Melanospora (Sordariomycetes, Ascomycota) and its relatives

Abstract The order Melanosporales comprises a large group of ascomycetes, most of them mycoparasites, characterized by the production of usually ostiolate, translucent ascomata, unitunicate asci, and unicellular, pigmented ascospores with germ pores or germ slits. The most studied taxa are Melanospora and Sphaerodes, but the boundaries with other morphologically closely related genera are not well resolved. In this study, the taxonomy of Melanospora and related taxa have been re-evaluated based on the analysis of nuclear rDNA, actin and elongation factor genes sequences of fresh isolates and numerous type and reference strains. The genus Melanospora has been restricted to species with ostiolate ascoma whose neck is composed of intermixed hyphae, and with a phialidic asexual morph. Microthecium has been re-established for species of Melanospora and Sphaerodes without a typical ascomatal neck or, if present, being short and composed of angular cells similar to those of the ascomatal wall, and usually producing bulbils. Three new genera have been proposed: Dactylidispora, possessing ascospores with a raised rim surrounding both terminal germ pores; Echinusitheca, with densely setose, dark ascomata; and Pseudomicrothecium, characterized by ascospores with indistinct germ pores. Dichotomous keys to identify the accepted genera of the Melanosporales, and keys to discriminate among the species of Melanospora and Microthecium, as well as a brief description of the accepted species of both genera, are also provided.


Introduction
Pustulipora, distinguished by its ascospores with a germ pore at each end surrounded by a blistered, rarely cushion-like structure showing an irregular pustulate appearance (Cannon 1982); Rhytidospora, characterized by non-ostiolate ascomata with a cephalothecoid ascomatal wall (Krug and Jeng 1979); Scopinella, producing brown, cuboid-ellipsoidal ascospores with two prominent longitudinal germ slits (Cannon and Hawksworth 1982); Setiferotheca, which produces ascospores similar to those of Arxiomyces and ascomata with a crown of dark brown setae surrounding the ostiole (Matsushima 1995); Syspastospora, possessing ascomata with a long neck composed of parallel arranged hyphae and cylindrical ascospores with a large terminal slightly sunken germ pore at each end (Cannon and Hawksworth 1982); and Vittatispora, which produces ascomata similar to those of Syspastospora and citriform ascospores with a longitudinal, thick, hyaline ridge (Chaudhary et al. 2006). Practically all taxonomic studies on these fungi have been based exclusively on the morphological characterization of the reproductive structures of preserved fungarium specimens, since unfortunately due to their mycoparasitism, many of these fungi do not grow in pure culture or do not produce ascomata in absence of their hosts. On the other hand, obtaining reliable nucleotide sequences from members of the Melanosporales is also difficult because of the usually large amount of DNA of their hosts. Based on the study of several freshly-isolated soil-borne fungi and of reference and type strains obtained from various culture collections, we have re-examined the phylogenetic relationships of the most relevant genera of the Ceratostomataceae. Consequently, the genus Melanospora has been redefined, Microthecium has been re-established, and three new genera have been proposed.

Fungal isolates
The strains included in this study are listed in Table 1. Fresh isolates were obtained from samples following previously described procedures for the activation of dormant ascospores in soil using acetic acid and phenol solutions (Stchigel et al. 2001, García et al. 2003. Ascomata were transferred to 55 mm diam. Petri dishes containing oatmeal agar (OA; oatmeal flakes, 30 g; agar-agar, 20 g; distilled water, 1 L) using a sterile needle, which were then incubated at 15, 25 and 35 °C.

Morphological study
For cultural characterization, isolates were grown for up to 30 d on OA, potato carrot agar (PCA; grated potatoes, 20 g; grated carrot, 20 g; agar-agar, 20 g; L-chloramphenicol, 100 mg; distilled water, 1 L), and potato dextrose agar (PDA; Pronadisa, Madrid, Spain) at 5, 10, 15, 20, 25, 30, 35 and 40 °C. Color notations in parentheses are from Kornerup and Wanscher (1984). Vegetative and reproductive structures were examined under an Olympus BH-1 brightfield microscope by direct mounting in lactic acid and water of the ascomata and/or microcultures grown on OA and PDA. Pictures were obtained with a Zeiss Axio Imager M1 brightfield microscope. The samples for scanning electron microscopy (SEM) were processed according to Figueras and Guarro (1988), and SEM micrographs were taken at 15 keV with a Jeol JSM 840 microscope.

Molecular study
The DNA of the fungal isolates (Table 1) was extracted and purified directly from the colonies according to the Fast DNA Kit protocol (MP Biomedicals, Solon, Ohio). The amplification of the small subunit (SSU), the D1−D3 domains of the large subunit (LSU) and the internal transcribed spacer region (ITS) of the nuclear rDNA, and the fragments of actin (act) and translation elongation factor 1-α (tef1) genes were performed according to White et al. (1990) (SSU), Vilgalys and Hester (1990) (LSU), Cano et al. (2004) (ITS), Voigt and Wöstermeyer (2000) (act) and Houbraken et al. (2007) (tef1). A BigDye Terminator 3.1 cycle sequencing kit (Applied Biosystems Inc., Foster City, California) was used to sequence both strands with a combination of the same primers used in the amplification. PCR products were purified and sequenced at Macrogen Europe (Amsterdam, The Netherlands) with a 3730XL DNA analyzer (Applied Biosystems), and the consensus sequences were obtained using SeqMan (version 7.0.0; DNASTAR, Madison, WI, USA). A phylogenetic study based on the analysis of SSU sequences of the isolates and type and reference strains of the Melanosporales and of some members of the Chaetosphaeriales, Coniochaetales, Coronophorales, Hypocreales, Microascales, Sordariales and Xylariales, using Thelebolus ellipsoideus (Thelebolales) as outgroup, was performed to confirm the taxonomic placement of our isolates. A subsequent study, carried out to infer the phylogenetic relationships among members of the Melanosporales, was based on the analysis of a combined data set including the ITS, LSU, act and tef1 sequences of our isolates and of type and reference strains of a large number of the Melanosporales, including Nectria cinnabarina and Pseudallescheria fusoidea as outgroups. The Maximum-Likelihood (ML) and Bayesian Inference (BI) methods were used in phylogenetic analyses as described by Hernández-Restrepo et al. (2016). Bootstrap support (BS) ≥70 and posterior probability values (PP) ≥0.95 were considered significant. The sequences generated in this study were deposited in GenBank (Table 1 and Fig. 1) and the alignments used in the phylogenetic analyses were deposited in TreeBASE (http://purl.org/phylo/treebase/ phylows/study/TB2:S17079). Sequences retrieved from GenBank and NBRC included in the SSU and combined analyses are shown in Fig. 1 and Table 1, respectively.

Results
The SSU phylogenetic study was based on an alignment of 1023 bp and produced a single ML tree (Fig. 1) inferred from a RAxML analysis. The members of the Melanosporales including our isolates were placed in a highly supported main clade (100 % BS / 0.99 PP), and the isolate CBS 137837, whose morphological features did not match any previously described taxon, occurred as a basal branch clearly separated from the other Melanosporales, which grouped together with a high support (95 % BS / 0.98 PP) and separated into three subclades. The lengths of the individual alignments used in the combined data set were 802 bp (LSU), 535 bp (ITS), 727 bp (act) and 846 bp (tef1), respectively, and the final total alignment was 2910 bp. In the ML tree derived from the RAxML analysis of the combined data set (Fig. 2), the Melanosporales were highly supported (100 % BS / 1 PP) and subdivided into seven lineages. The first clade (89 % BS / 1 PP; Clade Microthecium) grouped all our isolates, with the exception of CBS 137837, and type or reference strains of Melanospora fimbriata, M. fusispora, M. levita, M. zobelii, Papulaspora sepedonioides, Pteridiosperma ciliatum, Sphaerodes compressa, S. fimicola, S. retispora, S. quadrangularis and S. tenuissima. All the fungi belonging to this clade have non-ostiolate ascomata, or when a neck is present, it is short and composed of angular cells similar to those of the ascomatal wall. Also, bulbils (microsclerotial-like asexual propagules) are present in most of these species. In spite of the high morphological variability shown by members of this clade, the loci used in the phylogenetic analysis were not able to separate the species from each other. The second clade (100% BS / 1 PP; Clade Melanospora) comprised the type species of Melanospora, M. zamiae, the type strain of M. verrucispora and a reference strain of M. damnosa. The members of this clade produce ostiolate ascomata with a long neck composed of hyphae irregularly arranged and ending in a crown of setae. In addition, an asexual morph is commonly present, which is characterized by solitary, sessile, flask-shaped phialides producing from rounded to ellipsoidal conidia. The third lineage comprised only   Table 1. Type strains of the different species are indicated with T . a reference strain of Melanospora kurssanoviana, which failed to sporulate in pure culture. The fourth clade (90 % BS / 0.98 PP; Clade Dactylidispora) was composed of the type strains of Sphaerodes ellipsospora and S. singaporensis, both characterized by ascospores with a raised rim surrounding the germ pores. Finally, the isolate CBS 137837 and the type strains of Melanospora subterranea and Vittatispora coorgii formed three independent branches. The isolate CBS 137837 produces globose, non-ostiolate, densely setose, dark ascomata and smooth-walled ascospores with a depressed germ pore at each end, while the other two species of this clade also possess morphological features unique in the Melanosporales, e.g. ascospores with indistinct germ pores in M. subterranea and with a longitudinal, thick, hyaline ridge in V. coorgii.
Etymology. From Greek δακτυλίδης-, ring, and from Latin -spora, spore, due to the raised rim that surrounds the germ pores of the ascospores.
Notes. The most distinctive characteristic of Dactylidispora is the production of smooth-walled ascospores with a germ pore at each end surrounded by a raised rim. Vittatispora, proposed as a new genus by Chaudhary et al. (2006), also produces a raised rim surrounding the germ pores. However, both genera can be easily distinguished by the nature of the ascomatal neck, which is composed of angular cells in Dactylidispora and of parallel arranged hyphae in Vittatispora; and by the presence of a hyaline ridge running the entire vertical length of the ascospore between the germ pores in Vittatispora. Moreover, in our phylogenetic study (Fig. 2), Vittatispora also constituted a lineage independent from the other members of the Melanosporales. Pustulipora is also morphologically similar to Dactylidispora being characterized by blistered, rarely cushion-like structures surrounding the germ pore (Cannon 1982). However, unfortunately, Pustulipora could not be included into this phylogenetic study since living cultures were not available.
Etymology. From Latin echinus-, sea urchin, and from Greek -τείχος, wall, because of the ascomata resemblance to a sea urchin, due to the abundance of setae.
Notes. This genus is characterized by dark, strongly setose, non-ostiolate ascomata. Apart from Echinusitheca, the other genera of the Melanosporales characterized by the production of dark semi-translucent ascomata are Arxiomyces and Scopinella, but both genera differ from Echinusitheca by the production of long ascomatal necks. Moreover, Scopinella can be easily distinguished from Echinusitheca by its cuboidellipsoidal ascospores with two prominent longitudinal germ slits, and Arxiomyces by its ellipsoidal ascospores that are rounded at the apex and truncated at the base, and with a broad germ pore that bears a mucilaginous and collapsing appendage. Description. Colonies on PDA attaining a diam. of 70-75 mm after 14 d at 35 °C, cottony and granulose due to the presence of a large number of ascomata, white with grey to black dots, depressed at the centre and margins fringed; reverse yellowishwhite to pale yellow (4A2 to 4A3) and with olive brown (4F2) dots. Colonies on OA attaining a diam. of 50-60 mm in 14 d at 35 °C, cottony and granulose due to the presence of numerous ascomata, margins arachnoid, white to orange white (5A2) with brownish grey dots (5F2); reverse yellowish-white to golden grey (4A2 to 4C2). Minimum, maximum, and optimum temperature of growth are 20, 40 and 35 °C, respectively. Mycelium composed of hyaline to pale yellow, septate, branched, smoothwalled hyphae, 1-3 μm diam. Ascomata non-ostiolate, immersed into the mycelium, solitary or gregarious, globose, 130-280 μm diam., setose, semi-translucent, pale brown to brown, appearing black when ascospores are mature; setae straight, becoming sinuous toward apex, 20-200 μm long, 5-20 μm wide at base, tapering gradually to a rounded tip of 2-5 μm diam., pale brown to brown, non-septate or rarely 1-septate, thick-walled, verrucose to tuberculate, sometimes branched at apex; ascomatal wall membranaceous, 30-40 μm thick, composed of 5-6 layers of flattened cells of 5-30 μm diam. of textura angularis to textura globulosa. Asci 8-spored, globose to subglobose, 20-25 × 15-20 μm, soon evanescent, non-stipitate, without apical structures, irregularly disposed at the centrum. Ascospores irregularly arranged in the asci, onecelled, at first hyaline, becoming brown to dark brown when mature, smooth-and thick-walled, ellipsoidal, 20-27 × 10-15 μm, with one germ pore at each end; germ pores 0.75-2 μm diam., depressed. Asexual morph absent.
Notes. This genus is distinguished by translucent ascomata with a neck composed of intermixed hyphae and with an apical crown of setae, smooth or ornamented ascospores with an apiculate germ pore at each end, and a phialidic asexual morph. The neck of Melanospora spp. is morphologically similar to those of Syspastospora and Vittatispora, which are also composed of hyphae. Syspastospora was introduced in 1982 by Cannon and Hawksworth to accommodate Melanospora parasitica, with three additional species described later (S. boninensis, S. cladoniae and S. tropicalis). This genus differs from Melanospora in the production of cylindrical to barrel-shaped ascospores with a large, slightly sunken germ pore at both ends (ellipsoidal, citriform or fusiform, having much smaller, apiculate or depressed germ pores in Melanospora). Vittatispora can be distinguished from Melanospora by the production of ascospores with a thick, hyaline, longitudinal ridge and a raised rim surrounding the germ pores. Moreover, Syspastospora and Vittatispora differs from Melanospora in the structure of the ascomatal neck, which is composed of hyphae in a parallel arrangement in both genera (interwoven hyphae in Melanospora).
Melanospora is now restricted to species with ascoma bearing a neck composed of interwoven hyphae and mostly ending in a crown of setae. This kind of neck differentiates this genus from Microthecium, which has a neck composed of angular cells similar to those of the ascomatal wall and possessing a crown of setae surrounding the ostiole rather than disposed at apex of the neck. The only exception is Melanospora mycoparasitica that does not have this sort of neck, being short, cellular and without the crown of setae at the top of this, although this could be due to the fact that it was described and illustrated at an early stage of ascomal development. In a study on the development and cytology of Melanospora tiffanii, Kowalski (1965) illustrated early stages of development with the neck appearing similar to that of M. mycoparasitica.
Long hyphal necks are produced in Melanospora arenaria, Melanospora caprina, Melanospora chionea, Melanospora langenaria, Melanospora longisetosa and Melanospora washingtonensis; therefore, these have been kept in the emended genus Melanospora, although they were not included in the phylogenetic study.
Key to the species of Melanospora Notes. This species is characterized by white, tomentose ascomata and discoid, smoothwalled ascospores with depressed germ pores. Notes. Melanospora damnosa is distinguised by the production of ascomata with a short neck and citriform to rhomboidal, smooth-walled ascospores with a slightly apiculate germ pore at each end. Notes. Melanospora lagenaria is similar to M. caprina, but the former has less tomentose ascomata with shorter necks ending in a poorly developed crown of setae. This species is also similar to M. arenaria. For morphological comparison see Notes of the latter species. Notes. This species is characterized by the formation of 4-spored asci and discoid, smooth-walled ascospores. Notes. This species is distinguished by its fusiform, slightly reticulate ascospores. Notes. Melanospora zamiae is characterized by the production of ellipsoidal to citriform, smooth-walled ascospores with a depressed germ pore at each end. Doguet (1955) described the presence of bulbils; however, later studies did not mention the presence of such sort of propagules (Calviello 1973, Cannon andHawksworth 1982), which rarely occur in the genus.
Notes. Cultures of this species are not available, but it was originally described as producing small asci (18-21 × 7-8 μm) and ascospores (4-6 × 3-4 μm). This species produced ostiolate ascomata without a neck, typical of Microthecium; however, such small ascospores have never been seen in Microthecium. Notes. Cultures are not available, and no illustrations were included in the protologue. It was reported as morphologically similar to Melanospora rhizophila [now considered a synonym of Melanospora zamiae (Doguet 1955)] when it was first described (Woronichin 1924).
Notes. This species possesses cylindrical asci and hyaline ascospores, features never seen in Melanospora. It was previously excluded from Melanospora by Doguet (1955). Notes. This species shows morphological features never observed in Melanospora, e.g. small asci (10-14 × 5-6.5 μm) and olivaceous ascospores (5-5.5 × 3-3.5 μm). The original description is not detailed enough to ascertain its possible taxonomical placement. Notes. This species is excluded from Melanospora due to its dark brown, nontranslucent, setose ascomata and its small ascospores (4.5-12 × 4-7 μm), which seem to indicate a closer relationship with Chaetomium. Notes. Cultures are not available, and the original description does not mention asci and ascospores. Therefore, we agree with Doguet (1955) in the exclusion of this fungus from Melanospora. Notes. In our phylogenetic study, M. kurssanoviana was placed in an independent lineage far from Melanospora. Unfortunately, the only living culture available is sterile.

Melanospora exsola
We did not find any distinctive morphological feature to differentiate this species from other members of the Melanosporales in the original description and in the drawing to introduce it as a new genus.
Notes. Doguet (1955) excluded this species due to the production of very large cylindrical asci (480-500 × 33-36 μm) and ascospores (42-52 × 28-35 μm), morphological features not observed in any other member of the Melanosporales. Notes. This species is transferred to Scopinella due to the morphology of its ascospores, i.e. octahedral ascospores with two prominent longitudinal germ slits. Notes. This species is excluded from Melanospora since its neck is cellular or absent, instead it is characterized by a dark ring-like structure around the germ pores of the ascospores (Stchigel et al. 1999). This fungus could represent a new genus since such structure is unique in the Melanosporales, and these kind of structures resulted in being phylogenetically informative, as in the case of Dactylidispora, which is distinguished by its ascospores with a raised rim around the germ pores. The type strain of this specimen was contaminated with another fungus and it could not be included in the molecular study. Notes. This species is excluded from Melanospora since it produces cylindrical asci with the ascospores uniseriately disposed, a feature never observed in this genus. Notes. Doguet (1955) excluded this species due to its oblong, pale yellow ascospores. Description. Ascomata ostiolate or not, superficial or immersed, globose to subglobose or pyriform, yellowish-orange, orange-brown or reddish, tomentose or glabrous; necks short or absent, conical, composed of angular cells similar to those of the ascomatal wall, usually with a crown of hyaline, septate, smooth-and thickwalled setae around the ostiole; ascomatal wall membranaceous, translucent, of textura angularis. Periphyses present. Paraphyses absent. Asci 8-spored, clavate, rounded at apex, without apical structures, thin-walled, evanescent. Ascospores one-celled, at first hyaline, becoming brown to dark brown when mature, ellipsoidal, fusiform, navicular, citriform, plataniform or spindle-shaped, smooth, reticulate, pitted or wrinkled, with a terminal apiculate or depressed germ pore at each end. Asexual morph phialidic, hyaline. Bulbils usually produced, pale orange to reddish-orange.

Melanospora octahedrica
Notes. Microthecium has translucent ascomata of textura angularis, cellular necks short or absent, ascospores smooth-walled or ornamented with a depressed or apiculate germ pore at each end, often producing bulbils and a phialidic asexual morph. Dactylidispora, Pustulipora and Pseudomicrothecium produce ascomata similar to Microthecium. However, the two first genera can be distinguished by the presence of a raised rim and blistered structure surrounding the germ pores of the ascospores, respectively, while Pseudomicrothecium differs in the production of 2-spored asci and ascospores with indistinct germ pores.
The species Mi. africanum, Mi. beatonii, Mi. brevirostratum, Mi. episphaerium, Mi. foveolatum, Mi. geoporae, Mi. hypomyces, Mi. internum, Mi. lenticulare, Mi. marchicum, Mi. masonii, Mi. micropertusum, Mi. moureai, Mi. nectrioides, Mi. pegleri and Mi. perplexum were not included in the phylogenetic study because we could not locate any specimens since the holotypes or living cultures of most of them are not available. However, these species were transferred to Microthecium based on their complete and well-illustrated descriptions.  Notes. Microthecium africanum is characterized by ostiolate ascomata and punctate, ellipsoidal ascospores. Two asexual morphs with different conidia have been reported: (i), 1-4(-5)-celled, globose and smooth-walled at first but becoming cylindrical and coarsely verrucose later; (ii), 1-2-celled, large, usually cylindrical and smooth-walled (Krug 1988). However, the type strain was probably not a pure culture because the SSU and LSU sequences match with different species of Fusarium and the pictures of the conidia type (i) resemble the chlamydospores produced by several species of this genus. Notes. This species is characterized by non-ostiolate ascomata, 4-spored asci and very coarsely reticulate, citriform ascospores. These morphological features are also observed in Microthecium perplexum, but this species produces ascospores with only a third of the surface coarsely reticulate while the rest remains smooth-walled. Microthecium episphaerium and Mi. retisporum differ from Mi. beatonii in the production of 8-spored asci. Moreover, Mi. retisporum produces a phialidic asexual morph and bulbils, which are absent in the other mentioned species, and smaller ascospores (17-20 × 10-12 × 7-9 μm) than in Mi. beatonii [28-34(-40) × 14-18(-20)  Notes. Microthecium brevirostrum, Mi. fallax and Mi. fimbriatum produce ostiolate ascomata and ellipsoidal to citriform, often plataniform, smooth-walled ascospores with an apiculate germ pore at each end. Microthecium fimbriatum is easily distinguished by its smaller (100-110 μm diam.), reddish ascomata, while Mi. fallax differs in the production of bulbils. Notes. This species is characterized by non-ostiolate ascomata and ellipsoidal to fusiform ascospores ornamented with wing-like appendages and wrinkles, and the production of a phialidic asexual morph and bulbils. Microthecium lenticulare and Mi. foveolatum also present ascospores with wing-like appendages, but these are pitted and not wrinkled (as in Mi. ciliatum), and neither species produces bulbils. Microthecium foveolatum and Mi. ciliatum are characterized by non-ostiolate ascomata and the production of a phialidic asexual morph, whereas Mi. lenticulare has ostiolate ascomata and lacks an asexual morph. Notes. This species is distinguished by the production of non-ostiolate ascomata and citriform, bilaterally flattened ascospores, with the narrow faces coarsely reticulate and the widest faces smooth or nearly so, along with the production of a phialidic asexual morph. Notes. This species is characterized by ostiolate ascomata, ellipsoidal to citriform, often plataniform, smooth-walled ascospores, and production of bulbils. [Basionym]

Microthecium compressum
Notes. This species is characterized by ostiolate ascomata, ellipsoidal to navicular or citriform, smooth-walled ascospores, and production of bulbils. Notes. Microthecium fimbriatum produces ostiolate ascomata, and citriform to plataniform, smooth-walled ascospores with a strongly apiculate and tuberculate germ pore at each end. Although the ascomata was described as small and reddish in the protologue, the strain included in this study (NBRC 8523) shows larger (250-380 μm diam.), orange-brown ascomata. Moreover, our isolate produces bulbils. Notes. Microthecium fimicola is characterized by ostiolate ascomata and coarsely reticulate ascospores with strongly apiculate germ pores at both ends. The other species with ostiolate ascomata and reticulate ascospores are Mi. internum and Mi. quadrangulatum. The main differences among them are the shape and size of the ascospores, being citriform in Mi. fimicola, spindle-shaped in Mi. internum and fusiform in Mi. quadrangulatum. The production of bulbils has only been observed in our fresh isolates of Mi. fimicola, although this was not previously reported. Notes. This species is easily distinguished by its non-ostiolate ascomata, ellipsoidal to fusiform ascospores ornamented with small pores and thick wing-like ridges usually longitudinal but often oblique, and production of phialidic asexual morph. For Notes. Microthecium masonii is characterized by ostiolate ascomata and ellipsoidal to fusiform, faintly striate-reticulate ascospores. The same type of ascospore ornamentation is also observed in Mi. micropertusum, but this latter species is easily distinguished by the presence of inconspicuous ridges forming a very coarse reticulum and a phialidic asexual morph. Description. Ascomata non-ostiolate, globose, translucent, pale brown to brown, appearing dark brown when the ascospores are mature, glabrous or setose; ascomatal wall membranaceous, of textura angularis. Asci 2-spored, clavate, short-stipitate, without apical structures, evanescent. Ascospores one-celled, at first hyaline, becoming dark brown to blackish when mature, ellipsoidal to citriform, umbonate and truncate at both ends, with a terminal indistinct germ pore at each end. Asexual morph absent.

Microthecium internum
Etymology. The name refers to the morphological resemblance to Microthecium. Notes. The new genus Pseudomicrothecium is proposed here to accommodate Melanospora subterranea because it constitutes a separate lineage in our phylogenetic study. This genus is characterized by its non-ostiolate ascomata, similar to those of Microthecium, 2-spored asci and smooth-walled ascospores with an indistinct germ pore at each end. Asci containing two ascospores have only been observed in some species of Scopinella (i.e. Scopinella gallicola and S. sphaerophila). However, Scopinella can be easily distinguished from Pseudomicrothecium by the production of ostiolate ascomata with long necks and cuboid-ellipsoidal ascospores with two prominent longitudinal germ slits.

Discussion
We have revised the taxonomy of relevant members of the family Ceratostomataceae based on the analyses of the SSU, LSU, ITS, act and tef1 nucleotide sequences. This study strongly supported the order Melanosporales proposed by Blackwell in 2007 (Hibbett et al. 2007). The phylogenetic inference showed seven lineages corresponding to the genera Dactylidispora, Echinusitheca, Melanospora, Microthecium, Pseudomicrothecium and Vittatispora, and to Melanospora kurssanoviana. Our results agree with previous studies Blackwell 2002, Fan et al. 2012) which already suggested and demonstrated that the ornamentation of the ascospores under SEM, a feature traditionally used to delimit most of the genera in the Melanosporales, is not useful for estimating phylogenetic relationships among these fungal taxa. Similarly, the morphology of the ascospores is of weak taxonomic value and a poor predictor for the generic delimitation of members of the family Sordariaceae, resulting in the synonymy of two relevant genera, i.e. Gelasinospora and Neurospora (Dettman et al. 2001, García et al. 2004, Nygren et al. 2011). In our study, two of the largest genera of the Melanosporales, Melanospora and Microthecium, grouped species with both smooth and ornamented ascospore walls. By contrast, a phylogenetic study of the Lasiosphaeriaceae (Miller and Huhndorf 2005) revealed that the morphology of the ascomatal wall was more phylogenetically informative than that of the ascospores, with several new genera proposed (i.e. Immersiella) or emended (i.e. Lasiosphaeria, Lasiosphaeris and Schizothecium) Huhndorf 2004, Cai et al. 2005). Here, the erection of the new genus Echinusitheca is a clear example of the relevance of the ascomatal morphology in the taxonomy of these fungi, and in fact this taxon together with Arxiomyces and Scopinella are the only genera in the Melanosporales that show dark semi-transluscent ascomata. In this context, although Echinusitheca has ascospores similar to those of Melanospora and Microthecium, this genus constitutes one of the lineages phylogenetically most distant within this order.
Another lineage considerably distant from the other members of the Melanosporales is constituted by the clade represented only by the species Melanospora kurssanoviana, suggesting that this fungus could represent a new genus. However, this new taxon is at this moment not proposed because its colonies, in spite of attempts to induce sporulation, remain sterile and a detailed morphological study was not possible. The infertility of the cultures is probably due to the fact that an important part of the members of this fungal group show a peculiar habitat developing a certain degree of mycoparasitism and requiring the presence of the host to complete the biologic cycle and develope reproductive structures. The mycoparasitism of Melanospora, Syspastospora and the species previously placed in Persiciospora and Sphaerodes has already been demonstrated by numerous authors (Doguet 1955, Calviello 1973, Jordan and Barnett 1978, Harveson and Kimbrough 2000, and this ability has been exploited in the biocontrol of phytopathogenic fungi (Vujanovic and Goh 2009, Goh and Vujanovic 2010, Kim and Vujanovic 2016. The genus Sphaeronaemella, which is characterized by pale and translucent ascomata, was thought to be related to Melanospora (Cannon and Hawksworth 1982). However, we do not agree with this relationship because it differs from the Melanosporales in the production of hyaline ascospores, as opposed to the pigmented ones in the members of that order. By contrast, our results correlate with those of other authors that demonstrated a closer phylogenetic relationship of this genus with the members of the order Microascales Blackwell 1994b, Hausner andReid 2004). In fact, our SSU tree seems to indicate that Sphaeronaemella could represent a new family of the Microascales; however, further studies including more taxa and additional genes are needed to more accurately confirm its taxonomic status.
The placement of our isolate of Persiciospora japonicum in the Microthecium clade once more demonstrated that the ornamentation of the ascospores, which is pitted in Persiciospora spp., is of poor taxonomic value, and consequently all the species of Persiciospora should be transferred to Microthecium. As it was above mentioned, the species of this latter genus show a typical cellular ascomatal neck which is also present in Persiciospora and constitutes a common feature in both genera. Surprisingly, in some previous phylogenetic studies, the species of Persiciospora were placed in the Hypocreales, closely related to Nectria (Zhang and Blackwell 2002, Maharachchikumbura et al. 2015, Schultes et al. 2017). However, this could be probably explained by a possible contamination of the cultures of Persiciospora spp. with an hypocrealean host (Fan et al. 2012). The same situation may have occurred with the cultures of Scopinella and Syspastospora, which led to a possible erroneous classification of both taxa in the Hypocreales (Zhang and Blackwell 2002, Chaudhary et al. 2006, Fan et al. 2012, Maharachchikumbura et al. 2015, Schultes et al. 2017. Pteridiosperma ciliatum, a member of the Melanosporales with ascospores ornamented with longitudinal wing-like ridges that anastomose each other to form a well defined reticulum (a distinctive feature of Pteridiosperma), was also found in the Microthecium clade, proving once again that the ascospore ornamentation is not phylogenetically informative. Consequently, we have synonymyzed the genus Pteridiosperma with Microthecium since Pteridiosperma spp. show non-ostiolate ascomata, or if ostiolate, they show a short neck composed of angular cells, which are typical morphological characteristics of Microthecium.
Another genus that our results demonstrated should be synonymized and included in Microthecium is Sphaerodes because its type species, S. episphaerium, shows morphological features (non-ostiolate ascomata) that fit with the current circumscription of that emended genus. Most of the species of Sphaerodes, with the exception of S. ellipsospora and S. singaporensis, which are now located in the new genus Dactylidispora, and S. mycoparasitica, which is now placed in Melanospora, are also transferred to Microthecium since these produce non-ostiolate or ostiolate ascomata without a neck, or less frequently with a short neck composed of angular cells similar to the ascomatal ones. Another relevant feature of the genus Microthecium is the production of bulbils. These propagules are typical of Papulaspora, an anamorphic genus that encompasses more than 40 species. Although it was initially accepted as a genus without a sexual morph (Hotson 1912), its link with species of Melanospora and Chaetomium has been reported (Roll-Hansen 1948, Zhang et al. 2004). In our phylogenetic study Papulaspora sepedonioides, the type species of the genus, was nested in the Microthecium clade, and therefore transferred to this genus. The relationship of this species with the Melanosporales had already previously been demonstrated by Davey et al. (2008) and Li et al. (2016). However, it has been demonstrated that Papulaspora is a polyphyletic genus, and other species of the genus have been reported as belonging to the classes Leotiomycetes and Sordariomycetes (Ascomycota). Therefore, the other species of Papulaspora not linked to the species of Microthecium should be transferred to other taxonomic groups. The relationship of some species of Papulaspora with the Melanosporales is also suggested by the production of similar phialidic synanamorphs (Van Beyma 1931, Hotson 1942. The most recent new combination performed in Sphaerodes, S. inferior, was done to accommodate S. retispora var. inferior since it was not clustering with S. retispora var. retispora (Schultes et al. 2017). However, we suspected that the sequences of S. retispora var. retispora deposited in GenBank were contaminated with the hypocrealean host. In order to corroborate it, we studied such sequenced strain demonstrating that it was effectively contaminated. Therefore, S. inferior is here considered a synonym of Mi. retisporum since the morphological differences are insufficient to recognize this variety as a different species.
There are important morphological differences among the strains of Microthecium that suggest the presence of several additional cryptic species in the genus; however, our phylogenetic study, in spite of having used five loci, was not able to resolve the boundaries among them.