Molecular systematics of Barbatosphaeria (Sordariomycetes): multigene phylogeny and secondary ITS structure

Abstract

Thirteen morphologically similar strains of barbatosphaeria- and tectonidula-like fungi were studied based on the comparison of cultural and morphological features of sexual and asexual morphs and phylogenetic analyses of five nuclear loci, i.e. internal transcribed spacer rDNA operon (ITS), large and small subunit nuclear ribosomal DNA, β-tubulin, and second largest subunit of RNA polymerase II. Phylogenetic results were supported by in-depth comparative analyses of common core secondary structure of ITS1 and ITS2 in all strains and the identification of non-conserved, co-evolving nucleotides that maintain base pairing in the RNA transcript. Barbatosphaeria is defined as a well-supported monophyletic clade comprising several lineages and is placed in the Sordariomycetes incertae sedis. The genus is expanded to encompass nine species with both septate and non-septate ascospores in clavate, stipitate asci with a non-amyloid apical annulus and non-stromatic ascomata with a long decumbent neck and carbonised wall often covered by pubescence. The asexual morphs are dematiaceous hyphomycetes with holoblastic conidiogenesis belonging to Ramichloridium and Sporothrix types. The morphologically similar Tectonidula, represented by the type species T. hippocrepida, grouped with members of Barbatosphaeria and is transferred to that genus. Four new species are introduced and three new combinations in Barbatosphaeria are proposed. A dichotomous key to species accepted in the genus is provided.

Keywords: Ramichloridium; Sporothrix; Tectonidula; phylogenetics; sequence analysis; spacer regions.

Fig. 1

Multilocus phylogenetic analysis of the nuc18S-nuc28S-rpb2 sequences of Barbatosphaeria and members of the Sordariomycetes. Phylogram inferred from the ML analysis with RAxML using a GTRCAT model of evolution. Only high branch support is shown at the nodes, maximum likelihood bootstrap support (ML BS) ≥ 85% and Bayesian posterior probability (PP) ≥ 95%). Branches with a black circle symbol indicate ML BS = 100%, PP values = 1.0. Taxa in bold refer to new combinations or new taxonomic treatments; ‘T’ refers to ex-type strains of species of Barbatosphaeria

Fig. 2

Phylogenetic analysis of the combined ITS rDNA and β-tubulin sequences of Barbatosphaeria. Phylogram inferred from the ITS rDNA sequences with ML analysis using a GTRCAT model of evolution. Legend as in Fig. 1.

Fig. 3

A partial secondary structure of ITS1 rRNA molecule of B. barbirostris showing three helices (H1–H3). All substitutions recorded among members of Barbatosphaeria are mapped on the 2D model. Parts of hairpin loops and three-way junctions highlighted with grey colour represent regions with variable number of nucleotides. Taxa highlighted with green colour refer to members of the B. arboricola clade.

Fig. 4

A partial secondary structure of ITS2 rRNA molecule of B. barbirostris showing four helices (H1–H4). All substitutions recorded among members of Barbatosphaeria are mapped on the 2D model. Parts of hairpin loops highlighted with grey colour represent regions with variable number of nucleotides. Legend to symbols and colours as in Fig. 3.

Fig. 5

Barbatosphaeria arboricola. a–c. Circular groups of ascomata, arrow indicates upper part of necks covered with pubescence; d. vertical section of the ascomatal wall; e. asci; f. ascospores; g. paraphyses (a–c. M.R. 3085; d–g. holotype PRM 924373). — h–l: Culture of sporothrix-like asexual morph; h. colony; i. conidia; j–l. conidiophores (ex-type culture CBS 127689, all on PDA at 25°C); d–f, l. DIC; g, i–k. PC. — Scale bars: a–c = 1000 μm, d = 50 μm, e–g, j–l = 10 μm, i = 5 μm.

Fig. 6

Barbatosphaeria barbirostris. a–c. Ascomata under periderm, arrow indicates upper part of the neck covered with pubescence; d. asci; e, f. ascospores (a, b from M.R. 3766; c from M.R. 649; d–f from M.R. 3767); d, f: DIC; e: PC. — Scale bars: a, b, c = 1000 μm, d = 10 μm, e, f = 5 μm.

Fig. 7

Barbatosphaeria dryina. a. Group of ascomata growing between periderm and wood on old stromata; b. vertical section of the ascomatal wall; c. ascospores; d. paraphyses; e, f. asci (a, b. M.R. 3717; c. M.R. 3111; d–f. M.R. 3072). — g–j: Culture of sporothrix-like asexual morph. g. colony; h–j. conidiophores; k. conidia (CBS 127691, all on PDA at 25°C); b, c, f. DIC; d, e, h–k. PC. — Scale bars: a = 1000 μm, b = 50 μm, c–k = 10 μm.

Fig. 8

Barbatosphaeria fimbriata. a, b. Ascomata growing between periderm and wood, young (a) densely covered with pubescence and old (b) glabrous; c. vertical section of the ascomatal wall, arrow indicates a superficial layer of interwoven hyphae; d. young asci with swollen bulbous base attached to ascogenous hyphae; e. asci; f. ascospores; g. paraphyses (a, c, d–g. holotype PRM 924374; b. M.R. 3694); c, e. DIC; d, f, g. PC. — Scale bars: a, b = 1000 μm, c = 50 μm, d–g = 10 μm.

Fig. 9

Barbatosphaeria neglecta. a. Ascomata; b. vertical section of the ascomatal wall; c, d. asci; e. young asci with swollen bulbous base attached to ascogenous hyphae; f. paraphyses; g. ascospores (holotype PRM 924375). — h–l: Culture of sporothrix-like asexual morph. h. colony; i, j. conidia; k, l. conidiophores (ex-type CBS 127693, all on PDA at 25°C); b, c, i, k. DIC; d–f, g, j, l. PC. — Scale bars: a = 1000 μm, b = 50 μm, c–f, k, l = 10 μm, g, i, j = 5 μm.

Fig. 10

Barbatosphaeria spp. a–i: Barbatosphaeria varioseptata. a. Ascomata growing between periderm and wood; b. vertical section of the ascomatal wall; c, d. ascospores; e. paraphyses with young asci attached to the ascogenous hyphae; f. asci (holotype PRM 924376). — g–i: Culture of sporothrix-like asexual morph. g. colony; h. conidiophores; i. conidia (ex-type CBS 137797, all on PDA at 25°C). — j–l: Barbatosphaeria sp. j, k. asci; l. ascospores (M.R. 3730). b–d, f, k, l. DIC; e, h–j. PC. — Scale bars: a = 1000 μm, b = 50 μm, c–f, h, j–l = 10 μm, i = 5 μm.