Introduction

The taxonomy of genera of foliar epiphytes is poorly known as they have not been well-studied. No molecular data is available for most genera and therefore an understanding of the higher level classification of these fungi is rather inadequate. We have, therefore, initiated a research program to collect and study these important taxa using morphology and phylogeny. Our initial study (Chomnunti et al. 2012) resulted in the transfer of the genus Trichomerium, previously placed in Capnodiaceae to Chaetothyriaceae in Chaetothyriales. We have also provided an account of Microthyriaceae (Wu et al. 2011) and are presently studying other genera of foliar epiphytes. Examples of foliar epiphyte genera with a sooty mold-like appearance are Aithaloderma, Capnodaria, Phragmocapnias and Scorias. Chomnunti et al. (2011) gave an account of the genera in Capnodiaceae, while Chomnunti et al. (2012) dealt with species of Chaetothyriaceae. The genus Trichomerium was placed in Chaetothyriaceae but no further data was provided (Chomnunti et al. 2011). Hughes and Seifert (2012) provided notes on the taxonomy and nomenclature of sooty mould names, but further work is required to resolve their interrelationship, especially at the molecular level.

Trichomerium was introduced by Spegazzini (1918) based on Trichomerium coffeicola (Puttemans) Speg. (1918) and it is estimated that the genus now includes 23 species (Kirk et al. 2008). Thirty-one names are listed in Index Fungorum. Trichomerium species are all foliar epiphytes, with superficial, setiferous, uniloculate ascostromata surrounded by loosely interwoven mycelium, with bitunicate asci and hyaline, septate ascospores (Spegazzini 1918). Batista and Ciferri (1963), Hughes (1976), Reynolds (1982), Reynolds and Gilbert (2005), Kwee (1988), Thaung (2006) and Chomnunti et al. (2011) have also commented on this genus.

Batista and Ciferri (1963) provided a key, descriptions and illustrations for several Trichomerium species and placed them in Capnodiaceae. They characterized the fruiting body (perithecium) as globose, long and sessile with scattered setae, aparaphysate and with 8-spored asci. Unfortunately, the key is hard to follow and the illustrations are sketchy and therefore it is very hard to understand Batista and Ciferri’s concept for the genus and its species. Hughes (1976) later transferred Trichomerium to Triposporiopsidaceae, but subsequent workers did not follow this arrangement. Triposporiopsidaceae is based on the genus Triposporiopsis which is now considered to be a species of Phragmocapnias and thus a synonym of Capnodiaceae. Reynolds (1982) re-examined all available collections and literature on Trichomerium and placed all species names as synonym under T. grandisporum (Ellis & G. Martin) Bat. & Cif., thus treating the genus as monotypic. The generic type, T. coffeicola was included as a synonym although it is not clear if Reynolds (1982) had examined the type material which has smaller ascospores than T. grandisporum. Recently, Chomnunti et al. (2011) transferred Trichomerium from Capnodiaceae to Chaetothyriaceae based on the morphology of the ascostromata and possession of trans-septate hyaline ascospores.

In this study, we re-describe the genus Trichomerium based on six specimens collected and examined from northern Thailand, including combined LSU and ITS rDNA sequence analysis. We have also examined type material of T. coffeicola var. macrosporum and describe three new species.

Material and methods

Isolates and morphology

Specimens of Trichomerium sp. on living leaves were collected from various localities and plants in northern Thailand, taken to the laboratory in zip-lock plastic bags and examined under the microscope for morphological characters. Single spore isolates were obtained following the method of Chomnunti et al. (2011) and colonies maintained on potato dextrose agar (PDA) at 28 °C. Cultures were used for a molecular study and deposited at Mae Fah Luang University Culture Collection (MFLUCC), BIOTEC Culture Collection (BCC) and International Fungal Research & Development Centre (IRFDC culture collection), the latter under material transfer agreement (MTA) No. 3/2010. The herbarium specimens are deposited at the Mae Fah Luang University (MFLU) Herbarium, Chiang Rai, Thailand.

DNA isolation, amplification and sequencing

Genomic DNA was extracted from fungal mycelium using Biospin Fungus Genomic DNA Extraction Kit (BioFlux®) according to the manufacturer’s protocol for maximum yield. Partial 28S rDNA and 5.8S rDNA regions were amplified using primers LROR and LR6 and ITS5 and ITS4, respectively (Vilgalys and Hester 1990, White et al. 1990). Amplification reaction mixtures contained 50 ng of template DNA, 1X PCR buffer, 0.5 μM of each primer in a 25 μL volume, 0.5 U of Taq DNA Polymerase, 400 μM of each dNTP, 3 mM of MgCl2. The PCR conditions were an initial denaturation at 94 °C for 5 min, followed by 35 cycles each consisting of 1 min denaturation at 94 °C, annealing for 30 s at 55 °C and extension at 72 °C for 1 min and a final extension at 72 °C for 5 min. PCR products were checked on 1 % agarose electrophoresis gels stained with ethidium bromide. The purified PCR products were then sequenced using the ABI-PRISM3730 DNA Analyzer (Applied Biosystems).

Phylogenetic analysis

DNA sequences were analyzed with available sequences of the Capnodiaceae, Chaetothyriaceae and Herpotrichiellaceae obtained from GenBank. Sequences were aligned using BioEdit (Hall 1999) and Clustal X v.1.83 (Thompson et al. 1997) and phylogenetic analysis were performed using PAUP* v. 4.0b10 (Swofford 2002). Ambiguous regions in the alignments were excluded from the phylogenetic analyses, gaps were treated as missing data. Maximum parsimony (MP) was performed with the heuristic search option on and addition of sequence using 1000 random with a stepwise starting tree, tree bisection and reconnection (TBR) as the branch-swapping algorithm. The parsimony scores including tree length (TL), consistency index (CI), retention index (RI) and homoplasy index (HI) were calculated. Clade stability was estimated in bootstrap (BT) analysis with 1000 replicates (Hillis and Bull 1993). Model of substitution used for Bayesian analyses was using MrModeltest 2.2 (Nylander 2004). The Bayesian analyses were performed in MrBayes 3.04b (Huelsenbeck and Ronquist 2001). Bayesian analyses were conducted with the Markov chains run from random starting tree for 1 000 000 generations and trees were sampled every 100 generations. The Markov Chain Monte Carlo (MCMC) algorithm was used to estimate posterior probabilities (PP) and obtained for each clade. Trees were visualized in TreeView (Page 1996). Details of sequences used are presented in Table 1. The LSU and ITS region were used in the phylogenetic analysis to determine generic or family placements. Venturia inaequalis was selected as outgroup. Sequences data are deposited in GenBank.

Table 1 LSU and ITS rDNA sequences included in this analysis, which were obtained from GenBank
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Results

Molecular phylogeny

The phylogenetic analysis includes representative sequences of Capnodiaceae and Chaetothyriaceae; the alignment of combined partial LSU and ITS rDNA comprised 73 taxa and 199 base pairs were excluded, the remaining 1131 included characters used in analysis, 767 characters were constant, 56 were variable characters are parsimony-uninformative and 308 were parsimony informative. A heuristic search found 100 equally parsimonious tree with the length (TL) of 1191 steps (CI = 0.472, RI = 0.846, RC = 0.399, HI = 0.528). All trees were similar in topology and not significantly different. A best scoring Maximum Parsimony tree is shown in Fig. 1. The phylogenetic tree obtained from Bayesian and maximum likelihood analyses is in agreement with a previous study based on MP analysis (Voglmayr et al. 2010). The six new Trichomerium strains formed a monophyletic group and clustered with nine strains of Chaetothyriales sp. associated with ants with 98 % of bootstrap support, but received a 100 % posterior probability (PP) in the Bayesian analysis (Clade A). The Trichomerium strains included the new species T. deniquelatum, T. foliicola and T. gloeosporum which clustered with high bootstrap support (89 % bootstrap support and 100 % PP). Other sequences of Chaetothyriales spp. associated with ants (CR08/2-2, CR08/2-1, CR07/3-2, M-Mo2) also grouped within clade A with 97 % of bootstrap support, but received a 100 % PP.

Fig. 1
figure 1

A Maximum parsimony tree obtained from a data set of 73 taxa including representatives of Chaetothyriales and Capnodiales, comprising two genes (LSU, ITS). The first set of numbers above the nodes are bootstrap values over 50 % and the second represents Bayesian posterior probabilities of more than 90 % and expressed as percentages. New sequences and types are in bold

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GenBank sequences for Coniosporium perforans, Glyphium elatum and Knufia cryptophialidica clustered in Clade B with strong support (100 % bootstrap support and 100 % PP). Species in Clade C are mostly teleomorphic genera of Chaetothyriales and form a monophyletic cluster (78 % bootstrap support and 100 % PP). Taxa in Clade D are members of Herpotrichiellaceae (anamorphic Chaetothyriales), some being human pathogens or rock inhabiting fungi. Clade E comprised 15 taxa of Capnodiaceae with five strains of Capnodiales isolated from ants (Voglmayr et al. 2010) and cluster together with strong support (100 % bootstrap support and 100 % PP). Phylogenetic dataclearly shows that Trichomerium belongs in Chaetothyriales and incorporates a strongly supported new family, Trichomeriaceae which is introduced below.

Trichomeriaceae Chomnunti & K.D. Hyde, fam. nov.

MycoBank 800935

Epiphytes on living trees or saprobes on honey dew insect excretions. The colonies are often mixed together with capnodiaceous taxa. Thallus comprised of mycelium on host surface with septate, brown hyphae. Ascostromata arise from mycelium mass, which is a subiculum, and are sessile, spherical, brown, uniloculate, ostiolate, surrounded by setae. Setae brown to dark brown or olivaceous, erect, straight or curved, septate or continuous. Peridium pale brown to brown or olivaceous, comprising several layers of cells of textura angularis. Asci apparently bitunicate, with an apical ring, cylindrical to clavate, with overlapping ascospores and an apical ring. Ascospores hyaline, septate, fusiform, round at ends, with or without a mucilaginous sheath.

Family type: Trichomerium Speg.

Trichomerium Speg. Physis, B. Aires 4: 284 (1918).

MycoBank 5560

Foliar epiphytes on living leaves or saprobes on honey dew insect excretions. Colonies often mixed with capnodiaceous taxa. Thallus arising from the compacted mycelium on the host surface, composed of septate, cylindrical, pale brown to brown hyphae. Ascostromata arise from the mycelial mass, which is a subiculum, and are sessile, globose to subglobose, brown, uniloculate, with a central ostiole, with setae surrounding the upper part. Setae brown to dark brown or olivaceous, erect, straight or curved, septate or continuous. Peridium pale brown to brown or olivaceous, comprising 2–3 layers of cells of textura angularis. Pseudoparaphyses indistinct. Asci bitunicate, with an apical ring, cylindrical to clavate, with overlapping ascospores. Ascospores hyaline, septate, fusiform, narrowly rounded at both ends, widest in the centre, with or without a mucilaginous sheath.

Anamorph: possibly Tripospermum Speg. (1918) (Kirk et al. 2008).

Typification details:

Trichomerium coffeicolum (Puttemans) Speg., Physis, B. Aires 4: 284 (1918).

≡ Limacinia coffeicola Puttemans, Cryptog. Mycol. 20: 163 (1904).

Trichomerium coffeicolum (≡ Limacinia coffeicola) was described by Puttemans (1904) from living leaves of Coffea arabica. A translation of the species diagnosis reads “Perithecium black, ovoid, truncate at the neck, covered by dark-sooty, simple, continuous, long and tapered setae, aparaphysate. Asci diversified, often elongated, 50–70 × 15–20 μm, with hyaline, sub-fusoid, 2-septate, 3-guttulate, 15–18 × 5–6 μm, irregularly arranged ascospores” (Fig. 2).

Fig. 2
figure 2

a–c Trichomerium coffeicola Puttemans. a Ascostromata with apical setae. b Asci. c 2-septate ascospores. (Redrawn from M.A. Puttemans 1904)

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Puttemans (1904) added, “…this species was common on the surface of the leaves. The mycelium is developed and surrounded with conidia and this seems identical to the mycelium and conidial forms of Capnodium. The genera lacked Triposporium and Limacinia forms. I still cannot confirm the relationship with Capnodium”.

We have tried to locate the type material of this species from URM and P but were not successful. Reynolds (1982) studied many specimens of Trichomerium Speg., including the type and concluded that the genus comprised a single species T. grandisporum and T. coffeicola was listed as a synonym. The description and drawings provided by Puttemans (1904) are informative and the generic concept for Trichomerium based on these illustrations of T. coffeicola is clear. In the protologue of Puttemans (1904) the ascospores are also smaller than those reported for T. grandisporum (15–18 × 5–6 versus 18–32 × 5–10 μm) and we doubt these taxa are the same species.

Here we therefore treat Trichomerium in the sense of T. coffeicolum based only on the protologue description and drawing. In this treatment T. deniquelatum, T. foliicola and T. gloeosporum are typical of Trichomerium and we suggest that it would be pragmatic to list T. foliicola as type of the genus in the Lists of Accepted Names to be developed by the subcommittee dealing with Dothideomycete names to be approved by the General Committee on Nomenclature (GCN) (Hawksworth 2012). This will ensure stability in application of the generic name. Trichomerium foliicola is supported by both herbaria and living material and, in addition, molecular sequence data. Of course if fresh collections of Trichomerium coffeicolum were found and sequenced this would not be necessary.

Trichomerium foliicola Chomnunti & K.D. Hyde, sp. nov. (Figs. 3a–l)

MycoBank 801117

Etymology: from the Latin foliicola meaning on living leaf, referring to various living host.

Black sheets of mycelia cover the leaves of the host and support dark brown superficial, gregarious ascostromata. The hyphae are septate, cylindrical, pale brown to brown with constrictions at the septa, mostly narrow; 3.5–7 μm wide (\( \overline x = 5.5\mu m \), n = 20). Ascostromata initials arising from a small irregular group of cells formed by the repeated division of a few hyphal cells, usually at a hyphal branches and becoming dense. Ascostromata 133–179 μm diam, 140–181 μm high (\( \overline x = 158 \times 162\mu m \), n = 20), subglobose to globose, brown, with abundant straight, aseptate to septate, dark brown to brown setae, up to 15 setae, mostly on the upper half of the ascostroma, (44–) 61–118 × 4–7 μm (\( \overline x = 83 \times 6\mu m \), n = 20) present on a single ascostromata. Ostiole central, ostiolar canal 32 μm wide at the base, 39 μm high, with an apical ring, with periphyses. Ascostroma wall 17–24 μm wide (\( \overline x = 20\mu m \), n = 20), thick-walled, inwardly hyaline, pale brown and brown towards the outside, comprised of 2–3 layers of textura angularis. Asci (47–) 63–70 × (14–) 20–26 μm (\( \overline x = 65 \times 22\mu m \), n = 10), 8-spored, ellipsoid to clavate, some obovoid, apparently bitunicate, with apical ring, aparaphysate. Ascospores 19–22 × 6–7 μm (\( \overline x = 21 \times 7\mu m \), n = 20) tri-seriate, hyaline with 2–3 septa, fusoid, with narrowly rounded ends.

Culture characteristics

Ascospores germinating on PDA within 12 h and germ tubes produce from both end cells and from the middle cell. Colonies growing slowly on PDA, reaching a diam of 2.5 cm. after 14 days at 28 °C. Mycelium initially black with dark green margin visible from both sides of the dish, colony on PDA velvety, radial towards the edge.

Material examined

THAILAND, Chiang Rai Province, Mae Ka Jan, on living leaf of Murraya paniculata, 11 October 2009, Putarak Chomnunti, DPC 042 (MFLU10−0007, holotype), ex-type living culture in MFLUCC10-0078= BCC40643; Ibid., 16 December 2009, Putarak Chomnunti (MFLU10-0683); Ibid., 7 March 2010, Putarak Chomnunti (MFLU10-0987). Ibid., Baan Du, on living leaf of Mangifera indica. 8 July 2009, Putarak Chomnunti, DPC 015 (MFLU09-0651), living culture in MFLUCC10-0054= BCC38853; Ibid., 2 September 2009, Putarak Chomnunti (MFLU09-0684); Ibid., 24 May 2011, Putarak Chomnunti (MFLU11-1151); Ibid., on living leaf of Psidium guajava, 25 October 2009, Putarak Chomnunti, DPC 020 (MFLU10-0002); living culture in MFLUCC10-0073= BCC41091; Ibid., 22 April 2010, Putarak Chomnunti (MFLU10-0988); Ibid., on living leaf of Phoenix dactylifera, 11 August 2009, Samantha Chandranath Karunarathna, DPC 023 (MFLU09-0656); living culture in MFLUCC10-0058= BCC39630; Ibid., 4 May 2010, Putarak Chomnunti (MFLU10-0989); Ibid.,20 August 2010, Putarak Chomnunti (MFLU10-0990); BRAZIL, Recife, Pernambuco, on leaves of Didymopanax morotoni, 4 February 1956, Severino José da Silva, URM 5302 (holoype); CUBA, Santiago de las Vesgas, in fragments of leaves of an unidentified host, 7 May 1913, F.L. Stevens (URM 13335); Ibid., on leaves of Sanchezia nobilis Hook., 19 January 1922, Charles and Ballon (URM 13483).

Notes: Trichomerium foliicola is similar to T. didymopanacis described from Didymopanax morototoni by Batista and Ciferri (1963) and referred to Capnodiales. Trichomerium didymopanacis was described as having epiphyllous colonies with superficial, black, septate mycelium, with non-setose, narrowed, globose, membranous ascostromata, and with a central ostiole. Ascostromata have numerous setae which are erect, straight or curved, brown, and with obtuse tips. Asci are ellipsoid, sessile, and aparaphysate. Ascospores are fusoid, with rounded ends, 1–3 septate and hyaline (Batista and Ciferri 1963). The drawing provided in the protologue however, is not detailed. We examined the type of T. didymopanacis (URM 5302), however the material was not in good condition and we could not find ascostromata. Although, the ascostroma, asci and ascospores in the description of T. didymopanacis provided in Batista and Ciferri (1963) overlap with those of T. foliicola (Table 2), it is not equivocal that these are the same species. We prefer to introduce a new species to avoid any confusion.

Table 2 Synopsis of characters of Trichomerium species mentioned in this study.
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Trichomerium deniqulatum Chomnunti & K.D. Hyde, sp. nov. (Figs. 4a–j)

MycoBank 800933

Etymology: from the Latin ‘denique’ meaning short, referring to short setae.

Black sheets of mycelia cover the leaves of the host which produce dark brown superficial, gregarious ascostromata. Mycelium superficial, septate, cylindrical, pale brown to brown hyphae, constricted at the septa; 5–7 μm wide (\( \overline x = 6\,\mu m \), n = 20). Ascostromata initials arise from a small irregular group of cells formed by the repeated division of a few hyphal cells young ascomata are brown and mixed with the hyphae. Ascostromata 154–175 μm diam, 163–180 μm high (\( \overline x = 165 \times 168\mu m \), n = 10), subglobose to globose, sessile, brown, with up to 5 setae around the upper half of the perithecium; setae sparse, indistinct, aseptate to septate, brown, straight, with cylindrical cells, (28–)32–54 × 3.7–6 μm (\( \overline x = 43 \times 5\mu m \), n = 20), aparaphysate. Ascostroma wall 12–15 μm wide (\( \overline x = 13\mu m \), n = 20) thick-walled, inwardly hyaline, brown towards the outside, comprised 2–3-layers of textura angularis. Asci 47–60 × 22–31 μm (\( \overline x = 54 \times 25\mu m \), n = 10), 8-spored, ellipsoid to clavate, some subglobose, apparently bitunicate with an apical ring, aparaphysate. Ascospores 18–25 × 6–8 μm (\( \overline x = 22 \times 7\mu m \), n = 20), tri-seriate, hyaline, fusoid, 3-septate, some with longitudinal septa, constricted at the septa, middle two cells wider and obliquely septate.

Culture characteristics

Ascospores germinating on PDA within 12 h and germ tubes arise from both end cells (Fig. 4j). Colonies growing slowly on PDA, reaching a diam of 4 cm after 14 days at 28 °C, velvety, radiating towards the edge. Mycelium initially black and dark green at the margin.

Fig 3
figure 3

a–l Trichomerium foliicola (holotype). a–c Ascostromata with ostiole and setae. d–f Vertical section through ascostromata. g Ascospores. h Ostiolar canal. i Peridium. j–l Asci and ascospores. Bars: a–f = 100 μm. k, l = 50 μm, h, i = 20 μm, g = 10 μm

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Material examined

THAILAND, Chiang Rai Province, Mae Fah Luang University, on living leaf of Psidium guajava. 12 September 2009, Putarak Chomnunti, DPC 039 (MFLU11-1150, holotype), ex-type living culture in MFLUCC10-0084= BCC40712; Ibid., 28 June 2010, Putarak Chomnunti (MFLU11-1152); Ibid., 11 October 2010, Putarak Chomnunti (MFLU11-1153)

Notes: Batista and Ciferri (1963) described several species of Trichomerium with sparse setae mostly on the upper part of ascomstromata including T. coffeicola, T. crotonis Bat., T. plumieriae Bat. & Cif., T. stuhlmannianum (Henn.) Bat. & Cif. and T. stuhlmannianum var. biseptatum Bat. & Cif. Trichomerium deniqulatum has few setae on the ascostromata and differs from others in size of setae and ascospores, the number of septa on the ascospores. T. deniqulatum differs from T. coffeicola in having shorter setae (43 × 5 versus 100 × 3–4 μm). In T. crotonis ascospores are 3–5 septate, while in T. deniqulatum ascospores are 2–3 septate. In T. plumieriae ascospores are slightly smaller (20 μm long versus 22 μm long) and in T. stuhlmannianum var. biseptatum ascospores are not more than 2-septate, while in T. deniqulatum, ascospores are 2–3-septate and with or without longitudinal septa. We considered short setae and ascospores with longitudinal septa as major characters to recognize the new species.

Trichomerium gloeosporum Chomnunti & K.D. Hyde, sp. nov. (Figs. 5a–m)

MycoBank 800934

Etymology: from the Latin gloeoid meaning slimy, referring to ascospore outer sheath.

Black sheets of superficial of mycelia cover the surface of leaves of the host. The hyphae are septate, cylindrical, pale brown to brown, constricted at the septa, 4–7 μm wide (\( \overline x = 5\mu m \), n = 20). Ascostromata initials arise from a small group of irregular cells formed by repeated division of a few hyphal cells, when young brown and mixed with hyphae. Ascostromata 116–140 μm diam, 113–150 μm high (\( \overline x = 128 \times 138\mu m \), n = 10), subglobose to globose, sessile, brown, with abundant setae, up to 10 setae around the upper half of the ascostromata; setae septate, brownish to dark brown or olivaceous, straight, with cylindrical cells, 71–121 × 4–7 μm (\( \overline x = 100 \times 6\mu m \), n = 20). Ascostroma wall 2–3 layered, 15–21 μm wide (\( \overline x = 18\mu m \), n = 20), thick walled, hyaline at the inner layers, brown at the outer layers, comprised of cells textura angularis. Asci 62–86 × 18–23 μm (\( \overline x = 75 \times 21\mu m \), n = 20), 8-spored, ellipsoidal to cylindrical, with short pedicel, apparently bitunicate, with an apical ring, aparaphysate. Ascospores 17–26 × 5–7 μm (\( \overline x = 22 \times 6\mu m \), n = 25), bi-seriate, hyaline, fusoid, 2–3-septate, not constricted at the septa, narrowly rounded at the ends, with a conspicuous mucilaginous sheath.

Culture characteristics

Ascospores germinating on PDA within 12 h and germ tubes produced from both end cells. Colonies growing slowly on PDA, reaching a diam of 3 cm after 14 days at 28 °C. Mycelium initially black with dark green margins, colony on PDA velvety, radial toward to the edge.

Fig. 4
figure 4

a–j Trichomerium deniqulatum (holotype). a–b Mycelium with immature ascostromata. c Vertical section through ascostromata. d Peridium. e–g Asci with apical ring in g (right ascus). h–i Ascospores. j Germination of ascospore. Bars: a = 100 μm. b, c, e = 50 μm, d, f, g, h = 20 μm, i, j = 10 μm

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Material examined

THAILAND, Chiang Rai Province, Muang District, near Baan Du, Ban Kua Krae, Ficus sp. tree in rice field, on living leaf, 4 October 2010, K.D. Hyde, DPC 051 (MFLU10-0016, holotype), ex-type living culture in MFLUCC10-0087; Ibid., 30 January 2011, Putarak Chomnunti (MFLU11-1154).

Notes: Trichomerium gloeosporum is distinct from hitherto described species in the genus in having ascospores with a distinct mucilaginous sheath. Conidia of a Trichomerium sp. were associated with the sooty mould, but we could not establish that they were related to the sexual morph. We collected material of a Trichomerium sp. from Phoenix dactylifera but unfortunately the specimen had too few of ascostromata to derive any valid morphological description. However, we derived a pure culture of the fungus from the specimen and carried out phylogenetic analysis. The culture did not produce any reproductive structures and therefore, at this stage, only the culture and sequence data are available. The sequence data is 95 % similar to T. gloeosporum and also very close to T. foliicola, hence accommodated under this species but further collections are needed to establish its identity.

Fig. 5
figure 5

a–m Trichomerium gloeosporum (holotype). a Sooty mould on living leaf of Ficus sp. b Ostiole. c Vertical section through ascostromata. d Peridium. e Setae. f Conidia. g–i Asci. j–m Ascospores. Bars: c, d, g = 50 μm, b, e, f, h, i = 20 μm, j–m = 10 μm

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Discussion

In this study, we report on the morphology and sequence data for six freshly collected strains of Trichomerium isolated from Thailand. The strains are described as the new species T. foliicola (4 strains), T. deniqulatum (1 strain) and T. gloeosporum (1 strain), based on phylogenetic and morphological data. The phylogenetic data show that the three species belong to Chaetothyriales and cluster with Chaetothyriales spp. from ant nests chambers (Voglmayr et al. 2010). They are not closely related to Chaetothyriaceae, Herpotrichiellaceae or Capnodiaceae. Consequently, a new family, Trichomeriaceae, typified by sessile, setiferous ascostromata, with ostiolate, aparaphysate ascostromata, bitunicate asci with an apical ring and 2–3–septate to trans-septate, hyaline, ascospores with or without a sheath, is introduced. Ascostromata with a mycelial cover, asci with an apical ring and trans-septate and sheathed ascospores are not known in Capnodiaceae. We treat Trichomerium in the sense of T. foliicola as the holotype specimen of T. coffeicola, the generic type is unavailable and also no molecular data exists for this species.

Reynolds (1982) examined several species of Trichomerium, clumped them under T. grandisporum and considered the genus to be monotypic. We have not followed Reynolds (1982) approach, as we believe the concept for Trichomerium as based on the generic type of Limacinia coffeicola Puttemans (1904) is clear. Furthermore, we accept four species in the present study and we believe that further studies of types and fresh collections will show the genus to be more speciose. Reynolds and Gilbert (2005) assigned the genus Trichomerium based on his concept of Trichomerium grandisporum (Ellis & G. Martin) Bat. & Cif. to Capnodiaceae using molecular sequence data (unpublished), which otherwise clustered with a black yeast clade (Berbee 1996). Recently, Chomnunti et al. (2011) excluded Trichomerium from Capnodiaceae on the basis of ascostromata and trans-septate hyaline ascospores and transferred the genus to Chaetothyriaceae. In this study, we diagnose Trichomerium based on morphological characteristics and DNA sequence data and accommodate the genus in a new family Trichomeriaceae in Chaetothyriales.