﻿Thyridium revised: Synonymisation of Phialemoniopsis under Thyridium and establishment of a new order, Thyridiales

﻿Abstract The genus Thyridium, previously known as a saprobic or hemibiotrophic ascomycete on various plants, was revised taxonomically and phylogenetically. Sequences of the following six regions, that is, the nuclear ribosomal internal transcribed spacer (ITS) region, the large subunit (LSU) of rDNA, the second largest RNA polymerase II subunit (rpb2) gene, translation elongation factor 1-alpha (tef1) gene, the actin (act) gene, and the beta-tubulin (tub2) gene, were generated for molecular phylogenetic analyses of species of this genus. Phialemoniopsis, a genus encompassing medically important species, is synonymised with Thyridium based on molecular evidence and morphological similarities in their asexual characters. The generic concept for Thyridium is expanded to include species possessing both coelomycetous and hyphomycetous complex asexual morphs. In addition to type species of Thyridium, T.vestitum, nine species were accepted in Thyridium upon morphological comparison and molecular phylogenetic analyses in this study. All seven species of Phialemoniopsis were treated as members of the genus Thyridium and new combinations were proposed. A bambusicolous fungus, Pleosporapunctulata, was transferred to Thyridium, and an epitype is designated for this species. A new species, T.flavostromatum, was described from Phyllostachyspubescens. The family Phialemoniopsidaceae, proposed as a familial placement for Phialemoniopsis, was regarded as a synonym of Thyridiaceae. A new order, Thyridiales, was established to accommodate Thyridiaceae; it forms a well-supported, monophyletic clade in Sordariomycetes.

Molecular information on Thyridium species is available only for two non-type strains (CBS 113027, CBS 125582) of the type species T. vestitum (Lutzoni et al. 2004;Spatafora et al. 2006;Vu et al. 2019); however, the phylogenetic relationships among species of this genus are unclear. A recent study on the phylogeny of Sordariomycetes has shown that T. vestitum is closely related to two Phialemoniopsis spp. (P. endophytica and P. ocularis), but their phylogenetic and taxonomic relationships have not been clarified (Dong et al. 2021;Hyde et al. 2021).
The genus Phialemoniopsis was placed in Phialemoniopsidaceae (Diaporthomycetidae family incertae sedis, Sordariomycetes; Hyde et al. 2021). Species of this genus are widely distributed in various environments and substrates, including industrial water, plant materials, raw sewage, and soil (Gams and McGinnis 1983;Halleen et al. 2007;Su et al. 2016). Several species have been reported from parts of the human body, such as blood, eye, toenail, skin, and sinus (Perdomo et al. 2013;Tsang et al. 2014), and some of them have also been isolated from patients with keratomycosis and phaeohyphomycosis (Perdomo et al. 2013;Desoubeaux et al. 2014). All species in this genus are known to be asexual.
In our ongoing taxonomic study of sordariomycetous fungi in Japan, several new specimens of Thyridium-like sexual morphs were collected. Single ascospore isolates from these specimens formed typical Phialemoniopsis-like asexual morphs in culture, suggesting that both genera are closely related. This study aims to reveal the taxonomic and phylogenetic relationships between Thyridium and Phialemoniopsis, and to clarify their ordinal position in Sordariomycetes.

Isolation and morphological observation
All materials were obtained from Japan. Morphological characteristics were observed in preparations mounted in distilled water by differential interference and phase contrast microscopy (Olympus BX53) using images captured with an Olympus digital camera (DP21). All specimens were deposited in the herbarium at Hirosaki University (HHUF), Hirosaki, Japan. Single spore isolations were performed from all specimens. Colony characteristics were recorded from growth on potato dextrose agar (PDA), malt extract agar (MEA), and oatmeal agar (OA) from Becton, Dickinson and Company (MD, USA), after a week at 25 °C in the dark. Colony colours were recorded according to Rayner (1970). To observe the asexual morphs in culture, 5 mm squares of mycelial agar were placed on water agar containing sterilised plant substrates such as rice straws and banana leaves. Then these plates were incubated at 25 °C for 2 weeks in the dark. When the substrates were colonised, the plates were incubated at 25 °C under black light blue illumination for 1-2 weeks to observe sporulation.

Phylogenetic analyses
DNA was extracted from four isolates using the ISOPLANT II kit (Nippon Gene, Tokyo, Japan) following the manufacturer's instructions. The following loci were amplified and sequenced: the internal transcribed spacer (ITS) region with primers ITS1 and ITS4 (White et al. 1990), the large subunit nuclear ribosomal DNA (LSU) with primers LR0R (Rehner and Samuels 1994) and LR5 or LR7 (Vilgalys and Hester 1990), the second largest RNA polymerase II subunit (rpb2) gene with primers fRPB2-5F and fRPB2-7cR (Liu et al. 1999), the translation elongation factor 1-alpha (tef1) gene with primers 983F and 2218R (Rehner and Buckley 2005), the actin (act) gene with primers Act-1 and Act-5ra (Voigt and Wöstemeyer 2000) and the beta-tubulin (tub2) gene with primers TUB-F and TUB-R (Cruse et al. 2002). PCR products were purified using the FastGene Gel/PCR Extraction Kit (Nippon Gene, Tokyo, Japan) following the manufacturer's instructions and sequenced at SolGent (South Korea). Newly generated sequences were deposited in GenBank (Table 1).
Primary analysis of LSU-rpb2-tef1 sequences from 88 strains of Sordariomycetes (Table 1) was conducted to clarify the ordinal/familial placement of Thyridium (or Phialemoniopsis) species. Barrmaelia rhamnicola and Entosordaria perfidiosa (Xylariomycetidae) were used as outgroups. As a secondary analysis, single gene trees of ITS, act and tub2, and a combined tree of these three loci were generated to assess the species boundaries of 17 strains within Thyridium/Phialemoniopsis (Table 2). All sequence alignments (LSU, ITS, rpb2, tef1, act and tub2) were produced using the server version of MAFFT (http://www.ebi.ac.uk/Tools/msa/mafft), checked and refined using MEGA v. 7.0 (Kumar et al. 2016).

Phylogeny
For primary analysis, ML and Bayesian phylogenetic trees were generated using an aligned sequence dataset comprising of LSU (1,205 base pairs), rpb2 (1,059 bp) and tef1 (954 bp). Of the 3,218 characters included in the alignment, 1,478 were variable and 1,686 were conserved. This combined dataset provided higher confidence values for ordinal and familial classification than those of individual gene trees, with 25 orders and three families (order unknown) being reconstructed in Sordariomycetes ( Fig. 1). ML analysis of the combined dataset was conducted based on the selected substitution model for each partition (GTR+G for LSU, J2+G for the first and third codon positions of rpb2, J1+G for the second codon positions of rpb2, F81+G for the first codon positions of tef1, JC69+G for the second codon positions of tef1, and J2+G for the third codon position of tef1). The ML tree with the highest log likelihood (-43687.562) is shown in Fig. 1. Topology recovered by Bayesian analysis was almost identical to that of the ML tree. All species previously described as Phialemoniopsis (marked with blue circle in Fig. 1), one species of "Linocarpon", two species of "Neolinocarpon" and four strains newly obtained in this study formed a monophyletic clade with the type species of Thyridium (T. vestitum). Their monophyly was completely supported (100% ML BS/1.0 Bayesian PP; Fig.1). The family Thyridiaceae was found to be related to Annulatascales and Myrmecridiales but did not cluster with any existing order in Sordariomycetes. For secondary analysis, ML and Bayesian phylogenetic trees were generated using sequences of ITS (483 bp), act (646 bp), tub2 (375 bp), and a combined dataset of these three regions (1,504 bp). The selected substitution models for each region were as follows: J2ef+G for ITS, F81+H for the first and second codon positions of act, J2+G for the third codon position of act, K80+H for the first codon positions of tub2, JC69+H for the second codon position of tub2 and TN93+H for the third codon position of tub2. The ML trees with the highest log likelihood (-1172(- .0198 in ITS, -1196 in act, -859.37115 in tub2 and -3315.7254 in ITS-act-tub2) are shown in Fig. 2. Our results confirmed close phylogenetic relationships between Thyridium and Phialemoniopsis ( Fig. 2A-D). Except for act (Fig. 2B) and tub2 (Fig. 2C), where sequence data of T. vestitum were unavailable, the existence of ten distinct species was suggested ( Fig. 2A, D). The following three lineages were found in our four strains ( Fig. 2A-D): 1) a bambusicolous lineage (KT 3891) close to T. curvatum and T. limonesiae, 2) a fungus on Betula maximowicziana (KT 3803) nested with T. pluriloculosum, which was previously reported from clinical sources (Perdomo et al. 2013), and 3) another bambusicolous lineage represented by two strains (KT 1015 and KT 3905).

Taxonomy
A new order, Thyridiales, is introduced to accommodate Thyridiaceae because its lineage is phylogenetically and morphologically distinct from any known orders in Sordariomycetes. We concluded Thyridium and Phialemoniopsis to be congeneric based on their morphological similarities and phylogenetic relatedness. An expanded generic circumscription of Thyridium that integrates the generic concept of Phialemoniopsis is provided below. One new species and eight new combinations of Thyridium are proposed. Notes. Thyridiaceae has been treated as incertae sedis in Sordariomycetes (Yue and Eriksson 1987). Members of Thyridiaceae differ from Myrmecridiales by having pycnidial conidiomata, becoming cup-shaped in the coelomycetous state and micronematous conidiophores with monophialidic conidiogenous cells in the hyphomycetous state. Myrmecridiales have brown thick-walled conidiophores with polyblastic conidiogenous cells (Crous et al. 2015a). Annulatascales have relatively massive refractive, well-developed, conspicuous apical annulus in asci (Wong et al. 1999;Campbell and Shearer 2004;Dong et al. 2021). In contrast, those of members of Thyridiaceae are compact and inconspicuous. Therefore, a new order, Thyridiales, is introduced for this lineage. Type genus. Thyridium Nitschke, Pyrenomyc. Germ. 1: 110 (1867). Notes. Phialemoniopsidaceae is considered a synonym of Thyridiaceae because Phialemoniopsis, the type genus of Phialemoniopsidaceae, was revealed congeneric with Thyridium and is placed in the synonymy of the latter genus in this study. The type genera of both families, that is, Thyridium and Phialemoniopsis, share many morphological features in their asexual states, as noted below. Sexual morph. Stromata scattered to grouped, subepidermal to erumpent, yellowish to dark brown, red in KOH or not changing. Ascomata perithecial, subglobose to ampulliform, single to grouped, immersed in stromata to erumpent through host surface. Ascomatal wall composed of several layers of polygonal, dark brown cells. Ostiolar neck cylindrical, short or long, separated or convergent in upper stromata, periphysate. Paraphyses numerous, septate, unbranched, cylindrical, hyaline. Asci unitunicate, cylindrical, broadly rounded at the apex, with a pronounced non-amyloid apical annulus, pedicellate. Ascospores obovoid or ellipsoid, smooth, pale brown to brown, with several transverse and 0-3 longitudinal or oblique septa.
Notes. The newly obtained Thyridium collections formed synasexual morphs, coelomycetous and hyphomycetous, in culture that were similar to those of Phialemoniopsis, having coelomycetous and/or hyphomycetous conidial states in culture (Perdomo et al. 2013). In this study, Phialemoniopsis is treated as a synonym of Thyridium because of their morphological similarities in asexual morphs and phylogenetic relatedness. The genus Pleurocytospora has been proposed as a synonym of Thyridium by culture studies (Leuchtmann and Müller 1986). We agree that the morphological features of Pleurocytospora, such as phialidic conidiogenous cells and hyaline, ellipsoidal conidia formed from both coelomycetous and hyphomycetous states (Leuchtmann and Müller 1986), are almost identical to those of the generic concept of Thyridium emended here.
Notes. The conidia from aerial hyphae of strain KT 3803 were larger (3-9 × 1-2.5 µm) in culture than those of the original description of Thyridium pluriloculosum (3-5 × 1-2.5 µm; Perdomo et al. 2013). However, we identified this new collection on Betula maximowicziana as T. pluriloculosum, based on the high sequence homology of three loci with ex-type culture of this species (CBS 131712; 99.6% in ITS, 99.2% in act, and 99.5% in tub2). The sexual-asexual relationship of T. pluriloculosum was verified in this study. Although this species has been reported from clinical sources as an asexual morph (Perdomo et al. 2013), the recently collected material represents a sexual morph on plant material.
Other specimen examined. Notes. This species has been described from Phyllostachys nigra var. henonis, as a species of Pleospora (Dothideomycetes; Hino 1961). Our phylogenetic analysis (Fig. 1) shows that this species is a member of the genus Thyridium (Sordariomycetes). The morphological features of this species are consistent with those of the genus Thyridium, including immersed to erumpent, single to grouped, perithecial ascomata with a cylindrical ostiolar neck, unitunicate asci and muriform, pigmented ascospores (Eriksson and Yue 1989). Therefore, we propose a new combination, T. punctulatum, for Pleospora punctulata.     (2002).

Discussion
We show that the asexual genus Phialemoniopsis (established by Perdomo et al. 2013) is a synonym of the sexual genus Thyridium (established by Nitschke 1867). We found a new species of Thyridium (T. flavostromatum), transferred Pleospora punctulata into Thyridium, and proposed seven new combinations in Thyridium for strains previously treated in Phialemoniopsis. We provided a revised generic circumscription of Thyridium based on both sexual and asexual characteristics and revealed the phylogenetic relationships of species within this genus. The genus Thyridium has been defined mainly on the basis of sexual characters (Nitschke 1867;Eriksson and Yue 1989). Currently, 33 species are recorded in this genus (http://www.indexfungorum.org, 2021). Asexual morphs are unknown in most species of Thyridium, with the exceptions of T. flavum and T. vestitum, in which asexual morphs have been recorded based on sexual-asexual association on the same specimen (Petch 1917) and on the basis of culture study (Leuchtmann and Müller 1986, this study), respectively. In contrast, the genus Phialemoniopsis has been defined based only on asexual characters (Perdomo et al. 2013). Its ordinal affiliation within Sordariomycetes has not been resolved, but recent phylogenetic analyses of this class suggest that Phialemoniopsis is close to Thyridium . In our phylogenetic analysis, all species previously described as Phialemoniopsis (marked with blue circle; Fig. 1) were clustered in a single clade, including the type species of Thyridium (T. vestitum), as well as two new strains proposed here (T. flavostromatum and T. punctulatum). Both genera have similar asexual morphs, which have conidiophores bearing small groups of phialides, hyaline phialidic conidiogenous cells, and ellipsoidal or allantoid, hyaline conidia in both coelomycetous and hyphomycetous states (Petch 1917;Leuchtmann and Müller 1986;Perdomo et al. 2013). Morphological and molecular phylogenetic evidence clearly shows that Phialemoniopsis is congeneric with Thyridium.
Synonymising Phialemoniopsis under Thyridium expanded information about the asexual morphs of Thyridium. In this genus, only T. vestitum has been demonstrated to have asexual morphs by culture studies (Leuchtmann and Müller 1986). It has both coelomycetous and hyphomycetous complex asexual morphs, which have phialidic conidiogenous cells with collarette and ellipsoidal to allantoid hyaline conidia (Leuchtmann and Müller 1986). Members of Phialemoniopsis also have coelomycetous and/or hyphomycetous conidial states (Perdomo et al. 2013;Tsang et al. 2014;Su et al. 2016;Martinez et al. 2021). The close relationship of Phialemoniopsis and Thyridium suggests that such complex asexual morphs may be common within Thyridium species.
In Thyridium, T. endophyticum and T. curvatum have been isolated from both plants and animals (Gam and McGinnis 1983;Halleen et al. 2007;Perdomo et al. 2013;Su et al. 2016;Ito et al. 2017). There are several examples of fungal species, including human pathogens, detected from various substrates. For example, Phaeoacremonium minimum is a pathogen on grapevines, where it forms both sexual and asexual morphs (Crous et al. 1996;Pascoe et al. 2004), but it has also been reported as a causative agent of subcutaneous phaeohyphomycosis in humans as asexual morph (Choi et al. 2011). Other species of Thyridium may also have cryptic life cycles and can colonise each host substrate at different reproductive stages. An example of this prediction can be found in T. pluriloculosum. This species was originally found in human nails as an asexual fungus (Perdomo et al. 2013), and its sexual state was rediscovered on twigs of Betula maximowicziana in our study.
Epitypification of the type species of Thyridium (T. vestitum) will be a necessary issue in the future. We used sequences from two non-type strains (CBS 113027, CBS 125582) of this species for phylogenetic analyses but they did not form a monophyletic clade (Fig. 1). Sequence differences between these two strains were found at 34 positions with four gaps in the LSU. These results indicate that the strains obtained from Acer pseudoplatanus (CBS 113027) and no host information (CBS 125582) in Austria are not conspecific. A fresh collection of T. vestitum on original host plant from the type locality (Ribes rubrum, Sweden; Fries 1823) and its phylogenetic analysis are required to fix generic circumscription of Thyridium.
Thyridiales established here may encompass other genera and families with morphologies distinct from the genus Thyridium (Thyridiaceae). Some species of "Linocarpon" and "Neolinocarpon" are nested within the Thyridiales (Fig. 1). Linocarpon and Neolinocarpon sensu stricto belong to Linocarpaceae (Chaetosphaeriales) and are morphologically distinct from Thyridium in having filiform, straight or curved, unicellular, hyaline, or pale-yellowish ascospores (Huhndorf and Miller 2011;Konta et al. 2017). The "Linocarpon" and "Neolinocarpon" species phylogenetically unrelated to Linocarpon and Neolinocarpon sensu stricto may be new lineages in Thyridiaceae or belong to its own new undescribed family. However, we cannot clarify the phylogenetic/taxonomic relatedness of these atypical Linocarpon-like species because none of them are ex-types and their morphological information are unavailable. Further molecular phylogenetic study of these fungi based on protein-coding sequences and finding additional specimens/isolates of "Linocarpon" and "Neolinocarpon" species related to Thyridium will be necessary to clarify their taxonomic affiliation and better understand the concept of Thyridiales.