a novel coniothyrium-like genus in Coniothyriaceae ( Pleosporales) from salt marsh ecosystems in Thailand

In coastal ecosystems, salt marsh habitats are common and consist of diverse halophytes, grasses, herbs, and shrubs, as well as microorganisms ^{[ 1, 2]} . These species-rich ecosystems are highly productive, and investigating fungal diversity in these habitats is important while many areas are still being explored ^{[ 2, 3]} . Researchers are currently studying the taxonomy of fungi in marine and semi-marine environments and increasing the number of known taxa recorded ^{[ 4− 6]} . Ascomycota was identified as the dominant group in world salt marsh ecosystems, including the highest diversity in Pleosporales, Dothideomycetes ^{[ 2]} . Coniothyriaceae is a pleosporalean family with a large number of terrestrial taxa, while taxa associated with salt marsh vegetation have rarely been reported ^{[ 2]} .

Coniothyriaceae was established by Cooke ^{[ 7]} to accommodate Coniothyrium species. The type genus and species of Coniothyriaceae are Coniothyrium Corda and C. palmarum Corda, respectively ^{[ 8− 10]} . This family was previously linked to Leptosphaeriaceae ^{[ 11]} and this was followed by several authors ^{[ 12− 16]} . Subsequently, molecular data analyses for phoma-like asexual morphs were performed by de Gruyter et al. ^{[ 17]} based on LSU and ITS sequence data and revealed that C. palmarum is phylogenetically distant from Leptosphaeriaceae and closely related to Coniothyriaceae. de Gruyter et al. ^{[ 17]} reinstated Coniothyriaceae as a distinct family in Pleosporales and transferred several Phoma and Pyrenochaeta species into this family. Thus, the morphological variations of Coniothyrium species were expanded by the addition of more characters, such as setose pycnidia and conidiogenesis with elongated conidiophores ^{[ 17]} . Several authors later updated the placements of many Coniothyrium species with generic level changes and novel genera placed in different families ^{[ 18, 19]} .

Verkley et al. ^{[ 19]} studied morphology and phylogenetic relationships of coniothyrium-like and closely related taxa. These species are coelomycetous and characterized in having pycnidial or stromatic conidiomata and small, subhyaline to pigmented, 1- or 2-celled conidia ^{[ 19]} . The phenotypic plasticity of these coelomycetous species has made their taxonomic placements uncertain and, thus the majority of them have been placed in Coniothyrium ^{[ 19− 21]} . Both Coniothyrium and coniothyrium-like species were identified as polyphyletic within Pleosporales and recent taxonomic treatments were mainly treated with combined morphology and molecular data analyses ^{[ 12, 16, 17, 19, 21− 32]} . Coniothyrium sensu stricto is characterized by 1-septate conidia and grouping in Coniothyriaceae ^{[ 17, 19− 21, 33]} . Currently, Coniothyriaceae consists of five genera, such as Coniothyrium, Foliophoma Crous , Neoconiothyrium Crous, Ochrocladosporium Crous & U. Braun and Staurosphaeria Rabenh. (≡ Hazslinszkyomyces Crous & R.K. Schumach.) ^{[ 10, 34]} .

Coniothyriaceae members have been identified as pathogens that cause necrotrophic and leaf spots on leaves, and saprobes on dead branches ^{[ 10, 17]} . The sexual morph is characterized in having cucurbitaria-like, black, globose ascomata, short central ostiole, textura angularis peridium cells, branched, septate, cellular pseudoparaphyses, 8-spored, cylindrical, bitunicate asci and muriform, ellipsoidal ascospores that are initially hyaline and brown at maturity ^{[ 10]} . Asexual morphs are coelomycetous and sometimes differentiated with phoma-like, camarosporium-like, coniothyrium-like, or cladosporium-like asexual characters. They are characterized in having dark, globose, pycnidial conidiomata, with central, sometimes papillate ostiole, cells of textura angularis or textura globulosa in the conidiomatal wall, hyaline macroconidiogenous and microconidial cells and conidia. Conidial morphology is varied as macroconidia and microconidia. Macroconidia are ellipsoid, red-brown, and septation is from the central transverse septum to muriformly septate, while microconidia are hyaline, globose to ellipsoid and aseptate ^{[ 9, 10, 24, 27, 35, 36]} .

The aim of the study

In this study, we aim to expand the taxonomy of fungi associated with dead plant hosts in salt marsh ecosystems. We investigate salt marsh habitats in Thailand to collect fungal specimens and isolate them to find out the taxonomic novelties. Morphological illustrations, comprehensive descriptions, and multi-gene phylogenetic analyses are provided to confirm the placement of new findings.

Fungal specimens were collected from salt marsh habitats in Pranburi Province, Thailand, 2021. Samples were preserved in sterile Ziploc bags in the laboratory and incubated at room temperature 25 °C. Rehydrated specimens were observed to identify fungal fruiting bodies and macro-morphology was observed by using a Motic SMZ 168 compound stereomicroscope. Micro-morphologies (e.g., conidiomata, conidiogenous cells, conidia) were examined from hand-sectioned structures using a Nikon ECLIPSE 80i compound stereomicroscope, equipped with a Canon 600D digital camera. The measurements of photomicrographs were obtained using Tarosoft (R) Image Frame Work version 0.9.7. Images were edited with Adobe Photoshop CS6 Extended version 13.0.1 software (Adobe Systems, San Jose, California, USA).

Single-spore isolation was carried out as described by Senanayake et al. ^{[ 37]} . Germinated spores were aseptically transferred into fresh malt extract agar medium (MEA) prepared in 50% or 100% concentrations of sterilized natural seawater ^{[ 38]} . Culture plates were incubated at 25 °C for six weeks and inspected every week. Herbarium specimens are preserved at Mae Fah Luang University Herbarium (MFLU) in Chiang Rai, Thailand. All living cultures are deposited at Mae Fah Luang Culture Collection (MFLUCC). Facesoffungi and Index Fungorum numbers for new taxa were obtained ^{[ 39, 40]} .

DNA extraction, PCR amplification, sequencing

The methodologies for DNA extraction, PCR, gel electrophoresis, and sequencing were followed, as detailed in Dissanayake et al. ^{[ 41]} . The genomic DNA was extracted from fresh mycelium using the E.Z.N.A Fungal DNA Mini Kit- D3390-02 (Omega Bio-Tek, USA) following the guidelines of the manufacturer. DNA sequences were obtained for the internal transcribed spacer region (ITS1, 5.8S, ITS2), the small subunit (SSU), and the large subunit (LSU) of the nuclear ribosomal RNA gene. PCR thermal cycle programs for each locus region are presented in Table 1. Purification and sequencing were outsourced to the Bio Genomed Co. LTD laboratory (Biogenomed Co., Thailand). Newly generated sequences were submitted to NCBI GenBank ( www.ncbi.nlm.nih.gov/genbank).

BioEdit v 7.0.9.0 program ^{[ 45]} was used to check the quality of the newly generated sequence chromatograms. For primary identification, contig sequences were checked with BLAST searches in NCBI. Sequences for phylogenetic analyses were downloaded from GenBank ( Table 2) following Hyde et al. ^{[ 25]} . Each gene matrix was aligned with MAFFT version 7 ^{[ 46]} with default parameters and manually adjusted for improvement where necessary using BioEdit v. 7.2 ^{[ 45]} . The trimAl v1.4 software was used for the automated removal of spurious sequences or poorly aligned regions in each single gene alignment, and gappyout was selected as the automated trimming method ^{[ 47]} . Two separate phylogenetic analyses were conducted: Maximum Likelihood (ML) and Bayesian Inference (BI). LSU, SSU, and ITS concatenated dataset was analyzed for Coniothyriaceae and selected families in Pleosporales.

Taxon treatments

Phylogenetic analyses

In the phylogenetic analyses, maximum likelihood (ML) was executed using IQ-Tree web server ( http://iqtree.cibiv.univie.ac.at/) with bootstrap support obtained from 1,000 pseudoreplicates ^{[ 48, 49]} . Bayesian Inference (BI) analysis was performed on the CIPRES Science Gateway portal under MrBayes on XSEDE (3.2.7a) ^{[ 50]} . Six simultaneous Markov chains were run for 1,000,000 generations, and trees were sampled every 1,000 ^th generation, ending the run automatically when the standard deviation of split frequencies dropped below 0.01. The best nucleotide substitution models for each genetic marker were evaluated using jModelTest2 on XSEDE in the online CIPRES Portal ( www.phylo.org/portal2) ^{[ 51, 52]} . The best-fit models under the AIC criterion were revealed to be GTR+I+G for ITS and LSU regions while GTR+I for SSU region. Phylogenetic trees were visualized with FigTree version 1.4.0 ^{[ 53]} and edited in Microsoft PowerPoint (2019).

The combined LSU, SSU, and ITS alignment was used to construct the final phylogenetic analysis ( Fig. 1) of maximum likelihood (ML) and Bayesian inference (BI).

Figure 1. 

Phylogram generated from maximum likelihood analysis based on combined LSU, SSU, and ITS sequenced data. Fifty-eight strains were included in the combined sequence analyses, which comprised 2251 characters with gaps (LSU = 800, SSU = 948, ITS = 503). Single gene analyses were also performed, and topology and clade stability were compared from the combined gene analyses. Ascochyta pisi Lib. (CBS 126.54) and Didymella azollae E. Shams, F. Dehghanizadeh, A. Pordel & M. Javan-Nikkhah (A1) were used as the outgroup taxa. The final ML optimization likelihood is -10163.644. The matrix included 494 distinct alignment patterns including undetermined characters. Estimated base frequencies were obtained as follows: A = 0.245, C = 0.219, G = 0.274, T= 0.262; substitution rates AC = 2.73290, AG = 3.93954, AT = 2.73290, CG = 1.0, CT = 7.93321, GT = 1.0 and the gamma distribution shape parameter α = 0.439534. Bootstrap support values for ML (first set) equal to or greater than 75% and BYPP equal to or greater than 0.95 are given above or below the nodes. The strains from the current study are in red bold and the type strains are in black bold. The scale bar represents the expected number of nucleotide substitutions per site.

Morphological analyses

Coniothyrioides Wijes., M.S. Calabon, E.B.G. Jones & K.D. Hyde, gen. nov.

Index Fungorum number: 555045; Facesoffungi number: 13901 Fig. 2

Etymology – Resembling Coniothyrium taxa

Saprobic on a submerged decaying wood in salt marsh ecosystems. Sexual morph: Undermined. Asexual morph: Coelomycetous. Forming conspicuous, round to irregular, black pycnidia. Conidiomata semi-immersed, erumpent through the host substrate, globose to subglobose, solitary, scattered to aggregated, uni-loculate, ostiolate, covered in setae, rigid when dehydrated, black. Setae originated from the outermost layers of conidiomatal wall, divergent, brown, with hyaline apex, septate, smooth-walled, uniformly wide from base to apex. Conidiomatal wall composed of several layers, from outer to inner layers black, dark brown, pale brown to hyaline cells of textura angularis. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner cavity, doliiform to subcylindrical, smooth-walled, hyaline, enteroblastic, phialidic conidiogenesis with periclinal thickening at the apex. Conidia solitary, ellipsoidal to obovoid, rounded at the apex, aseptate, initially hyaline, becoming pale to dark brown at maturity, smooth-walled, sometimes finely verruculose, with smaller guttules at young and indistinct at maturity.

Figure 2. 

Coniothyrioides thailandica sp. nov. (MFLU 22-0276, holotype). (a) & (b) Appearance of conidiomata on a submerged decaying woody substrate. (c) Longitudinal section of conidioma. (d) Conidiomatal wall. (e) The appearance of setae. (f) & (g) Conidiogenous cells with developing conidia. (h) Conidia. Scale bars: a = 200 μm, b = 100 μm, c = 50 μm, d = 20 μm, e, h = 10 μm, f, g = 5 μm.

Type species – Coniothyrioides thailandica

Note – Coniothyrioides gen. nov. is a monotypic genus associated with decaying woody substrates in salt marsh habitats in central Thailand. This genus is characterized in having pycnidial conidiomata with the cells of textura angularis wall surrounded by distinct setae, doliiform to subcylindrical, hyaline conidiogenous cells, and ellipsoidal to obovoid, aseptate and hyaline to brown conidia. Based on some conidial characteristics such as aseptate, hyaline to brown conidia the genus shares similar morphologies to coniothyrium-like taxa ^{[ 19]} , by ellipsoidal to subcylindrical conidia sharing similar characters to Coniothyrium and Neoconiothyrium ^{[ 9, 16, 20, 24]} . However, other accepted genera in Coniothyriaceae differ from this genus in conidial morphologies: Foliophoma has only hyaline conidia except for F. camporesii D. Pem & K.D. Hyde; Hazslinszkyomyces has muriformly septate conidia ^{[ 27]} ; Ochrocladosporium has cladosporium-like conidia ^{[ 35]} . Moreover, phylogenetically Coniothyrioides forms a distinct lineage within Coniothyriaceae ( Fig. 1). Coniothyrium carteri (Gruyter & Boerema) Verkley & Gruyter (LG1401_MS6E) was the closest species based on BLAST result of ITS (94.33% similarity) and C. telephii (Allesch.) Verkley & Gruyter (UTHSC:DI16-189) was the closest species LSU sequence data (99.31% similarity) and sequences are lacking for SSU in the GenBank. The genus is known from its asexual morph and the sexual morphology was not observed.

In our phylogenetic analyses, Foliophoma species were grouped outside of Coniothyriaceae with closer to Libertasomycetaceae and Pleosporaceae species. Foliophoma was introduced by Crous & Groenewald ^{[ 27]} to accommodate F. fallens (Sacc.) Crous, in Coniothyriaceae based on the parsimony analyses of single LSU and ITS sequence data. Foliophoma camporesii was later introduced based on morphology and maximum likelihood analyses of LSU-SSU- ITS sequence data by Hyde et al. ^{[ 25]} . Based on morphology, Foliophoma species share similar characteristics to the species of Coniothyriaceae in having dark brown conidiomata, conidial wall with textura angularis cells, phialidic conidiogenesis sometimes with periclinal thickening or percurrent proliferation, and mainly ellipsoidal shaped conidia. However, the type species of the genus, F. fallens differs other Coniothyriaceae taxa in having eustromatic conidiomata. Based on this taxonomic uncertainty, more fresh collections with additional coding genes are required to clarify the accurate placement of Foliophoma.

Coniothyrioides thailandica Wijes., M.S. Calabon, E.B.G Jones & K.D. Hyde, sp. nov.

Index Fungorum number: 555050; Facesoffungi number: 13902

Etymology – The name reflects the county Thailand, from where the species was isolated.

Saprobic on a submerged and decaying woody substrate. Sexual morph: Undermined. Asexual morph: Coelomycetous. Conidiomata 150–200 μm high, 100–150 μm diam. (x̄ = 160 × 130 µm), pycnidial, semi-immersed, erumpent through the host substrate, globose to subglobose, solitary, scattered to aggregated, uni-loculate, ostiolate, covered by setae, rigid when dehydrated, black. Setae 3–5 µm wide, originating from the outermost layers of conidiomatal wall, divergent, brown, with hyaline apex, septate, smooth-walled, uniformly wide from base to apex. Conidiomatal wall 15–20 µm wide, equally thickened, composed of several layers, outermost layers dark brown to black, towards inside pale brown to hyaline cells of textura angularis, surrounded by setae. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 4–5 μm long × 2.5–3.5 μm wide, lining the inner cavity, doliiform to subcylindrical, smooth-walled, hyaline, enteroblastic, phialidic conidiogenesis with periclinal thickening at the apex. Conidia 3–5 × 2.5–3 μm ( $\overline x $ = 4.5 × 2.7 µm, n = 20), solitary, ellipsoidal to obovoid, rounded at the apex, aseptate, initially hyaline, becoming pale to dark brown at maturity, smooth-walled, sometimes finely verruculose, with smaller guttules at young and indistinct at maturity.

Culture characteristics – On MEA, colony circular with a filamentous margin, reaching 40–45 mm diam. in 25 d at 25 °C, light gray from above, brown from center becoming light gray in the margin below, surface rough, dry, flat, with dense mycelia, edge filiform.

Material examined – Thailand, Pranburi Province, on a submerged decaying wood, 23 March 2021, Mark S. Calabon, SPAR26 (MFLU 22-0276, holotype), ex-type living cultures, MFLUCC 22-0193.

GenBank numbers – ITS = OQ023276, LSU = OQ023277, SSU = OQ025050.

Notes – Coniothyrioides thailandica sp. nov. shares morphological characters with other representatives in Coniothyriaceae in having pycnidial, globose, uni-locular conidioma with a central ostiole, peridial wall with the cells of textura angularis, and doliiform to subcylindrical conidiogenous cells, phialidic conidiogenesis with a periclinal thickening at the apex. The synopsis of asexual morphological characters for the generic types of the family including their hosts and localities is presented in Table 3. Based on the presence of conidiomatal setae, our species (MFLU 22-0276) resembles Neoconio thyrium ^{[ 24]} . In addition, our species resembles Foliophoma camporesii (MFLU 17-1006) in having hyaline to brown and aseptate conidia but differs in having larger conidiomata (150–200 × 100–150 vs 40–47 × 40–69 μm) and the presence of setae on the wall ( Table 3). Phylogenetically, our strain (MFLUCC 22-0193) formed an independent lineage within Coniothyriaceae with 94% ML and 0.99 BI statistical support ( Fig. 1). The base pair differences between our stain and the strains represent type species of other genera in Coniothyriaceae are listed ( Table 4). Thus, the evidence based on both morphology and phylogeny, we establish Coniothyrioides as a new genus in Coniothyriaceae with C. thailandica as the type species.

Members of Coniothyriaceae have high morphological plasticity and it is not adequate to use only morphology for identification at the genus level. Coniothyrium dolichi (Mohanty) Verkley & Gruyter (≡ Pyrenochaeta dolichi Mohanty, CBS 124140) and C. glycines (R.B. Stewart) Verkley & Gruyter (≡ P. glycines R.B. Stewart, CBS 124455) form a separate clade within Coniothyriaceae ( Fig. 1). Based on the morphology observed from corn meal agar medium (CMA) by Grondona et al. ^{[ 54]} , C. dolichi differs to our species by having two types of conidiogenous cells including discrete, ampulliform conidiogenous cells and integrated, cylindrical conidiogenous cells on filiform, septate conidiophores in the same conidioma, while our species has doliiform to subcylindrical conidiogenous cells and conidiophores are reduced to conidiogenous cells ^{[ 54]} . The pycnidial conidiomata and the ostiole of C. dolichi covered by dark brown, septate setae resembles our species and conidia are aseptate, ellipsoid, and hyaline with more or fewer guttules while our species has brown conidia at maturity ^{[ 54]} . The original description of C. dolichi, mentioned that conidia were greenish-yellow in mass similar to coniothyrium-like conidia, as well as to our species ^{[ 55].} Also, a monodictys-like synanamorph was reported in C. dolichi based on its dark brown to black, dictyosporous conidia by differs from our species ^{[ 17, 54, 56]} . Coniothyrium glycines produces monophialidic, ampulliform, conidiogenous cells and aseptate, ellipsoidal conidia (4–8 × 1–3 µm), while our species has doliiform to subcylindrical conidiogenous cells ^{[ 57, 58]} . The unique character of C. glycines is well-defined, dark brown to black, melanized sclerotia covered with setae which differs from our species and other Coniothyrium taxa. Based on the multi-gene phylogeny provided by de Gruyter et al. ^{[ 17]} and the results of our study, the placements of these two species were confirmed in Coniothyriaceae. Also, Coniothyrium triseptatum Dayar., Thyagaraja & K.D. Hyde (MFLU 19-0758) creates a separate lineage in Coniothyriaceae ( Fig. 1) and only sexual morph was reported for this fungus. Therefore, we could not compare the morphology of C. triseptatum with our species ^{[ 31]} .

In this study, we introduced the novel genus Coniothyrioides in Coniothyriaceae, with C. thailandica as the type species, following the guidelines and major criteria for defining generic and species boundaries in Dothideomycetes by Chethana et al. ^{[ 59]} and Pem et al. ^{[ 60]} . The coelomycetous asexual morph of Coniothyrioides was associated with the decaying and submerged wood in the salt marsh habitats. Traditionally, morphology is used to delimit coelomycetes by considering the characteristics of conidiomata, conidiophores, conidiogenesis, and conidia including host associations ^{[ 20, 32, 61]} . However, accurate taxonomy of most coniothyrium-like species is challenging because of their simplicity, plasticity, and morphological variations ^{[ 19]} . In our study, genera in Coniothyriaceae differ in some conidial morphologies. For instance, Coniothyrium is characterized by aseptate to 1-septate, ellipsoidal to clavate or cylindrical, brown conidia ^{[ 9, 17, 62]} , Foliophoma with aseptate, ovoid ellipsoidal, only hyaline or hyaline-brown conidia, and Hazslinszkyomyces ellipsoidal to obovoid, transversely and muriformly septate, uniformly brown conidia ^{[ 27]} . Neoconiothyrium species have aseptate or 1-septate, ellipsoid to subclavate or subcylindrical, hyaline to medium brown conidia ^{[ 24]} while Ochrocladosporium species have cladosporium-like pale brown, aseptate or 1-septate conidia that occurring in branched chains ^{[ 35]} . Coniothyrioides thailandica is characterized by aseptate, ellipsoidal to obovoid, hyaline to pale or dark brown conidia. However, the phylogenetic analyses in this study reveal these morphological differences are not strong enough for generic delimitation of the family. Some characteristics of Coniothyrioides overlap with those of other accepted genera in the family, such as the conidiogenous cell morphology of Coniothyrium and Foliophoma which have doliiform to cylindrical or subcylindrical, hyaline, phialidic conidiogenesis with periclinal thickening and conidia show aseptate, ellipsoid-associated shapes, and hyaline to brown pigmentation ( Table 3).

The number of fungi was estimated at between 2.2 to 3.8 million ^{[ 63]} , with about 100,000-150,000 known species and fungus-like taxa ^{[ 64 − 66]} . There are 151,834 species listed in Species Fungorum ^{[ 67]} . An up-to-date online database ( https://coelomycetes.org/) for coelomycetes is being implemented ^{[ 21, 32]} . As coniothyrium-like taxa are frequently collected and morphologically similar, it is likely that they will remain unidentified. Therefore, it is to be expected that if molecular data are incorporated in morpho-taxonomic studies of these groups, will help identify many more novel taxa. This has occurred in other genera, which are plant pathogens and ecologically more important. According to Bhunjun et al. ^{[ 65]} Coniothyrium is one of the most speciose genera listed in Species Fungorum in 2021 and studies of coniothyrium-like taxa may yield more novel species. A few records of Coniothyriaceae taxa have been identified in salt marsh ecosystems, such as Coniothyrium obiones Jaap (India and Portugal) and as unidentified Coniothyrium species (USA) ^{[ 2]} . However, Wanasinghe et al. ^{[ 29]} referred the placement of C. obiones in Neocamarosporiaceae based on multi-gene phylogeny. In marine habitats, C. cerealis E. Müll. was isolated as an alga-derived fungus in the Baltic Sea by Elsebai et al. ^{[ 68, 69]} .

In this study, we discussed the morphology and multi-gene phylogenetic analyses results of our new collection to verify its identity and phylogenetic placement in Coniothyriaceae. Based on ecological and geographical data on salt marsh fungi, we noted lack records of Coniothyriaceae worldwide (see Calabon et al. ^{[ 2]} ). Thus, we propose that additional collections be conducted in order to identify other Coniothyriaceae taxa and improve our understanding of fungal diversity in salt marsh ecosystems, a topic that is currently understudied.