Amphirosellinia Gallaeciana Sp. Nov. (Xylariaceae), a New Species from Spain

Amphirosellinia gallaeciana sp. nov. (Xylariaceae), a new species from Spain

Jacques FOURNIER Abstract: The new species Amphirosellinia gallaeciana is described and illustrated based on material José Manuel CASTRO MARCOTE collected on branchlets of Castanea and Quercus in Galicia (Spain). The material collected is assigned to Am- Enrique RUBIO phirosellinia based on macro- and micromorphological characters and comparison of the ITS sequence with Christian LECHAT those of two species of Amphirosellinia, including the type species. The new name is proposed based on morphological diﬀerences with other known species that are discussed in detail. Keywords: Ascomycota, Galicia, pyrenomycetes, saproxylic fungi, taxonomy, Xylariales. Ascomycete.org, 10 (5) : 200–204 Mise en ligne le 04/11/2018 Resumen: Se describe e ilustra la nueva especie Amphirosellinia gallaeciana en base a material recolectado 10.25664/ART-0242 sobre ramitas de Castanea y Quercus en Galicia (españa). este material ha sido asignado al género Amphiro- sellinia teniendo en cuenta sus caracteres macro y micromorfológicos y la comparación de la secuencia de la región ITS con la de dos especies de Amphirosellinia, incluyendo la especie tipo. Se propone este nuevo nombre en base a las diferencias morfológicas con otras especies conocidas, diferencias que detalladamente se discuten. Palabras clave: Ascomycota, Galicia, hongos xilosaprobios, pyrenomycetes, taxonomía, Xylariales.

Résumé : la nouvelle espèce Amphirosellinia gallaeciana est décrite et illustrée à partir de matériel récolté sur des branchettes de Castanea et Quercus en Galice (espagne). les récoltes sont attribuées au genre Am- phirosellinia en se fondant sur les caractères macro- et micromorphologiques et la comparaison d’une sé- quence ITS avec celles de deux espèces d’Amphirosellinia, dont l’espèce type. le nouveau nom est proposé en se fondant sur les diﬀérences morphologiques avec les espèces connues, qui sont discutées en détail. Mots-clés : Ascomycota, champignons saproxyliques, Galice, pyrénomycètes, taxinomie, Xylariales.

Introduction for ITS. The product was then employed as template in two semi- nested PCRs using primers ITS1F-ITS2 and ITS3-ITS4. PCR reactions were performed under a program consisting of a hot start at 95 ºC We report herein a xylariaceous fungus repeatedly collected in for 5 min, followed by 35 cycles at 94 ºC, 54 ºC and 72 ºC (45, 30 and the vicinity of A Coruña in the autonomous community of Galicia in 45 s respectively) and a final 72 ºC step for 10 min. PCR products Spain by one of us (JMCM). Its stromata are pustulate, multiperithe- were checked in 1% agarose gels, and positive reactions were se- ciate, slightly erumpent from the bark of branchlets and twigs of quenced with one or more PCR primers. Chromatograms were Castanea and Quercus, rupturing the periderm in a stellate pattern, checked searching for putative reading errors, and these were cor- exposing flattened polygonal discs roughened by stout ostiolar rected. A consensus sequence was assembled using the chro- necks. Microscopical observation shows unitunicate asci with a mas- matograms from both semi-nested PCRs. Phylogenetic and sive apical apparatus bluing in Melzer’s reagent and blackish brown, molecular evolutionary analyses were conducted using MeGA ver- ellipsoid, unicellular ascospores with a germ slit; these characters sion 6 (TAMuRA et al., 2013). are typical of the Xylariaceae Tul. & C. Tul. The macro- and micromor- phology, in particular the spiralling germ slit of ascospores, led us to consider it a species of the genus Amphirosellinia Y.-M. Ju, J.D. Taxonomy Rogers, H.-M. Hsieh & Vasilyeva. This placement is supported by comparison between its ITS sequences and those of two species in Amphirosellinia gallaeciana J. Fourn. & J.M. Castro Marcote, sp. that genus, including the type species A. nigrospora Y.-M. Ju, J.D. nov. – Mycobank Mb 827962. Plates 1–2 and Fig. 1. Rogers et H.-M. Hsieh. The morphological comparison of our collections with the five known species of Amphirosellinia showed that Diagnosis: Differs from Amphirosellinia nigrospora, the most sim- they represent a distinct species, for which we introduce the new ilar species as to shape and colour of ascospores, primarily by sig- name A. gallaeciana, in reference to the autonomous community of nificantly smaller ascospores 19–21.5 × 8.2–10.9 µm vs. 26.5–32 × Spain where the material was collected. 11.5–13.5 µm and multiperitheciate stromata with orange brown Galicia is in the westernmost region of Spain with a strong oceanic tissue around the perithecia. influence, enjoying mild winters and substantial precipitation, as reported by FouRnIeR et al. (2016). These climatic conditions are highly Typification: SPAIn: Galicia: A Coruña, Cee, Toba, ca. 20 m a.s.l, on favourable to fungi and especially to Xylariaceae, of which three rare a dead corticated branchlet of Castanea sativa Mill. (Fagaceae) ca. species were recently reported from this region (FouRnIeR et al., 2016; 1.5 cm diam, 15 May 2017, leg. J.M. Castro Marcote, PR10505171324 RubIo & De lA PeñA, 2016; RubIo et al., 2016) and several rare or poten- (AH, Holotype; lIP, Isotype). ITS Genbank MH379801. tially new species are still under investigation. Etymology: From latin Gallaecia, ancient Iberian Roman province, now called Galicia, the westernmost region of Spain where Materials and methods the fungus was collected.

Refer to FouRnIeR et al. (2016) for information on microscopical ob- Stromata corticolous, pustulate, largely immersed to slightly servations and photographic methods. The holotype is deposited erumpent, 1.2–1.6 mm high (including the ostiolar necks), rupturing in the herbarium of Alcalá de Henares (AH) and an isotype is de- the periderm and appearing on host surface as polygonal, often tri- posited in lille (lIP). DnA extraction, amplification, and sequencing angular or trapezoid mounds 1–3.6(–5.7) mm in their greatest di- were performed by AlVAlAb (Santander, Spain): Total DnA was ex- mension, containing (1–)2–10(–17) perithecia; upper surface tracted from dry specimens employing a modified protocol based appearing as a flat, dark brown to blackish brown disc, with perithe- on MuRRAY & THoMPSon (1980). PCR amplification was performed with cial contours unexposed or rarely slightly exposed, roughened by the primers ITS1F and ITS4 (WHITe et al., 1990; GARDeS & bRunS, 1993) stout, black ostiolar necks; outer crust 80–100 µm thick beneath the

200 disc, ca. 40 µm thick at sides, black, leathery, easily cut with a razor Known distribution: Spain (Galicia). blade, extending downwards through the bark tissue to join the un- derlying wood; soft, brownish orange, pithy, persistent tissue pre- Other specimens examined (paratypes): SPAIn: Galicia: A sent in places between perithecia. Perithecia subglobose to slightly Coruña, Cee, Toba, ca. 20 m a.s.l, on a dead corticated branchlet of depressed or laterally flattened, 0.9–1.1 mm high × 0.85–1.1 mm Quercus robur l. (Fagaceae), 24 Feb. 2007, leg. J.M. Castro Marcote, diam. Ostioles central or slightly eccentric, strongly papillate, open- PR1240207251 (AH); A Coruña, Cee, Canosa, ca. 120 m a.s.l, on a ing through obtusely rounded ostiolar necks 0.12–0.21 mm high × dead fallen corticated branchlet of Quercus robur l. (Fagaceae) ca. 0.2–0.25 mm diam at base. 0.8 cm diam, 3 Sept. 2017, leg. J.M. Castro Marcote & R. Montes Asci slightly clavate, long stipitate, with (4–)8 obliquely uniseri- Papín, PR10309171360 (AH); A Coruña, Corcubión, Redonda, 100 m ately overlapping ascospores, frequently irregularly biseriate in a.s.l, on a dead fallen corticated branchlet of Castanea sativa Mill. ascus apex, the spore-bearing parts 124–160 × 14–15.5 µm, the (Fagaceae) ca. 2.5 cm diam, 22 Feb. 2018, leg. J.M. Castro Marcote, stipes 90–140 µm long, with a short-cylindrical, slightly barrel- PR12302181366 (AH). shaped apical apparatus with obtuse angles, 7.5–9 µm high × 6.5– Comments: The fungus that we describe herein as A. gallaeciana 7.3 µm wide (Me = 8.4 × 6.9 µm, n = 30), bluing in Melzer’s reagent. is characterized by corticolous, immersed, multiperitheciate stro- Paraphyses copious, hyphal, hyaline, thin-walled, septate, remotely mata rupturing the periderm to expose a flat disc with stout ostiolar branched, much longer than asci, 5–9.5 µm wide at base, gradually papillae, slightly clavate unitunicate asci with a massive apical ap- tapering to 2 µm wide above asci, minutely guttulate, discretely em- paratus staining blue in Melzer’s reagent, and dark brown, one- bedded in mucilaginous material. Ascospores (17.8–)18.9– celled ellipsoid-inequilateral ascospores featuring a spiralling germ 21.7(–22.4) × (7.8–)8.2–10.9(–11.6) µm, Q = (1.7–)1.9–2.5(–2.8), n = slit on the ventral side. This set of characters matches well with the 120 (Me = 20.2 × 9.4 µm, Qe = 2.1), ellipsoid-inequilateral with definition of Amphirosellinia but does not key out to any of the five broadly to more rarely narrowly rounded ends, at times slightly ven- species so far accepted in the genus (Ju et al., 2004). It differs from trally concave, unicellular, blackish brown, with a long, almost spore- most species of Amphirosellinia that are one- to few-peritheciate by length, spiralling germ slit on the flattened side, not reaching the its multiperitheciate stromata, the most similar in this regard being ends, sometimes surrounded with gelatinous material when freshly A. tennesseensis Vasilyeva, J.D. Rogers, Y.-M. Ju et H.-M. Hsieh, which released from the ascus, visible in India ink, forming an evanescent is reported to feature 3–10 perithecia per stroma (Ju et al., 2004). irregular sheath, but no appendages or persistent sheath detected; However, besides its different geographical origin, A. tennesseensis epispore smooth. is different in having white fungal material between perithecia vs. Asexual morph on the natural substrate not seen. orange brown, longer and narrower apical apparati 11–15(–18) × 6–

Fig. 1 – Maximum likelihood phylogeny (-lnl = 2279.09315) of Amphirosellinia gallaeciana inferred by Tamura-nei model from a 658 bp matrix of ITS sequences, rooted to Hypoxylon rubiginosum.

Ascomycete.org 201 Plate 1 – Amphirosellinia gallaeciana PR1/505171324 (Holotype). A: Habit of stromata on host surface; b-D: Stromata in top view showing a blackish brown surface, stout, black ostiolar papillae and ruptured, inrolled peridem at margins; e-H: Stromata in vertical section showing variously arranged perithecia, ruptured periderm (white arrows), a thin black outer crust (red arrows) and orange brown tissue embedding the perithecia (green arrows). Scale bars: A = 2 mm; b-H = 0.5 mm.

202 Ascomycete.org Plate 2 – Amphirosellinia gallaeciana PR1/505171324 (Holotype). A: Two adjacent immature asci, one showing a subclavate outline, in diluted blue Pelikan® ink; b: Immature and mature asci showing variable number and arrangement of ascospores, in diluted blue Pelikan® ink; C: long-stipitate ascus, in diluted blue Pelikan® ink; D: base of paraphyses, in black Pelikan® ink; e: Ascal apical apparati, in Melzer’s reagent; F: Ascospores with broadly rounded ends, in 1% SDS; G: Freshly ejected mature ascospore surrounded by gelatinous material, in India ink; H: Immature guttulated ascospore lacking appendage or sheath, in black Pelikan® ink; I: barely mature ascospores in side view showing germ slits, in black Pelikan® ink; J: As- cospore in side view showing a concave ventral side, in black Pelikan® ink; K: Mature ascospores showing germ slits, in PVA-lactophenol. Scale bars: A-C = 50 µm; D-K = 10 µm.

Ascomycete.org 203 7 µm vs. 7.5–9 × 6.5–7.3 µm and paler, narrower ascospores (18–)19– Acknowledgements 23(–27) × 7.5–8.5 µm vs. (17.8–)18.9–21.7(–22.4) × (7.8–) 8.2–10.9(– 11.6) µm. In colour and shape, the ascospores of A. gallaeciana We gratefully acknowledge Prof. Jack Rogers (Pullman, uSA) and strikingly resemble those of A. nigrospora Y.-M. Ju, J.D. Rogers et H.- Dr. Yu-Ming Ju (Academia Sinica, Taiwan) for having critically read M. Hsieh, the type species known from Taiwan. They primarily differ our manuscript and having provided helpful comments, corrections in the former being significantly smaller, 18.9–21.7 × 8.2–10.9 µm and suggestions to improve this article. vs. 26.5–32 × 11.5–13.5 µm. Moreover, stromata of A. gallaeciana differ in being consistently multiperitheciate, in having orange brown References tissue between perithecia, and in having smaller and differently shaped ascal apical apparati. FouRnIeR J., DelGADo M.-A. & CASTIllo J. 2016. — Xylaria xylarioides (Xy- Amphirosellinia evansii (læssøe & Spooner) Y.-M. Ju, J.D. Rogers et lariaceae), a subtropical species reported for the first time from H.-M. Hsieh was the only species previously known from europe (uK) europe. Ascomycete.org, 8 (5): 221–226. doi: 10.25664/art-0189 and thus should be compared with the new species from Galicia. GARDeS M. & bRunS T.D. 1993. — ITS primers with enhanced specificity The stromata of both species are erumpent from the bark, splitting for basidiomycetes – application to the identification of mycor- the periderm into a stellate pattern; those of other known species rhizae and rusts. Molecular Ecology, 2 (2): 113–118. largely remain immersed in bark tissue (Ju et al., 2004). The stromata HSIeH H.-M., lIn C.-R., FAnG M.-J., RoGeRS J. D., FouRnIeR J., leCHAT C. & Ju of A. evansii are typically uniperitheciate and globose with strongly Y.-M. 2010. — Phylogenetic status of Xylaria subgenus Pseudoxy- exposed contours, which led to its first placement in Rosellinia De laria among taxa of the subfamily Xylarioideae (Xylariaceae) and not. by læSSøe & SPooneR (1994). In contrast, those of A. gallaeciana phylogeny of the taxa involved in the subfamily. Molecular Phylo- are multiperitheciate and appear as flat discoid structures with un- genetics and Evolution, 54: 957–969. doi: 10.1016/j.ympev.2009.12.015 exposed perithecial contours, only roughened by stout ostiolar Ju Y.-M., RoGeRS J.D., HSIeH H.-M. & VASIlYeVA l. 2004. — Amphirosellinia necks. other differential characters between the two species are the gen. nov. and a new species of Entoleuca. Mycologia, 96 (6): 1393– presence of synnematous conidial structures around stromata of 1402. doi: 10.1080/15572536.2005.11832889 læSSøe T. & SPooneR b.M. 1994. — Rosellinia & Astrocystis (Xylariaceae): A. evansii and its longer and more cylindrical ascospores 22–27 × new species and generic concepts. Kew Bulletin, 49 (1): 1–70. doi: 8.5–10 µm. 10.2307/4110199 Molecular data are available for A. fushanensis Y.-M. Ju, J.D. Rogers MuRRAY M.G & THoMPSon W.F. 1980. — Rapid isolation of high molec- et H.-M. Hsieh and A. nigrospora and both species were included in ular weight plant DnA. Nucleic Acids Research, 8 (19): 4321–4325. multigene phylogenies with congruent results showing that they RubIo e. & De lA PeñA S. 2016. — Annulohypoxylon michelianum, una clustered on a separate branch within the Po clade of Xylaria and rara especie laurícola recolectada en el noroeste español. As- were distant from genera like Entoleuca J.D. Rogers & Y.-M. Ju and comycete.org, 8 (1): 25–29. doi: 10.25664/art-0167 Rosellinia with which affinities could have been considered (HSIeH et RubIo e., CASTRo MARCoTe J.M. & CoSTA J.M. 2016. — nuevas aporta- al., 2010; u’Ren et al., 2016). ciones al conocimiento de Xylaria crozonensis (Ascomycota, Xylar- We compared the ITS sequence from the holotype of A. gallae- iaceae). Ascomycete.org, 8 (2): 73–76. doi: 10.25664/art-0173 ciana with those of A. fushanensis and A. nigrospora generated from TAMuRA K., STeCHeR G., PeTeRSon D., FIlIPSKI A. & KuMAR S. 2013. — the holotypes of these species, available on Genbank (Fig. 1). our MeGA6: Molecular evolutionary Genetics Analysis version 6.0. analysis showed that A. gallaeciana clustered with A. fushanensis, Molecular Biology and Evolution, 30 (12): 2725–2729. doi: with a 91% similarity, while A. nigrospora was on a more distant 10.1093/molbev/mst197 u’ Ren J.M., MIADlIKoWSKA J., ZIMMeRMAn n.b., luTZonI F., STAJICH J.e & branch with an 89% similarity with A. fushanensis. This supports the ARnolD A.e. 2016. — Contributions of north American endophytes placement of the new species in Amphirosellinia and confirms that to the phylogeny, ecology and taxonomy of Xylariaceae (Sordari- ITS sequences only, on account of their high variability, do not pro- omycetes, Ascomycota). Molecular Phylogenetics and Evolution, 98: vide a good phylogenetic resolution, which could be attained by 210–232. doi: 10.1016/j.ympev.2016.02.010 using sequences of certain protein-coding loci. WHITe T.J., bRunS T., lee S. & TAYloR J. 1990. — Amplification and direct All collections of A. gallaeciana were made on dead twigs and sequencing of fungal ribosomal RnA genes for phylogenetics. In: branchlets of Castanea and Quercus that were apparently recently InnIS M.A., GelFAnD D.H., SnInSKY J.J. & WHITe T.J. (eds). PCR Protocols: fallen onto the ground; this suggests that it is an endophyte or an a guide to methods and applications. new York, Academic Press: early colonizer of dying branchlets probably with an aerial lifestyle. 315–322. ef

1 2 3 4

1: J. Fournier – Las Muros, 09420 Rimont, France – [email protected] 2: J.M. Castro Marcote – Rúa de Arriba 1, 15270 Cee, Spain – [email protected] 3: E. Rubio – José Cueto 3 5ºB, 33407 Avilés, Spain – [email protected] 4: C. Lechat – 64 route de Chizé, 79360 Villiers-en-Bois, France – [email protected]

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