Fresh samples of decaying fruits and twigs from Dipterocarpaceae sp. were collected at the Mushroom Research Center, Chiang Mai, Thailand, in 2019. Samples were observed using a stereomicroscope (Motic SMZ-171). The detailed method of collection, observation of specimens, and isolation were carried out as references in the study by Senanayake et al. (2020) and Tang et al. (2022). The Tarosoft (R) Image Frame Work application (IFW 0.97 version) was used to take measurements, and the photoplates were made by Adobe Photoshop CS6 (Adobe Systems, USA). The type specimens were deposited in the Mae Fah Luang University Herbarium (MFLU), Chiang Rai, Thailand, and the ex-type cultures were deposited in the Culture Collection at Mae Fah Luang University (MFLUCC). Index Fungorum (2023) and Faces of Fungi numbers were acquired as detailed by Jayasiri et al. (2015). New species are established as recommended by Chethana et al. (2021a), and the records of new taxa in the Greater Mekong Subregion were uploaded to the GMS database (Chaiwan et al., 2021).

Fresh mycelia were prepared from the living culture that grew for 28 days and stored in the refrigerator at −20°C. DNA extraction, polymerase chain reaction (PCR) amplifications, sequencing, and phylogenetic analyses were carried out following the study by Tang et al. (2022). The manufacturer's instructions were followed while using the genomic DNA extraction kits [Sangon Biotech (Shanghai) Co., Ltd., China], in order to obtain DNA. The genes and primers used in this study were as follows: for internal transcribed spacer region (ITS), ITS5 and ITS4 (White et al., 1990); 28S large subunit rDNA region (LSU), LR0R, and LR5 (Vilgalys and Hester, 1990; Cubeta et al., 1991); translation elongation factor 1-alpha (tef1-α), EF1-728F, and EF2 (O'Donnell et al., 1998; Carbone and Kohn, 1999); and for RNA polymerase II second largest subunit (rpb2), frpb2-5f , and frpb2-7cr (Liu et al., 1999) genes. The PCR was carried out in a volume of 50 μl. The reagents that were used in the polymerase chain reaction were as follows: the DNA template (2 μl), forward primers (2 μl), reverse primers (2 μl), 2 ×Taq PCR Master Mix (25 μl), and 19 μl of ddH₂O (double-distilled water). The annealing temperature was set to 52°C for 1 min and extension at 72°C for 90 s in LSU and ITS, followed by 35 cycles; 56°C for 1 min and extension at 72°C for 90 s in tef1-α, followed by 35 cycles; and 55°C for 1 min and extension at 72°C for 90 s in rpb2 followed by 35 cycles. The products of PCR were checked on 1% agarose gels and sent to Sangon Biotech (Shanghai) Co., Ltd., China for sequencing.

The forward and reverse primers of the newly generated sequence were assembled by the Contig Express v3.0.0 application, and the most similar taxa were found by BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) in NCBI. A combination of sequence data (ITS, LSU, tef1-α, and rpb2) of Cryphonectriaceae and Coryneaceae in GenBank (Tables 1, 2) was downloaded for phylogenetic analyses. Sequence data of each region were aligned by the online version of MAFFT v. 7 (https://mafft.cbrc.jp/alignment/server/index.html) (Katoh et al., 2017), through the “auto” option. Multiple genes were combined by SequenceMatrix (Vaidya et al., 2011). The aligned sequences were trimmed by manually adjusting and using trimAl v 1.2, with the “-gt 0.6” option (Capella-Gutiérrez et al., 2009). The phylogenetic analyses in this study were based on the maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI), by using a combined sequence dataset of ITS, LSU, tef1-α, and rpb2. The analysis of maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) was processed in the CIPRES web portal (Miller et al., 2010) using the “RAxML-HPC v.8 on XSEDE” tool, “PAUP on XSEDE” tool, and “MrBayes on XSEDE” tool, respectively (Huelsenbeck and Ronquist, 2001; Swofford, 2002; Stamatakis et al., 2008; Ronquist et al., 2012).

For ML analysis, the GTRGAMMA+I-Invar model of nucleotide evolution was used, and RAxML rapid bootstrapping was set to 1,000 bootstrap replicates (Stamatakis et al., 2008).

For MP analysis, 1,000 random taxa addition was used to infer trees. With branches of zero length collapsed and all multiple parsimonious trees saved, the value of Maxtrees was set to 5,000. For trees produced using various optimal criteria, parsimony score values for tree length (TL), consistency index (CI), retention index (RI), and homoplasy index (HI) were determined. To evaluate the clade stability, 1,000 iterations of the Bootstrap (BT) method were utilized, each comprising 100 trials of random stepwise addition of taxa (Hillis and Bull, 1993).

For BI, MrModeltest v2 was used for the selection of the best-fit model for each gene region. The Markov chain Monte Carlo (MCMC) algorithm was launched with four chains running concurrently from a random tree topology. When the divided frequencies' average standard deviation dropped below 0.01, the procedure was immediately terminated. The burn-in factor was set at 25%, and the sampling interval for trees was set to every 1,000th generation. The posterior probabilities (PP) for the remaining trees were computed (Dissanayake et al., 2020). Adobe Illustrator version 51.1052.0.0 and FigTree version 1.4.0 were further used to view trees (Adobe Inc., San Jose, California, United States).

For the phylogenetic analyses, a combined dataset of ITS, LSU, tef1-α, and rpb2 sequences was used. The dataset of Cryphonectriaceae included 70 taxa, with Foliocryphia eucalypti (CBS 124779) and Foliocryphia eucalyptorum (CBS 142536) as outgroups. The data matrix comprised 2,860 total characteristics, including gaps (ITS: 1–481 bp, LSU: 482–1,290 bp, tef1-α: 1,291–1,858 bp, and rpb2: 1,859–2,564 bp). Phylogenetic reconstructions with broadly comparable topologies were produced by the combined dataset of ML, MP, and BI analyses. The top-scoring ML tree with a final ML optimization likelihood value of −16,383.140512 (ln) is shown in Figure 1. In the ML analysis, the GTRGAMMA + I-Invar model was used, and the results showed 1,022 unique alignment patterns and 27.97% of indeterminate characteristics or gaps. Base frequency estimates were as follows: A = 0.229377, C = 0.266423, G = 0.271764, and T = 0.232436; substitution rates were as follows: AC = 1.760988, AG = 4.032209, AT = 1.914644, CG = 1.261342, CT = 8.527324, and GT = 1.000000; gamma distribution shape parameter alpha = 0.176927; and the tree length was 1.784127. The findings of the MP analysis showed that 2,564 characteristics remained unchanged, 103 were changeable but parsimoniously uninformative, and 733 were parsimoniously informative. The following values were displayed by the most parsimonious tree: TL = 2693, CI = 0.494, RI = 0.779, RC = 0.385, and HI = 0.506. The best-fit models for the BI analysis were GTR + I + G for ITS, LSU, tef1-α, and rpb2. With a final average standard deviation of split frequencies of 0.009895, Bayesian posterior probabilities (BYPP) from MCMC were analyzed. A new taxon correlated with the Cryphonectriaceae clade and is sister to Chrysomorbus. It is distinct from all other Cryphonectriaceae genera sampled herein, although with no support (Figure 1).

For the tree of Coryneaceae, the combined sequence dataset of 33 taxa was used with Neopestalotiopsis protearum (CBS 114178) and Neopestalotiopsis rosae (CBS 101057) as the outgroups. The data matrix comprised 2,977 total characteristics, including gaps (ITS: 1–597 bp, LSU: 598–1,426 bp, tef1-α: 1,427–2,123 bp, and rpb2: 2,124–3,151 bp). Based on the results of phylogenetic analysis, the top-scoring RAxML tree with a final ML optimization likelihood value of −19,448.697623 (ln) is shown in Figure 2. The GTRGAMMA + I-Invar model was applied to the RAxML analysis, and the findings revealed 1,332 distinct alignment patterns and 33.88% of ambiguous characteristics or gaps. The following were the base frequency estimates: A = 0.237835, C = 0.267649, G = 0.278605, and T = 0.215911; the substitution rates: AC = 1.607401, AG = 1.967526, AT = 1.403753, CG = 1.150806, CT = 5.717313, and GT = 1.000000; the gamma distribution shape parameter alpha = 0.260733; and the tree length = 3.464265. The results of the MP analysis revealed that 3,151 characteristics remained constant, 271 were variable and parsimoniously uninformative, and 1,142 were parsimoniously informative. The most frugal tree resulted in TL = 3,542, CI = 0.636, RI = 0.684, RC = 0.435, and HI = 0.364 as its values. For the BI analysis, the best-fit models were GTR+G for ITS, tef1-α, and rpb2 and SYM + I + G for LSU. The BYPP from MCMC were examined with a final average standard deviation of split frequencies of 0.009847. Based on the results of phylogenetic analysis of the combined ITS, LSU, tef1-α, and rpb2 sequencing data, the new taxon is related to Coryneum, Hyaloterminalis, and Talekpea within Coryneaceae, with statistical support of 72% ML and 1 BYPP. It differs from any other Coryneaceae genus sampled here (Figure 2).

Sexual morph see Jiang et al. (2020). Asexual morph Conidiomata semi-immersed to erumpent on the substrate, solitary, subglobose to pulvinate, pyriform, uni- to multiloculate, yellow, orange to fuscous black; necks absent or present with one to several attenuated necks. Conidiophores sometimes reduced to conidiogenous cells, cylindrical, hyaline, septate, or not. Conidiogenous cells hyaline, smooth, phialidic, ampulliform, inconspicuous, lining the inner cavity of conidiomata, with attenuate or truncate apices. Conidia hyaline, cylindrical, minute, seldom sigmoid, or slightly curved, aseptate (Jiang et al., 2020).

Notes: Cryphonectriaceae was described by Gryzenhout et al. (2006) to accommodate the Cryphonectria-Endothia complex based on LSU sequence data, and it mainly comprises plant pathogens (Vermeulen et al., 2011). Recently, Jiang et al. (2020) reevaluated this family based on morphology and combined ITS, LSU, tef1-α, and rpb2 multi-gene phylogenetic analysis. It now contains 22 genera and 56 species (Jiang et al., 2020; this study).

Type genus: Cryphonectria (Sacc.) Sacc. & D. Sacc.

Pulvinaticonidioma X. Tang, Jayaward, J.C. Kang & K.D. Hyde, gen. nov.

Saprobic on Dipterocarpaceae sp. Sexual morph not observed. Asexual morph Coelomycetous. Conidiomata immersed to semi-immersed in the substrate, solitary, glabrous or rough, pycnidial, subglobose, unilocular, thick-walled, ostiolate, brown to dark brown. Ostiole central, single with slightly protruding ostiolar papilla. Conidiomata wall composed of thick-walled, pale brown to dark brown cells of textura angularis at the exterior, and convex and pulvinate at the base. Conidiophores hyaline reduced to conidiogenous cells. Conidiogenous cells phialidic, cylindrical to ampulliform, determinate, smooth-walled, hyaline. Conidia hyaline, cylindrical, with obtuse ends, straight, unicellular, aseptate, thick- and smooth-walled.

Notes: Pulvinaticonidioma is characterized by solitary, subglobose, pycnidial conidiomata, phialidic, conidiogenous cells, and aseptate hyaline conidia. This matches with the morphological characteristics of Cryphonectriaceae (Jiang et al., 2020). Phylogenetically, Pulvinaticonidioma clusters with Chrysomorbus (Figures 3, 4). Both Pulvinaticonidioma and Chrysomorbus have a coelomycetous asexual morph (Chen et al., 2018). The former differs from the species in Chrysomorbus in having unilocular, glabrous or rough, thick-walled, ostiolate conidiomata with hyaline cells of textura angularis at the exterior, convex and pulvinate at the base; aseptate, straight, cylindrical, unicellular, and hyaline conidia with obtuse ends. After the comprehensive consideration based on the morphological and phylogenetic analysis, we, herein, introduce Pulvinaticonidioma as a new genus in Cryphonectriaceae, with Pulvinaticonidioma hyalinum as the type.

Saprobic on Dipterocarpaceae sp. Sexual morph not observed. Asexual morph Coelomycetous. Conidiomata 297–473 × 211–316 μm (x̄ = 375 × 267 μm, n = 20), immersed to semi-immersed in substrate, solitary, glabrous or rough, pycnidial, subglobose, unilocular, thick-walled, ostiolate, brown to dark brown. Ostiole 51–65 × 34–48 μm (x̄ = 58 × 42 μm, n = 10), central, single with slightly protruding ostiolar papilla. Conidiomata wall 50–88 μm (x̄ = 70 μm, n = 20) wide, composed of thick-walled, pale brown to dark brown cells of textura angularis at the exterior, convex and pulvinate at the base 103–202 μm high (x̄ = 144 μm, n = 20). Conidiophores hyaline, reduced to conidiogenous cells. Conidiogenous cells 6–11.5 × 1.8–3.4 μm (x̄ = 7 × 2.5 μm, n = 20), phialidic, cylindrical to ampulliform, determinate, smooth-walled, hyaline. Conidia 15–20 × 2–3 μm (x̄ = 17 × 2.5 μm, n = 20) hyaline, cylindrical, with obtuse ends, straight, unicellular, aseptate, thick- and smooth-walled.

Culture characters: Conidia germinated on PDA within 24 h, and germ tubes produced from one end. The culture was incubated at room temperature. Colonies reached 45 mm diameter after 15 days, flat, spreading, fluffy colonies, circular with irregular lightly orange outer ring, cottony. The surface is lightly rough, with orange-red colonies, cream-colored hyphae attached to the center of the colony, with an irregular orange-yellow edge. The reverse orange-red, more orange-yellow at the margins, not pigmented.

Material examined: Thailand, Chiang Mai province, Mae Taeng District, on the fruits (pericarp and wings of the pod) of Dipterocarpaceae, 8 August 2019, Xia Tang, Dip17 (MFLU 23-0052, holotype; ex-type living culture, MFLUCC 23-0002), on the fruits of Dipterocarpaceae, 23 October 2020, Xia Tang, Dip41 (MFLU 23-0053, paratype; ex-paratype living culture, MFLUCC 23-0004).

Notes: The two Pulvinaticonidioma hyalinum collections, showing similar morphology clustered together with ML = 100, MP = 100, and BYPP = 1 support (Figure 1). The base pair differences between the two strains were as follows: ITS = 0.7% (4/557), LSU = 0% (0/811), tef1-α = 6.2% (38/613), and rpb2 = 1% (11/983), respectively, and we identified them as the same species following the guidelines for species delineation proposed by Chethana et al. (2021a). Pulvinaticonidioma hyalinum matches the characteristics of Cryphonectriaceae and is similar in having unilocular conidiomata without necks and conidiomata walls made of cells of textura globulosa (Jiang et al., 2020). However, P. hyalinum differs from the type species of Cryphonectriaceae, Chrysomorbus lagerstroemiae in their fruiting body, conidiomata wall, conidiophores, and conidia. Pulvinaticonidioma hyalinum has brown to dark brown conidiomata with slightly protruding ostiolar papilla, hyaline cells of textura angularis at the exterior, interior layers that are convex and pulvinate at the base, and unbranched conidiophores, while Ch. lagerstroemiae has uni- to multilocular, conidiomata lacking ostioles, with convoluted locules, and occasionally aseptate conidia with separating septa and branching conidiophores. The conidiogenous cells in P. hyalinum are phialidic, cylindrical to ampulliform with hyaline, straight, aseptate, unicellular, conidia with obtuse ends, while Ch. lagerstroemiae has flask-shaped conidiogenous cells with attenuated apices and minute, cylindrical conidia with obtuse ends, that are hyaline, fusoid to oval, aseptate, and exuded as orange droplets (Chen et al., 2018). The phylogenetic analysis of the combined ITS, LSU, tef1-α, and rpb2 sequence data showed that P. hyalinum belongs to Cryphonectriaceae and forms a separate lineage sister to Chrysomorbus. Although the bootstrap values are low, the phylogenetic analysis supports the placement of our new taxa in Cryphonectriaceae, as well as the possibility of other close relatives that have not yet been discovered; hence, their placement within the family is subjected to change. The base pair differences between P. hyalinum and the type species of Chrysomorbus, viz. Ch. lagerstroemiae were as follows: ITS = 5% (27/539), LSU = 1.4% (11/811), and tef1-α = 26.5% (151/569), respectively. Based on the phylogenetic analysis and morphological comparison of the nearest genus, we, herein, introduce Pulvinaticonidioma as a new genus to accommodate the new collection, P. hyalinum.

Saprobes and pathogens exist on dead wood and living plants, respectively. Sexual morph: Stromata erumpent, solitary, comprising pseudoparenchymatous cells. Ectostromatic comprising small cells of textura prismatica, brown to black, disk well or poorly developed. Ascomata brown to black, ostiolate, aggregated, immersed, arranged in valsoid configuration, perithecial, coriaceous, globose to subglobose, papillate. Papilla central, upright, sometimes converging, broad, comprising brown cells of textura porrecta. Peridium thick-walled, comprising outer, brown cells of textura angularis and inner, thick-walled, hyaline, compressed cells of textura angularis. Paraphyses attached to the base, cellular, broad, septate, longer than asci. Asci ellipsoid to cylindrical, unitunicate, 8-spored, pedicellate, rounded at the apex with a J-, apical ring. Ascospores hyaline or initially hyaline, brown at maturity, overlapping uni- to biseriate, irregularly fasciculate, ellipsoid, 1–3-septate, fusoid or elongate, sometimes end-cells pointed, often distoseptate, pale brown or hyaline end-cells, straight or curved not constricted at the septa, guttulate, smooth-walled (added from Hyde et al., 2020b). Asexual morph: see Hyde et al. (2020b) and Rathnayaka et al. (2020).

Type genus: Coryneum Nees

Notes: Coryneaceae was described by Corda (1839) to accommodate Coryneum as the type genus. Rathnayaka et al. (2020) amended the description of this family to accommodate these genera based on their morphological characteristics and treated Talekpea and Hyaloterminalis in Coryneaceae. Until now, there are three genera included in Coryneaceae, viz. Coryneum (Nees von Esenbeck, 1816), Hyaloterminalis, and Talekpea (Rathnayaka et al., 2020; Wijayawardene et al., 2022).

Saprobic on Dipterocarpaceae sp. Asexual morph Not observed. Sexual morph Ascomata perithecial, erumpent, scattered, solitary, coriaceous, immersed, globose to subglobose, papillate, ostiolate, dark brown to black. The Ostiole canal narrowing toward the base, internally covered by hyaline periphyses, cells around the base small, thick-walled, and brown. Peridium comprising brown, compressed, cells of textura angularis. Hamathecium composed of cylindrical, unbranched, straight to flexible, smooth, hyaline, septate paraphyses slightly constricted at the septa, tapering toward to end, longer than asci. Asci 8-spored, unitunicate, clavate to broadly fusoid, short pedicellate, apex blunt, with an indistinct, J- apical ring, evanescent. Ascospores overlapping uniseriate to biseriate, biturbinate to subellipsoidal, 1-septate, slightly constricted at the septa, guttulate, smooth, hyaline to pale brown.

Notes: Subellipsoidispora share characteristics with Coryneaceae, such as perithecial, coriaceous, ostiolate, brown to black ascomata; with thick-walled peridium having outer and inner brown cells of textura angularis and hyaline, compressed cells of textura angularis, respectively; paraphyses are longer than asci; clavate to broadly fusoid, 8-spored asci with J- apical ring; guttulate and smooth, hyaline to pale brown and straight ascospores (Hyde et al., 2020b). Coryneaceae contains three genera, viz. Coryneum, Hyaloterminalis, and Talekpea (Rathnayaka et al., 2020). Both Subellipsoidispora and Coryneum have the ascomycetous sexual morph, while Talekpea and Hyaloterminalis have a hyphomycetous asexual morph (Senanayake et al., 2017, 2018). Subellipsoidispora differs from the species in Coryneum in having scattered, solitary ascomata; a thick-walled ostiolar canal narrowing toward the base, internally covered by hyaline periphyses, a peridium of brown-walled, compressed, cells of textura angularis, clavate to broadly fusoid, short pedicellate asci and biturbinate to subellipsoidal, 1-septate, guttulate ascospores, slightly constricted at the septa. In the phylogenetic analysis, Subellipsoidispora clusters in Coryneaceae and forms a separate lineage sister to Hyaloterminalis and Talekpea (Figure 2). Based on its unique morphology (Figure 5) and phylogenetic evidence (Figure 1), Subellipsoidispora is introduced as a new genus of Coryneaceae, and the sexual morph is described in this study, awaiting the discovery of its asexual morph.

Saprobic on dead barks of Dipterocarpaceae sp. Sexual morph Ascomata 117–270 × 71–155 μm (x̄ = 199 × 105 μm, n = 20), immersed, scattered, solitary, globose to subglobose, dark brown to black, coriaceous, ostiolate, papillate. The Ostiole canal narrowing toward the base, internally covered by hyaline periphyses, cells around the base small, thick-walled, and brown. Peridium 8–28 μm wide (x̄ = 18 μm, n = 20), comprising brown, compressed, cells of textura angularis. Paraphyses 3–6 μm wide (x̄ = 5.5 μm, n = 30), cylindrical, unbranched, straight to flexible, smooth, hyaline, septate, slightly constricted at the septa, tapering toward to end, longer than asci. Asci 67–90 × 13–24 μm (x̄ = 79 × 19 μm, n = 20), 8-spored, unitunicate, clavate to broadly fusoid, short pedicellate, apex blunt, with an indistinct, J- apical ring, evanescent. Ascospores 13–16 × 5–9 μm (x̄ = 14 × 7 μm, n = 20), overlapping uniseriate to biseriate, biturbinate to subellipsoidal, 1-septate, slightly constricted at the septa rounded at both ends, guttulate, smooth-walled, hyaline to pale brown. Asexual morph not observed.

Culture characters: Colonies grown on PDA and incubated at 25°C reached a diameter of 40 mm after 2 weeks, flat, spreading, fluffy, with a pale brown ring interlaced in the colonies. Surface lightly rough with brown mycelium, colonies somewhat raised in the middle, and with an irregular edge. The reverse side dark brown with an irregular, penniform, brown edge, and not pigmented.

Material examined: Thailand, Chiang Mai Province, Mae Taeng district, on dead bark of Dipterocarpaceae, 15 July 2020, Xia Tang, Dip25 (MFLU 23-0054, holotype; ex-type living culture, MFLUCC 23-0003).

Notes: Subellipsoidispora guttulata is similar to Coryneum umbonatum in having immersed, coriaceous, brown to black ascomata, and unitunicate asci with an indistinct J- apical ring. However, S. guttulata differs from C. umbonatum in having clavate to broadly fusoid, short pedicellate asci and subellipsoidal, 1-septate, guttulate, hyaline to pale brown ascospores, while C. umbonatum has ellipsoid to cylindrical, stalk pedicellate asci, and ellipsoid, fusoid or elongate, distoseptate, straight or curved spores that are brown at maturity (Senanayake et al., 2018). Phylogenetic analysis showed that S. guttulata belongs to Coryneaceae and forms a basal lineage sister to Coryneum, an ascomycetous genus, Hyaloterminalis and Talekpea, a hyphomycetous and monotypic genus. The base pair differences between S. guttulata and C. umbonatum were as follows: ITS = 7.7% (45/581), LSU = 3.2% (26/842), and rpb2 = 21.7% (223/1029), and the differences between S. guttulata and Talekpea foeticia were as follows: ITS = 12.5% (65/520) and LSU = 2% (17/843). Based on its phylogenetic and morphological analyses, we place S. guttulata as the type species of Subellipsoidispora in Coryneaceae.