=72.4%) that it evolved from an ancestral parasite on termites (Fig. 8 node E). Ophiocordyceps salganeicola/blattae was retrieved as a sister group to a clade composed mostly by termite (Blattodea, Termitidae) parasites with 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 originated from an ancestor infecting termites, instead of beetle larvae as previously proposed (Araújo and Hughes 2019).

Ecology and natural history of Salganea-Ophiocordyceps relationships
The insect cuticle represents a formidable barrier to infection by bacterial and fungal pathogens with relatively few having managed to cross it. Once inside the insect, innate immunity provides further challenges to an invading pathogen (Evans 1988). However, once these have been overcome the insect body provides a stable environment for their development. This is particularly true for colonial cockroaches that spend most of their lives protected inside nests, for example some wood-feeding species within the families Cryptocercidae and Blaberidae, specifically the subfamily Panesthiinae (Panesthiini, Ancaudeliini, Caepariini, and Salganeini). Among those groups, one of the most well-known social cockroaches is the genus Salganea, comprised of about 50 species (Beccaloni 2007;Bell et al. 2007;Wang et al. 2014). All the known species within the genus live within and feed on decaying wood, building chambers and galleries inside hardwood or coniferous logs that may take decades to degrade (Maekawa et al. 2008), providing long-term stable homeostatic conditions. Such a protected environment certainly benefits indirectly the fungal parasites that are already inside the host body. An exposed cadaver on the forest floor would be much more susceptible to being scavenged by animals or consumed by other microorganisms.
Salganea species form social groups, composed mostly of biparental families, consisting of a male-female and their offspring (Maekawa et al. 2008). Sociality endows insects with advantages such as increased efficiency of brood care, foraging and anti-predator defenses. However, infectious diseases can potentially spread more easily within a colony because of their high densities, frequent social contact and also because group members are often close relatives and thus susceptible to the same parasitic infections (Cremer et al. 2007). Therefore, it is surprising that only three species of Ophiocordyceps, a common and widespread genus of entomopathogenic fungi, have been recorded infecting the equally diverse and globally distributed cockroaches (Bourguignon et al. 2018). Ophiocordyceps species infecting social insects, notably ants, are one of the most broadly distributed and ubiquitous entomopathogenic fungi in tropical forests worldwide (Araújo et al. 2015(Araújo et al. , 2018. They often form epizootic events, in which hundreds of infected ants can be found in a small patch of forest (Evans and Samson 1982;Pontoppidan et al. 2009). On the other hand, however, Ophiocordyceps on social cockroaches are rare in Japan and only one or two infected individuals are collected in the same log, despite the ubiquity and abundance of hosts in one area. Based on our extensive field surveys we found that ascomata of O. salganeicola in Okinawa start to emerge in decaying logs in early April. However, they seem to require at least a few months to become mature and develop the sexual morph in the field. This development occurs in parallel with the mating season of the host cockroaches from April to July when newly emerged adults leave their logs and parents, fly, mate and burrow into a new nest (Osaki Haruka, personal communication). Presumably, these young adults might become infected by the ascospores/conidia of O. salganeicola during colonization of a new log. The host is then later killed and consumed by the fungal parasite, eventually producing new fruiting bodies in the next mating season. On the other hand, there has been no record of an infected nymph in 26 fungal specimens observed in Okinawa, but only a single infected nymph (see Fig. 2e) out of more 27 specimens in Yakushima between 2015 and 2019, suggesting the outbreak of this fungus within an established colony is rather rare and the primary targets are likely newly emerged adults. However, this proposed life-cycle of O. salganeicola is only hypothetical and requires periodical field observations in the same ecological habitat along with host insect behaviors (Maekawa et al. 2008), in order to determine how and when the fungus infects and kills the host.
Does O. salganeicola manipulate host behavior before death?
The behavior manipulation caused by Ophiocordyceps fungi on their hosts is a striking phenomenon, especially in species associated with ants, the so-called "zombie-ant fungi" (Evans et al. 2011;Araújo et al. 2018). It has been posited that species within the Ophiocordyceps unilateralis core clade infecting Camponotini ants evolved such an ability as a response to the strong social immunity displayed by ant societies that prevents fungal transmission and development inside the colony (Araújo and Hughes 2019). Conversely, as far as we know, there is no evidence of social cockroaches recognizing the infected members of their colony, except for the parental and sibling's grooming behavior that might fend off superficial parasites. Thus, fungal infection, development and transmission could potentially occur in the same log where other members of the colony still inhabit, in which no drastic behavior manipulation is needed in order to remove the host from its nest and thus complete the parasite's life-cycle. However, there is a possibility of a subtle manipulation.
Salganea cockroaches burrow and nest deep inside the trunk, only becoming exposed to the external environment in the mating season, whereas the Ophiocordycepsinfected ones are found only a few centimeters below the wood surface (Fig. 2). Thus, we posit that the fungus might potentially be able to manipulate host's behavior by leading to a migration towards a more superficial layer inside the log. This host migration could be stimulated by the need for a more oxygen-and water-rich stratum and/or attraction to the light coming from an opening, through which the fungal ascoma emerge and disperse its spores ( Fig. 2a-b). Further studies are needed to test this hypothesis.

Host association, speciation, and distribution
While some distinct, mostly macro-, morphological features can be observed in O. salganeicola infecting both host species that diverged from a single ancestor around 4-5 mya (Maekawa et al. 1999;Maekawa and Matsumoto 2003), nucleotide sequences of fungal specimens are highly conserved among diverse strains in wide range of geographic regions and islands. The number of polymorphic sites within all aligned five gene sequences from multiple samples (Table 3) was only two out of 4202 and both were synonymous. Thus, geographic and reproductive isolation of the fungal strains may have not yet resulted in the allopatric speciation of O. salganeicola. The long branch length and high host specificity to the genus Salganea indicate a long co-evolutionary relationship with the host populations and hence unlikelihood of recent host jumping (Fig. 5 node B). However, we still do not know whether the single parasite strain can only persist in one geographic region and/or island infecting the same host populations over generations, or jump across multiple closely related host populations and species horizontally even after such a long geographic isolation of the Ryukyu Islands. In some of these islands such as Amamioshima and Tokunoshima in Kagoshima and Ishigaki-jima and Iriomote-jima in Okinawa, there has been no collection record of O. salganeicola, suggesting their current absence. These questions on the evolution of host associations of O. salganeicola in the Japanese archipelago of Ryukyus deserve particular attention for studying host-parasite co-evolution in the context of island biology, which possibly can be tested by artificial infection experiments using the field-collected fungal ascoma and laboratory-reared Salganea colonies from different islands. Furthermore, S. taiwaensis and the other Salganea cockroach species are distributed not only in Japan, but also in wide greographic regions in South, Southeast and East Asia (Wang et al. 2014). Additional screening of Salganea and related host species for entomopathogens in Asia-Pacific regions might unravel the phylogeography and evolutionary origins of cockroach-Ophiocordyceps associations.
Ophiocordyceps cf. blattae Ophiocordyceps blattae was described by Petch (1924) and was collected at Hakgala (Sri Lanka, formerly Ceylon) only twice (Fig. 1). A rare species, it was originally collected on cockroaches attached by fungal structures to the underside of leaves, exhibiting a cylindric, grey to lavender ascoma. The specimen we used in this study exhibited many similarities with the type specimen. For example, it also infects a very similar species of cockroach, kills its hosts on the underside of leaves, attached by fungal structures, and the ascomata emerge laterally from the host's thorax. Furthermore, the ascomata are very similar in macro-morphological features. Unfortunately, we could not assess the micro-morphological features in this study and thus, to avoid any ambiguities, we are calling our material O. cf. blattae. We made efforts to sequence the original material from 1924, but all our attempts failed. As this is the type species of the genus Ophiocordyceps, further efforts are needed to fix the phylogenetic placement of the type species and thus, rediscuss the systematics of the whole genus. We have considered to propose the specimen used in this study as the epitype of O. blattae, however, since it was collected in Thailand, not Sri Lanka as it was originally found by Petch, future efforts might address this issue. However, herein we provide a good perspective for future efforts and revealed another clade likely bearing cryptic species within Ophiocordyceps on cockroaches.
CONCLUSION Japan may harbour one of the richest reservoirs of entomopathogenic fungi in the world. In no other country is there an amateur society devoted to collecting and illustrating them. We still know very little about these organisms and their ecological roles in the environment and dynamic associations with host insects, including blattodean-associated ones such as O. salganeicola. Therefore, it is crucial to understand their true diversity with the invaluable help by such amateur and professional mycologists. As we move forward and describe more and more species through microscopic and molecular tools, new insights into the evolutionary origins of these organisms are being revealed, as well as their ecological associations with the insect hosts. Currently, there is still a substantial gap in our knowledge about insect ecology and fungal biology within the context of host-parasite interactions and their life-cycles. In this study, our goal was to describe an ecologically rare fungal species parasitizing unique social insects, and to provide some insights into their evolution by considering natural histories of both the parasite and its host. Thereby, our study contributes to the understanding of one of the most prolific and diverse groups of entomopathogenic fungi, the genus Ophiocordyceps, and incidentally shed new light on the origins of the economically important O. sinensis.