Xylaria iriomotensis sp. nov. from termite nests and notes on X. angulosa

Abstract

Background

Fungus gardens of the termite Odontotermes formosanus, excavated from Iriomote Island, Okinawa Prefecture, Japan, were subsequently incubated under laboratory conditions. A Xylaria species emerging from these fungus gardens was initially identified as X. angulosa, a species originally described from North Sulawesi, Indonesia. The Iriomote fungus is now described as a distinct species, X. iriomotensis.

Results

Xylaria iriomotensis is peculiar in producing the teleomorph in culture but lacking an anamorph. Cultures of X. angulosa were obtained from two Taiwan specimens, which agree with the holotype from BO and the isotypes from NY and WSP in their stromata being repeatedly dichotomously branched and possessing a black core. In contrast to X. iriomotensis, X. angulosa does not form the teleomorph in culture but a typical Xylaria anamorph with conidiophores densely arranged in palisades. The ITS sequence obtained from the WSP isotype shared high similarities with those two Taiwan specimens as well as an Indian specimen, reconfirming the latter three specimens as X. angulosa. These four specimens shared 98.28–99.66% similarities at ITS sequences among themselves but only 84.25–85.01% similarities with X. iriomotensis. Molecular phylogenetic studies based on sequences of multiple protein-coding loci indicate that, while X. iriomotensis is grouped with three soil-dwelling species of the X. guepini cluster, X. angulosa belongs to the X. nigripes cluster, which includes all known species capable of producing massive sclerotia.

Conclusion

Xylaria iriomotensis has the teleomorph known only in culture, remaining to be rediscovered in its natural habitat where the stromatal morphology may be somewhat varied. The geographic distribution of X. angulosa, previously known only in Indonesia, has been expanded to Taiwan and India. Xylaria angulosa grouping with the X. nigripes cluster in our phylogenetic analyses indicates its potential to form massive sclerotia within termite nests.

Background

Xylariaceae Tul. & C. Tul. (Xylariales, Sordariomycetes, Ascomycota) contains more than a thousand of described species that are characterized by mid- to large-sized stromata and display diversified life styles (Rogers 1979, 2000; Suwannasai et al. 2023; Whalley 1985, 1996). Xylaria Hill ex Schrank, commonly found in the tropics and subtropics (Ju and Rogers 1999; Rajtar et al. 2023; Rogers et al. 1987, 1988; San Martín and Rogers 1989; Van der Gucht 1995; Vandegrift et al. 2023), is a highly diversified genus in the Xylariaceae, where species are characterized by dark, one-celled ascospores with a germ slit and unitunicate asci topped with a ring-like apparatus staining blue in an iodine reagent. Most Xylaria species are found on dead wood (Fournier et al. 2018, 2019, 2020), and fewer species grow on fallen fruits and seeds (Ju et al. 2018), dead leaves and petioles (Ju and Hsieh 2023), and termite nests and soil (Hsieh et al. 2024; Ju and Hsieh 2007; Ju et al. 2022, 2023; Rogers et al. 2005; Wangsawat et al. 2021).

Xylaria species inhabiting termite nests and soil belong to the subgenus Pseudoxylaria, where approximately 40 species are included. Most of the members are associated with termite nests (Ju and Hsieh 2007; Ju et al. 2022, 2023; Rogers et al. 2005; Wangsawat et al. 2021) and only four are associated with soil (Chou et al. 2017; Hsieh et al. 2022; Ju et al. 2011; Kim et al. 2016). While the stromata of these species display a wide range of variation: delicate to stout, with an acute to rounded apex, stipitate to sessile, unbranched to branched, smooth to roughened, their ascospores are quite consistent in being small, shorter than 8 μm in most cases, and their ostioles are coarsely conic-papillate.

Okane and Nakagiri (2007) reported two xylariaceous fungi from termite nests of Odontotermes formosanus in Iriomote Island, Okinawa Pref., Japan: Geniculisynnema termiticola Okane & Nakagiri and X. angulosa J. D. Rogers, Callan & Samuels. These two species produced stromata from the excavated nests, which were brought back to the laboratory and incubated in moist chambers. Geniculisynnema termiticola is an anamorphic Xylaria species and was recombined with the genus Xylaria to form X. termiticola (Okane & Nakagiri) Y.-M. Ju (Réblová et al. 2016) to comply with the change to the International Code of Nomenclature for algae, fungi, and plants (McNeill et al. 2012), where two or more names for different morphs of the same taxon are no longer allowed. The material identified as X. angulosa was based on the teleomorph produced on agar media, where an anamorph is lacking.

In this study, we compared the Iriomote material with the type material of X. angulosa from North Sulawesi, Indonesia (Rogers et al. 1987) and found it distinctive from the latter. As the Iriomote material does not fit a known species, we thus describe it as a new species, X. iriomotensis. We also provide teleomorphic, anamorphic, and culture descriptions of X. angulosa and inferred its phylogenetic relationships with X. iriomotensis in the context of numerous other Xylaria species using sequences of three protein-coding loci.

Methods

Fungal material and observation

While stromata of X. angulosa came from field-collected as well as loaned herbarium specimens, those of X. iriomotensis were formed under laboratory conditions by incubating fungus gardens of Odontotermes formosanus in a moist chamber. The fungus gardens, from which X. iriomotensis emerged, were collected from Iriomote Island, Okinawa Pref., Japan, as documented in Okane and Nakagiri (2007). The brown stromata of X. iriomotensis were formed among other whitish synnemata within a week. To obtain cultures of X. iriomotensis and X. angulosa, the stromatal tissue of these two species was placed on oatmeal agar (OMA), potato dextrose agar (PDA), and 2% malt extract agar (MEA) without adding peptone (Kenerley and Rogers 1976). Resulting colonies were transferred to 9-cm plastic Petri dishes containing 2% OMA for culture descriptions and incubated at 20 °C under 12 h fluorescent light. Asci, ascospores, conidiogenous cells, and conidia were examined using differential interference contrast microscopy (DIC) and bright field microscopy (BF). Material was mounted in water and Melzer’s iodine reagent for examination by DIC and BF. The cultures of X. angulosa and X. iriomotensis are available at Bioresource Collection and Research Center in Taiwan (BCRC) and Biological Resource Center of National Institute of Technology and Evaluation in Japan (NBRC), respectively. The holotype of X. iriomotensis was harvested from the stromata produced on OMA.

Analyzing ITS sequences

Sequences of nuclear rDNA internal transcribed spacers (ITS = ITS1-5.8 S-ITS2) were obtained according to Hsieh et al. (2009). To reconfirm that X. iriomotensis is not conspecific with X. angulosa, we compared its ITS sequence with that from the isotype of X. angulosa deposited at WSP. We then analyzed these sequences with the ITS sequences of various species from termite nests and soil that we had obtained (the italicized ITS sequences listed in Table 1) using Maximum-Likelihood (ML) and Bayesian Inference (BI) phylogenetic methods, with ML and BI trees generated using RAxML analysis ver. 8.2.10 (Stamatakis 2014) and MrBayes ver. 3.2.6 (Ronquist et al. 2012), respectively, as detailed in Ju et al. (2023). The outgroup was X. hypoxylon (L.) Grev.

Table 1 Sequences of taxa and isolates included in the ML and BI analyses

To calculate the similarities between pairs of ITS sequences from different collections of X. angulosa, we used DNADIST from the PHYLIP version 3.6 phylogenetic inference package (Felsenstein 2005).

Phylogenetic analyses

Sequences of the loci for β-tubulin (β-TUB) and α-actin (α-ACT) were obtained from the studied Xylaria species following Hsieh et al. (2005), while those of the loci for the second largest subunit of RNA polymerase II (RPB2) was obtained following Hsieh et al. (2010).

The concatenated sequences of α-ACT, RPB2, and β-TUB from X. angulosa and X. iriomotensis were incorporated into the RPB2-TUB-ACT dataset as presented by Ju et al. (2023). This dataset includes the taxa with available sequences listed in Table 1. These concatenated sequences encompass various species of Xylaria and its closely related genera. The outgroup used was Biscogniauxia arima F. San Martín, Y.-M. Ju & J. D. Rogers. The resulting RPB2-TUB-ACT dataset was aligned using Clustal X 1.81 (Thompson et al. 1997) with “gap penalty” set to 10 and “gap extension penalty” set to 0.2, and was manually improved. It was then subjected to ML and BI analyses. Models of evolution for both ML and BI trees were defined by MrModeltest 2.4 (Nylander 2004), and consensus trees were viewed in FigTree ver. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

Results

Reconfirmation of X. angulosa and X. iriomotensis being different species through a comparison of their ITS sequences

The ITS sequences from the isotype of X. angulosa deposited at WSP and three specimens identifiable as X. angulosa—two from Taiwan and one from India—exhibited high similarities ranging from 98.28 to 99.66%. These sequences were grouped together using BI and ML phylogenetic methods. This grouping is depicted in Fig. 1, where X. iriomotensis was not included in the same group as X. angulosa. The similarities between the ITS sequences of X. iriomotensis and X. angulosa were relatively low, ranging from 84.25 to 85.01%.

Fig. 1

Phylogenetic tree generated by ML analysis from the ITS dataset with the sequence from the WSP isotype of X. angulosa included. The species with sequences generated in the present study are in boldface. Numbers at internodes represent bootstrap values and are immediately followed by the posterior probability values greater than 50% with BI analysis. Xylaria hypoxylon is the outgroup taxon

Molecular phylogenetic analyses conducted based on the RPB2‑TUB‑ACT dataset

Both BI and ML analyses based on the RPB2-TUB-ACT dataset were conducted and yielded congruent results. Therefore, only the ML tree is presented (Fig. 2). The results indicate that both X. angulosa and X. iriomotensis belong to the TE clade (Hsieh et al. 2010; U’Ren et al. 2016). This clade is equivalent to the subgenus Pseudoxylaria, which encompasses Xylaria species associated with termite nests and soil. While X. iriomotensis was grouped within the X. guepini (Fr.) Fr. cluster with three soil-dwelling species—X. coprinicola Y.-M. Ju, H.-M. Hsieh & X.-S. He, X. guepini, and X. terricola Y.-M. Ju, H.-M. Hsieh & W.-N. Chou, X. angulosa belonged to the X. nigripes cluster, which includes all known sclerotium-forming species (Ju et al. 2022). Despite X. angulosa exhibiting repeatedly dichotomously branched stromata, a characteristic commonly observed in the species of the X. furcata cluster (Ju et al. 2023), it did not show a close relationship with X. furcata or its resembling species.

Fig. 2

Phylogenetic tree generated by ML analysis from the RPB2-TUB-ACT dataset. The subclades of the TE clade including the X. guepini cluster and the X. nigripes cluster are in shade. The species newly described in the present study are in boldface. Numbers at internodes represent bootstrap values and are immediately followed by the posterior probability values greater than 50% with BI analysis. Biscogniauxia arima is the outgroup taxon

Taxonomy

Xylaria iriomotensis I. Okane, H.-M. Hsieh & Y.-M. Ju, sp. nov. Figure 3.

Fig. 3

Xylaria iriomotensis (from the holotype). A, B Colony on 9-cm Petri plate containing OA at 2 and 4 wk, respectively. C, D Fresh stromata produced in culture. E Surface of a dried stroma showing inconspicuous to conspicuous perithecial mounds and conic-papillate ostioles. F Vertical section of a stroma. G Asci and a paraphysis. H Ascal apical rings and ascospores. I Ascospores, with some showing a germ slit. Bars in C, D = 1 mm; E, F = 0.25 mm; G–I = 5 μm

MycoBank MB856549.

Typification. JAPAN. Okinawa Pref., Iriomote Island, emerging from incubated termite nests of Odontotermes formosanus, 18 Oct 2001, Okane, I. (cultured), as X. angulosa (holotype of X. iriomotensis HAST 146312), culture accession number: NBRC 33288.

Etymology. Referring to Iriomote Island where the termite nests were collected.

Colonies on OMA reaching the edge of 9-cm Petri dish in 3 wk, white initially, becoming light grayish brown, appressed to slightly cottony, zonate, with diffuse margins. Reverse uncolored. Stromata arising from concentric zones, cylindrical at fertile part, unbranched or branched occasionally, with an apiculate to acicular apex, on a short stipe or nearly sessile, 0.5–2 cm long in total length × 0.6–1.5 mm diam; surface dark copper brown to dark brown, with inconspicuous to conspicuous perithecial mounds when dried, lacking an outer layer, underlain with a thin, soft layer 20–30 μm thick, concolorous with the surface; interior whitish, homogeneous, soft. Perithecia spherical, 150–250 μm broad. Ostioles conic-papillate, 80–100 μm high × 110–140 μm broad at base. Asci with eight ascospores arranged in uniseriate to partially biseriate manner, cylindrical, 45–55 μm total length, the spore-bearing part 25–45 μm long × 3–4.5 μm broad, with an apical ring staining light 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 to short fusoid, slightly inequilateral, with narrowly rounded ends, smooth, (3.5–)4.0–4.5(–5.0) × 2.0–2.5(–3.0) µm (4.3 ± 0.3 × 2.4 ± 0.2 μm, N = 80), with a straight germ slit slightly less than spore-length to nearly spore-length on the ventral side, lacking a hyaline sheath; epispore smooth. Paraphyses hyaline, tapering upwards, extending much beyond hymenial layer, unbranched.

Anamorph not produced

Notes. Xylaria iriomotensis is characterized by dark brown, cylindrical stromata, which are topped with an apiculate to acicular apex and lack an outer layer, and the lack of an anamorph. Its teleomorph, initially emerging from fungus combs incubated under laboratory conditions, is readily produced on agar media (Okane and Nakagiri 2007). In our phylogenetic analyses based on the RPB2-TUB-ACT dataset (Fig. 2), X. iriomotensis was grouped with the X. guepini cluster, to which three soil-dwelling species—X. coprinicola, X. guepini, and X. terricola—also belong. Xylaria coprinicola and X. guepini are similar to X. iriomotensis in ascospore morphology, in lacking a stromatal outer layer, and in being capable of producing the teleomorph in culture, but they mainly differ in possessing an anamorph and being soil-dwelling. Xylaria terricola is an anamorphic species and thus morphologically incomparable with X. iriomotensis.

The material of X. iriomotensis was previously identified as X. angulosa by Okane and Nakagiri (2007). Both species have a sterile stromatal apex, a dark brown stromatal surface, and similar ascospore features. However, X. iriomotensis has unbranched or occasionally branched stromata with a homogeneous, whitish interior and inconspicuous to conspicuous perithecial mounds on the stromatal surface. In contrast, X. angulosa has frequently dichotomously branched stromata with a heterogenous interior, which is white at perithecial layer but black at core, and half-exposed perithecial mounds.

Xylaria angulosa J. D. Rogers, Callan & Samuels, Mycotaxon 29: 149. 1987. Figures 4 and 5.

Fig. 4

Xylaria angulosa (A–C from the NY isotype, D, E, H–J from HAST 146295, F, G from HAST 146296). A, D, F Stromata. B, C, E, G Stromatal surfaces; the cut in G shows the dark core. H Vertical section of a stroma. I Ascal apical rings and ascospores. J Ascospores, with some showing a germ slit. Bars in F = 1 cm; A, D, F = 5 mm; B, C, E, G = 0.5 mm; H = 0.25 mm; I, J = 5 μm

Fig. 5

Xylaria angulosa (from HAST 146295). A, B Colony on 9-cm Petri plate containing OA at 2 and 3 wk, respectively. C Stromatal surface overlain with pale mouse gray conidial masses. D, E Conidiophores in densely arranged palisades, hyaline initially, becoming yellowish later. F Conidia. Bars in C = 2.5 mm; D–F = 5 μm

Stromata antler-like at fertile part, dichotomously branched one to three times in broad angles or occasionally unbranched or trichotomously branched, with sterile apices short to long, abruptly narrowed when young, broken off when mature, on a glabrous stipe, with a tortuose rooting base, 3.5–5.5 cm long above ground, 1–3 cm long × 2.3–3 mm diam at fertile part; surface dark brown, becoming black, with half-exposed perithecial mounds, lacking an outer layer, underlain with a thin, black layer ca. 10 μm thick; interior white at perithecial layer, with a black core, coriaceous. Perithecia spherical to subspherical, 200–350 μm broad × 200–400 μm high. Ostioles conic-papillate, 50–70 μm high × 90–120 μm broad at base. Asci with eight ascospores arranged in uniseriate manner, cylindrical, 55–75 μm total length, the spore-bearing part 30–40 μm long × 3.5–5 μm broad, with an apical ring staining blue in Melzer’s iodine reagent, inverted hat-shaped, 1–1.5 μm high × 1.5 μm broad. Ascospores brown to dark brown, unicellular, ellipsoid to short fusoid, slightly inequilateral, with narrowly rounded ends, smooth, (4–)4.5–5.5(–6) × (2–)2.5–3(–3.5) µm (5.0 ± 0.3 × 2.6 ± 0.2 μm, N = 110), with a straight germ slit spore-length or nearly so on the ventral side, lacking a hyaline sheath; epispore smooth.

Cultures and anamorph. Colonies reaching the edge of 9-cm Petri dish in 3 wk, whitish, mostly submerged, azonate, with diffuse margins. Reverse pale tan-colored. Stromata arising close to the center of the colonies, antler-like, branched several times, up to 6 cm long × 1.2–1.6 mm diam, white, immediately becoming black at base and pale mouse gray on nearly entire surface due to production of conidia. Anamorph produced on the stromatal surface. Conidiophores in upright, densely arranged palisades, dichotomously branched several times from base, smooth, hyaline, becoming yellowish. Conidiogenous cells terminal, cylindrical, 10–15 × 4–5 μm, smooth, bearing one to several terminal poroid conidial secession scars. Conidia produced holoblastically, hyaline, smooth, subglobose to ellipsoid, (6.5–)7–8.5(–10) × (5–)5.5–7.5(–8.5) µm (7.8 ± 0.9 × 6.5 ± 0.9 μm, N = 40), with a flattened base indicating former point of attachment to conidiogenous cell.

Specimens examined. INDIA. Dehra Dun, on termite nests, Lehmann, J., as X. furcata (WSP ex Rogers herb.). INDONESIA. North Sulawesi, Eastern Dumoga-Bone National Park, vic. “Hog’s Back Camp”, on ground, 30–31 Oct 1985, Samuels, G. J. GS2465 (holotype of X. angulosa BO 19889, isotypes NY, WSP ex Rogers herb). TAIWAN. I-lan Co., Yuan-shan, Fu-shan, on ground, 27 Sep 2014, Ke, Y.-H. 103092701 (cultured from stroma YMJ3267) (HAST 146295); Tainan City, Shen-hua District, Liu-fen-liao, on ground, 28 Jun 2010, Chou, K.-H. 99062804 (cultured from stroma YMJ1199) (HAST 146296), largely immature.

Notes. Xylaria angulosa was originally described from North Sulawesi, Indonesia (Rogers et al. 1987). Its stromata are stout and frequently branched dichotomously in broad angles, and have a black core inside, unlike those of X. furcata and its resembling species (Ju et al. 2023), where stromata are delicate and branched in sharp angles, and lack a black core inside. With the ITS sequence available from the WSP isotype, we reconfirmed that three specimens, two from Taiwan and one from India, are X. angulosa despite their stromata being less robust and reduced in size (Fig. 4). The anamorph was observed from the surface of the stromata formed in culture, with conidiophores produced in upright, densely arranged palisades, as opposed to the mononematous conidiophores produced by X. furcata and its resembling species.

Phylogenetic analyses based on the RPB2-TUB-ACT dataset (Fig. 2) indicated that X. angulosa grouped within the X. nigripes cluster, most species of which are capable of forming massive sclerotia (Ju et al. 2022). More field collecting is required to find out if X. angulosa is another species capable of forming massive sclerotia.

The NY isotype contains two species; besides X. angulosa, it also contains three stromata of X. brunneovinosa Y.-M. Ju & H.-M. Hsieh (Hsieh et al. 2020; Ju and Hsieh 2007), which can be distinguished from the former species by its vinaceous-tinged stromatal surface, lack of a dark core inside the stromata, and larger ascospores.

Discussion

The original material of X. iriomotensis, induced from fungus gardens under laboratory conditions in 2001, was reported as X. angulosa by Okane and Nakagiri (2007). It remains the only source for X. iriomotensis, which is yet to be rediscovered in its natural habitat.

The ITS sequences from the holotype of X. iriomotensis and the WSP isotype of X. angulosa allowed us to reconfirm them as two distinct species (Fig. 1). Phylogenetic analyses using sequences of three protein-coding loci, α-ACT, RPB2, and β-TUB, revealed that they are not closely related species. In the phylogenetic tree, X. iriomotensis is grouped with three soil-dwelling Xylaria species of the X. guepini cluster: X. coprinicola, X. guepini, and X. terricola, while X. angulosa is placed in the X. nigripes cluster, which includes species known for producing massive sclerotia within termite nests (Fig. 2).

The grouping of X. iriomotensis, which lacks an anamorph, with three soil-dwelling species is interesting due to the latter three species possessing highly diversified anamorphs. Xylaria terricola, an anamorphic species, produces flabellate stromata resembling those of the Xylocoremium state of X. flabelliformis (Schwein.) Berk. & M. A. Curtis (Chou et al. 2017). Its conidiophores are loosely arranged, rather than in densely arranged palisades found in most Xylaria species. In contrast, X. coprinicola and X. guepini produce mononematous conidiophores, each terminating into an unswollen or swollen top, from which multiple crowded branches arise.

Our phylogenetic analyses suggested that X. angulosa is a member of the X. nigripes cluster. The anamorphs of the species in the X. nigripes cluster are fairly consistent, having their conidiophores in densely arranged palisades, and X. angulosa is no aberration. The phylogenetic analyses also implied that X. angulosa may have the potential to form massive sclerotia within termite nests.