Exploring diversity rock-inhabiting fungi from northern Thailand: a new genus and three new species belonged to the family Herpotrichiellaceae

Members of the family Herpotrichiellaceae are distributed worldwide and can be found in various habitats including on insects, plants, rocks, and in the soil. They are also known to be opportunistic human pathogens. In this study, 12 strains of rock-inhabiting fungi that belong to Herpotrichiellaceae were isolated from rock samples collected from forests located in Lamphun and Sukhothai provinces of northern Thailand during the period from 2021 to 2022. On the basis of the morphological characteristics, growth temperature, and multi-gene phylogenetic analyses of a combination of the internal transcribed spacer, the large subunit, and the small subunit of ribosomal RNA, beta tubulin and the translation elongation factor 1-a genes, the new genus, Petriomyces gen. nov., has been established to accommodate the single species, Pe. obovoidisporus sp. nov. In addition, three new species of Cladophialophora have also been introduced, namely, Cl. rupestricola, Cl. sribuabanensis, and Cl. thailandensis. Descriptions, illustrations, and a phylogenetic trees indicating the placement of these new taxa are provided. Here, we provide updates and discussions on the phylogenetic placement of other fungal genera within Herpotrichiellaceae.

Rocks are one of the interesting natural habitats of fungi.Rockinhabiting fungi were divided into two groups on the basis of their ecology and taxonomy.The first group comprises hyphomycetes of soil and epiphytic fungi (such as Aureobasidium or Phoma), whereas the second group includes melanized cell-walled fungi that exhibit slow growth, meristematic growth, or the production of yeast-like cells, typically belonging to the orders Capnodoales, Chaetothyriales, and Dothideales (Wollenzien et al., 1995;Gorbushina, 2007;Coleine and Selbmann, 2021).There are numerous reports on the biotechnological capabilities of rockinhabiting fungi in astrobiology, radioprotection, biomedical, and bioremediation fields (Aureli et al., 2020;Lin and Xu, 2020;Tran-Ly et al., 2020;Coleine and Selbmann, 2021;Mattoon et al., 2021;Cassaro et al., 2022;Liu et al., 2022).Several species of herpotrichiellaceous fungi were previously isolated from rocks, such as Cladophialophora nyingchiensis, Cl. tumulicola, Exophiala bonariae, Ex. clavispora, and Ex.siamensis (Isola et al., 2016;Kiyuna et al., 2018;Sun et al., 2020;Thitla et al., 2022).In addition, numerous prior reports have highlighted Thailand as a hot spot for discovering new fungal species (Hyde et al., 2018;Khuna et al., 2022).Nonetheless, information on the rock-inhabiting fungi in Thailand is still limited.Thus, the main objective of this study is to study the diversity of rock-inhabiting fungi in Thailand.During our investigation, we identified 12 herpotrichiellaceous fungi, viz., 10 strains of Cladophialophora and two strains of unrecognized fungal taxa.Morphology, growth temperature, and multi-gene phylogenetic analyses indicate that four herpotrichiellaceous fungi are novel in Herpotrichiellaceae.In addition, we have updated the existing reference data on the members of Herpotrichiellaceae.

Sample collection and fungal isolation
Rock samples appearing black fungal mycelia were collected from a dipterocarp forests in Lamphun (18°32′13″N 99°07′31″E, elevation at 432 m; and 18°32′11″N 99°07′22″E, elevation at 444 m) and Sukhothai (17°32′58″N 99°29′49″E, elevation at 153 m) provinces, Thailand, in 2021−2022.During the collection period, Lamphun province had daily rainfall of 6.5 mm, whereas Sukhothai province received daily rainfall of 2.3 mm.Temperatures in Lamphun province ranged from 22°C to 36°C, whereas temperatures in Sukhothai province ranged from 24°C to 38°C.Rock samples were obtained following the method described by Thitla et al. (2022).Fungi were isolated using an adaptation of the technique reported by Selbmann et al. (2014).Rock samples were cleaned in 1% sodium hypochlorite for 10 min before being rinsed five times with sterile and deionized water to eliminate any trace of detergent.To obtain the fungal strain, pounding rock samples and seeding rock shards were sprinkled onto malt extract agar (MEA; Gibco, Life Technologies Corporation, USA) and dichloran-rose bengal agar (DRBC; Difco, Becton, Dickinson and Company, USA) supplemented with chloramphenicol 100 mg/L.Plates were incubated at 25°C for 4 weeks, with daily inspections.Dark mycelia fungi were aseptically transferred to a MEA plates.Pure fungal strains were kept in 15% glycerol and deposited in the Culture Collection of Sustainable Development of Biological Resources Laboratory (SDBR), Faculty of Science, Chiang Mai University, Chiang Mai, Thailand.In addition, new fungal taxa were registered in the MycoBank database (MycoBank, 2023).

Morphology and growth temperature
Macro-morphologies, including colony characterization, pigment production, and colony diameter, were investigated on potato dextrose agar (PDA; Condalab, Laboratorios Conda S.A., Spain), MEA, and oatmeal agar (OA; Difco, Becton, Dickinson and Company, USA) at 25°C in the dark for 28 days.To examine the fungal growth at different temperatures, the colony diameter was measured on MEA at 4°C, 10°C, 15°C, 20°C, 25°C, 28°C, 30°C, 35°C , 37°C, and 40°C for 4 weeks in darkness.Three replicates were performed for each fungal strain at each temperature.Micromorphological features were investigated using a light microscope (Nikon Eclipse Ni-U, Japan).The Tarosoft (R) Image was used to calculate the size of fungal structures (e.g., hyphae, conidiophore, conidiogenous cell, conidia, and chlamydospore).

DNA extraction, amplification, and sequencing
Fungal genomic DNA of each fungal strain was extracted from mycelia grown on MEA at 25°C for 3 weeks, using s DNA extraction kit (FAVORGEN, Ping-Tung, Taiwan).The internal transcribed spacer (ITS), large subunit (nrLSU), and small subunit (nrSSU) of ribosomal RNA, beta tubulin gene (tub2), and the translation elongation factor 1-a (tef1-a) genes were amplified by polymerase chain reaction (PCR) using ITS5/ITS4 (White et al., 1990), LROR/ LR5 (Vilgalys and Hester, 1990;Rehner and Samuels, 1994), NS1/ NS4 (White et al., 1990), Bt2a/Bt2b (Glass and Donaldson, 1995), and EF1-728F/EF1-986R (Carbone and Kohn, 1999) primers, respectively.PCR amplifications were performed using 20 µL of reaction mixtures, consisting of 1 µL of genomic DNA, 1 µL of each primer, 10 µL of Quick TaqTM HS Dye-Mix (TOYOBO, Japan), and 7 µL of deionized water.The conditions of PCR reactions consisted of a first denaturation step performed at 95°C for 5 min and denaturation step at 95°C for 30 s; then, to amplify the ITS region, an annealing step was performed at 55°C for 30 s, an annealing step of nrSSU and nrLSU region was performed at 52°C for 45 s, whereas annealing step of the tub2 gene was performed at 52°C for 30 s; an elongation step were performed at 72°C for 1 min; lastly to amplify the tef1-a gene, annealing step at 57°C for 1 min and an elongation step at 72°C for 1.30 min were was performed.
Cycles were performed 35 times, with a final extension at 72°C for 10 min on a peqSTAR thermal cycler (PEQLAB Ltd., Fareham, UK).PCR products were checked on 1% agarose gel electrophoresis and measured quantity with NanoDrop OneC (Thermo Scientific, USA).Then, the PCR clean-up Gel Extraction NucleoSpin ® Gel and the PCR Clean-up Kit (Macherey-Nagel, Düren, Germany) were used to purify PCR products.Purified PCR products were sequenced by 1st BASE Company (Kembangan, Malaysia).

Sequence alignment and phylogenetic analyses
The ITS, nrLSU, nrSSU, tub2, and tef1-a sequence data were assembled using the software Sequencher 5.4.6 (Nishimura, 2000).The consensus sequences were blasted in the BLAST search tool via NCBI website.Two datasets were prepared to construct the phylogenetic trees for clarifying the family Herpotrichiellaceae (Table 1) and genus Cladophialophora (Table 2).Multiple sequence alignment was performed by MUSCLE using MEGA 6 (Edgar, 2004) and adjusted manually in BioEdit v.7.2.5 (Hall, 2004).Phylogenetic analysis of the family Herpotrichiellaceae (analysis I) was carried out on the basis of only the ITS, nrLSU, and SSU sequences because the amount of available sequence data in the tub2 and tef1-a genes is practically limited.To construct a phylogenetic tree of Cladophialophora (analysis II), five gene datasets (ITS, nrLSU, nrSSU, tub2, and tef1-a) were used.Maximum likelihood (ML) analysis was generated with 25 categories and 1,000 bootstrap (BS) replications under the GTRCAT model using the RAxML-HPC2 on XSEDE (v.8.2.12) in the CIPRES web portal (Felsenstein, 1985;Stamatakis, 2006;Miller et al., 2009).Bayesian inference (BI) analysis was performed using MrBayes v.3.2.6 (Ronquist and Huelsenbeck, 2003).Bayesian posterior probability (PP) was determined by Markov chain Monte Carlo (MCMC) sampling.Six simultaneous Markov chains were run for 5 million generations for analysis I and 2 million generations for analysis II with random initial trees, wherein every 100th generations were sampled.The first 20% of generated trees representing the burn-in phase of the analysis were discarded, whereas the remaining trees were used for calculating PP in the majority-rule consensus tree.Branches with BS support and PP values of more than or equal to 75% and 0.95, respectively, were deemed to have been substantially supported.The tree topologies were visualized in FigTree v1.4.0 (Rambaut, 2019).

Results
Fungal isolation, morphological study, and growth temperature A total of 12 fungal strains were obtained from different rock samples.Ten fungal strains (SDBR-CMU446, SDBR-CMU447, SDBR-CMU448, SDBR-CMU449, SDBR-CMU450, SDBR-CMU451, SDBR-CMU452, SDBR-CMU453, SDBR-CMU476, and SDBR-CMU477) exhibited similar characteristics by appearing  one-celled and hyaline and by forming conidial chains.Initially, these 10 fungal strains were identified as Cladophialophora species according to their micromorphological features.However, they were divided into three different groups on the basis of their colony characteristics on culture media, micromorphological, and growth temperature profiles (Table 3).In addition, two fungal strains (SDBR-CMU478 and SDBR-CMU479) appeared similar in their characteristics with produced sympodial conidial formation on conidiogenous loci and by appearing one-celled, hyaline to subhyaline, and obovoidal conidia.On the basis of morphological characteristics, these two fungal strains could not be assigned to any genera.Therefore, multi-gene phylogenetic analyses were used to identify their species-level and phylogenetic placement.The observation of fungal growth at various temperatures (4°C −40°C) revealed that temperature had a strong influence on the fungal growth of the obtained fungi.The average colony diameter of each fungal strain is presented in Table 3.The results indicate that all 12 fungal strains could not grow at 4°C, 37°C, and 40°C.Three strains (SDBR-CMU446, SDBR-CMU447, and SDBR-CMU448) could not grow at 10°C but grew at temperatures ranging from 25°C to 30°C.Five strains (SDBR-CMU449, SDBR-CMU450, SDBR-CMU451, SDBR-CMU452, and SDBR-CMU453) exhibited the highest average colony diameter at 28°C.Two strains (SDBR-CMU476 and SDBR-CMU477) could not grow at 10°C and 35°C, yet they showed the greatest average colony diameter at 28°C.Two fungal strains (SDBR-CMU478 and SDBR-CMU479) grew at temperatures ranging from 10°C to 30°C and grew well at 28°C.

Phylogenetic study
For analysis I, a phylogenetic tree of the family Herpotrichiellaceae was constructed from a combined ITS, nrLSU, and nrSSU sequence dataset.The dataset comprised 159 sequence strains from representatives in the families Herpotrichiellaceae and Trichomeriaceae, including the new strains that were proposed in this study.Epibryon interlamellare CBS 126286 and Ep.turfosorum CBS 126587 (famliy Epibryaceae) were selected as the outgroup.The concatenated dataset comprised 3,022 positions (ITS, 1−928 base pair (bp); nrLSU, 929−1,842 bp; and nrSSU, 1,843−3,022 bp) including gaps.RAxML analysis of the integrated dataset yielded the best scoring tree with a final ML optimization likelihood value of −45,154.8753.The matrix contained 1,610 distinct alignment patterns with 38.67% undetermined characters or gaps.The estimated base frequencies were recorded as follows: A = 0.2716, C = 0.2009, G = 0.2559, and T = 0.2714; whereas the substitution rates were established as AC = 0.9883, AG = 1.7181,AT = 0.9613, CG = 0.7406, CT = 2.9999, and GT = 1.0000.The gamma distribution shape parameter alpha value was equal to 0.1267, whereas the tree length was equal to 17.4438.At the end of the total MCMC generations, the final average standard deviation of the split frequencies was calculated to be 0.009866 through BI analysis.According to the topological results, ML and BI phylogenetic analyses produced similar topologies.Therefore, only the phylogenetic tree constructed from the ML analysis is shown in Figure 1.The phylogenetic tree clearly separates the family  Herpotrichiellaceae from the family Trichomeriaceae with strong support (100% BS and 1.00 PP).In this study, all fungal strains obtained belonged to the family Herpotrichiellaceae and were separated from the previously known species (Figure 1).Cladophiophora rupestricola (SDBR-CMU446, SDBR-CMU447, and SDBR-CMU448) forms a distinct lineage sister clade to Cl. sribuabanensis (SDBR-CMU476 and SDBR-CMU477) with 100% BS and 1.00 PP support values.Accordingly, five fungal strains (SDBR-CMU449, SDBR-CMU450, SDBR-CMU451, SDBR-CMU452, and SDBR-CMU453) of Cl. thailandensis form a distinct lineage closely related to Cl. inabaensis (EUCL1) and Cl.lanosa (KNU 16032).In addition, two fungal strains of Petriomyces obovoidisporus (SDBR-CMU478 and SDBR-CMU479) formed a well-resolved clade (100% BS and 1.00 PP; Figure 1) in Herpotrichiellaceae, with Atrokylindriopsis setulose (HMAS245592) and Exophiala siamensis (SDBR-CMU417) as the sister clade.
In our phylogenetic tree for clarifying the family Herpotrichiellaceae (Figure 1 Trichomeriaceae with strong support of 100% BS and 1.00 PP (Figure 1).
For analysis II, the phylogenetic placement of the genus Cladophialophora within Herpotrichiellaceae was established by combining five gene sequence datasets (ITS, nrLSU, nrSSU, tub2, and tef1-a) from a total of 71 taxa.The concatenated dataset comprised 3,931 positions including gaps (ITS, 1−692 bp; nrLSU, 693−1,512 bp; nrSSU, 1,513−3,185 bp; tub2, 3,186−3,698; and tef1a, 3,699−3,931 bp).RAxML analysis of the integrated dataset yielded the best scoring tree with a final ML optimization likelihood value of −21,115.8911.The matrix contained 1,300 distinct alignment patterns with 48.15% undetermined characters or gaps.The estimated base frequencies were recorded as follows: A = 0.2256, C = 0.2775, G = 0.2336, and T = 0.2632; whereas the substitution rates were established as AC = 1.0546,AG = 2.0969, AT = 0.9937, CG = 0.5923, CT = 3.1988, and GT = 1.0000.The gamma distribution shape parameter alpha value was equal to 0.4693, whereas the tree length was equal to 4.6134.The final average standard deviation of split frequencies at the end of total MCMC generations was calculated as 0.007796 through the BI analysis.The phylogram demonstrated that all three new species of Cladophialophora discovered in this study were distinctly separate from the previously known species.Cladophialophora rupestricola and Cl.sribuabanensis remained a sister group with strong support (100% BS and 1.00 PP), which was consistent with the findings of analysis I. Furthermore, Cl. thailandensis was a distinct lineage that is still related to Cl. inabaensis and Cl.lanosa.
Habitat and distribution: sandstone on natural forest; known from Sukhothai province, Thailand.
Growth temperature: minimum at 15°C, optimum at the range of 25°C−30°C, maximum at 35°C, and no growth at 10°C and 37°C.
Habitat and distribution: sandstone; collected from Lamphun province, Thailand.
Growth temperature: minimum at 15°C, optimum at the range of 28°C, maximum at 30°C, and no growth at 10°C and 35°C.
Habitat and distribution: sandstone; collected from Lamphun province, Thailand.
Growth temperature: minimum at 10°C, optimum at 28°C, maximum at 35°C, and no growth at 4°C and 37°C.
Habitat and distribution: sandstone; known from Lamphun province, Thailand.

Discussion
Herpotrichiellaceae is one of the well-known families in the order Chaetothyriales (Wijayawardene et al., 2020;Tian et al., 2021;Bezerra et al., 2022;Wijayawardene et al., 2022).Presently, four genera in Herpotrichiellaceae have been discovered on rocks, viz., Cladophialophora, Exophiala, Phialophora, and Rhinocladiella (Sun et al., 2020;Liu et al., 2022;Thitla et al., 2022).In this study, we introduced new taxa of rock-inhabiting fungi in the family Herpotrichiellaceae that were collected from northern Thailand.These taxa comprised a novel genus, named Petriomyces and three new species of Cladophialophora.The genus Petriomyces has been introduced on the basis of its fungal habitat, morphological characteristics, growth temperature, and phylogenetic analysis.Consequently, the number of rock-inhabiting fungal genera in the family Herpotrichiellaceae has increased to five genera.
Prior to this study, 20 genera (Aculeata, Atrokylindriopsis, Brycekendrickomyces, Capronia, Cladophialophora, Exophiala, Fonsecaea, Marinophialophora, Melanoctona, Metulocladosporiella, Minimelanolocus, Neosorocybe, Phialophora, Pleomelogramma, Rhinocladiella, Sorocybe, Thysanorea, Uncispora, Valentiella, and Veronaea) have been accepted into the family Herpotrichiellaceae (Wijayawardene et al., 2020;Tian et al., 2021;Bezerra et al., 2022;Wijayawardene et al., 2022).However, the number of genera in Herpotrichiellaceae has been a subject of debate in previous studies due to the variability of their morphological characteristics (Quan et al., 2020;Tian et al., 2021).Therefore, molecular phylogeny has been recognized as a valuable tool for researchers in the identification of fungi within Herpotrichiellaceae. Consequently, the aim of this study was to inspect and update the members of this family through molecular phylogeny using combined ITS, nrLSU, and nrSSU sequences.In our phylogenetic tree (Figure 1) indicated that species of herpotrichiellaceous fungi are paraphyletic and polyphyletic within the family Herpotrichiellaceae and Trichomeriaceae and this is concurred with previous studies (Quan et al., 2020;Wan et al., 2021).
A new genus (Petriomyces) obtained from this study formed within Herpotrichiellaceae (Figure 1).Regarding species Brycekendrickomyces and Metulocladosporiella, which were previously placed in the family Herpotrichiellaceae (Crous et al., 2006;Crous et al., 2009;Wijayawardene et al., 2020;Tian et al., 2021;Bezerra et al., 2022;Wijayawardene et al., 2022), our phylogenetic analysis placed their type sequences within the family Trichomeriaceae.Morphologically, these two species were reported from asexual characters, whereas the members of family Trichomeriaceae are predominantly known for their sexual characters.Although, there have been reports of asexual reproduction in certain species within family Trichomeriaceae, the specific asexual characters vary within the family.Because of the variability of asexual characters within the family Trichomeriaceae, we have tentatively transferred genera Brycekendrickomyces and Metulocladosporiella to the family Trichomeriaceae based solely on their phylogenetic placement.However, further studies, including additional collections and the linkage of sexual and asexual morphs for these two species, are necessary to confirm their phylogenetic placement and to gain a better understanding of the diversity of asexual characters within the family Trichomeriaceae.Furthermore, Neosorocybe and Sorocybe were also previously classified into Herpotrichiellaceae (Crous et al., 2020b;Wijayawardene et al., 2022).However, the phylogenetic analysis in this study revealed that these genera formed a unique clade to be clearly distinct from Herpotrichiellaceae and Trichomeriaceae (Figure 1).These two genera should be excluded from the family Herpotrichiellaceae.Therefore, we propose that the family Herpotrichiellaceae should contain 17 fungal genera (Table 4).However, there is still a need for the molecular data of the genus Pleomelogramma (Spegazzini, 1909) to confirm its phylogenetic placement.Most herpotrichiellaceous fungi still have a broadly paraphyletic and polyphyletic relationship.Therefore, further resolutions and characterizations are required to achieve a better understanding of their taxonomic classification within Herpotrichiellaceae.
In this study, the phylogenetic results indicate that Cladophialophora is a polyphyletic group, as is consistent with the findings of previous studies that reported the polyphyletic nature of the Cladophialophora species (Badali et al., 2008;Teixeira et al., 2017;Quan et al., 2020).Our three new species of Cladophialophora (Cl.rupestricola, Cl. sribuabanensis, and Cl.thailandensis) were established on the basis of comprehensive analyses of morphological characteristics, growth temperature, and multi-gene phylogeny.Furthermore, the nucleotide comparisons of the ITS, tef1a, or tub2 genes with phylogenetically related species revealed nucleotide differences that were greater than 1.5%.This is in line with the suggestion made by Jeewon and Hyde (2016) that nucleotide differences above 1.5% are necessary to justify the recognition of a new species.Therefore, Cl. rupestricola, Cl. sribuabanensis, and Cl.thailandensis can be considered new species, whereas the total number of accepted Cladophialophora species globally has increased to 52.On the basis of our phylogenetic analysis, a total of 38 Cladophialophora species were classified as members of Herpotrichiellaceae.Three species (Cl.eucalypti, Cl. proteae, and Cl. The fungal family is represented by a color symbol; Herpotrichiellaceae is shown by ◼, Trichomeriaceae is indicated by •, incertae sedis in class Chaetothyriales is indicated by ▴, and not reported is indicated by "NR."  et al., 2020;Crous et al., 2021).However, the phylogenetic placements of the remaining four species (Cl.cladoniae, Cl. hawksworthii, Cl. megalosporae, and Cl.normandinae) are uncertain and the acquisition of further molecular data will be necessary.An investigation of fungal grown at variant temperatures indicates that our fungi could grow under a wide range of temperature conditions.Three new species of Cladophialophora could thrive at temperatures ranging from 10°C to 35°C.Moreover, a new genus Pe. obovoidisporus could thrive at temperatures ranging between 10°C and 30°C.Accordingly, these temperatures were within a range of 10°C−38°C in the dipterocarp forest in Lamphun and Sukhothai provinces, Thailand, respectively.As has become evident, both fungi have adapted to survive in their respective environments.Remarkably, growth temperature is one of the factors that can limit fungal ability to infect human and animal bodies due to their high body temperatures (Garcia-Solache and Casadevall, 2010).Some herpotrichiellaceous fungi that are known to infect humans and animals can grow at high temperatures.For example, Cl. arxii, Cl. carrionii, Phia.chinensis, and Phia.expanda can grow at temperatures of 37°C or higher (de Hoog et al., 1995;Li et al., 2017).The growth temperature of herpotrichiellaceous fungi is often associated with clinical predilections with the species growing at 40°C often causing systemic infections (de Hoog et al., 2000;Badali et al., 2008).Three new species of Cladophialophora and Petriomyces identified in this study may not be the source pathogen in humans and animals because they cannot grow at temperatures above 37°C.However, their ability to be the causal agents of infections in humans and animals needs to be further investigated.Currently, global temperatures are rising because of climate change, which can affect all aspects of a given ecosystem (Gadre et al., 2022).As a result, this environment may have an impact on the global diversity and distribution of herpotrichiellaceous fungi.Some herpotrichiellace fungi can survive and grow at high temperatures, but others cannot survive at higher temperatures and die.
In conclusion, we investigated the rock-inhabiting fungi belonged to the family Herpotrichiellaceae collected from northern Thailand.Three new species of Cladophialophora (Cl.rupestricola, Cl. sribuabanensis, and Cl.thailandensis) and a new genus (Petriomyces) were identified on the basis of the relevant morphological characteristics, growth temperature, and multi-gene phylogeny.Furthermore, the relevant data on the genera in Herpotrichiellaceae have been updated.In this study, we propose that Herpotrichiellaceae consists of 17 genera, including Aculeata, Atrokylindriopsis, Capronia, Cladophialophora, Exophiala, Fonsecaea, Marinophialophora, Melanoctona, Minimelanolocus, Petriomyces, Phialophora, Pleomelogramma, Rhinocladiella, Thysanorea, Uncispora, Valentiella, and Veronaea.However, the classification of several genera in this family is still problematic due to variabilities in some of the morphological characteristics, their existing polyphyletic relationships, and the limited amount of molecular data, especially protein coding genes.Therefore, further studies involving morphology, ecology, and molecular investigations, as well as in-depth evolutionary studies, should be conducted to further clarify the family Herpotrichiellaceae.
), 15 previously known herpotrichiellaceous genera (Aculeata, Atrokylindriopsis, C a p r o n i a , Cl a d o ph i a l o ph o r a , E x o p h i a l a , F o n s e c a e a , Marinophialophora, Melanoctona, Minimelanolocus, Phialophora, Rhinocladiella, Thysanorea, Uncispora, Valentiella, and Veronaea) and a new genus (Petriomyces) identified in this study were assigned to the Herpotrichiellaceae clade.The genera Brycekendrickomyces and Metulocladosporiella were placed in the Trichomeriaceae clade.The clade of genera Neosorocybe and Sorocybe was clearly separated from the clade of the families Herpotrichiellaceae and

FIGURE 1
FIGURE 1 Phylogenetic tree generated from maximum likelihood analysis of 159 fungal strains based on a combined ITS, nrLSU, and nrSSU sequence dataset.Epibryon interlamellare CBS 126286 and Ep.turfosorum CBS 126587 were used as the outgroup.The numbers above branches show bootstrap percentages (left) and Bayesian posterior probabilities (right).Bootstrap values ≥75% and Bayesian posterior probabilities ≥0.95 are shown.The scale bar reflects the estimated number of nucleotide substitutions per site.The newly generated sequences are in blue.The ex-type species are in bold.

FIGURE 4
FIGURE 4 Phylogenetic relationships of Cladophialophora within the family Herpotrichiellaceae reconstructed by maximum likelihood analysis based on a combined dataset of ITS, nrLSU, nrSSU, tub2, and tef1-a genes.Bradymyces alpinus CCFEE 5493 and Br.oncorhynchi CCF 4369 were used as the outgroup.The values presented above branches represent the bootstrap percentages (left) and Bayesian posterior probabilities (right).Bootstrap values ≥75% and Bayesian posterior probabilities ≥0.95 are displayed.The scale bar indicates the estimated number of nucleotide substitutions per site.The newly generated sequences are in blue, whereas the ex-type species are indicated in bold.

TABLE 1
GenBank accession numbers of herpotrichiellaceous fungi used in the molecular phylogenetic analysis.Species obtained in this study are in bold.Superscript "T" represents ex-type species."−" represents the absence of sequence data in GenBank database.

TABLE 2
GenBank accession numbers of Cladophialophora in the family Herpotrichiellaceae used in the molecular phylogenetic analysis.

TABLE 2 Continued
Species obtained in this study are in bold.Superscript "T" represents ex-type species."−" represents the absence of sequence data in GenBank database.

TABLE 3
Colony diameter of obtained fungi cultured on MEA at various temperatures for 28 days in the dark.
*The results are average colony diameter ± standard deviation, and "−" represents no growth.

TABLE 4
Comparison of fungal genera belonging to the family Herpotrichiellaceae from previous reports and this study. .