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. 2, which showed X. insolita clustering with X. sp. 5 and X. ochraceostroma Y.-M. Ju and H.-M. Hsieh and X. subescharoidea clustering with X. escharoidea.
Taxonomy
Xylaria insolita Y.-M. Ju, H.-M. Hsieh et J.-C. Chou, sp. nov. Figs. 3, 4.
MycoBank MB 834498.
Etymology. Denoting the highly variable palmate stromata.
Stromata palmate at fertile part, 2–15-digitate, with tapering sterile apices, substipitate or sessile, 2–4 cm in total length above ground, 1–2.5 mm diam at clavae; surface dull grayish brown with a yellow tinge, rugulose, with conspicuous to half-exposed perithecial mounds unevenly aggregated or evenly distributed, overlain with a grayish brown outer layer gradually ruptured by perithecial mounds into flaky remnants and sloughing off afterwards, underlain with a thin, soft, black layer ca. 10 µm thick; interior white, soft, homogeneous. Perithecia spherical, 300–400 µm diam. Ostioles coarsely conic-papillate, ca. 100 µm broad at base. Asci with eight ascospores arranged in uniseriate manner, cylindrical, 105–135 µm total length, the spore-bearing part 40–50 µm long × 4–5 µm broad, with an apical ring staining blue in Melzer’s iodine reagent, inverted hat-shaped, 1.6–2.2 µm high × 1.6–2.5 µm broad. Ascospores brown to dark brown, unicellular, ellipsoid-inequilateral, laterally compressed, with one end narrowly rounded and slightly beaked and the other end broadly rounded, smooth, (5.2–)5.6–6.2 (–6.7) × (3.3–)3.5–3.9 (–4.0) × (2.5–)2.6–2.8 (–3.0) µm (5.9 ± 0.3 × 3.7 ± 0.2 × 2.7 ± 0.1 µm, N = 40), with a straight germ slit spore-length or nearly so on the dorsal side, lacking a hyaline sheath; epispore smooth.
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.
Typification. TAIWAN. Hua-lien County, Ji-an Township, Fu-hsin Village, from termite nests underground, 3 Sep 2010, Chou, J.-C. 99090301 (cultured) (holotype HAST 144970), GenBank accessions: ITS = MN655979, rpb2 = MN656981, β-tub = MN656983, α-act = MN656985.
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).
Xylaria subescharoidea Y.-M. Ju, H.-M. Hsieh et J.-C. Chou, sp. nov. Figs. 5, 6.
MycoBank MB 834499.
Etymology. Referring to its stromata resembling those of X. escharoidea in gross morphology.
Stromata cylindrical to cylindric-fusoid at fertile part, unbranched, with a narrowly rounded to mucronate apex, on a long, glabrous stipe, with a tortuous rooting base, 4.5–11.5 cm long above ground, 3.5–9.5 cm long × 3–6 mm diam at fertile part; surface pale brown to ochraceous when fully mature, with conspicuous perithecial mounds and tuberculate between perithecial mounds, lacking an outer layer, underlain with a layer of black ellipsoidal granules between ostioles; interior white, hard, brittle, with a black core. Perithecia obovoid, 300–500 µm diam × 600–800 µm high. Ostioles papillate, ca. 100 µm broad at base. Asci with eight ascospores arranged in uniseriate manner, cylindrical, 50–65 µm total length, the spore-bearing part 25–33 µm long × 3–4 µm broad, with an apical ring staining blue in Melzer’s iodine reagent, inverted hat-shaped, 1–1.5 µm high × 1–1.5 µm broad. Ascospores brown to dark brown, unicellular, ellipsoid, nearly equilateral, with narrowly rounded ends, smooth, (4.0–)4.3–4.7 (–4.9) × (2.3–)2.5–2.9 (–3.0) µm (4.5 ± 0.2 × 2.7 ± 0.2 µm, N = 40), with a median, pore-like germination site, lacking a hyaline sheath; epispore smooth.
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 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.
Typification. TAIWAN. Hua-lien County, Ji-an Township, Fu-hsin Village, from termite nests underground, 4 Jun–14 Jul 2010, Chou, J.-C. 99060401 (cultured) (holotype HAST 144971), GenBank accessions: ITS = MN655980, rpb2 = MN656982, β-tub = MN656984, α-act = MN656986.
Additional specimen examined. Tainan City, Nan-hsi District, on ground of mango orchard, 23 May 2006, Chou, K.-H. 95052301 (cultured), as X. sp. 2 in Hsieh et al. (2010) (HAST), immature, GenBank accessions: ITS = GU324754, rpb2 = GQ853025, β-tub = GQ502708, α-act = GQ853043.
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 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.
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.