683 MycoKeys MycoKeys 116: 275-301 (2025) DOI: 10.3897/mycokeys.116.150635 Research Article Two new species of Penicillium and a new genus in Xylariomycetidae from the forest dump-sites in Chiang Mai, Thailand Tanapol Thitla“®, Jutamart Monkai?**®, Weiqian Meng'®, Surapong Khuna™, Ning Xie™®, Sinang Hongsanan'®, Saisamorn Lumyong?*5© on fF wo NY — Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Science and Oceanography, Shenzhen University, Shenzhen 518060, China Office of Research Administration, Chiang Mai University, Chiang Mai 50200, Thailand Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Center of Excellence in Microbial Diversity and Sustainable Utilization, Chiang Mai University, Chiang Mai 50200, Thailand Academy of Science, The Royal Society of Thailand, Bangkok 10300, Thailand Corresponding authors: Saisamorn Lumyong (scboi009@gmail.com); Sinang Hongsanan (sinang333@gmail.com) OPEN Qaceess This article is part of: Exploring the Hidden Fungal Diversity: Biodiversity, Taxonomy, and Phylogeny of Saprobic Fungi Edited by Samantha C. Karunarathna, Danushka Sandaruwan Tennakoon, Ajay Kumar Gautam Academic editor: Samantha C. Karunarathna Received: 18 February 2025 Accepted: 5 April 2025 Published: 29 April 2025 Citation: Thitla T, Monkai J, Meng W, Khuna S, Xie N, Hongsanan S, Lumyong S (2025) Two new species of Penicillium and a new genus in Xylariomycetidae from the forest dump-sites in Chiang Mai, Thailand. Mycokeys 116: 275-301. https://doi. org/10.3897/mycokeys.116.150635 Copyright: © Tanapol Thitla et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract Waste accumulation in forest regions can have a severe impact on the soil mycobiome. However, research on soil fungi inhabiting forest disposal sites remains limited. Therefore, this study focused on the taxonomy and phylogeny of ascomycetes isolated from soil in forest dump-sites in Chiang Mai, Thailand. The fungal strains were identified using morphological characterisations and multigene phylogenetic reconstruction. A new genus, Pseudoleptodontidium, typified by Ps. chiangmaiense sp. nov. (Amphisphaeriales genera incertae sedis, Xylariomycetidae), along with two new species, Penicillium chiangmaiense (series Janthinella, section Lanata-Divaricata) and P. terrae (series Erubescentia, section Exilicaulis) (Aspergillaceae, Eurotiales), are described in detail and compared with closely-related species. Our discovery offers valuable insights into the soil ascomycetes associated with forest disturbances. Key words: Eurotiomycetes, new taxa, Pseudoleptodontidium, soil fungi, Sordariomy- cetes, taxonomy Introduction The disposal of waste materials through open burning, landfilling and dumping in land areas or water resources contributes to environmental issues, such as air pollution (PM, .), as well as water and soil pollution, which can endanger the health and livelihood of humans, animals, plants and other organisms (Lin et al. 2020; Wanthongchai et al. 2021). Soil serves as a natural habitat for a wide range of fauna and flora, including fungi. Fungi are a major component of soil ecosystems, playing crucial roles in the cycling of nutrients and the de- composition of organic materials (Frac et al. 2018; Coleine et al. 2022). The most abundant soil fungi belong to the Ascomycota, which includes the class- es Arthoniomycetes, Dothideomycetes, Eurotiomycetes, Leotiomycetes and Sordariomycetes (Tedersoo et al. 2021; Gomes de Farias et al. 2023). Amongst * These authors have contributed equally to this work. ZED Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae these, Fusarium, Penicillium and Phoma are the most frequently isolated genera (Tedersoo et al. 2021; Yasanthika et al. 2023). However, contamination with pollutants may adversely affect their diversity, population and ecological func- tions (Frac et al. 2018; Schloter et al. 2018; Coleine et al. 2022). The ability to synthesise a wide range of enzymes for breaking down various substrates enables soil fungi to adapt and thrive in diverse environments and harsh condi- tions (Singh et al. 2021; Coleine et al. 2022; Sun et al. 2024). Extensive studies have focused on isolating and characterising soil fungi from contaminated areas, landfills and urban dump-sites (Sangale et al. 2019; Verma and Gupta 2019; Ren et al. 2021; Khan et al. 2022; Gong et al. 2023; Sathiyabama et al. 2024; Sun et al. 2024). These studies have revealed diverse soil fungal communities and identified numerous new fungal taxa and strains from these polluted habitats. Moreover, they have demonstrated a significant potential for biodegradation and bioremediation. For example, Yasanthika et al. (2021) studied soil ascomycetes in China and reported a new species, Jux- tiphoma yunnanensis, as well as two new records, Lecanicillium dimorphum and Scopulariopsis brevicaulis, from urban-industrialised soils. Ren et al. (2021) iso- lated 29 fungal strains from soils contaminated with explosive materials in Chi- na. Amongst them, the isolate of Fusarium solani demonstrated the ability to decompose alkyne-terminated polybutadiene with urethane segments (PUPB) (Ren et al. 2021). Similarly, Sangale et al. (2019) obtained 109 fungal isolates from the dumping sites of mangrove rhizosphere soil and revealed that the strains of Aspergillus terreus and A. sydowii were the most effective in breaking down polythene. Additionally, the strain of Penicillium citrinum, isolated from municipal landfill soils in Bhopal, India, has demonstrated efficacy in degrading low-density polyethylene (LDPE) without prior pretreatment (Khan et al. 2022). Dump-sites, especially those located within forested areas, represent an un- derexplored yet ecologically significant niche. Forest dump-sites provide a dis- tinctive habitat, characterised by decreased soil nutrients, fluctuating tempera- ture and moisture levels and potential exposure to pollutants (Kooch et al. 2023; Sun et al. 2024). It is essential for exploring novel soil fungi from this habitat in order to determine fungal diversity and investigate their biodegradation strate- gies. Therefore, the present study aims to isolate and identify soil ascomycetes from disposal sites located in forests of northern Thailand. The topsoil samples from forest dump-sites in Chiang Mai Province were collected and isolated for fungi, leading to the discovery of five novel Ascomycota strains. Based on mo- lecular analyses and morphological characteristics, two new species of Penicil- lium and a new genus in Xylariomycetidae were introduced and described. Materials and methods Fungal isolation Soil samples (0-10 cm depth) were collected from three forest dump-sites in June 2024 in Chiang Mai Province, Thailand: (1) Papae, Mae Taeng District, (2) Suthep, Muang Chiang Mai District and (3) Mae Sa, Mae Rim District (Fig. 1). The collection details were noted (Rathnayaka et al. 2024) and the soil samples were placed in plastic bags and taken to the Sustainable Development of Biological Resources Laboratory (SDBR), at the Department of Biology, Faculty of Science, Chiang Mai Mycokeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 276 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae .. >} J ’ \ ' j : A i j fi é Rae) bs j RY ~ a re a ” i “| f ie a 4 “. > “a Figure 1. Forest dump-sites used for soil fungal isolation in this study A Papae, Mae Tang District, Chiang Mai Province B Suthep, Muang Chiang Mai District, Chiang Mai Province C Mae Sa, Mae Rim District, Chiang Mai Province. University, Thailand. Upon arrival, soil fungi were isolated immediately using the serial dilution plating method with three serial dilutions in sterile water (Yasanthika et al. 2022). After dilution, 100 ul of the soil suspension was dropped and spread on potato dextrose agar (PDA; CONDALAB, Spain) supplemented with 100 g/ml of streptomycin. The isolation plates were incubated at 25 °C in the dark for 5 days. The appearing fungal colonies were transferred to fresh PDA using the hyphal tip method (Korhonen and Hintikka 1980). The pure cultures were deposited and per- manently preserved in a metabolically inactive state at the Culture Collection of Microbial Shenzhen University (MBSZU), Shenzhen University, China. Morphological characterisation The morphological characteristics of the obtained fungi were observed in both macro-morphology and micro-morphology, with different details depending on each fungus. To investigate the morphology of Penicillium (comprising MBSZU 24-007 to MBSZU 24-010), the colony characteristics, growth rate, pigment production, sporulation or related features were investigated on Blakeslee’s Malt extract agar (MEAbI), creatine sucrose agar (CREA), Czapek yeast autolysate agar with 5% NaCl (CYAS), Czapek’s agar (CZ), Dichloran 18% glycerol agar (DG18), malt extract agar (MEA), oatmeal agar (OA), PDA and yeast extract sucrose agar (YES) at 25 °C in darkness for 7 days. The experiment was also performed on Czapek yeast autolysate agar (CYA) at 25, 30 and 37 °C in darkness for 7 days to characterise the macro-morphology (Visagie et al. 2014; Khuna et al. 2023). Mi- cro-morphologically, the characteristics of conidiophores, stipes, conidiogenous cells, conidia or other structures were observed under a light microscope (Nikon DS-Ri2; Nikon, Japan), using fungal colonies grown on MEA at 25 °C in darkness for 7 days. Size data were evaluated by at least 50 measurements per structure. The colony characteristics, growth rate and pigment production of Pseudo- leptodontidium (MBSZU 25-005) were studied on PDA and MEA at 25 °C in dark- ness for 14 days. Micro-morphology was observed under a light microscope us- ing a fungal colony grown on PDA at 25 °C in darkness for 14-21 days. The size of each morphological structure was measured at least 50 times per structure. MycoKeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 277 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae DNA extraction, amplification and sequencing Fungal genomic DNA from each strain was extracted from the fungal myce- lium, which had grown on PDA at 25 °C for a week, using an E.Z.N.A® Tissue DNA Kit (Omega, USA). The polymerase chain reaction (PCR) technique was used to amplify each region. Each target locus was amplified using the specific primers (Table 1). PCR amplifications were performed in 20 ul reaction mix- tures, consisting of 1 pl of genomic DNA, 1 ul of each primer, 10 pl of 2x Phanta Max Master Mix (Dye Plus) (Vazyme, China) and 7 ul of deionised water. The PCR amplification was performed using a T100 Thermal Cycler (BIO-RAD, USA), with an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of denaturation, annealing and elongation steps. The denaturation and elongation steps were performed at 95 °C for 30 s and 72 °C for 60 s, respectively. The annealing step was performed at different conditions depending on each target locus (Table 1). A final elongation step was performed at 72 °C for 10 minutes. The success or failure of the PCR product was determined through 1% agarose gel electrophoresis, followed by purification of the product using the E.Z.N.A® Gel Extraction Kit (Omega, USA). The quality and quantity of the purified PCR products were assessed using 1% agarose gel electrophoresis and a Nanodrop 2000 Spectrophotometer (Thermo Scientific, USA). Subsequently, the products were sequenced by BGI-Shenzhen Company (Shenzhen, Guangdong, China). The bidirectional sequence data were assembled using the software Se- quencher 5.4.6 (Nishimura 2000). The consensus sequence data were searched for sequence similarity via the Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI) website. Table 1. The specific primer and annealing condition of each locus used in this study. Loci* ITS LSU CAM RPB2 TUB Primer ITS4/ITS5 LROR/LR5 CF1/CF4 Cmd5/Cmd6 fRPB2-5F/ fRPB2-7cR Bt2a/Bt2b T1/Bt2b Annealing condition Temperatures (°C) Annealing period (s) References 52 30 | White et al. (1990) 52 30 Vilgalys and Hester (1990); Rehner and Samuels (1994) 51 60 Peterson et al. (2005) 58 30 Hong et al. (2006) 56 60 Liu et al. (1999) 52 30 Glass and Donaldson (1995) 55 45 Glass and Donaldson (1995); O'Donnell and Cigelnik (1997) * ITS — Internal Transcribed Spacer region of the rRNA; LSU — 28S large subunit of the nuclear rRNA; TUB — beta-tubulin gene; CAM —- calmodulin gene; RPB2 — RNA polymerase II second largest subunit genes. Phylogenetic analysis The multi-loci phylogenetic dataset was obtained, based on previous studies of Penicillium section Exilicaulis (Ansari et al. 2023; Liu et al. 2023; Wang et al. 2023b; Visagie et al. 2024a, 2024b), Penicillium section Lanata-Divaricata (Lenz et al. 2022; Liu et al. 2023; Wang et al. 2023b; Araujo et al. 2024; Vis- agie et al. 2024b) and Xylariomycetidae (Samarakoon et al. 2022; Crous et al. 2023; Li et al. 2024; Samarakoon 2024) (Suppl. material 1: tables S1—S3). Mycokeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 278 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae The sequence data for each locus were individually aligned using MUSCLE through the software MEGA 6 (Edgar 2004) and manually adjusted in BioEd- it v.7.2.5 (Hall 2004). The concatenation of the ITS, TUB, CAM and RPB2 loci was performed for the phylogenetic analysis of Penicillium; in contrast, the combined ITS, LSU, RPB2 and TUB loci were used for the analysis of Xylariomycetidae. Maximum Likelihood (ML) and Bayesian Inference (BI) analyses were applied to generate a phylogenetic tree. The ML analysis was conducted with 25 categories and 1,000 bootstrap (BS) replications under the GTRCAT model using RAXxML-HPC2 on XSEDE (v.8.2.12) in the CIPRES web portal (Felsenstein 1985; Stamatakis 2006; Miller et al. 2009). The best- fit models of nucleotide substitution for individual locus were determined by using MrModelTest v.2.3 based on the Akaike Information Criterion (AIC) (Nylander 2004). The GTR+I+G substitution model was the best fit for all loci. The BI analysis was performed using MrBayes v.3.2.6 (Ronquist and Huelsenbeck 2003). Bayesian Posterior Probability (PP) was examined by Markov Chain Monte Carlo (MCMC) sampling. Six simultaneous Markov chains were run with random initial trees, wherein every 100" generation was sampled. The first 20% of generated trees, representing the burn-in phase of the analysis, were discarded, while the remaining trees were used to calculate PP in the majority-rule consensus tree. The tree topologies were visualised in FigTree v.1.4.0 with BS support and PP values equal to or higher than 75% and 0.95, respectively, in branches (Rambaut 2019). The final alignment and phylogram were submitted to TreeBASE (http://purl.org/ phylo/treebase/phylows/study/TB2:S32075, accessed 19 March 2025). Results Phylogenetic analysis Phylogenetic analysis of 72 taxa from Penicillium, section Exilicaulis (includ- ing P. terrae MBSZU 24-007 and MBSZU 24-008) was performed using a com- bined ITS, TUB, CAM and RPB2 sequence dataset. Penicillium janthinellum CBS 340.48 and P limosum CBS 339.97 were selected as the outgroup. The com- bined dataset comprised 2,630 characters (ITS, 1-564 bp; TUB, 565-1,102 bp; CAM, 1,103-1,701 bp; RPB2, 1,702-2,630 bp), including gaps. RAxML analysis of the integrated dataset yielded the best-scoring tree with a final ML optimisa- tion likelihood value of -26380.0905. The matrix contained 1,279 distinct align- ment patterns, with 13.06% of the characters being undetermined or gaps. The estimated base frequencies were recorded as follows: A = 0.2238, C = 0.2765, G = 0.2706 and T = 0.2291, while the substitution rates were as follows: AC = 1.0947, AG = 3.5202, AT = 1.1705, CG = 0.7818, CT = 5.4306 and GT = 1.0000. The gamma distribution shape parameter alpha value was equal to 0.2342, while the tree length was equal to 2.4771. The final average standard deviation of the split frequencies at the end of the total MCMC generations was comput- ed as 0.003644 via BI analysis. Phylogenetic analysis of 111 taxa from Penicillium section Lanata-Divar- icata (including P. chiangmaiense MBSZU 24-009 and MBSZU 24-010) was performed using a combined ITS, TUB, CAM and RPB2 sequence dataset. MycoKkeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 279 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae Penicillium alogum CBS 140996 and P. stolkiae CBS 315.67 were select- ed as outgroups. The combined dataset comprised 2,549 characters (ITS, 1-563 bp; TUB, 564-1,114 bp; CAM, 1,115-1,794 bp; RPB2, 1,795-2,549 bp), including gaps. RAXML analysis of the integrated dataset yielded the best scoring tree with a final ML optimisation likelihood value of -35195.9174. The matrix contained 1,381 distinct alignment patterns with 12.17% un- determined characters or gaps. The estimated base frequencies were recorded as follows: A = 0.2214, C = 0.2908, G = 0.2615 and T = 0.2263, while the substitution rates were as follows: AC = 1.1361, AG = 3.5568, AT = 1.5061, CG = 0.7521, CT = 5.3860 and GT = 1.0000. The gamma distri- bution shape parameter alpha value was equal to 0.2744, while the tree length was equal to 3.5928. The final average standard deviation of the split frequencies at the end of the total MCMC generations was computed as 0.005628 via BI analysis. Phylogenetic analysis of species in subclass Xylariomycetidae was performed using a combined ITS, LSU, RPB2 and TUB sequence dataset of MBSZU 25-005 (proposed as Pseudoleptodontidium chiangmaiensis), together with 118 taxa of the subclass. Achaetomium macrosporum CBS 532.94, Chaetomium elatum CBS 374.66 and Sordaria fimicola CBS 723.96 were selected as outgroups. The combined dataset comprised 3,560 char- acters (ITS, 1-693 bp; LSU, 694-1,592 bp; RPB2, 1,593-2,656 bp; TUB, 2,657-3,560 bp), including gaps. RAxML analysis of the integrated dataset yielded the best scoring tree with a final ML optimisation likelihood value of -83630.121273. The matrix contained 2,615 distinct alignment patterns with 39.42% undetermined characters or gaps. The estimated base frequencies were recorded as follows: A = 0.256414, C = 0.231937, G = 0.280501 and T = 0.231149, while the substitution rates were as follows: AC = 0.888171, AG = 2.661198, AT = 1.161270, CG = 0.868099, CT = 3.494813 and GT = 1.000000. The gamma distribution shape parameter alpha value was equal to 0.351763, while the tree length was equal to 15.592567. The final average standard deviation of the split frequencies at the end of the total MCMC generations was computed as 0.009989 via BI analysis. Topologically, the ML and BI phylogenetic trees of all fungal species had sim- ilar results; therefore, only the ML phylogram was demonstrated in this study. The phylogram of Penicillium section Exilicaulis showed that two new strains (MBSZU 24-007 and MBSZU 24-008) separated from other recognised species with 100% BS and 1.00 PP supports (Fig. 2). These fungal strains formed a sister clade with P. laeve DTO270G8 (BS 99% and PP 1.00) and belonged to the series Erubescentia. While the phylogram of Penicillium section Lanata-Divaricata exhibited that MBSZU 24-009 and MBSZU 24-010 formed a distinct clade, clearly separated from other taxa with significant support (BS 100% and PP 1.00; Fig. 3). These strains also formed a sister clade with P. brefeldianum CBS 235.81 (BS 100% and PP 1.00) within the Series Janthinella clade. The phylogram of Xylariomycetidae showed that MBSZU 25-005 clustered amongst families and taxa in Amphisphaeriales. This strain also formed a sis- ter clade to Neoleptodontidium aciculare CBS 123.86 and N. aquaticum CBS 149455 (BS 96% and PP 1.00; Fig. 4). Mycokeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 280 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae Penicillium aotearoae CBS 140999 Penicillium melinii CBS 218.30 aun Penicillium xanthomelinii CBS 139163 Penicillium diabolicalicense CBS 140967 felt 99/1.00) Penicillium velutinum CBS 2530.32 ithe 99/100 Penicillium maclennaniae CBS 198.81 i Penicillium hemitrachum CBS 139134 a atl Penicillium smithii CBS 276.83 Ser. Lapidosa Penicillium raciborskii NRRL 2150 Penicillium atrosanguineum CBS 380.75 Penicillium terrenum CBS 313.67 Penicillium burgense CBS 325.89 Penicillium lapidosum CBS 343.48 100/100. PeHIC Penicillium jant 0.04 Figure 2. Phylogram generated from Maximum Likelihood analysis of 72 specimens belonging to the genus Penicillium sec- tion Exilicaulis, using the combined ITS, TUB, CAM and RPB2 genes. Penicillium janthinellum CBS 340.48 and P. limosum CBS 339.97 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 fungal strains in this study are blue. Type species are bold. Taxonomy Penicillium terrae Thitla, Monkai, Lumyong & Hongsanan, sp. nov. MycoBank No: 857423 Etymology. The specific epithet terrae refers to the soil substrate, from which this species was isolated. Holotype. THAILAND * Chiang Mai Province, Mae Taeng District, Papae, on soil in the forest dump-sites, 20 June 2024, T. Thitla & J. Monkai; VRO40 (SZU25- 005, holotype); ex-type living culture, MBSZU 24-008, dried culture permanently preserved in a metabolically inactive state, SZU25-005. MycoKeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 281 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae 100/1.09) Penicillium heveae COAD3467 861.00) * Penteillium yuyongnianti CGMCC 3.25187 Penicillium taii CGMCC 3.25176 ee Penicillium globosum CGMCC 3.183800 94/100) Penicillium cataractarum CBS 140974 Penicillium newtonturnerae BRIP 74909a Penicillium guangxtense CGMCC 3.18793 Penicillium echinulonalgiovense CBS 328.59 Penicillium griseoflavum CGMCC 3.18799 = Penicillium mariae-crucis CBS 271.83 96/1.00p= Penicillium onobense CBS 174.81 82/100 Penicillium uttarakhandense NFCCI 4808 Penicillium paraherquet CBS 338.59 5911 DO) | Penicillium brasilianum CBS 253.55 el Penicillium skrjabinii CBS 439.75 Penicillium alagoense URM 8086 = Meret 100/1.00-— Penicillium pedernalense CBS 140770 Ser. Simplicissima Penicillium infrabuccalum CBS 140983 ete 100/100) Penicillium laevigatum CGMCC 3.18801 100/1.00 r1| Penicillium wandoense CNUFC-WT31-1 Penicillium jinyunshanicum CGMCC 3.25162 Penicillium araracuarense CBS 113149 94/1.00 il Penicillium wotroi CBS 118171 Cy Penicillium spinuliferum CGMCC 3.18807 aa Penicillium vickeryae BRIP 72552a 86/0.99 m Penicillium panissanguinenm CBS 140989 99/1.00 i Penicillium subfuscum CBS 147455 Penicillium simplicissimum CBS 372.48 Penicillium fengjieense C@GMCC 3.25157 10.97 Penicillium tanzanicum CBS 140968 Penicillium diatemitis CCF 3904 100/1.00 Penicillium soosanum CCF 3778 een Penicillium hepuense AS 3.16039 Ser. Oxalica 1901.00 Penicillium jiaozhouwanicum AS 3.16038 Penicillium oxalicum CBS 219.30 Penicillium austrosinense CGMCC 3.18797 ri 100/100 Penicillium guaibinense CCDCA 11512 a Penicillium marvkayhuntiae BRIP 74934a 100/1.00 Penicillium singorense CBS 138214 100/1,00 Penicillium pauciramulum CGMCC 3.25164 91/1.00 -—__ | Penicillium ausonanum FMR 16948 100/1.00 Penicillium viridissimum CGMCC 3.18796 Penicillium amphipolaria CBS 140997 75/099 Penicillium vanderhamment CBS 126216 Penicillium penarojense CBS 113178 Penicillium stangiae URM 8347 100/1.00 (J Penicillium griseopurpureum CBS 406.65 100/1.00 EE Penicillium jianfenglingense CGMCC 3.18802 Penicillium rubriannulatem CGMCC 3.18804 Penicillium daleae CBS 211.28 100/1.00 Penicillium limpopoense CMV 012F9 Penicillium abidjanum CBS 246.67 Penicillium zonatum CBS 992.72 4093;}-— Penicillium soli KUMC' ~yoor1.o0) Ey Penicillium zthanji Penicillium Indwigit Penicillium cremeogrise { Penicillium ashbyae MST-F 22310 Penicillium ortum CBS 135669 76048) 100/1.007— Penicillium koreense CBS 141338 ale Penicillium tengii CGMCC Be ‘= Penicillium donggangicum Ct C ‘ool bs Penicillium raperi CBS 281.58 100/1.00 100/1.00 100/1.00 100/100 Penicillium curticaule CE Penicillium potchefstroomes Penicillium lineolatum CBS 188.77 Penicillium setosum CBS 144865 -10.95] Penicillium silvertonense CMV 007TE3 ee Penicillium javanicum CBS 341.48 37/099 Penicillium nordestinense URM 8423 75/0.99-F] Penicillium malacosphaerulum CBS 135120 91/1.00} | 100/1.00) f Penicillium reticulisporum CBS 122.68 Penicillium caperatum CBS 443.75 Penicillium elleniae CBS 118135 Penicillium uruguayense CBS 143247 100/100) Penicillium coeruleum CBS 141.45 84/1.00 Penicillium levitum CBS 343.48 98/100} | 100/100— Penicillium siccitolerans FMR 17381 Penicillium meloforme CBS 445.74 Penicillium ehrlichii CBS 324.48 —_ Penicillium melanosporum FMR 17424 1001.00 — Penicillium subrutilans C@GMCC 3.25174 Penicillium camponotum CBS 140982 1001.00 Penicillium rolfsii CBS 368.48 P tay Penicillium amazonense COAD 3476 Penicillium piscarium CBS 362.48 Penicillium terrarumae CBS 131811 99/100 Penicillium rotoruae CBS 145838 LO0/1.00) 99/1.00 Penicillium ochrochloron CBS 357.48 Penicillium svalbardense CBS 122416 Penicillium flaviroseum CGMCC 3.18805 11.00) Penicillium subrubescens CBS 132785 Penicillium puivillorum CBS 280.39 Penicillium salifarme CGMCC 3.18806 Penicillium excelsum TBT 31516 ivan) Penicillium annulatum CBS 135126 Penicillium bissettii CBS 140972 Penicillium fructuariae-cellae CBS 145110 Penicillium hainanense CGMCC 3.18798 Penicillium vasconiae CBS 339.79 Penicillium coffeatum CGMCC 3.25152 Penicillium stolkiae CBS 313.67 Penicillium alogum CBS 140996 ~/1.00 Outgroups (Sect. Stolkia) Figure 3. Phylogram generated from Maximum Likelihood analysis of 111 specimens belonging to the genus Pen- icillium section Lanata-Divaricata using the combined ITS, TUB, CAM and RPB2 genes. Penicillium alogum CBS 140996 and P. stolkiae CBS 315.67 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 fungal strains in this study are red. Type species are bold. MycoKeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 282 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae 100/1.00 Sareea 75/098 eimatosporium rosa 7110.98 Strickeria kochii CBS 5 100/100, Seiridium cupressi CBS | 100/1.00 Seiridium marginatum CBS 100/1.00 Parabartalinia late. = Truncateila an, /1.00 Paraphysalospora euc Clypeophysalospora Bagadiella lunata CBS 12 100/1.00 Neophysalospora eucalypti CBS Plectasphaera eucalypti CBS 12 98/0.99, Castanediella brevis KUMIEC 0132 90/1.00} Castanediella malaysiana ae 2 99/1.00 Castanediella eucal; 79/0.90 Synnemadiella euc Hates 27 Castanediella acaciae CBS 139896 _ AGES Beltrania rhombica strain CBS | Beltraniella endiandrae CBS ie 75/1.00 Beltraniopsis longiconidiop Ane ae UC a arap sassy" eciopsis caespitosa E Amph icphaerin thailandica MFLU 1 pe eee hydei CMUB 400 isphaeria sambu : Nemuicieiata trachycarpi C ae Neoarthrinium mise 164.80 PidoplitchKovielia terricola CBS 180.77 100/1.00 Neoleptodontidium aciculare ae Neoleptodontidium aquaticum Pseudole, ree oe Cylindrium aigarvense CB Cylindrium aeruginosum CBS 693.83 Cylindrium pnrgamentum CPC 29580 um grande CBS 145655 Phiogicylindrium uniforme CB 100/100 aN hatin ani Apiospora dichotomant! 100/- ree camelliae- 100/1.00 94/1.00 100/1.00 Wieraouee ise CBS ee 100/1.00, Neoamphisphe -/1.00 Neoamphisp Appendicospora hongkong 76i- 94/0.99 Leiosphaerella praeciara Leiosphaerella lvcopodina CBS 1 = yponectria 100/1.00 Dactytaria retrophylli CBS 148271 = _ Dactylaria acaciae CPC 29771 100/1.00 Vialaea inst ie Pseudotruncatella bolusanthi CBS1 145532, Pseudotruncatella arezzoensis ie 100/1.00 rae, Ps lesiniaceae 100/1.00 fodosphaeria Jodosphaeria h g6/., Oxydothis paimicola MFLU 100/1.00 Oxydothis metroxylicola MI Oxydothis rh ee MFLUC 100/1.00, Melogramma campylosporum MELU 18-0778 babii i campylos, wii ie 17-0348 100/1.00 Eutypa cerasi ia Diatrype disci URLS mis CBS 197 100/1.00 Gyrothrix euci 98/1.00 Wasanoeoeee fici een ) 100/1.00, Neogyrothrix oleae C Neogyrothrix oleae CBS 14 99/'1.00 99/1.00 100/1.00[—_ ; Anthostomella fo: 99/1. Psendoanthostome Hi Pseudoanthostomella en Melanographium phoenic: Mclanoarapnenae ee i = Nigro, 100/1.00 : Topadostoma abiness sp pecans turgidum CBS 133207 100/1.00 Pirozynskiomyces sinensis UAMH 81/1.00 Coniocessia anai 100/1.00 Hansfordia pulvinata CBS Hansfordia pruni CBS 19 100/1.00 ——_— ae 100/1.00 Barrmaelia macrospora CBS 6 91/1.00 Barrmaetia rhamnicola CBS412772 / 99/1.00 “Entosordaria perfidiosa CBS 142773 poe Entosordaria quercina CBS 142774 | Castellaniomyces rosae MFLU Induratia apiospora A’ Xylariales 81/0.98, i Neobarrinaelia h Catenuli d ra 100/1.00 Vamsapriya bambusicol Vamsapriya breviconidi 100/1.00 Graphostroma pi _ Biscogniauxia nu 98/1.00 Nemania abortiva Xylaria bami 11.00 ‘Anthostomelloides krabie 100/1.00 = e Daldinia dennisii CBS 114 41 Daldinia grandis CBS 114736 Pee as petriniae CBS 114746 7 lonicicola siamense strain MFLUCC 15-0670 ne inuelleri CBS 1436 100/1.00 100/1.00 tomium m orum CB Chagientiien Denes CBS 374.66 . Sordaria fimicola CBS 723.96 Outgroups (Sordariales) 0.1 Figure 4. Phylogram generated from Maximum Likelihood analysis of 119 specimens belonging to the subclass Xylar- iomycetidae of the combined ITS, LSU, RPB2 and TUB genes. Achaetomium macrosporum CBS 532.94, Chaetomium elatum CBS 374.66 and Sordaria fimicola CBS 723.96 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 fungal strains in this study are red. Type species are bold. MycoKeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 283 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae Colony diam. (in mm) 7 days, 25 °C: CREA 8-11, CYA 13-18, CYAS 7-9, CZ 11-15, DG18 12-16, MEA 15-19, MEAbI 16-19, OA 13-19, PDA 12-15 and YES 9-13. 7 days, 30 °C: CYA 10-15. 7 days, 37 °C: CYA no growth. Culture characteristics. Colonies at 25 °C for 7 days on CREA thin colonies; acid production absent (Fig. 5A). Colonies on CYA circular, convex, wrinkled texture, entire margin; white mycelia; soluble pigment absent; reverse yellow- ish-brown (Fig. 5B). Colonies on CYAS barely growing, circular, raised, wrinkled texture, undulate margin; white mycelia; soluble pigment absent; reverse white (Fig. 5C). On CZ thin colonies, circular, flat, entire margin; white mycelia; solu- ble pigment absent; reverse white (Fig. 5D). On DG18 circular, flat, wrinkled at the centre, margin smooth and entire; grey mycelia at the centre, white myce- lia at the margin; soluble pigment absent; reverse greenish-grey to light yellow (Fig. 5E). Colonies on MEA circular, flat, smooth texture, entire margin; light grey mycelia; soluble pigment absent; reverse light yellow to white (Fig. 5F). On MEAbI circular, flat, wrinkled at the centre, margin smooth and entire; light grey at the centre, white at the margin; soluble pigment absent; reverse yellow- ish-brown (Fig. 5G). On OA circular, flat, smooth textured, entire margin; light brown mycelia at the centre, white mycelia at the margin; soluble pigment ab- sent; reverse white (Fig. 5H). Colonies on PDA circular, flat, wrinkled texture, entire margin; white mycelia; soluble pigment absent; reverse white to light yel- low (Fig. 51). Colonies on YES circular, convex, wrinkled texture, entire margin; white mycelia; soluble pigment absent; reverse light brown (Fig. 5J). Sporula- tion abundantly produces on all media. Micromorphology. Conidiophores mononematous, growing out at right an- gles from hyphae, unbranched, smooth, hyaline, 3-14 x 1-3 um (Fig. 5K—P). Phialides solitary, terminal, ampulliform, smooth, hyaline, 5-12 x 1-4 um (Fig. 5K-P). Conidia globose to subglobose, 2-4 um diam., smooth, hyaline (Fig. 5K-N, Q). Sclerotia not observed. Sexual morph absent. Additional strain examined. THAILAND * Chiang Mai Province, Mae Taeng District, Papae, on soil in the forest dump-sites, 20 June 2024, T. Thitla & J. Monkai; CMUVRO039; living culture, MBSZU 24-007, dried culture permanently preserved in a metabolically inactive state, CMUVRO39. Habitat and distribution. Soil; only known from Chiang Mai Province, Thailand. Notes. Penicillium section Exilicaulis was first established by Pitt (1980), with P restrictum as the type species. This section was initially proposed to accom- modate Penicillium species characterised by monoverticillate conidiophores and non-vesiculated stipes. Subsequently, phylogenetic studies expanded the section to include species with bi-verticillate conidiophores and those with con- idiophores bearing solitary phialides (Houbraken and Samson 2011; Visagie et al. 2016a, b; da Silva et al. 2023). Species of the sect. Exilicaulis have been iso- lated from diverse environments, including soil, marine ecosystems, air, plants and insects (Ansari et al. 2023). Currently, this section comprises over 60 spe- cies across six series: Alutacea, Citreonigra, Corylophila, Erubescentia, Lapidosa and Restricta (Ansari et al. 2023; Visagie et al. 2024a). Penicillium terrae is classified within section Exilicaulis, series Erubescen- tia. Phylogenetically, this species is closely related to P. laeve and P. ovatum (Fig. 2). However, P. laeve and P. ovatum were unable to grow on CREA and CYAS media, while P. terrae can grow on these media. Regarding growth rates, P. laeve exhibited slower growth than P. terrae, including CYA (8-9 mm), DG18 Mycokeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 284 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae CZ, DG18, MEA, MEAbI, OA, PDA and YES, respectively K-P conidiophores, phialides and conidia Q conidia. Scale bar: 2 cm (A-J); 10 um (K); 5 um (L-Q). MycoKeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 285 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae (5-7 mm), OA (7-8 mm) and YES (8-9 mm) at 25 °C, as well as CYA at 30 °C (4-5 mm) (Visagie et al. 2016a). Similarly, P. ovatum also demonstrated slow- er growth compared to P. terrae on CYA (10-11 mm), DG18 (9-11 mm), MEA (7-8 mm) and OA (10-11 mm) at 25 °C (Visagie et al. 2016a). Micromorpho- logically, the phialides of P. laeve (4-6 um x 2-3 um) and P. ovatum (4.5-7 um x 2-3 um) were shorter than P. terrae (Visagie et al. 2016a). In terms of conidia, P. terrae produced globose to subglobose conidia with 2-4 um, while P. leave produced globose conidia measuring 2.5-3 um diam. and P. ovatum produced ellipsoidal conidia with 2-3 x 1.5-2 um (Visagie et al. 2016a). Furthermore, a pairwise nucleotide comparison between P. terrae and P. laeve showed differ- ences of 0.86% (5/581 bp, including gaps) in ITS, 2.87% (13/453 bp, including gaps) in TUB, 2.62% (13/497 bp, including gaps) in CAM and 1.39% (13/938 bp, including gaps) in RPB2. Similarly, the comparison between P. terrae and P. ovatum revealed nucleotide differences of 2.64% (15/569 bp, including gaps) in ITS, 14.41% (65/451 bp, including gaps) in TUB, 17.74% (91/513 bp, including gaps) in CAM and 12.37% (116/938 bp, including gaps) in RPB2. Penicillium chiangmaiense Thitla, Monkai, Lumyong & Hongsanan, sp. nov. MycoBank No: 857424 Etymology. The specific epithet “chiangmaiense’ refers to the type locality “Chi- ang Mai Province, Thailand”. Holotype. THAILAND * Chiang Mai Province, Mae Rim District, Mae Sa, on soil in the forest dump-sites, 27 June 2024, T. Thitla & J. Monkai; VROO5 (SZU25- 006, holotype); ex-type living culture, MBSZU 24-009, dried culture permanently preserved in a metabolically inactive state, SZU25-006. Colony diam. (in mm) 7 days, 25 °C: CREA 40-44, CYA 50-52, CYAS 35-38, CZ 48-49, DG18 34-39, MEA 47-51, MEAbI 51-53, OA 53-54, PDA 49-50 and YES 32-38. 7 days, 30 °C: CYA 59-61. 7 days, 37 °C: CYA 55-56. Culture characteristics. Colonies at 25 °C for 7 days on CREA thin colo- nies; acid production absent (Fig. 6A). Colonies on CYA and CYAS wrinkled texture, velvety, circular, flat, entire margin; white mycelia; soluble pigment absent; reverse light brown (Fig. 6B, C). On CZ, thin colonies, circular, flat, filamentous margin; white mycelia; soluble pigment absent; reverse white (Fig. 6D). On DG18, wrinkled texture, velvety, circular, flat, entire margin; white mycelia; soluble pigment absent; reverse white to pale yellow (Fig. 6E). Colonies on MEA and MEAbI smooth texture, circular, flat, entire margin; pale yellow at the centre, white at the margin; soluble pigment absent; re- verse pale brown to white (Fig. 6F, G). On OA, smooth textured, velvety, circu- lar, flat, entire margin; white mycelia; soluble pigment absent; reverse light yellow to white (Fig. 6H). Colonies on PDA circular, flat, smooth texture, entire margin; white mycelia; soluble pigment absent; reverse white to light yellow (Fig. 61). Colonies on YES circular, flat, wrinkled texture, velvety, en- tire margin; white mycelia; soluble pigment absent; reverse brownish-yellow (Fig. 6J). Sporulation abundantly produces on DG18, MEA and MEAbI me- dia. Sclerotia produces MEA, MEAbI and OA (Fig. 6P). Micromorphology. Conidiophores monoverticillate, sometimes divaricate. Stipes hyaline, smooth-walled, 80-270 x 2-3 um (Fig. 6K-N). Phialides Mycokeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 286 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae Figure 6. Penicillium chiangmaiense (MBSZU 24-009, ex-type living culture) A-J colonies at 25 °C for 7 days on CREA, CYA, CYAS, CZ, DG18, MEA, MEAbI, OA, PDA and YES, respectively K-N conidiophores, phialides and conidia O conidia P sclerotia produced on culture media. Scale bar: 2 cm (A-J); 10 pm (K-O); 100 um (P). terminal, ampulliform, hyaline, smooth—walled 6-17 x 2-3.5 um (Fig. 6K—N). Conidia globose to subglobose, 2-4 um diam., smooth, hyaline (Fig. 6K—-O). Sclerotia pale brown to brown, globose to irregular, 180-260 um diam. (Fig. 6P). Sexual morph absent. Additional strain examined. Thailand » Chiang Mai Province, Mae Rim District, Mae Sa, on soil in the forest dump-sites, 27 June 2024, T. Thitla & J. Monkai; CMUVROO5-2; living culture, MBSZU 24-010, dried culture permanently preserved in a metabolically inactive state, CMUVROO05-2. Habitat and distribution. Soil; only known from Chiang Mai Province, Thailand. MycoKeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 287 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae Notes. Penicillium section Lanata-Divaricata was established by Thom (1930) to include species with biverticillate conidiophores, which usually contain a main conidiophore axis and metulae that diverge (referred to as divaricate conidio- phores), as well as broadly spreading colonies (Houbraken and Samson 2011; Pangging et al. 2021). Species within this section have been isolated from vari- ous sources, including soil, air, fluvial sediments and plants (Nobrega et al. 2024). Currently, the section is divided into five series: Dalearum, Janthinella, Oxalica, Rolfsiorum and Simplicissima (Ansari et al. 2023; Visagie et al. 2024a). Penicillium chiangmaiense is classified within section Lanata-Divaricata, se- ries Janthinella. Inthe phylogenetic tree (Fig. 3), the new species is closely related to P. brefeldianum, P. limosum and P. michoacanense. However, P. brefeldianum produces sexual structures on cornmeal agar and P. limosum produces on CZ, MEA and OA, while P. chiangmaiense does not exhibit any sexual features (Dodge 1933; Ueda 1995). Furthermore, the growth rate of P. limosum on MEA (42 mm in 14 days) was slower than that of P. chiangmaiense (47-51 mm in 7 days) (Ueda 1995). In the case of P. michoacanense, the stipes (15-60 x 1-1.5 um) and phialides (4—5 x 1.5 um) were shorter than those of P. chiangmaiense (sti- pes 80-270 x 2-3 um; phialides 6-17 x 2-3.5 um) (Rodriguez-Andrade et al. 2021). Moreover, P. michoacanense produced weak acid on CREA, while P. chi- angmaiense does not produce it (Rodriguez-Andrade et al. 2021). Additionally, the pairwise nucleotide comparison of P. chiangmaiense with related species revealed significant differences. The comparison of P. chiangmaiense to P brefeldianum showed 0.90% (5/556 bp) difference in ITS, 4.73% (21/444 bp) in TUB, 4.28% (24/561 bp) in CAM and 1.46% (11/755 bp) in RPB2, including gaps. Differences in P. chiangmaiense and P. limosum were 1.09% (6/548 bp) in ITS, 5.00% (22/440 bp) in TUB, 6.28% (35/557 bp) in CAM and 0.93% (7/755 bp) in RPB2, including gaps. In comparison between P. chiangmaiense and P. micho- acanense, the differences were 0.73% (4/548 bp) in ITS, 2.84% (11/388 bp) in TUB, 8.35% (34/407 bp) in CAM and 1.61% (12/745 bp) in RPB2, including gaps. Pseudoleptodontidium Thitla, Monkai, Lumyong & Hongsanan, gen. nov. MycoBank No: 857466 Etymology. The name refers to its morphological similarity to Leptodontidium. Classification. Sordariomycetes, Xylariomycetidae, Amphisphaeriales, incer- tae sedis. Asexual morph: Mycelium composed of hyaline to black, thin- to thick-walled, smooth, branched, septate. Conidiophores arising from hyphae, solitary, erect, cylindrical, pale brown to dark brown, thick-walled, occasionally roughened on lower part, septate. Conidiogenous cells terminal and intercalary on con- idiophores, occasionally lateral on hyphae, obclavate, sympodially proliferate, denticulate, hyaline to pale brown, smooth, septate. Conidia hyaline, smooth, aseptate, subglobose to ellipsoidal, slightly curved. Chlamydospores solitary, terminal on hyphae, medium brown to dark brown, smooth, thick-walled, asep- tate, subglobose. Sexual morph: absent. Type species. Pseudoleptodontidium chiangmaiense Thitla, Monkai, Lumyong & Hongsanan, sp. nov. Mycokeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 288 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae Notes. Herndndez-Restrepo et al. (2017) established Leptodontidium in Leptodontidiaceae (Helotiales, Leotiomycetes), characterised by erect conid- iophores and conidiogenous cells with a long rachis bearing denticles, as well as the presence of a Beauveria-like synasexual morph. Neoleptodontidium was introduced by Crous et al. (2023) due to its morphological resemblance to Lept- odontidium, but it differs in having minute, terminal and lateral exophiala-like phialides. Based on LSU phylogeny, the type species of Neoleptodontidium (N. aquaticum) clustered with Leptodontidium aciculare (Crous et al. 2023). Hence, Crous et al. (2023) transferred L. aciculare to Neoleptodontidium as N. aciculare by the morphological and phylogenetic congruence. Pseudoleptodontidium is morphologically similar to Neoleptodontidium, sharing septate, subcylindrical conidiophores, terminal and lateral phialidic conidiogenous cells and aseptate subcylindrical conidia (Crous et al. 2023). However, Pseudoleptodontidium can be distinguished from Neoleptodontid- ium by its obclavate, sympodially proliferating, denticulate conidiogenous cells and subglobose to ellipsoidal conidia. The phylogeny, based on a com- bined ITS, LSU, RPB2 and TUB dataset, revealed that Pseudoleptodontidi- um forms an independent lineage, sister to Neo/eptodontidium with signifi- cant support (BS 96% ML and PP 1.00; Fig. 4). Although Crous et al. (2023) placed Neoleptodontidium in Xylariales genera incertae sedis, our phyloge- ny indicates that Pseudoleptodontidium and Neoleptodontidium are closely related to the Amphisphaeriaceae, Cylidriaceae, Phlogicylindriaceae and Amphisphaeriales genera incertae sedis (Neoarthrinium, Pidoplitchkoviella) (Fig. 4). Therefore, due to their distinct morphology and phylogeny, Pseudo- leptodontidium is introduced as a genus incertae sedis in Amphisphaeriales, with Ps. chiangmaiense designated as the type species. Pseudoleptodontidium chiangmaiense Thitla, Monkai, Lumyong & Hongsanan, sp. nov. MycoBank No: 857467 Etymology. The specific epithet chiangmaiense refers to the type locality, Chi- ang Mai Province, Thailand. Holotype. Thailand*Chiang Mai Province, Mueang Chiang Mai District, Su Thep, on soil in the forest dump-sites, 21 June 2024, T. Thitla & J. Monkai; VR044 (SZU25-007, holotype); ex-type living culture, MBSZU 25-005, dried cul- ture permanently preserved in a metabolically inactive state, SZU25-007. Colony diam. (in mm) 14 days, 25 °C: PDA 36-40 and MEA 31-38. Culture characteristics. Colonies at 25 °C for 14 days on PDA velvety, circular, flat, entire margin; dark green at the centre, greenish-yellow at the middle, white at the margin; soluble pigment absent; reverse dark green to pale yellow, white at the margin (Fig. 7A). Colonies on MEA velvety, circular, flat, entire margin; dark green to black at the centre, yellowish-green to white at the margin; soluble pigment absent; reverse dark green at the centre, pale yellow to white at the margin (Fig. 7B). Micromorphology. Mycelium composed of hyaline to black, thin- to thick- walled, smooth, branched, septate, 2-4.5 um diam. hyphae (Fig. 7C—-L). Con- idiophores arising from hyphae, solitary, erect, cylindrical, pale brown to dark Mycokeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 289 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae Figure 7. Pseudoleptodontidium chiangmaiense (MBSZU 25-005, ex-type living culture) A, B colonies from surface and reverse view at 25 °C for 14 days on PDA and MEA, respectively C-J conidiophores, conidiogenous cells and conidia K conidia L chlamydospore. Scale bar: 2 cm (A, B); 10 um (C-L). brown, thick-walled, occasionally roughened on lower part, septate, 7-70 x 2.5-5 um (Fig. 7C—G). Conidiogenous cells terminal and intercalary on conidio- phores, occasionally lateral on hyphae, obclavate, sympodially proliferate, den- ticulate, hyaline to pale brown, smooth, 0-1 septate, 7.5-26 x 3-5 um (Fig. 7C- J). Conidia hyaline, smooth, aseptate, subglobose to ellipsoidal, slightly curved, 3-7.5 x 1.5-4 um (Fig. 7K). Chlamydospores solitary, terminal on hyphae, me- dium brown to dark brown, smooth, thick-walled, aseptate, subglobose, 6-8 x 4.5-6 um (Fig. 7L). Sexual morph absent. Habitat and distribution. Soil; only known from Chiang Mai Province, Thailand. MycoKeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 290 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae Notes. Pseudoleptodontidium chiangmaiense has aclose relationship with Neo- leptodontidium aciculare and N. aquaticum (Fig. 4). However, their morphological characteristics are distinct: Ps. chiangmaiense has broader conidiogenous cells (7.5-26 x 3-5 um) than N. aciculare (15-30 x 2-3 um) and N. aquaticum (10-30 x 2-2.5 um) and larger conidia (3-7.5 x 1.5—-4 um) than N. aciculare (3-4 x 1-2 um) and N. aquaticum (3-4 x 1.5 um) (Rao and De Hoog 1986; Hernandez-Re- strepo et al. 2017). The pairwise nucleotide comparison between Ps. chiang- maiense and N. aciculare revealed differences of 16.38% (95/580 bp, including gaps) in the ITS region and 4.92% (40/813 bp, including gaps) in the LSU region. Additionally, the comparison between Ps. chiangmaiense and N. aquaticum re- vealed differences of 16.23% (87/536 bp, including gaps) in the ITS region and 4.80% (39/813 bp, including gaps) in the LSU region. Discussion This study identifies a new genus in Xylariomycetidae, namely Pseudoleptodon- tidium, accommodating Ps. chiangmaiense sp. nov., along with two new spe- cies of Penicillium: P. terrae in section Exilicaulis and P. chiangmaiense in sec- tion Lanata-Divaricata. These species were isolated from soil collected in forest dump-sites in Chiang Mai Province, Thailand. They were characterised through morphological observations and multigene phylogenetic analyses (Figs 2-6). Penicillium is a highly impactful genus, with species ranging from mycotox- in-producing plant pathogens and opportunistic animal and human pathogens to valuable sources of enzymes, antibiotics and bioactive compounds (Oshika- ta et al. 2013; Perrone and Susca 2017; Costa et al. 2019; Toghueo and Boyom 2020; Wolski 2023; Suwannarach et al. 2024). The genus was proposed by Link (1809) and currently comprises two subgenera, 34 sections, 102 series and 535 accepted species (Visagie et al. 2024a). Penicillium was traditionally iden- tified, based on macro-morphology (such as colony characteristics and pig- ment production) and micro-morphology (including conidiophores, branches, metula, phialides and conidia) (Khuna et al. 2023). However, relying solely on morphological characteristics has proven insufficient for accurate identifica- tion. Consequently, an integrated approach combining morphology, molecular data and extrolite analysis is currently used to identify species within the genus Penicillium (Visagie et al. 2014; Labuda et al. 2021; Nguyen and Pham 2022; Visagie et al. 2024a, 2024b). In section Exilicaulis, key genetic data for species identification include the internal transcribed spacer region (ITS), beta-tubulin (TUB), calmodulin (CAM) and RNA polymerase II subunit (RPB2) genes (Visagie et al. 2016c). Initially, PR Jaeve and P ovatum were introduced under the genus Torulomyces as T. laevis and T. ovatus, respectively (Ando et al. 1998). Subse- quently, phylogenetic analyses using RNA polymerase II largest subunit (RPB7), RPB2, the protein required for processing of 20S pre-rRNA in the cytoplasm (Tsr1) and the subunit of the cytosolic chaperonin Cct ring complex (Cct8) led to transfer to Penicillium section Torulomyces (Houbraken and Samson 2011). Visagie et al. (2016a) reclassified these species into section Exilicaulis using ITS, TUB, CAM and RPB2 sequence data. Currently, P Jaeve and P ovatum be- long to the series Erubescentia, characterised by species with monoverticillate conidiophores, short stipes and the ability to grow at 37 °C (Houbraken et al. MycoKkeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 291 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae 2020). However, both P laeve and P. ovatum, along with P. terrae, produce con- idiophores with solitary phialides and were unable to grow at 37 °C. Addition- ally, the phylogenetic clade of these species formed a basal clade with other species in this series with strong support (BS 97% and PP 1.00) (Fig. 2). In our opinion, this distinct clade may represent a potential new series within section Exilicaulis and should be further studied in the future. Prior to this study, P laeve was the only species in section Exilicaulis reported from Thailand (Ando et al. 1998). The discovery of P terrae from soil in Thailand marks the second species from this section identified in the country. Furthermore, this new species represents the 69" global species in section Exilicaulis, as shown in Suppl. material 1: table S1, excluding P janthinellum and P limosum. In addition, this study proposed a new species, P chiangmaiense in section Lanata-Divaricata, which is the second species recorded in Thailand from this section, following the first species (P. singorense) described by Visagie et al. (2014b). Additionally, this new species represents the 108" global species in section Lanata-Divaricata, as outlined in Suppl. material 1: table S2, excluding P alogum and P stolkiae. Ecologically, Penicillium species have been isolated from different environ- ments (Ansari et al. 2023; Nobrega et al. 2024). For instance, P chiangmaiense and its closely-related species, including P. brefeldianum, P. limosum and P. mi- choacanense, have been found in the human digestive tract, marine sediments and soil (Dodge 1933; Ueda 1995; Rodriguez-Andrade et al. 2021). Similarly, P terrae and its relatives, including P /aeve and P ovatum, have primarily been reported from soil, with P laeve specifically found in forest soils in Thailand (Ando et al. 1998). These findings highlight the ecological plasticity of Peni- cilllum species, which can potentially thrive in disturbed ecosystems. Future studies examining their functional traits and metabolic profiles could further enhance better understanding of their ecological significance. Xylariomycetidae is a large subclass within Sordariomycetes comprising nu- merous taxa that are polyphyletic and paraphyletic (Wendt et al. 2017; Daranag- ama et al. 2018; Konta et al. 2020; Samarakoon et al. 2022). The taxonomic classification of Xylariomycetidae has undergone considerable change (Ma- harachchikumbura et al. 2016; Samarakoon et al. 2016, 2022). Earlier, Amphis- phaeriales was considered a synonym of Xylariales (Maharachchikumbura et al. 2016). However, based on morphology, molecular data, divergence estimates and ancestral state reconstruction, Samarakoon et al. (2016, 2022) subse- quently reclassified Amphisphaeriales, Delonicicolales and Xylariales in Xylar- iomycetidae. Molecular phylogeny, based on concatenated ITS, LSU, RPB2, TUB and TEF-1a sequence data, demonstrated the placement of Amphisphaeriales in a sister clade to Xylariales (Samarakoon et al. 2022), which is consistent with our study (Fig. 4). However, we did not incorporate TEF-7a into the phylogenetic tree, as the number of taxa with available sequence data was low. The classifi- cation of taxa within Xylariomycetidae remains ambiguous, as more than 50 in- certae sedis genera await taxonomic resolution (Samarakoon et al. 2022). Like- wise, our study was unable to assign the novel genus Pseudoleptodontidium to any family within the Xylariomycetidae (Fig. 4). The new lineage of Pseudo- leptodontidium and Neoleptodontidium also lacks significant statistical support for placement within other taxa and families in Amphisphaeriales, though it is likely linked to Amphisphaeriaceae, Cylidriaceae and Phlogicylindriaceae (Fig. 4). Further taxonomic and phylogenetic studies, including the collection of new Mycokeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 292 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae specimens and the examination of additional isolates, are necessary to confirm the familial placement of Pseudoleptodontidium and Neoleptodontidium. Members of Xylariomycetidae have a worldwide distribution and occupy var- ious ecological niches, including saprobes, endophytes and pathogens (U’Ren et al. 2016; Daranagama et al. 2018; Sugita et al. 2022; Samarakoon 2024). Re- cently, several new taxa have been reported as saprobes on dead plant materials from Thailand (Monkai et al. 2022; Afshari et al. 2023; Samarakoon et al. 2023; Karimi et al. 2023, 2024; Samarakoon 2024; Thakshila et al. 2024). In this study, Pseudoleptodontidium was isolated from soil associated with a forest dump-site in Thailand, whereas Neoleptodontidium species have been found in hydroponic water and decomposing wood in the USA and India (Rao and De Hoog 1986; Hernandez-Restrepo et al. 2017). This demonstrates that these taxa have a broad distribution range, highlighting their adaptability in diverse environments. These findings significantly contribute to our understanding of fungal diver- sity and ecology, particularly within the Ascomycota and highlight the richness and diversity of soil fungal communities in Thailand. Penicillium and some Xy- lariomycetidae taxa, such as Amphisphaeria, Annulohypoxylon and Hypoxylon are recognised for possessing a wide variety of secondary metabolites, which have prospective agricultural and therapeutic uses (Toghueo and Boyom 2020; Becker and Stadler 2021; Wang et al. 2023a; Wolski 2023). The discovery of novel fungi in forest dump areas presents an opportunity to explore and charac- terise these fungi for various applications. Therefore, further research is neces- sary to evaluate the capabilities of new fungal strains for extracellular enzyme production and the degradation of synthetic materials. Acknowledgements This research work is partially funded by Chiang Mai University. Shaun Penny- cook is thanked for the nomenclatural advice. Sinang Hongsanan would like to thank the National Natural Science Foundation of China (32400012), 2023 Shenzhen Pengcheng Distinguished Positions and scientific research funds for high-tech talents /high-level talents and Shenzhen University 2035 Program for Excellent Research, Grant No. 2024C006. Ning Xie would like to thank the Natural Science Foundation of Guangdong Province (2024B1515020034) and National Key R&D Program of China (2021YFA0910800). Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding This work was funded by the Natural Science Foundation of Guangdong Province (2024B1515020034), the International Research Fellowship (Visiting Researcher Pro- gram), Chiang Mai University (Grant No. 014/2567), and the National Key R&D Program of China (2021YFA0910800). Mycokeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 293 Tanapol Thitla et al.: Two new species of Penicillium and a new genus in Xylariomycetidae Author contributions Conceptualisation: Tanapol Thitla, Jutamart Monkai, Sinang Hongsanan, Saisamorn Lumyong. Collection and morphological examinations: Tanapol Thitla, Jutamart Monkai, Weigian Meng, Surapong Khuna. Molecular sequencing and phylogenetic analyses: Tanapol Thitla, Jutamart Monkai, Weiqian Meng, Surapong Khuna. Original draft prepa- ration: Tanapol Thitla, Jutamart Monkai, Surapong Khuna. Review and editing, supervi- sion: Tanapol Thitla, Jutamart Monkai, Weigian Meng, Surapong Khuna, Ning Xie, Sinang Hongsanan, Saisamorn Lumyong. All authors have read and agreed to the published version of the manuscript. Author ORCIDs Tanapol Thitla © https://orcid.org/0000-0003-3092-0679 Jutamart Monkai © https://orcid.org/0000-0001-6043-0625 Weigian Meng ® https://orcid.org/0009-0006-3840-0992 Surapong Khuna ® https://orcid.org/0000-0003-1999-4001 Ning Xie © https://orcid.org/0000-0002-5866-8535 Sinang Hongsanan ® https://orcid.org/0000-0003-0550-3152 Saisamorn Lumyong ® https://orcid.org/0000-0002-6485-414X Data availability The datasets generated during and/or analysed during the current study are available in the MycoBank repository (included in the manuscript) and GenBank (included in Suppl. material 1: tables S1-S3). Additionally, the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Afshari N, Karimi O, De Farias ARG, Suwannarach N, Bhunjun CS, Zeng X-Y, Lumyong S (2023) Additions to Diatrypaceae (Xylariales): Novel taxa and new host associations. 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GenBank accession numbers of Penicillium section Lanata-Divaricata used in multi-locus phylogenetic analysis. ta- ble S3. GenBank accession numbers of taxa in Xylariomycetidae used in multi-genes phylogenetic analysis. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.116.150635.suppl1 MycoKkeys 116: 275-301 (2025), DOI: 10.3897/mycokeys.116.150635 301