Xylaria insolita and X. subescharoidea: two newly described species collected from a termite nesting site in Hua-lien, Taiwan

Background A number of Xylaria species are exclusively associated with nests of macrotermitine termites. A nesting site of Odontotermes formosanus in eastern Taiwan, which is the only macrotermitine termite known on the island, had been inundated during the raining season of 2010, and hundreds of Xylaria stromata emerged from it thereafter. A thorough examination of these stromata showed that they represent a mixture of different species. Results Five Xylaria species were identified from the stromata collected from the nesting site, including two undescribed species, which are newly described as X. insolita and X. subescharoidea herein, and three known species X. brunneovinosa, X. escharoidea, and X. furcata. Conclusion Totally, there are 28 Xylaria species growing on termite nests or ground in the world. Although O. formosanus is the only macrotermitine species known in Taiwan, the Xylaria diversity associated with its nests is fairly high; the species number has reached 12 with X. furcata, X. insolita, and X. subescharoidea added to the Taiwan mycobiota.


Background
Xylaria species are characterized by upright massive stromata and high ascal apical rings, and geniculosporiumlike anamorphs. More than 20 Xylaria species are specifically associated with fungus gardens built within nests of macrotermitine termites (Ju and Hsieh 2007;Rogers et al. 2005). There is only one macrotermitine species Odontotermes formosanus Shiraki found in Taiwan , and nine Xylaria species have been collected from its nests (Ju and Hsieh 2007).
In 2010 we conducted a survey on Xylaria species at a backyard of a residence in Hua-lien located in eastern Taiwan, where a nesting site of O. formosanus had previously been inundated following a heavy rain, and numerous Xylaria stromata kept emerging from the nesting site after termite activities had ceased. Among these stromata, several Xylaria species were identified, and two of these are undescribed species.
In this study, we describe the two undescribed species as new, i.e., X. insolita and X. subescharoidea. Their ITS, β-tubulin, RPB2, and α-actin were sequenced and analyzed in the context of the dataset mainly from Hsieh et al. (2010) to infer its phylogenetic relationships within Xylaria. Xylaria diversity emerging from a macrotermitine termite nesting site has poorly been documented, and we thus take the opportunity to comment on the Xylaria species found at the Hua-lien termite nesting site.

Collecting, fungal observation, isolation, and culturing
Species of Xylaria emerging from a nesting site of black-winged subterranean termite were surveyed from the backyard of a residence in Fu-hsin Village, Ji-an Open Access  Taiwan during June and Sep tember, 2010. Stromata were photographed on site, and collected stromata were air-dried after culture isolation.
Material was mounted in water and Melzer's iodine reagent for examination of microscopic features by differential interference contrast microscopy and bright field microscopy.
Cultures were obtained by placing tissue from freshly collected stromata on SME medium (Kenerley and Rogers 1976). Resulting colonies were transferred to 9-cm plastic Petri dishes containing 2% Difco oatmeal agar (OA), from which the culture descriptions were made, and were incubated at 20 °C under 12 h fluorescent light.
The combined sequences of rpb2, β-tub and α-act of X. insolita and X. subescharoidea were added to the RPB2-TUB-ACT dataset in Hsieh et al. (2010) (Fournier et al. 2018). Also, Xylaria cubensis (Mont.) Fr. and X. laevis C. G. Lloyd in Hsieh et al. (2010) are replaced by X. flabelliformis (Schwein.) Fr. and X. cubensis, respectively (Ju et al. 2016). The resulting dataset contained 135 isolates of 117 taxa (Additional file 1: Table S1), where major genera of the subfamily Xylarioideae as well as representatives of various groups and species aggregates of Xylaria were included. Three out-group taxa were

Survey of the collecting site
More than 100 stromata were found at the collecting site ( Fig. 1a, b). Totally, five Xylaria species were identified, including X. brunneovinosa Y.-M. Ju and H.-M. Hsieh (Fig. 1c), X. escharoidea (Berk.) Sacc. (Fig. 1d), and anamorphic X. furcata Fr. (Fig 1e), and two undescribed species, which are described herein as X. insolita and X. subescharoidea. Stromata of these five Xylaria species were intermixed with one another, not being restricted to particular areas at the nesting site. Most of the stromata were of X. brunneovinosa and X. escharoidea, and only 14 stromata of X. insolita, nine of X. subescharoidea, and two of X. furcata were found.

Phylogenic analyses
With X. insolita and X. subescharoidea included in the phylogenetic analyses, the overall tree topologies resulting from BA (Additional file 2) and MP analyses were highly similar to those in Hsieh et al. (2010), having the two species grouped within the TE clade ( Fig. 2), to which all of the studied Xylaria species of the subgenus Pseudoxylaria belong. We present only the portion of the tree concerning subgenus Pseudoxylaria in Fig Etymology. Denoting the highly variable palmate stromata.
Cultures and anamorph. Colonies reaching the edge of 9-cm Petri dish in 5 week, yellowish, slightly cottony, zonate, with diffuse margins. Reverse fawn-colored. Stromata arising from concentric zones and strongly inclined outwards, cylindrical, tapering at top, unbranched or branched, 0.3-1.3 cm long × 0.5-1.2 mm diam, yellow grading to brown towards the base, white on the surface of upper part but becoming pale olivaceous gray due to production of conidia. Conidiophores in upright, densely arranged palisades, dichotomously branched several times from base, smooth, hyaline, grading to light brown downwards. Conidiogenous cells terminal, cylindrical, 6.5-12 × 2-3 µm, smooth, bearing terminal, slightly denticulate conidial secession scars. Conidia produced holoblastically in sympodial sequence, hyaline, smooth, obovoid to ellipsoid, (3.0-)3.5-4.7 (-6.4) × (2.5-)2.7-3.1 (-3.8) µm (4.1 ± 0.6 × 2.9 ± 0.2 µm, N = 40), with a flattened base indicating former point of attachment to conidiogenous cell. Notes. Xylaria insolita is peculiar among Xylaria species in having highly variable palmate stromata and laterally compressed, slightly beaked ascospores with the germ slit on the dorsal side. Unlike most of the Xylaria species where the teleomorph and anamorph are produced in different times or on different stromata, X. insolita can have the anamorph and teleomorph coexist on the same stromata at the same time, with mature perithecia produced at the lower part of stromata and conidiogenesis on the finger-like terminals. Perithecial contours are conspicuous to half-exposed, evenly distributed or unevenly clumped together. The outer stromatal layer is ruptured by developing perithecia into flaky remnants, which remain attached at maturity but are gradually worn off afterwards.
Colonies on OA are yellowish, with stromata produced in concentric zones. The stromata produced in cultures never reach maturity, having a yellow surface and producing pale olivaceous gray conidial masses and resembling much those immature stromata produced in nature.
Phylogenetic analyses clustered X. insolita together with X. ochraceostroma and X. sp. 5, a fungus known only in anamorph. Unlike X. insolita where the conidiophores are in densely arranged palisades, X. ochraceostroma has repeatedly dichotomously branched conidiophores that arise singly on the stromatal surface and render the surface a granular appearance (Ju and Hsieh 2007). The general appearance of the conidiophores of X. ochraceostroma resembles that of terverticillate penicilli characteristic of Penicillium Link subgenus Penicillium. Xylaria ochraceostroma also differs from X. insolita by lacking a black layer beneath the ochraceous stromatal surface and having the ascospore germ slit on the ventral side. Conidiophores of X. sp. 5 also arise singly and have a swollen top, thus resembling the vesiculate conidiophores of Aspergillus P. Micheli ex Haller (unpublished data of Y-MJ). Etymology. Referring to its stromata resembling those of X. escharoidea in gross morphology.
Cultures and anamorph. Colonies reaching the edge of 9-cm Petri dish in 3 week, whitish, immediately becoming blackish, mostly submerged, faintly zonate, with diffuse margins. Reverse uncolored. Stromata arising from concentric zones, cylindrical, tapering upwards, flexuous, unbranched, up to 4 cm long × 1.2-2.2 mm Fig. 3 Xylaria insolita (from the holotype). a-f Stromata in natural habit; stromata in e, f are immature, bearing conidia only. g Dried stromata. h Stromatal surface with outer stromatal layer ruptured by developing perithecia into flaky remnants. I. Outer stromatal layer worn off to reveal the rugulose surface. Bars in a-f = 1 cm; h, i = 0.5 mm diam, black at base, white on the surface of upper part but becoming pale mouse gray due to production of conidia. Conidiophores composed of upright conidiogenous cells only. Conidiogenous cells arising directly from stromatal surface, cylindrical, 8.5-17 × 3.5-5 µm, smooth, bearing one to several terminal denticulate conidial secession scars. Conidia produced holoblastically in sympodial sequence, hyaline, smooth, variable in shape, subglobose, obovoid to ellipsoid, equilateral or slightly to significantly oblique, (4.3-)5.0-7.2 (-8.9) × (3.3-)3.7-4.5 (-4.7) µm (6.1 ± 1.1 × 4.1 ± 0.4 µm, N = 40), with a minute flattened base indicating former point of attachment to conidiogenous cell. Notes. Xylaria subescharoidea is characterized by having pale brown to ochraceous, long cylindrical stromata, lacking an outer stromatal layer, lacking a continuous black layer immediately beneath the surface, having nearly equilateral ascospores that possess a pore-like germination site. Black ellipsoidal granules between ostioles form a layer below the surface and give rise to the tuberculate appearance of the stromatal surface. It remains Fig. 4 Xylaria insolita (from the holotype). a Ascal apical rings and ascospores. b Ascospores; the arrows point towards two ascospores showing a germ slit. c Vertical section of a perithecium. d, e Colony on 9-cm Petri plate containing OA at 2.5 week and 6 week, respectively. f Stromata produced in culture. g Conidiophores. h Conidia. Bars in a, b, g, h = 5 μm; c = 0.125 mm; f = 1 mm unknown as to whether these granules possess certain functions or represent aborted perithecia. Xylaria subescharoidea is closely related to X. escharoidea (Fig. 2), with which it shares a pore-like ascospore germination site and long cylindrical stromata that possess a dark core and lack an outer layer. Xylaria escharoidea differs from X. subescharoidea by strongly inequilateral ascospores that are laterally compressed, a dark gray to dull black surface when fully mature, and a continuous black layer beneath the surface.

Typification
Conidiophores in most Xylaria species are dichotomously branched several times and have the conidiogenous cells densely arranged in palisades. The conidiophores of X. subescharoidea, however, are highly reduced to mostly upright conidiogenous cells, which are loosely arranged. This sets a difference between X. subescharoidea and X. escharoidea. The difference between the two species also lies in their colony growth rates, with the colonies of X. escharoidea covering 9-cm Petri dishes in 5 days, much faster than those of X. subescharoidea.
Xylaria sp. 2 in Hsieh et al. (2010) is based on an immature specimen, which is proven the same as the present species by culture morphology, the anamorph, and DNA sequences.

Conclusion
Twelve out of 28 species of Xylaria known in the world have been recorded from termite nests or ground in Taiwan, with X. furcata, X. insolita, and X. subescharoidea included. Xylaria species growing on termite nests are primarily associated with macrotermitine termites, and Odontotermes formosanus is the only macrotermitine species known in Taiwan (Hsieh et al. 2010). Given the fact that the species number of macrotermitine termites in the world is approximately 330 (Kambhampati and Eggleton 2000), the global Xylaria diversity associated with termite nests is likely severely underestimated.
Additional file 1. List of taxa included in the present study.
Additional file 2. Overall tree topology resulting from BA analysis.

Fig. 6
Xylaria subescharoidea (from the holotype). a Ascal apical rings and ascospores. b Ascospores with a half of these showing a pore-like germination site. c, d Colony on 9-cm Petri plate containing OA at 1.5 week and 3 week, respectively. e Stromata produced in culture. f Conidiophores. g Conidia. Bars in a, b, f, g = 5 μm; e = 2 mm