The first Laboulbeniales (Ascomycota, Laboulbeniomycetes) from an American millipede, discovered through social media

Laboulbeniales are highly specialized arthropod-associated fungi. The majority of the almost 2200 known species live on insects, although they also occur on other arthropod hosts. Recently, the number of Laboulbeniales associated with millipedes has increased considerably. Here we describe the first species of a Laboulbeniales fungus, Troglomyces twitterisp. nov., from an American millipede. The new species was initially discovered on a photo of Cambala annulata (Say, 1821) from Ohio, USA, which had been shared on Twitter. A subsequent microscopic study of Cambala millipedes in museum collections in Denmark and France confirmed the discovery.


Introduction
Fungi of the order Laboulbeniales form a rather large group of ascomycetous fungi with around 2200 described species in 142 genera . They are obligatorily associated with living arthropods and spend their entire life cycle on their host (Blackwell et al. 2020). Traditionally they have been defined as parasites, with complex haustoria penetrating into the host (Jensen et al. 2019). However, the ab-sence of haustoria in most Laboulbeniales questions their parasitic nature (Tragust et al. 2016). The majority of Laboulbeniales hosts are insects, mostly Coleoptera (80% of described species) and Diptera (10%) (Weir and Hammond 1997), but also other arthropods have been reported as hosts: mites, millipedes and harvestmen, the latter with a single species (Santamaria et al. 2017).
Laboulbeniales have been long neglected both by mycologists and entomologists. The reason may be that entomologists are often unaware of their presence in part due to their small size and the lack of collaboration between entomologists and mycologists that have less access to the hosts on which these fungi depend. In addition, the study of Laboulbeniales was hindered by technical issues due to their size and difficulty to isolate DNA until recently (Haelewaters et al. 2015;Sundberg et al. 2017).
Research on Laboulbeniales has traditionally been taxonomic, with a recent emergence of molecular phylogenetic studies both at species-level and higher taxonomic levels (e.g., Sundberg et al. 2018;Haelewaters et al. 2019a, b). A few studies have provided insights into the interaction of Laboulbeniales and their hosts, especially in those parasitizing insects (Báthori et al. 2015Jensen et al. 2019), but very little is known about general Laboulbeniales biology (Tragust et al. 2016;Szentiványi et al. 2020).
During the last decade, the number of Laboulbeniales species associated with millipedes (Diplopoda) has grown significantly from eight prior to 2014 to a current count of 30 species (Santamaria et al. 2014(Santamaria et al. , 2016Enghoff and Santamaria 2015;Reboleira et al. 2018). These species have been collected in Europe, Macaronesia, the Middle East, Africa, SE Asia, Indonesia, Australia and New Zealand, but until now, no Laboulbeniales from American millipedes have been reported.
Millipede hosts of Laboulbeniales usually combine the following traits: i) successive generations of adults overlap in time; ii) their populations are large and stable, and iii) they inhabit moist environments (Santamaria et al. 2014). The transmission of the ascospores in millipede hosts most often occurs directly, by contacts of the hosts during copulation, hence this is why most thalli are found growing around the gonopode and gonopores .
After the observation of a shared photo of a North American Cambala annulata (Say, 1821) millipede on Twitter (Fig. 1), we identified the presence of Laboulbeniales on this specimen. Subsequently, we decided to screen Cambala millipedes in museum collections resulting in the discovery of an undescribed species in the laboulbenialean genus Troglomyces, which was found on several specimens. This new species is formally described here.

Methods
Specimens of Cambala spp. from the collections of the Natural History Museum of Denmark in Copenhagen (NHMD) and in the National Museum of Natural History in Paris (MNHN) were investigated for the presence of Laboulbeniales under a binocular stereomicroscope Leica M165C. The thalli of the fungus on the infected Cambala specimens were removed using an insect pin and mounted with lactophenol on a mi- croscope slide following the methodology of Santamaría et al. (2018). Specimens were studied using a Leica DMR microscope equipped with differential interference contrast (DIC) optics and photographed with a Jenoptik ProgRes 10 Plus digital camera. Diagnosis. Septa II-III and II-VI approximately at the same level. Dorsal and ventral margin of cell II of equal to subequal height, in contrast to all other Troglomyces, such that cell II is not adnate to either cell VI or the perithecium. Primary appendage branched. Perithecial apex bearing four slightly protruding lips, one of them being longer.

Order Laboulbeniales Lindau
Types Description. Thallus hyaline, except for the blackened foot. Basal cell of the receptacle (I) about twice as long as broad, enlarged distally. Suprabasal cell of the receptacle (II) pentagonal, isodiametric, up to 1.5 times as long as broad, margins parallel to somewhat broadened distally. Septa II-III and II-VI variably oblique, located approximately at the same level. Septum II-VI slightly longer than II-III. Cell III very narrow, up to 8 times longer than broad; adnate to the perithecium along half or three quarters of the latter's length. Primary appendage branched above the first or, more frequently, the second cell, into several simple or once ramified branches; surpassing the perithecial apex. Basal and suprabasal cells of appendage similar in size and shape; about two times as long as broad. Primary septum (Fig. 2C, "a") slightly constricted and strongly oblique. Only one antheridium has been seen in an immature thallus, as a simple phialid on a branch of the primary appendage (Fig. 2G, "an"). Perithecial stalk cell (VI) very inconspicuous, strongly flattened (Fig. 2B, 2F, "VI"). Perithecium ovoidal, broadest at the middle or third basal part, gradually tapering upwards. Apex bearing four not quite protruding lips, one of them slightly longer (Fig. 2E, arrow). A small tooth-like outgrowth on the outer side near the apex (Fig. 2F, arrow).
Etymology. Named after the social media platform Twitter, where it was observed for the first time.

Discussion
The most distinctive characteristic of Troglomyces twitteri vis-à-vis its congeners is found in the shape and location of cell II, which is bigger than in other species and does not extend laterally to cell VI or the perithecium. The strongly flattened and inconspicuous cell VI is shared with T. tetralabiatus Santam., Enghoff & Reboleira, probably the mostly similar species. Troglomyces twitteri differs from the other species as follows: Troglomyces dioicus Santam., Enghoff & Reboleira is dioecious, has a conspicuous spiny process and an unbranched appendage; T. tetralabiatus shows four very conspicuous and elongated perithecial lips; T. bilabiatus Santam., Enghoff & Reboleira has two elongated lips, an unbranched appendage, and the antheridia are placed directly on the lower cells of the appendage; T. pusillus Santam & Enghoff has an unbranched appendage and the second cell of this appendage functions as an intercalary antheridium; T. triandrus Santam & Enghoff has three superposed antheridia formed by the third, fourth and fifth cells of the appendage; T. botryandrus Santam., Enghoff & Reboleira has two groups of antheridia in bunches near the base of appendage; T. manfrediae S. Colla has an unbranched appendage and an antheridium on the corner of the appendage basal cell; T. rossii Santam., Enghoff & Reboleira has a bifurcate appendage with a characteristic trapezoidal, small cell in the bifurcation.
Arthropods of the class Diplopoda, commonly known as millipedes, play an important role in the decomposition of organic matter above and below the ground (Hopkin and Read 1992; David 2015; . Millipedes have poor dispersal abilities and consequently show high endemism patterns, converting them into excellent models for the study of Laboulbeniales biogeographical patterns (Santamaria et al. 2014(Santamaria et al. , 2016Reboleira et al. 2018). The gonopores of millipedes are situated on the third body ring from the front, and in the vast majority of millipedes, mating takes place by the introduction of modified appendages (gonopods) on the seventh body ring into the female gonopore; the distribution of laboulbenialean thalli on millipede hosts very often reflects this behavior (Enghoff and Santamaria 2015).
Species of Troglomyces have so far been found only on millipedes belonging to the orders Julida and Chordeumatida. The here reported find of T. twitteri on Cambala is not only a first record of Laboulbeniales from an American millipede, it also represents the first record of Troglomyces from the order Spirostreptida. Species of Spirostreptida are, on the other hand, hosts for many species of another Laboulbeniales genus: Rickia Cavara (Santamaria et al. 2016). Spirostreptidan hosts include one species of Cambalidae, Chiraziulus kaiseri (Mauriès, 1983), which is host to Rickia appendicifera Santam., Enghoff & Reboleira. The genus Cambala Gray, 1832 is endemic to North America. Cambala annulata (Say, 1821) and C. hubrichti Hoffman, 1958 are dominant members of the litter fauna in the southern Appalachian Mountains (Shelley 1979). Troglomyces twitteri is probably overlooked but widespread in this area, i.e. the potential geographic distribution of the fungus is likely to match the distribution of its hosts. Like most other millipedes, Cambala species secrete strongly smelling defensive chemicals from glands along their body. Eisner et al. (1965) identified 2-methyl-1,4-benzoquinone and 2-methyl-3-methoxy-1.4 benzoquinone in the secretion of C. hubrichiti.
The abundance of thalli on the host was reduced compared to some other species of millipedes that are known to have high load of Laboulbeniales. For example, thalli of Rickia gigas Santam., Enghoff & Reboleira on Archispirostreptus spp. were reported as "hairs" in internet fora by keepers of millipedes as pets (Santamaría et al. 2016). The distribution of T. twitteri thalli on the host body follows a transmission pattern that is associated with mating behavior, the fungi being mostly found around the gonopods/ gonopores of the millipedes (Santamaria et al. 2014(Santamaria et al. , 2016. However, the thalli observed on Twitter were on the dorsal side of the first two body rings. This suggests that, under higher thallus densities, thalli can spread from the genital areas of the millipedes to the back, i.e. higher than in the specimens studied. The use of social media is now a considerable part of how humans interact and perceive the news of a changing world. Photographs in online databases (e.g., Flickr and iNaturalist) and social media (e.g., Facebook and Instagram) have previously provided new species of insects and plants for science, and new hosts for parasites -after careful examination by taxonomists (Winterton et al. 2012;Gonella et al. 2015;Jaume-Schinkel et al. 2020). There is an increasing interplay between research and social media platforms, and many scientists use Twitter to promote and share research, a phenomenon also promoted by scientific publisher companies (Bik and Goldstein 2013). To our knowledge, this is the first time that a new species for science has been discovered on Twitter, as a result of a casual observation of a photo shared by a colleague. This, again, emphasizes the importance of such platforms for sharing research and making new discoveries. The circumstances of this species' discovery should encourage data sharing among amateur naturalists and professional scientists.