Reflections on Menisporopsis, Multiguttulispora and Tainosphaeria Using Molecular and Morphological Data

The genera Menisporopsis, Multiguttulispora and Tainosphaeria (Chaetosphaeriaceae) are saprobes inhabiting decaying plant material. This study is based on an integrated morpho-molecular characterisation to assess their generic concepts and explore phylogenetic relationships. Menisporopsis is revealed as polyphyletic, and species with 1-septate conidia and synnemata growing unilaterally along the seta are placed in the new segregate genus Arcuatospora. Codinaea dimorpha and C. triseptata are shown to be congeneric with Multiguttulispora sympodialis, the type species. Two new combinations are proposed: M. sympodialis is found conspecific with M. dimorpha. The Tainosphaeria complex is resolved into three genera. We found that the morphological separation of three groups within the genus is consistent with phylogenetic relationships. Tainosphaeria s. str. is accepted with five species. Tainosphaeria aseptata and T. lunata are transferred to the newly erected Phialoturbella, whereas T. obclavata is revealed as conspecific with Phialogeniculata guadalcanalensis, reducing it to a synonym. A new genus Flectospora is erected for a chloridium-like fungus nested in the Tainosphaeria clade. Based on molecular evidence, we show that asymmetrical, scolecosporous ascospores are a unique teleomorphic characteristic among family members. Therefore, we propose new combinations for Chaetosphaeria hispida in Paragaeumannomyces and Ch. spinosa in the new genus Ericiosphaeria, both exhibiting this rare morphotype.


Introduction
The present paper is concerned with the phylogeny and taxonomy of Menisporopsis [1], Multiguttulispora [2] and Tainosphaeria [3], classified in the Chaetosphaeriaceae. They occur mainly as dematiaceous hyphomycetous anamorphs, inhabiting leaf litter and decaying bark and wood. New morphological and DNA sequence data indicate that Menisporopsis and Tainosphaeria are associated with considerable morphological heterogeneity and genetic diversity. However, Multiguttulispora, though narrowly delimited, shows an undescribed affinity with known species.
The genus Menisporopsis, with M. theobromae as the type species, was established for fungi with pigmented, synnematous conidiophores growing around a central seta [1]. The conidiogenous cells are mono-or polyphialidic, producing aseptate, falcate, allantoid and fusiform hyaline conidia, with one to several setulae inserted at the ends or irregularly. Its members inhabit leaf litter, and occasionally decaying wood of various host plants in tropical and subtropical geographic areas [1,2,[4][5][6][7]. The teleomorph is known only for M. kobensis [7]. Although most of the 13 species described in Menisporopsis [8] are congeneric with the type species, two species deviate. Menisporopsis novae-zelandiae has 1-septate conidia and synnemata are formed unilaterally along the seta [9], while M. ludoviciana is characterised by bacilliform conidia without setulae and single or fasciculate setae with a Table 1. Taxa, isolate information and GenBank accession numbers for sequences. New sequences determined for this study and taxonomic novelties are given in bold. Morphological characteristics were acquired from fungi growing on natural substrates and in culture. Ascomata, conidiophores and conidia from the natural substrates were rehydrated with tap water and examined with an Olympus SZX12 dissecting microscope (Olympus America, Inc., Melville, NY, USA). Sections of ascomatal wall, asci, ascospores and paraphyses, and conidiophores and conidia were mounted in 90% lactic acid, water or Melzer's reagent. Measurements were taken in Melzer's reagent. Means ± standard deviation (SD) based on a minimum of 20-25 measurements were given for sizes of asci, ascospores and conidia. Microscopic structures were examined using an Olympus BX51 compound microscope with differential interference contrast (DIC) and phase-contrast (PC) illumination. An Olympus DP70 camera operated by Imaging Software CellˆD (Olympus) were used to capture images of microscopic structures. Macroscopic images of colonies were documented using a Canon EOS 77D digital camera with Canon EF 100 mm f/2.8L Macro IS USM objective (Canon Europe Ltd., Middlesex, UK) with daylight spectrum 5500K 16W LED lights. All images were processed with Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA).
Single and multiple ascospore and conidial isolates were obtained from fresh material with the aid of a single-spore isolator (Meopta, Přerov, Czech Republic) and incubated on water agar or Modified Leonian's agar (MLA) [23] at a temperature of 20-25 • C. For comparison, strains were inoculated in triplicate on cornmeal dextrose agar (CMD) (Oxoid Limited, Hampshire, UK; 2% dextrose), MLA, oatmeal agar (OA) and potato-carrot agar (PCA) [24]. Descriptions of colonies were based on 4-week-old cultures grown in darkness at 22-23 • C. Strains were also inoculated on cornmeal agar (CMA) with sterile stems of Urtica dioica and synthetic nutrient agar (SNA) with pine needles [24] to induce sporulation.
Total genomic DNA was extracted from mycelium removed from 3-4-wk-old cultures grown on MLA using the DNeasy ® UltraClean ® Microbial Kit (Qiagen GmbH, Hilden, Ger-many) following the manufacturer's protocol for filamentous fungi. All PCR amplifications were carried out in 25 µL volume reactions using a Q5 High Fidelity DNA polymerase kit (New England Biolabs Inc., Hitchin, UK) according to the manufacturer's protocol.
Primers V9G/LR8 [25,26] were used for amplification of the internal transcribed spacer region (ITS1-5.8S-ITS2) (ITS) and the nuclear large subunit 28S rDNA gene (28S) (approximately 1800 base pairs at the 5 -end). PCR was carried out in a BioRad C1000 thermal cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) as follows: 98 • C for 30 s; 40 cycles of denaturation (98 • C for 10 s), annealing (62 • C for 30 s) and elongation (72 • C for 90 s) and a final extension step (72 • C for 5 min). Amplicons were purified from agarose gels using a NucleoSpin ® Gel and PCR Clean-up Kit (Macherey-Nagel GmbH & Co. KG, Dueren, Germany) following the manufacturer's instructions, with an elution volume of 25 µL. The DNA concentration was assessed fluorimetrically using Quant-iT PicoGreen dsDNA Assay Kit and Qubit fluorometer (Invitrogen/Thermo Fisher Scientific Inc., Waltham, MA, USA) to assure the required sequencing concentrations adjusted for the length of amplicons/number of reads required.
Consensus secondary structure (2D) models for the ITS1 and ITS2 for members of the Chaetosphaeriaceae were built using the Ppfold program v3.0 [50]. The obtained 2D consensus models were further improved using the program Mfold [51] and RNAfold web server through the ViennaRNA Web Services [52,53] and adjusted manually if necessary, based on a comparison of homologous positions in the multiple sequence alignment. The predicted 2D RNA structures were obtained in a dot bracket notation and were visualized and drawn using the program VARNA: Visualization Applet for RNA [54].
The ITS-28S for newly sequenced taxa and those retrieved from GenBank and NBRC online catalogue were added to the combined ITS-28S alignment. Sequences were aligned manually in Bioedit v7.1.8 [55]. Consensus 2D structure models for the ITS1 and ITS2 were used to compare nucleotides at homologous positions (in helices and loops) in order to construct a reliable multiple sequence alignment. A predicted 2D model of the 28S of Saccharomyces cerevisiae [56] was used to improve the alignment of this gene. The models were highly consistent in all species. Initially, we considered applying Gblocks [57] to determine and remove putative ambiguous areas in the alignment. However, following a study of the Chaetosphaeriaceae by Réblová et al. [20], who tested performance of the alignment improved with 2D structure with and without Gblocks, we included the whole ITS-28S alignment except for 91 nucleotides (nt) at the 5 -end of 28S, because of the incompleteness in the majority of sequences.
The ITS and 28S datasets, for which we assumed rate heterogeneity, were evaluated using PartitionFinder2 [58], implemented in the CIPRES Science Gateway v3.3 [59,60], to find the best partitioning scheme for our datasets and to select best-fit models under corrected Akaike information criteria. The GTR+I+G model was selected for both partitions. The ITS and 28S data sets were concatenated, and the alignment (deposited in TreeBASE 28300) was subjected to phylogenetic analysis. The full dataset consisted of 2386 characters including gaps (ITS = 614 characters; 28S = 1772) and 1048 unique character sites (RAxML). Tracylla aristata and T. eucalypti (Tracyllales) were selected as outgroup taxa.
Phylogenetic reconstructions were performed using Bayesian Inference (BI) and Maximum Likelihood (ML) analyses through the CIPRES Science Gateway v.3.3. ML analysis was conducted with RAXML-HPC v8.2.12 [61] with a GTRCAT approximation. Nodal support was determined by non-parametric bootstrapping (BS) with 1000 replicates. BI analysis was performed in a likelihood framework as implemented in MrBayes v3.2.6 [62]. Two Bayesian searches were performed using default parameters. The B-MCMCMC analyses lasted until the average standard deviation of split frequencies was below 0.01 with trees saved every 1000 generations. The first 25% of saved trees, representing the burn-in phase of the analysis, were discarded. The remaining trees were used for calculating posterior probabilities (PP) of recovered branches. The BI and ML phylogenetic trees were compared visually for topological conflict among supported clades.

Phylogenetic Analyses
Phylogenetic analysis was based on the combined ITS-28S sequences of 109 members of the Chaetosphaeriaceae. The phylogenetic trees generated by BI and ML analyses were largely congruent; they only slightly varied in the topology of statistically unsupported clades. In the ML and BI analyses, nodes with support values of ≥75% ML BS and ≥0.95 BI PP were considered well-supported. The ML tree is shown in Figure 1. The Chaetosphaeriaceae included 50 lineages representing genera or natural groups of species. Menisporopsis was resolved as two separate, well-supported clades. The lineage containing M. theobromae and the other four Menisporopsis species represents the core of the genus (97/1.0). Two strains of Menisporopsis novae-zelandiae from Venezuela (CBS 109474, CBS 1094746), two morphologically similar isolates from Thailand (CBS 147509, CBS 147510) and strains from Japan (CBS 694.74) and Nepal (MUCL 43189) clustered as another lineage (100/1.0). They are proposed as the new genus Arcuatospora. The Tainosphaeria complex includes five lineages that correspond to particular morphologies, namely Anacacumisporium, Phialogeniculata, Tainosphaeria s. str., and two unnamed clades described as new genera, Phialoturbella and Flectospora, below. The Tainosphaeria s. str. subclade (100/1.0) includes T. crassiparies, T. jonesii, T. monophialidica, T. siamensis and a new species, T. cecropiae (CBS 101687). Two former Tainosphaeria species, T. aseptata and T. lunata, and the isolate ICMP 23826 formed a distinct subclade (90/1.0) that is introduced as Phialoturbella. Tainosphaeria obclavata (=Phialogeniculata guadalcanalensis) was resolved as a basal lineage to Phialoturbella. A chloridium-like dematiaceous hyphomycete represented by the strain CBS 112964 and another strain ICMP 23840 nested in the Tainosphaeria clade as monophyletic lineage (95/1.0), are introduced here as the new genus Flectospora. The genus Chloridium s. str. was resolved as a separate, well-supported clade (95/1.0). Two   Phylogenetic tree based on the combined ITS-28S rDNA sequences constructed by maximum likelihood (RAxML) of selected members of the Chaetosphaeriaceae. Species names given in bold and placed in green boxes are taxonomic novelties. T, E, I, N and P indicate ex-type, ex-epitype, ex-isotype, ex-neotype and ex-paratype strains; asterisk (*) indicates ex-type of Tainosphaeria obclavata (=Phialogeniculata guadalcanalensis). Thickened branches indicate branch support with ML BS = 100% and PP values = 1.0. Branch support of nodes ≥ 75% ML and ≥0.95 PP is indicated above or below branches. (B) Phylogenetic tree based on the combined ITS-28S rDNA sequences of the Chaetosphaeriaceae (continued). For legend refer to (A). Abbreviation: p.p. after a genus name (pro parte). Description: Colonies on the natural substrate effuse, hairy, mycelium superficial, composed of setose synnemata. Anamorph: Setae erect, straight, arise singly from a discoid, pseudoparenchymatous subiculum, dark brown to black, opaque, thick-walled, paler and thinner-walled at the apex, apex sterile, broadly rounded, occasionally terminating into a phialide. Conidiophores macronematous, synnematous, closely bound, parallel, unbranched, brown, synnemata arise around the base of the seta, surrounds the seta, diverge from it towards their apices and become unilateral. Conidiogenous cells integrated, terminal, mono-or polyphialidic, extending percurrently and sympodially, paler than the conidiophores; collarettes subhyaline, cup-shaped or funnel-shaped. Conidia falcate, slightly truncate at the base with a basal scar, 1-septate, hyaline, with a straight or gently curved setula at each end, inserted terminally at the apex, subterminally at the base, conidia accumulate in slimy fascicles. Teleomorph: not observed.
DNA sequence data of six strains initially identified as A. novae-zelandiae suggest it is a species complex with three cryptic species, i.e., A. seorsa, introduced for the long-spored variant, and two Arcuatospora spp. CBS 694.74 and MUCL 43189 treated as separate species. Arcuatospora novae-zelandiae with shorter conidia from Brazil [70], for which DNA data are not available, probably represents another undescribed species based on morphology. Based on many published records, A. novae-zelandiae appears to be a common species with a widespread geographical distribution in subtropical and tropical regions. Because of the discovered genetic variability of the A. novae-zelandiae species complex, these records need to be verified using molecular data.
Habitat and geographical distribution: Saprobe on leaf litter, known only in Japan. Note: The strain CBS 694.74 sporulated weakly only on Urtica stems on CMA. Due to the lack of morphological characteristics such as synnemata and setae, this strain is accepted as Arcuatospora sp. 1 and distinguished from other species by DNA data. In the phylogenetic tree, it grouped as a sister to the Arcuatospora sp. 2 MUCL 43189.
Habitat and geographical distribution: The substrate and host are unknown; the strain originates from Asia, Nepal.
Note: Although the strain MUCL 43189 shares with A. novae-zelandiae morphological characteristics of conidiophores, phialides and conidia, the phylogenetic analysis did not support their close relationship. When grown in culture, MUCL 43189 did not form setae, not even after prolonged incubation. In the conidial size, this strain corresponds to A. novae-zelandiae, although the conidia were slightly shorter and narrower in their lower range. In the absence of morphological traits such as setae, the isolate MUCL 43189 is referred to as Arcuatospora sp. 2 in this study. More material is needed to study this species under natural conditions.  [3]). Teleomorph: Ascomata perithecial, non-stromatic, superficial, solitary or in small groups, globose to ovoid, papillate, dark brown to nearly black, setose. Setae rigid, dark brown, opaque, simple, acute, aseptate, never conidiogenous. Ostiolar canal periphysate. Ascomatal wall fragile, carbonaceous, two-layered. Paraphyses persistent, branching, hyaline, septate, longer than asci. Asci unitunicate, cylindrical-clavate, with a non-amyloid apical annulus, eight-spored. Ascospores cylindrical to filiform, straight, sometimes bent to sigmoid, asymmetrical, rounded at the apical end, tapering towards the basal end, aseptate (probably transversely septate), hyaline, without mucilaginous sheath or appendages, arranged 3-4-seriately or in a fascicle within the asci.
Habitat and geographical distribution: Saprobes on decaying wood, known in the USA [3].
Note: The genus Ericiosphaeria is introduced for fungi with minute, dark ascomata covered by short, opaque, acute setae, two-layered ascomatal wall, scolecosporous, hyaline ascospores and anamorphs with phialidic conidiogenesis. The new genus is typified with E. spinosa, previously classified in Chaetosphaeria [3].
Ericiosphaeria is remarkably similar to Paragaeumannomyces [7,20] in the characteristics of asci, ascospores and setae, which are never conidiogenous, but differs in anatomy of the ascomatal wall. Ericiosphaeria has the wall two-layered, dark brown and carbonaceous compared to Paragaeumannomyces with three-layered ascomatal wall. The outer wall is usually coloured, ranging from white, yellow-white, ginger to reddish-brown or occasionally dark brown and is composed of globose to angular cells. The middle layer, on the other hand, is the typical 'chaetosphaeriaceous' ascomatal wall, which is melanized and composed of brown, brick-like cells. Both genera are comparable in the anamorphic characteristics, so far observed only in culture. Paragaeumannomyces has been linked with a craspedodidymum-like and chloridium-like synanamorphs [72], while Ericiosphaeria forms a chloridium-like anamorph [3]. Both genera were resolved as closely related taxa.  [3].
Habitat and geographical distribution: Saprobes on decaying wood, known so far in New Zealand and Thailand.
Flectospora laminata was initially considered the chloridium-like anamorph of Chaetosphaeria hispida [19], characterised by scolecosporous, septate, hyaline ascospores and ascomata covered with short, opaque spines. The holotype of Ch. hispida contains conidiophores of the chloridium-like fungus growing near the ascomata. Identical, sporulating conidiophores were obtained in culture (strain CBS 112964), from which the DNA sequences were derived. However, phylogenetic analysis of ITS-28S sequences, including the chloridium-like anamorph, E. spinosa, members of Paragaeumannomyces and other morphologically similar species, have raised suspicions that the chloridium-like fungus is not the anamorph but likely a contamination. The isolation into axenic culture occurred during the fieldwork in the Khao Yai forest in Thailand. Although the strain was derived from ascospores, the conidia were probably removed from the natural material by accident.
Although Ch. hispida, E. spinosa and Paragaeumannomyces are remarkably similar in characters to ascomata, asci and especially ascospores, they grouped in distantly related clades. The ex-type strain of E. spinosa S.M.H. 2754 and Paragaeumannomyces spp. clustered in one clade, while the ex-type strain of Ch. hispida CBS 112964 nested in the Tainosphaeria clade. It is difficult to reconcile such different teleomorphic features with the revealed phylogenetic relationships. Known teleomorphs of the Tainosphaeria clade have glabrous ascomata and symmetrical, ellipsoidal to fusiform, 0-several-septate ascospores ( [3], this study). The results of the phylogenetic analysis prompted the revision of the holotype of Ch. hispida and has been found to be a species of Paragaeumannomyces [7,20]. Based on the phylogenetic arguments and critical evaluation of teleomorphic and anamorphic characteristics, we conclude that the chloridium-like presumed anamorph of Ch. hispida does not belong to its life cycle. Therefore, the hyphomycete growing in the holotype of Ch. hispida is transferred to the new genus Flectospora as F. laminata and a new combination for Ch. hispida in Paragaeumannomyces is proposed below.
Characteristics in culture: On CMD colonies 20-22 mm diameter, circular, flat, margin rhizoidal, lanose, funiculose centrally, white-beige, with a pale ochre outer zone of submerged growth, reverse dark beige. On MLA colonies 22-27 mm diameter, circular, raised, margin entire, lanose, floccose, cobwebby towards the periphery, whitish to mouse grey with an ochre outer zone of submerged growth, pale ochre pigment diffusing into agar, reverse ochre. On OA colonies 12-13 mm diameter, circular, flat, margin entire to weakly undulate, cobwebby, white to ochre-beige with irregular dark grey spots due to aggregated sporulating conidiophores, with an olivaceous ochre outer zone of submerged growth, reverse pale ochre-beige. On PCA colonies 17-20 mm diameter, circular, circular, flat, margin entire, lanose to cobwebby, ochre-beige an olivaceous ochre outer zone of submerged growth, reverse of the same colours. Sporulation was abundant on MLA and PCA, sparse on CMD and OA.
Habitat and geographical distribution: Saprobe on decaying wood, known only in Thailand.
Note: This species is tentatively placed in Flectospora; in the phylogenetic analysis it grouped as a sister to F. laminata in a strongly supported clade. It is characterised by dark, glabrous, rostrate ascomata, ellipsoidal, hyaline, 0-1-septate ascospores and apricot pigment diffusing into the agar. Although anamorphs of the Tainosphaeria clade are conspicuous dematiaceous phialidic hyphomycetes such as Anacacumisporium [18], Phialogeniculata [16,17], Phialoturbella (this study) and Tainosphaeria [3], which readily sporulate in culture, the anamorph of the present species is unknown. On the natural substrate only ascomata occurred and the culture derived from ascospores remained sterile. The species is included in the study to further characterise the emerging Tainosphaeria clade. Emended description: Colonies on natural substrate effuse, hairy, mycelium partially superficial, partially immersed, composed of conidiophores. Anamorph: Setae absent. Conidiophores macronematous, mononematous, solitary or arise in groups of 2-3, straight or flexuous, unbranched, septate, smooth, dark brown, opaque and thick-walled, paler and thinner-walled towards the apex, with several lateral phialidic openings or fertile zones consisting of aggregated lateral openings along the conidiophore axis. Conidiogenous cells integrated, terminal, mono-and polyphialidic, extending sympodially over a short distance, cylindrical, paler than the conidiophore, often with persistent remnants of the collarettes; collarettes funnel-shaped, hyaline to subhyaline. Conidia ellipsoidal to oblong to ellipsoidal-fusiform, with an inconspicuous basal scar, transversely septate, hyaline, with a gently curved setula at each end, conidia accumulate in slimy fascicles. Synanamorph (formed only in culture): Setae absent. Conidiophores semi-macronematous, mononematous, pale brown, mostly reduced to single conidiogenous cells, which are monophialidic, integrated, terminal, pale brown, collarettes funnel-shaped. Conidia falcate-fusiform to navicular, with a basal hilum, aseptate, hyaline, without setulae, accumulate in colourless slimy masses. Teleomorph: not observed.
Habitat and geographical distribution: The holotype of M. dimorpha was isolated from the air in Japan, and other records confirm members of Multiguttulispora as saprobes on decaying plant material or endophytes. Species have a widespread geographical distribution in the tropical and temperate zones of Asia, Caribbean, Micronesia and North and South America [2,21,22,65,[74][75][76].
Note: Lin et al. [2] introduced Multiguttulispora, typified with M. sympodialis, for dematiaceous hyphomycetes with macronematous conidiophores terminating in polyblastic, sympodial conidiogenous cells and septate, hyaline conidia with setulae. In the present phylogeny, the genus forms a monophyletic lineage containing two species, M. dimorpha and M. triseptata. Comparison of morphological characters and DNA sequences revealed M. sympodialis conspecific with Codinaea dimorpha [21] and its close relationship to C. triseptata [22]. Based on the phylogenetic evidence and morphological data, both Codinaea species are transferred to Multiguttulispora, and M. sympodialis is reduced to the synonymy of M. dimorpha.
Multiguttulispora is delimited to fungi with ellipsoidal to oblong to ellipsoidal-fusiform, hyaline, 3-septate conidia with a basal hilum and a gently curved setula at each end. The phialides extend sympodially over a very short distance; the phialidic apertures become densely aggregated at the tip of the conidiogenous cells and give the phialide a somewhat geniculate appearance. This feature is prominent in M. dimorpha. Solitary lateral openings or small fertile zones comprising several clustered phialidic openings are spread in irregular intervals along the conidiophore axis. The collarettes are easily detached and can remain attached to the bottom of the released conidia. Colonies of the analysed strains on the four growth media are compared in Figure 8.    Characteristics in culture: On CMD colonies 25-30 mm diameter, circular, flat, margin fimbriate, cobwebby becoming mucoid, isabelline with a white tinge, with a prominent outer zone of submerged growth, reverse pale ochre-isabelline. On MLA colonies 45-47 mm diameter, circular, raised, margin fimbriate, velvety, floccose, wrinkled, appearing powdery, mucoid towards the margin, furrowed, white with irregular yellow-orange spots, with ochre to golden outer zone of submerged growth, pale yellow pigment diffusing into the agar, reverse yellow-orange with irregular cinnamon spots. On OA colonies 38-40 mm diameter, circular, flat, margin lobate, velvety, locally mucoid, zonate, deep orange-yellow with a pale peach tinge becoming peach-pink centrally, white towards the periphery, pale peach pigment diffusing into the agar, reverse peach. On PCA colonies 52-53 mm diameter, circular, flat, margin undulate, cobwebby becoming mucoid, pinkbeige, reverse pale pink-beige. Sporulation was absent on CMD, MLA, sparse on PCA and OA after prolonged incubation.
Habitat and geographical distribution: Saprobe on decaying wood, leaves or fruits of Inga sp. and other unknown hosts, also isolated from the air. It is known in Japan, Malaysia, Peru and Thailand [2,21,65,76].
Note: Lin et al. [2] described this species as M. sympodialis from a decaying fruit in Thailand, strain MFLUCC 18-0153. Another conspecific strain CBS 140002 examined in this study is from a twig of Eucalyptus sp. in Malaysia (as Dictyochaeta triseptata) [76]. The latter strain corresponds to the protologue of Codinaea dimorpha [21] in all details except that the conidiophores were longer due to numerous sympodial extensions. Codinaea dimorpha was originally isolated from the air in Kobe in Japan [21]. The type strain (NHL 2891) is not available, but another strain of C. dimorpha NBRC 33230 (=IFO 33230 = KIH C 0300) also isolated from the air was deposited by N. Toyazaki, one of the co-authors of C. dimorpha. Although the culture of NBRC 33230 is currently unavailable, its and 28S rDNA sequences publicly accessible [30], were included in our study. Based on the results of the phylogenetic analysis and comparative morphology, M. sympodialis (MFLUCC 18-0153) and C. dimorpha (NBRC 33230, CBS 140002) are revealed as conspecific. Therefore, a new combination is proposed for C. dimorpha in Multiguttulispora and M. sympodialis is reduced to synonymy. Conidia on the natural substrate were recorded shorter 15.8-20.9 × 6-8.3 µm (MFLUCC 18-0153, [2]) than conidia from the culture 22-27.5 × 6-7 µm (CBS 140002, this study) and 22-28 × 7-8 µm (NHL 2891 ex-type, [21]).
Multiguttulispora dimorpha produces brightly coloured pigments in vitro. On MLA, the species formed a conspicuous ochre to golden outer zone of submerged growth and yellow pigment diffused in the agar ( Figure 9J). On OA, peach-pink pigment was formed in older colonies ( Figure 9K). Distinct pigments produced in vitro have been reported by other authors. Toyazaki and Udagawa [21] observed deep yellow or deep orange pigment on OA. Crous et al. [76] [21]). Multiguttulispora dimorpha produces brightly coloured pigments in vitro. On MLA, the species formed a conspicuous ochre to golden outer zone of submerged growth and yellow pigment diffused in the agar ( Figure 9J). On OA, peach-pink pigment was formed in older colonies ( Figure 9K). Distinct pigments produced in vitro have been reported by other authors. Toyazaki and Udagawa [21] observed deep yellow or deep orange pigment on OA. Crous et al. [76] 8. 1986. Characteristics in culture: On CMD colonies 47-48 mm diameter, circular, flat, margin fimbriate, lanose, cobwebby at the margin, white with ochre tinge when aerial hyphae are sparse, with an orange-beige to olivaceous beige outer zone of submerged growth, pale ochre-pink pigment diffusing into the agar, reverse dark amber to dark brown. On MLA colonies 45-46 mm diameter, circular, flat with raised margin, margin entire to weakly fimbriate becoming curled, lanose, floccose, mucoid at the margin, finely furrowed, white with irregular yellow-orange patches, with an ochre to pale cinnamon outer zone of submerged growth, yellow-ochre pigment diffusing into the agar, reverse dark brown centrally, deep apricot towards the margin. On OA colonies 53-55 mm diameter, circular, flat, margin entire, velvety-lanose, white, olivaceous beige to olivaceous grey towards the periphery, white at the margin, locally with minute, ochre patches of aerial mycelium bearing ochre exudates, ochre to cinnamon pigment diffusing into the agar, reverse cinnamon to brown, ochre towards the margin. On PCA colonies 50-51 mm diameter, circular, flat, margin fimbriate, velvety, mucoid, smooth at the margin, white-grey, fawn towards the periphery ochre pigment diffusing into the agar, reverse dark brown. Sporulation was delayed (>8 weeks), moderate on MLA and PCA, absent on CMD and OA.
Habitat and geographical distribution: Saprobe on dead leaves and decaying fruits or as a leaf endophyte of Mucuna urens, Musa sp. and other unidentified hosts, known in Cuba, Guyana, Peru, Pohnpei and USA ( [22,74,75], this study).
Multiguttulispora triseptata closely resembles M. dimorpha in conidial characteristics. Their conidial size overlaps considerably, which makes identification based on this sole character challenging. Multiguttulispora triseptata differs especially in mono-sometimes polyphialidic conidiogenous cells with only a few apertures and the absence of fertile zones of aggregated phialidic openings. Instead, single intercalary phialidic apertures occur along the upper part of the conidiophore.
Specimen examined: THAILAND, Nakhon Nayok Province, Khao Yai National Park, Bung Phai trail ca. 5 km NW from Khao Yai forest Headquarters on a way to Pak Chong, alt. Habitat and geographical distribution: Saprobe on decaying wood, known only in Thailand.
Note: Re-examination of the holotype of Ch. hispida [19] revealed that the ascomatal wall is three-layered. The outer layer is partly deteriorated and it was initially overlooked. Based on the morphological study, this species is transferred to Paragaeumannomyces. The three-layered ascomatal wall with an outer layer composed of globose to angular cells is unique among species of the Chaetosphaeriaceae and is an important diagnostic trait in distinguishing between Paragaeumannomyces and Ericiosphaeria. Paragaeumannomyces garethjonesii [42] resembles P. hispidus in brown ascomata with short, acute spines and 7-septate ascospores, but differs in shorter asci (120-152 × 10.7-13.3 µm) and shorter ascospores (63.3-75 × 2.3-3.7 µm).
Catania et al. [78] described Ch. hispida var. podocarpi on bark and wood of Podocarpus parlatorei in Argentina. In morphological characteristics, it matches the genus Ericiosphaeria and is similar to E. spinosa, but differs in shorter ascospores (35.5-)40-49.5(-51) × 3-4 µm vs. 68-76 × 2-3 µm [3]. A dematiaceous hyphomycete growing in the juxtaposition to the ascomata was described as the anamorph. It has mononematous, simple, pigmented conidiophores with terminal conidiogenous cells and ellipsoidal-fusiform, 3-septate conidia with middle cells brown and end cells hyaline. The conidiogenous cells were depicted with several conidiogenous loci at the apex, but the mode of conidiogenesis was not described. The associated hyphomycete is reminiscent of Cacumisporium capitulatum, the anamorph of Ch. decastyla [79]. Interestingly, C. capitulatum is another member of the Paragaeumannomyces clade that includes teleomorphs with filiform to cylindrical, septate, asymmetrical ascospores. In C. capitulatum, the phialides extend above the collarette and have several subsequent narrow annellate proliferations; the conidia are formed successively on multiple conidiogenous loci and their arrangement at the tip of the phialide looks similar to that illustrated for the anamorph of Ch. hispida var. podocarpi. For now, we refrain from proposing a new combination at species rank for a taxon named as a variety of Ch. hispida. Its phylogenetic relationships should be resolved with molecular data. Habitat and geographical distribution: Saprobe on decaying stems of Musa paradisiaca and wood of unidentified hosts in freshwater and terrestrial habitats. The species is known in Malaysia, Solomon Islands and Thailand [15][16][17]80].
Habitat and geographical distribution: Saprobes on decaying bark and wood, known in China and New Zealand ( [2,15], this study).
Note: Tainosphaeria [3] grouped into three lineages in the ITS-28S phylogenetic tree ( Figure 1). Tainosphaeria crassiparies and four other species clustered in a monophyletic clade and represent the core of the genus characterised by macronematous, solitary, simple conidiophores, usually monophialidic conidiogenous cells and falcate, setulate conidia. However, three Tainosphaeria species were not resolved congeneric with T. crassiparies. Tainosphaeria aseptata, T. lunata, and a morphologically similar strain ICMP 23826 from New Zealand, clustered as a separate lineage, introduced as the new genus Phialoturbella (Ph.). This is characterised by macronematous, solitary or crowded, simple conidiophores with mono-occasionally polyphialidic conidiogenous cells and aseptate, falcate to lunate conidia without setulae. Tainosphaeria obclavata is transferred to Phialogeniculata; for a discussion see above.
Several morphologically similar species of Dictyochaeta can be considered relatives or possible candidates for inclusion in Phialoturbella, namely D. apiculata [71], D. botulispora [9], D. heteroderae [82], D. illinoensis [83] and D. occidentalis [84]. Unfortunately, none of these species has available DNA sequences or cultures. Recollecting these taxa and obtaining axenic cultures and DNA data is necessary to resolve this little-known complex of species. Habitat and geographical distribution: Saprobe on decaying wood, known only in China [2].
Note: For description and illustrations, refer to Lin et al. [2]. Phialoturbella aseptata has unbranched, solitary conidiophores, monophialidic conidiogenous cells extending percurrently and long fusiform, curved, aseptate conidia 14.4-18.9 × 3.3-4.4 µm [2]. On a photograph accompanying the protologue, a conidiophore is depicted with structures resembling persistent remnants of the collarettes, suggesting the conidiogenous cells can also form lateral openings and extend sympodially. The conidia are tapering apically and the basal end appears slightly truncate with an inconspicuous scar, which is visible on two images. Considering these characters, D. illinoensis [83] is highly similar to P. aseptata in features of conidia. The conidial size of both species overlaps, but the conidia of D. illinoensis are slightly longer in their upper range, 15.7-23.3 × 3.8-4.5 µm.
Habitat and geographical distribution: Saprobe on decaying petioles of Cecropia sp., known only in Puerto Rico.
Note: This species sporulated only on stems of U. dioica on CMA. When grown in culture, T. cecropiae formed two kinds of conidia, with and without setulae, of which the latter were more abundant. Although two synanamorphs with conidia without setulae were reported for T. crassiparies [3], they differed in shape and size from the falcate, setulate conidia of the anamorph. The size of setulate and non-setulate conidia of T. cecropiae overlaps considerably, suggesting that instead of the presence of a synanamorph, the setulae formation in some conidia of T. cecropiae may be delayed or the setulae easily detach. The conidiophores have the first 2-3 cells above the base noticeably darker than the rest of the conidiophore, giving them an almost bicolour appearance. Except for T. crassiparies, species of Tainosphaeria share similar conidial size (13.5-19 × 2-3.5 µm) and therefore, it is difficult to distinguish them based on this character alone. Tainosphaeria cecropiae differs from other members of the genus by characteristically pigmented conidiophores and formation of setulate and non-setulate conidia in culture.
The strain of T. cecropiae was originally deposited under the name Codinaea coffeae [85]. The latter species was isolated from nursery soil of Coffea arabica in Mexico and differs from T. cecropiae in shorter conidia (10.8-18 × 3.4-5 µm) and brown conidiophores becoming paler towards the apex without distinct dark and pale zones.

Discussion
New lines of evidence, based on results of phylogenetic analysis of the ITS-28S DNA sequences and comparative morphological studies of newly collected material, ex-type strains and other isolates, provided a sound basis for assessing generic concepts of Menisporopsis, Multiguttulispora and Tainosphaeria. Five species of Menisporopsis, representing the core of the genus, clustered in a monophyletic clade with M. theobromae. Menisporopsis has hyaline, aseptate conidia with one or more setulae inserted at each end or irregularly and synnemata surrounding the central, dark brown seta. The unique combination of these morphological traits makes Menisporopsis a well-recognisable genus. Our analysis confirms that the interspecific variation is based mainly on conidial shape and characters of setulae, i.e., their number and position on conidia. Identification key and synopsis of known species of Menisporopsis were published in Tsui et al. [5], Castañeda-Ruíz et al. [6] and Cruz et al. [70]. Menisporopsis novae-zelandiae and other isolates with 1-septate, setulate conidia and synnemata that grow unilaterally along the seta were resolved as a separate, strongly supported clade. They are accommodated in the new segregate genus Arcuatospora in this study. Menisporopsis ludoviciana [10,11] is another species that does not conform to the generic description. Castañeda-Ruíz et al. [13] transferred M. ludoviciana to Vermiculariopsiella (Vermiculariopsiellales) based on original description and illustration. However, its systematic placement awaits verification with molecular data.
The interspecific variability of Multiguttulispora was assessed using novel ITS and 28S rDNA sequences and a comparison of morphological data. Two species initially accommodated in Codinaea, C. dimorpha and C. triseptata [21,22], were resolved as being congeneric with the type species M. sympodialis, which is treated as a synonym of M. dimorpha. The conidial shape and septation, the extension of the phialides, and the arrangement of the collarettes along the robust, dark brown, almost opaque setiform conidiophores are the most valuable diagnostic features that distinguish Multiguttulispora from Codinaea. The latter genus is delimited to species with setae and conidiophores arising in tufts with monoor polyphialidic conidiogenous cells and hyaline, usually falcate, aseptate conidia with setulae [14]. In the phylogenetic tree, these genera formed distantly related lineages.
The Tainosphaeria complex has been revised. Based on anamorphic characteristics, species previously classified in Tainosphaeria can be distinguished into three morphological groups that are consistent with revealed phylogenetic relationships (Figure 1). These lineages include Tainosphaeria, Phialogeniculata and Phialoturbella. They differ mainly in the characteristics of conidia and conidiophores. Tainosphaeria contains five species, i.e., T. cecropiae, T. crassiparies, T. jonesii, T. monophialidica, and T. siamensis, and is delimited to fungi with mononematous, pigmented, simple conidiophores, mono-occasionally polyphialidic conidiogenous cells and falcate, hyaline, aseptate, conidia with setulae. The new segregate genus Phialoturbella contains two former Tainosphaeria species, such as Ph. aseptata, Ph. lunata and the new species Ph. calva. Their conidia are similar to those of Tainosphaeria, but lack setulae. Tainosphaeria obclavata is found conspecific with Phialogeniculata guadalcanalensis and is transferred to synonymy. Phialogeniculata differs from Phialoturbella and Tainosphaeria in septate, asymmetrical conidia and geniculate conidiophores with funnel-shaped and slightly stipitate collarettes, and accommodates four species. Although Kuthubutheen and Nawawi [80] transferred P. guadalcanalensis to Dictyochaeta, such a relationship could not be confirmed using molecular DNA data. Dictyochaeta is resolved as a well-supported lineage and delimited to species with setae accompanied by shorter conidiophores with mono-or polyphialidic conidiogenous cells and falcate to falcate-clavate, slightly asymmetrical conidia without septa and setulae [32,86].
In the phylogenetic tree based on the combined ITS-28S sequences, two other morphotypes were revealed in the Tainosphaeria clade. The genus Anacacumisporium with a single species, A. appendiculatum, was described for saprobic lignicolous fungi in China [18]. It can be recognised in having fusiform, septate conidia with middle cells brown and end cells hyaline, and a simple setula at each end, borne on terminal mono-or polyphialidic conidiogenous cells. Flectospora represents the fifth lineage with distinct morphology in the Tainosphaeria clade. It includes species with hyaline, aseptate, ellipsoidal to obovoid, slightly curved conidia borne on monophialides on pigmented conidiophores, superficial dark ascomata, short-stipitate asci with hyaline, ellipsoidal, septate ascospores. One of the species, F. laminata, is introduced for the alleged chloridium-like anamorph of Ch. hispida [19]. Revision of the holotype of Ch. hispida and phylogenetic analysis of members of the Chaetosphaeriaceae led to a new taxonomic treatment of Ch. hispida, morphologically similar to Ch. spinosa, and clarification of the anamorph of Ch. hispida. New combinations are proposed for both Chaetosphaeria species. Chaetosphaeria hispida is transferred to Paragaeumannomyces and Ch. spinosa is accepted in Ericiosphaeria. Chaetosphaeria s. str., represented by the type species Ch. innumera in the phylogenetic analysis, forms a separate lineage (Figure 1).
Although species of Chaetosphaeria have hyaline, transversely septate ascospores that are primarily symmetrical, ellipsoidal or fusiform, a handful of species have cylindrical, cylindricalfusiform to filiform, sometimes asymmetrical ascospores. Based on the results of ITS-28S phylogenetic analysis, species with cylindrical-filiform and asymmetrical ascospores grouped in one clade only. Apart from Ericiosphaeria, this clade also includes Paragaeumannomyces and other species such as Cacumisporium capitulatum, Catenularia cubensis, Ch. fusiformis, Ch. lignomollis, Exserticlava vasiformis and Stanjehughesia hormiscioides [3,7,20,79,87,88]. Of these taxa, Cacumisporium, Ericiosphaeria and Paragaeumannomyces possess asymmetrical ascospores. The similarity between Ericiosphaeria and Paragaeumannomyces in the ascospore and ascoma characteristics is undisputable; they differ mainly in the anatomy of the ascomatal wall. In the phylogenetic analysis, they are shown as sister taxa, although their relationship is statistically supported only in the ML analysis. A recent account of the morphology, taxonomy and phylogeny of Paragaeumannomyces has been published in Réblová et al. [20]. The authors [20] reported a correlation between ascoma morphology and ascospore reaction in Melzer's reagent. The ascospores of species of Paragaeumannomyces with glabrous ascomata, occasionally with ostiolar setae, exhibit a strong dextrinoid reaction in Melzer's reagent, while ascospores of species with setose ascomata have a negative or weak reaction. Our observations of P. hispidus are in agreement with Réblová et al. [20]; the ascomata are covered with acute setae over the whole surface and ascospores do not stain in Melzer's reagent. Taxonomy, systematics and relationships of Ch. fusiformis and Ch. lignomollis are more complex and beyond the scope of this study (Réblová et al., unpubl.).
In this study, we contributed to the delimitation of several monophyletic chaetosphaeriaceous genera and identification of unique sets of morphological characters that define them and are phylogenetically consistent. Our study provided new data and revealed new phylogenetic relationships in the Chaetosphaeriaceae.