«83 MycoKeys MycoKeys 113: 147-168 (2025) DOI: 10.3897/mycokeys.113.139427 Research Article Three new asexual Kirschsteiniothelia species from Guizhou Province, China Xing-Juan Xiao'23®, Ning-Guo Liu™, Jian Ma'®, Li-Juan Zhang'2©, Dan-Feng Bao‘, Song Bai®, Fatimah Al-Otibi2®®, Kevin D. Hyde?®®, Yong-Zhong Lu'® ee wo NY — Guizhou Province, China School of Food and Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang 550025, China Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand Engineering and Research Center for Southwest Biopharmaceutical Resource of National Education Ministry of China, Guizhou University, Guiyang, 550025, 5 Guizhou Industry Polytechnic College, Guiyang 550008, China 6 Department of Botany and Microbiology, College of Science, King Saud University, RO. Box 22452, Riyadh 11495, Saudi Arabia Corresponding authors: Song Bai (basonmail@163.com); Yong-Zhong Lu (yzlu@git.edu.cn) OPEN Qaccess 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: Danushka Sandaruwan Tennakoon Received: 16 October 2024 Accepted: 8 January 2025 Published: 3 February 2025 Citation: Xiao X-J, Liu N-G, Ma J, Zhang L-J, Bao D-F, Bai S, Al-Otibi F, Hyde KD, Lu Y-Z (2025) Three new asexual Kirschsteiniothelia species from Guizhou Province, China. Mycokeys 113: 147-168. https://doi. org/10.3897/mycokeys.113.139427 Copyright: © Xing-Juan Xiao 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 During our investigation of saprobic fungi in southwestern China, three micro-hypho- mycetous fungi were isolated from dead wood in freshwater and terrestrial habitats in Guizhou Province. Phylogenetic analyses of ITS, LSU, and SSU sequences, per- formed using Maximum Likelihood and Bayesian Inference methods, confirmed that these isolates belong to Kirschsteiniothelia. Based on distinct morphological char- acteristics and molecular phylogenetic evidence, we describe three new species: Kirschsteiniothelia guizhouensis, K. weiningensis, and K. xishu/ensis. Furthermore, the effectiveness of three DNA markers for species-level identification within Kirschstein- iothelia was evaluated using Assemble Species by Automatic Partitioning (ASAP) analysis, which identified the ITS nucleotide sequences as the most reliable marker for species differentiation within the genus. Key words: 3 new species, asexual morph, phylogeny, taxonomy Introduction Kirschsteiniothelia was introduced by Hawksworth (1985) to accommo- date the Microthelia incrustans-group (Dothideales), with K. aethiops as the type species. Hawksworth (1985) and Barr (1987) placed the genus in Pleosporaceae. Subsequently, Barr (1993) suggested that this genus should be transferred to Pleomassariaceae based on its host, morphology, and asexual form. However, Schoch et al. (2006) recommended that this genus should have its own family, as the type species K. aethiops did not show close associations with Pleosporaceae based on their phylogenetic analyses. Boonmee et al. (2012) proposed a new family, Kirschsteiniotheli- aceae to accommodate the taxa grouping with K. aethiops, and they trans- ferred K. elaterascus to Morosphaeria (Morosphaeriaceae) and K. maritima 147 Xing-Juan Xiao et al.: Three new asexual Kirschsteiniothelia species to Halokirschteiniothelia (Mytilinidiaceae). Kirschsteiniothelia has been linked with the asexual genus Dendryphiopsis, as confirmed by several studies based on morphology and phylogeny (Hughes 1953; Schoch et al. 2009; Boonmee et al. 2012). Wijayawardene et al. (2014) proposed using Kirschsteiniothelia over Dendryphiopsis and designated K. atra as the type species. Subsequently, Hernandez-Restrepo et al. (2017) established a new order, Kirschsteiniotheliales, to accommodate this family where it is now included (Hyde et al. 2024). The sexual morph of Kirschsteiniothelia is characterized by superficial, subglobose to globose, dark brown to black ascomata, with a peridium of tex- tura angularis, cylindrical-clavate asci which are apically rounded with a small ocular chamber, and ellipsoidal, 1-2 septate, smooth-walled, olive brown to dark brown, dull green ascospores (Pem et al. 2024). The asexual morph has long, straight to slightly curved, septate, apically branched or unbranched, dark brown conidiophores, and broadly obovoid, fusiform or obclavate, red- dish brown to dark brown or grayish brown, septate conidia (Hawksworth 1985; Boonmee et al. 2012; Mehrabi et al. 2017; Xu et al. 2023; Liu et al. 2024; Meng et al. 2024). Species of Kirschsteiniothelia are commonly found as saprobes from terrestrial and freshwater habitats in tropical or subtropical regions (Bao et al. 2018; Rodriguez-Andrade et al. 2019; Dong et al. 2020; Sun et al. 2021; Verma et al. 2021; Meng et al. 2024). In this study, we collected four hyphomycetous samples of dead wood from freshwater and terrestrial habitats in Guizhou Province, China. Three new spe- cies, namely Kirschsteiniothelia guizhouensis, K. weiningensis and K. xishuiensis, were identified based on morphological evidence and phylogenetic analyses of combined ITS, LSU, and SSU sequence data. This paper provides a polyphasic approach (molecular data, morphological characteristics and location informa- tion) for introducing the new species (Maharachchikumbura et al. 2021). Materials and methods Isolation and morphological observation Decaying wood samples were randomly collected from Guizhou Xishui Na- tional Nature Reserve, Xishui County, within 28°9'17"-28°33'50"N, 105°55'50"- 106°24'9"E; elevation 600-1,200 m, and Weining County, along the Wuji- angyuan River (26°52'33"-26°57'50"N, 104°22'18"-104°25'43"E; elevation 2,000-2,100 m). The fresh specimens were stored in sterile, damp plastic containers at room temperature for approximately 15 to 20 days. The fresh samples were examined using stereo microscopes (SMZ 745 and SMZ 800N, Nikon, Tokyo, Japan), and their micro-morphological characteristics were ob- served using an ECLIPSE Ni compound microscope (Nikon, Tokyo, Japan). The method for single spore isolation followed the procedure outlined by Senanayake et al. (2020). Purified cultures were maintained in an incubator at 28 °C under light conditions, and the morphological characteristics of the colonies were carefully observed and recorded. Dried specimens were depos- ited at the Herbarium of Kunming Institute of Botany, Chinese Academy of Sci- ences (Herb. HKAS), Kunming, China, as well as the Herbarium of the Guizhou MycoKeys 113: 147-168 (2025), DOI: 10.3897/mycokeys.113.139427 148 Xing-Juan Xiao et al.: Three new asexual Kirschsteiniothelia species Academy of Agriculture Sciences (GZAAS), Guiyang, China. The cultures were deposited in the Guizhou Culture Collection (GZCC), Guizhou, China. Index Fun- gorum (http://www.indexfungorum.org/names/names.asp) and Facesoffungi database (Jayasiri et al. 2015) numbers were obtained. DNA extraction, PCR amplification, and sequencing Mycelia from 30-day-old cultures were scraped from PDA plates using ster- ile toothpicks and placed into 1.5 mL microcentrifuge tubes. DNA was ex- tracted using the Ezup Column Fungi Genomic DNA Purification Kit, follow- ing the manufacturer's instructions. PCR amplifications were performed for three loci: internal transcribed spacer (ITS), ribosomal large subunit rDNA (LSU), and ribosomal small subunit rDNA (SSU), using the primer pairs ITS5/ ITS4 (White et al. 1990), LROR/LR5 (Vilgalys and Hester 1990; Rehner and Samuels 1994) and NS1/NS4, respectively. The thermal cycling conditions for ITS, LSU, and SSU followed the procedures detailed by Ma et al. (2023). After PCR amplification, the products were analyzed using 1% agarose gel electrophoresis. Purification and sequencing of PCR products were carried out by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). The newly obtained sequences were deposited in the GenBank database (https://ncbi. nlm.nih.gov/WebSub/). Phylogenetic analyses BioEdit version 7.0.5.3 (Hall 1999) was used to inspect the original sequenc- es for quality, including checking base-calling errors, and potential contami- nations or ambiguities in the nucleotide data. The SeqMan v. 7.0.0 (DNASTAR, Madison, WI, USA, Swindell and Plasterer 1997) was used to assemble forward and reverse sequences. The sequences used for phylogenetic analyses were obtained from GenBank (Table 1) and downloaded using the One-click Fungal Phylogenetic Tool (OFPT) (Zeng et al. 2023). For each locus, sequence align- ments were performed using the online multiple alignment tool MAFFT version 7, and the resulting alignments were refined with the trimAl tool (Capella-Gutiér- rez et al. 2009; Katoh and Standley 2013). The phylogenetic tree was construct- ed using the methods described by Su et al. (2022), which included Maximum Likelihood (ML) and Bayesian Inference (BI). The phylogenetic trees were edited using FigTree v1.4.0, and the final layout was completed using Adobe Photoshop 2018 and Adobe Illustrator 2021 (Ado- be Systems, San Jose, CA, USA). Analysis of matrix partitions by Assemble Species by Automatic Partitioning (ASAP) ASAP (Assemble Species by Automatic Partitioning) analysis was conducted using the ASAP online platform (https://bioinfo.mnhn.fr/abi/public/asap). The Kimura 2-Parameter model was selected to generate a list of partitions ranked by scores. To avoid unstable results due to gene deletion in some species, se- lect “*” species for analysis. MycoKeys 113: 147-168 (2025), DOI: 10.3897/mycokeys.113.139427 149 Xing-Juan Xiao et al.: Three new asexual Kirschsteiniothelia species Table 2. Synopsis of the morphological characteristics of asexual taxa of Kirschsteiniothelia species reported from China. No. 1 10 11 12 13 14 Species Kirschsteiniothelia aquatica K. atra K. bulbosapicalis K. cangshanensis K. dendryphioides K. dushanensis K. fluminicola K. guangdongensis K. guizhouensis K. longirostrata K. longisporum K. nabanheensis K. pini K. puerensis Distribution Yunnan Province Yunnan Province, Guizhou Province Hainan Province Yunnan Province Yunnan Province Guizhou Province Yunnan Province Guangdong Province Guizhou Province Hainan Province Sichuan Province yunnan Province Sichuan Province Yunnan Province Habitat Freshwater Freshwater, terrestrial Terrestrial Freshwater Freshwater Freshwater Freshwater Terrestrial Freshwater Terrestrial Terrestrial Terrestrial Terrestrial Terrestrial Host Dead wood Dead wood, Edgeworthia chrysantha Dead wood Dead wood Dead wood Dead wood Dead wood Submerged wood Dead wood Dead wood Pinus taeda Dead wood Pinus sp. Coffea sp. Conidiophores Conidiogenous cells Unbranched, Monoblastic, cylindrical, dark brown, | __ cylindrical, dark 114-151 x 7-8 um. brown. Branched, cylindrical, Doliiform or 5-10-septate, dark lageniform, brown, 245-355 x pale brown or 8-10 um. subhyaline. Unbranched, smooth, brown to dark brown, Cylindrical, brown to dark brown, 58-128 x 7.5- 6-17 x 7-10.5 um. 12.5 um. Unbranched, Monoblastic, cylindrical, pale cylindrical, pale brown, 105.5-135.5 x brown. 6-8 um. Branched, smooth, Cylindrical, brown to dark brown, 179-467 x 4.5-8 um. doliiform, pale brown to brown, 9-19 x 4-8 um. Unbranched, Monoblastic, cylindrical, verrucose, cylindrical or septate, dark brown, doliform, brown, 160-307 x 6.5-13 um. | 9-26 3-7 um. Unbranched, Monoblastic, cylindrical, smooth, cylindrical, dark dark brown to black, brown. 209-286 x 7-9 um. Unbranched, Monoblastic, cylindrical, septate, cylindrical to dark brown, 250-350 x ampulliform, 10-18 um. dark brown, 15- 18 x 9-12 um. Branched, cylindrical, Monoblastic, multi-septate, thick- branches, walled, dark brown, subcylindrical, 195-477 x 11-16 um. | light to dark brown, 5.5-16 x 4.5-8 ym. Unbranched, smooth, brown to dark brown, 80-252 x 4.5-9.5 um. Cylindrical, pale brown to brown, 6.5-16 x 5-9 um. Branched, solitary Cylindrical, or fasciculate, erect, verruculose, dark cylindrical, septate, brown. verruculose, dark brown to black, 115- 285 x 6.5-14 um. Irregular or Monotretic, subscorpioid cylindrical or branched, cylindrical, | doliiform, brown to smooth, septate, black, | dark brown, 20-24 brown to brown, 320- x 4-6 um. 588 x 8-12 um. Unbranched, Monoblastic, cylindrical, 3-8 cylindrical, pale brown, 15-21 x 3-5 um. septate, smooth, brown or dark brown, 69-124 x 3.5-7 um. Dark brown to black, smooth, 15-25 x 5-10 um. Unbranched, solitary or caespitose, cylindrical, smooth, 7-15 septate, dark brown, 100-250 x 5-12 um. MycoKeys 113: 147-168 (2025), DOI: 10.3897/mycokeys.113.139427 Conidia Obclavate, smooth, septate, dark brown, 35-46 x 7.5-8.5 um. Cylindrical, smooth, 3-4 septate, brown, 54-63 x 14-18 um. Cylindrical, ovoid to obclavate, rostrate, smooth, septate, olivaceous to reddish-brown to dark brown, with sheath, 118- 236.5 x 15-27 um. Obclavate, septate, pale brown to brown, with sheath, 33-43 x 7.5- 8.5 um. Cylindrical, oblong and occasionally clavate, smooth, guttulate, 2-4 septate, brown, 30-55 x 9-13.5 um. Rostrate, smooth, 5-11 septate, olivaceous brown to soot brown, with sheath, 62-81 x 12.5-18 um. Solitary to short-catenate, obclavate, rostrate, multi- septate, subhyaline to dark brown, guttulate, 47.5-86.5 x 8-10 um. Elongated, flask-shaped, smooth, 13 septate, blackish brown to black, with sheath, 290-300 um long, 42-50 um wide at base, 20-22 um wide at apex. Obclavate, rostrate, septate, olivaceous brown to brown, with sheath, 36.5-65 x 8-16.5 um. Cylindrical, obpyriform to obclavate, rostrate, smooth, septate, pale brown to brown, with sheath, 36.5-109 x 8-16 um. Cylindrical, obclavate, elongated, thick-walled, 3-15 septate, verruculose, brown, 35-130 x 8.5- 15 um. Obclavate or fusiform, 3-7 septate, smooth, dark brown to brown, 32-112 x 8-12 um. Obclavate, 3-6 septate, brown, 22-45 x 5-10 um. Obclavate, 5-12 septate, pale brown to brown, a hyaline sheath (some two globose sheaths), 60-140 x 5-20 um. References Bao et al. (2018) Su et al. (2016), Tang et al. (2024) Tang et al. (2024) Bao et al. (2018) Tang et al. (2024) Yang et al. (2023) Bao et al. (2018) Senanayake et al. (2023) This study Tang et al. (2024) Tian et al. (2024) Liu et al. (2023) Jin et al. (2024) Hyde et al. (2023) 150 Xing-Juan Xiao et al.: Three new asexual Kirschsteiniothelia species No. 15 16 17 18 19 20 21 Species K. ramus K. rostrata K. sichuanensis K. submersa K. weiningensis K. xishuangbannaensis K. xishuiensis Distribution Hainan Province Yunnan Province Sichuan Province Yunnan Province Guizhou Province Yunnan Province Guizhou Province Habitat Host Conidiophores Conidiogenous cells Conidia References Freshwater | Dead wood Simple or mostly Monotretic, Cylindrical, 2-3 septate, Zhang et al. apically branched, branches, pale verruculose, brown, 42-56 (2023) cylindrical, septate, brown to brown, x 15-22 um. brown, 102-248 x 18-27 x 6.5-9 um. 5-11 ym. Freshwater | Dead wood | Unbranched, smooth, Monoblastic, Obclavate, rostrate, Bao et al. septate, brown to cylindrical or smooth, 6-17 septate, (2018) dark brown, 90-120 x lageniform, olivaceous brown to 7.5-8.5 ym. smooth, mid to brown, 77.5-108.5 x dark brown. 17.5-20.5 um. Terrestrial | Dead wood Unbranched, Monoblastic, Obclavate, 2-7 septate, Jin et al. cylindrical, 4-8 cylindrical, pale smooth, brown, 34-54 x (2024) septate, smooth, brown, 10-22 x 8-14 um. brown or dark brown, 6-9 um. 82-194 x 5-10 um. Freshwater | Dead wood Unbranched, Monoblastic, Obclavate, 4-6 septate, Su et al. cylindrical, smooth, cylindrical, pale smooth, brown to pale (2016) multi-septate, blackish brown. brown, 37.5-51.5 x to brown, 220- 8.5-9.5 um. 280 x 6-7 um. Freshwater | Dead wood Unbranched, Monoblastic, Obclavate, rostrate, This study cylindrical, multi- cylindrical, mid septate, pale brown to septate, brown to to dark brown, brown, 20-45 x 6-10 um dark brown, 74-122 x 9-23 x 4-8 um. wide. 5-9 um. Terrestrial Hevea Septate, brown to Cylindrical or Obclavate, rostrate, Xu et al. brasiliensis | dark brown, 35-150 x lageniform, some have guttulate, 3-8 (2023) 5-15 um. smooth, brown to septate, yellow-brown dark brown, 10- to brown, with sheaths, 50 x 5-10 um. 30-150 x 5-20 um. Terrestrial | Dead wood Unbranched, Monoblastic, Obclavate, rostrate, This study cylindrical, multi- cylindrical, midto | septate, olivaceous brown septate, dark brown, dark brown, 13-__| to soot brown, with sheath, 170-280 x 8-13 um. 17.5 x 5.5-9 um. 35.5-67.5 x 11-20 um. Results Phylogenetic analysis The partial ITS, LSU and SSU nucleotide sequences were used to determine the phylogenetic positions of the new taxa, and the datasets consisted of 77 isolates representing 47 Kirschsteiniothelia species. Pseudorobillarda eu- calypti (MFLUCC 12-0422) and P. phragmitis (CBS 398.61) were selected as the outgroup taxa. The concatenated sequence matrix includes ITS (1-506 bp), LSU (507-1,360 bp) and SSU (1,361-2,378 bp). The final ML optimiza- tion likelihood value of the best RAxML tree (Fig. 1) was -19027.817769, and the estimated base frequencies were as follows: A = 0.228043, C = 0.257324, G = 0.302209, T = 0.212423; substitution rates AC = 1.135651, AG = 2.447765, AT = 0.902710, CG = 1.148377, CT = 5.581689, GT = 1.000000. Gamma distribu- tion shape parameter alpha is 0.279545. The concatenated ITS, LSU and SSU datasets were analyzed using ML and BI methods with similar tree topologies. The resulting multi-gene phylogenetic tree confirmed that our newly obtained strains Kirschsteiniothelia guizhouensis (GZCC 24-0034 and GZCC 24-0041), K. xishuiensis (GZCC 24-0052), and K. weiningensis (GZCC 24-0072) formed distinct clades within Kirschsteiniothelia (Fig. 1). Two isolates of K. guizhouen- sis (GZCC 24-0041 and GZCC 24-0034) clustered together and were sister to K. acutisporum (MFLU 21-0127) (100% MLB/1.00 BYPP). Kirschsteiniothelia xishuiensis (GZCC 24-0052) formed a distinct clade basal to K. submersa (S- 481, S-601, and MFLUCC 15-0427), K. sichuanensis (UESTCC 24.0127) and K. extensum (MFLU 21-0130) (100% MLB/1.00 BYPP). While K. weiningensis MycoKeys 113: 147-168 (2025), DOI: 10.3897/mycokeys.113.139427 151 Xing-Juan Xiao et al.: Three new asexual Kirschsteiniothelia species ane Kirschsteiniothelia guangdongensis ZHKUCC 22-0233 T Kirschsteiniothelia tectonae MFLUCC 23-0272 sx/.| Kirschsteiniothelia tectonae MFLUCC 23-0271 Kirschsteiniothelia tectonae MFLUCC 13-0470 Kirschsteiniothelia tectonae MFLUCC 12-0050" Kirschsteiniothelia spatiosum MFLU 21-0128 caine Kirschsteiniothelia agumbensis NFCCI 5714 of rita rostrata MFLU 15-1154" a Kirschsteiniothelia rostrata MFLUCC 15-0619T Kirschsteiniothelia rostrata MFLUCC 16-1124 Kirschsteiniothelia crustaceum MFLU 21-0129T Kirschsteiniothelia thujina JF13210 — Kirschsteiniothelia laojunensis KUN L 88727 =e Kirschsteiniothelia sp. UTHSCSA DI22-44 1 Kirschsteiniothelia sp. UTHSCSA DI22-45 Kirschsteiniothelia esperanzae T. Raymundo 6581" Kirschsteiniothelia sp. 7020611638 Kirschsteiniothelia dujuanhuensis KUNCC 22-12671 Kirschsteiniothelia guizhouensis GZCC 24-0041 Kirschsteiniothelia euizhonensis GECC 24-0034" Kirschsteiniothelia acutisporum MFLU 21-0127" Kirschsteiniothelia puerensis ZAKUCC 22-0271" Kirschsteiniothelia puerensis ZHKUCC 22-0272 Kirschsteiniothelia chiangmaiensis MFLU 23-0358 * 960,99 Airschsteiniothelia thailandica MELUCC 20-0116 T wo|' Kirschsteiniothelia thailandica MFLU 20-0263 Kirschsteiniothelia thailandica MFLUCC 22-0020 . Kirschsteiniothelia xishuangbannaensis MFLUCC 23-0273 Kirschsteiniothelia xishuangbannaensis MFLUCC 23-0274 90/1 80/1 Kirschsteiniothelia xishuangbannaensis ZH KUCC 22-0221 Kirschsteiniothelia nabanheensis HJIAUP C2004 Kirschsteiniothelia nabanheensis HJAUP C2006 Kirschsteiniothelia septemseptatum MFLU 21-0126 T Kirschsteiniothelia inthanonensis MFLUCC 23-0277" Kirschsteiniothelia submersa § 481 Kirschsteiniothelia submersa MFLUCC 15-0427? Kirschsteiniothelia sichuanensis UESTCC 24-0127 Kirschsteiniothelia extensum MFLU 21-0130 T Kirschsteiniothelia xishuiensis GZCC 24-0052 ! Kirschsteiniothelia attra DEN n-+ Kirschsteiniothelia atra § 783 Kirschsteiniothelia atra MFLUCC 16-1104 DIJAYJOINIAISYISALY Kirschsteiniothelia emarceis MFLU 10-0037 T Kirschsteiniothelia sp. KUNCC 10431 Kirschsteiniothelia atra MFLUCC 15 0424 Kirschsteiniothelia atra CBS 109. 53 Kirschsteiniothelia lignicola MFLUCC 10-0036 - Kirschsteiniothelia lignicola MFLUCC 10-0105 Kirschsteiniothelia ramus GLCC 23-0596 * Kirschsteiniothelia saprophytica MFLUCC 23-0276 Kirschsteiniothelia saprophytica MFLUCC 23-0275 B Kirschsteiniothelia zizyphifolii MFLUCC 23-0270 * oo. Kirschsteiniothelia ebriosa CBS H 233791 Kirschsteiniothelia ebriosa CBS 1438421 Kirschsteiniothelia vinigena CBS H 23378" 1o.| Kirschsteiniothelia sp. CSN604 Kirschsteiniothelia sp. CSN602 toon! Airschsteiniothelia arasbaranica IRAN 2509C Kirschsteiniothelia arasbaranica IRAN 2508C" Kirschsteiniothelia sp. KUNCC 23-13755 Kirschsteiniothelia pini UESTCC 24-0131 Kirschsteiniothelia sp. E38 Kirschsteiniothelia aquatica MFLUCC 16-1685? wy Kirschsteiniothelia sp. KUNCC 23-13756 Kirschsteiniothelia sp. KUNCC 23-14559 — Kirschsteiniothelia cangshanensis MFLUCC 16-1350 T Kirschsteiniothelia weiningensis GZ.CC 24-0072 Y Kirschsteiniothelia phoenicis MFLUCC 18-0216 Kirschsteiniothelia phoenicis MFLU 18-0153 Kirschsteiniothelia fluminicola MELUCC 16-1263 © Kirschsteiniothelia dushanensis GZCC 19-0415 Pseudorobillarda phragmitis CBS 398. 61 Pseudorobillarda eucalypti MFLUCC 12-0422 Out group 94/1 82/- L oa Figure 1. Maximum Likelihood (ML) majority rule consensus tree for the ITS LSU and SSU sequence data alignment of Kirschsteiniothelia and related taxa. Pseudorobillarda eucalypti (MFLUCC 12-0422) and P phragmitis (CBS 398.61) are the outgroup taxa. ML bootstrap support values (MLB = 75%) and Bayesian posterior probabilities (BYPP = 0.95) are indicated below or above the nodes. Ex-type strains are in bold and marked with T, and the new species are in red. MycoKeys 113: 147-168 (2025), DOI: 10.3897/mycokeys.113.139427 152 Xing-Juan Xiao et al.: Three new asexual Kirschsteiniothelia species (GZCC 24-0072) grouped with K. cangshanensis (MFLUCC 16-1350) and Kirschsteiniothelia sp. (KUNCC 23-13756 and KUNCC 23-14559), but in a dis- tinct lineage (96% MLB/0.98 BYPP). Assemble species by Automatic Partitioning (ASAP) results Three single-locus (ITS, LSU, SSU) datasets, comprising 41 strains were used for analysis. The ASAP analysis of ITS region identified 30 distinct groups with- in Kirschsteiniothelia. Similarly, the LSU region was classified into 27 groups, while the SSU region was divided into 22 groups. In the ASAP analysis, Kirschsteiniothelia longisporum (UESTCC 24.0190), K. pini (UESTCC 24-0131), Kirschsteiniothelia sp. (KUNCC 23-13756 and KUNCC 23-13755), and K. weiningensis (GZCC 24-0072) grouped togeth- er based on the SSU dataset. However, they were divided into five distinct groups in the ITS dataset and three groups in the LSU dataset. Kirschsteinio- thelia thailandica (MFLUCC 20-0116, MFLUCC 22-0020, and MFLU 20-0263) and K. xishuangbannaensis (ZHKUCC 22-0221, ZHKUCC 22-0220, MFLUCC 23-0273, and MFLUCC 23-0274) were treated as a single group based on the SSU dataset. However, in the ITS, LSU, and combined datasets, they were divided into two distinct groups. Kirschsteiniothelia xishuiensis (GZCC 24- 0052) and K. submersa (MFLUCC 15-0427) grouped together in the SSU dataset but were identified as separate species in the ITS and LSU datasets. Kirschsteiniothelia guizhouensis (GZCC 24-0034) consistently appeared as a single group across all single-marker analyses. Therefore, the ITS is currently considered the most reliable marker for iden- tifying Kirschsteiniothelia taxa at the species level, following the principle that “the smaller the ASAP score, the better” (Puillandre et al. 2021). Taxonomy Kirschsteiniothelia guizhouensis X.J. Xiao, Y.Z. Lu & K.D. Hyde, sp. nov. Index Fungorum: IF903149 Facesoffungi Number: FoF 16776 Fig: 3 Etymology. Referring to the collecting location at Guizhou Province in China. Holotype. HKAS 139503. Description. Saprobic on submerged decaying wood in a freshwater habitat. Sexual morph: Undetermined. Asexual morph: Colonies on natural substrate ef- fuse, dark brown to black with white tip, hairy. Mycelium immersed, composed of brown to dark brown, branched, septate, smooth hyphae. Conidiophores 195-477 x 11-16 um (X = 307 x 13.5 um, n = 20), macronematous, mononem- atous, erect, straight to slightly curved, apically branched, cylindrical, tapering towards the apex, dark brown, multi-septate, thick-walled. Conidiogenous cells 5.5-16 x 4.5-8 um (X = 11 x 6 um, n = 30), monoblastic, integrated and dis- crete, terminal at the apex of the stem and branches, subcylindrical, light to dark brown. Conidia 36.5—65 x 8-16.5 um (x = 50 x 12.5 um, n = 20), acroge- nous, solitary, dry, olivaceous brown to brown, pale brown to hyaline at the apex, obclavate, sometimes apical cell swollen to subglobose, rostrate, straight or MycoKeys 113: 147-168 (2025), DOI: 10.3897/mycokeys.113.139427 153 Xing-Juan Xiao et al.: Three new asexual Kirschsteiniothelia species Rank Nb subsets/asap score _e oO @ o (eo) [51/15 0) (2012.0) [26/10.0) (2577.5) [2216.5] (2172.5) [20/1.5) [1975.0] [17/10.0) (12/11.5)] hm a Ce ee a Kirschsteiniothelia acutisportim MFLU 21-0127T Kirschsteiniothelia aquatica MFLUCC 16-L685T I <0.001 <0.05 <0.1 >0.1 NIA ITS Kirschsteiniothelia sp. KUNCC 23-13756 Kirschsteiniothelia weiningensis GCC 24-0072 Kirschsteiniothelia sp. KUNCC 23-13755 Kirschsteiniothelia pini UESTCC 24-0131 Kirschsteiniothelia arasbaranica TRAN 2509C Kirschsteiniothelia arasharanica IRAN 2508CT Kirschsteiniothelia atra MFLUCC 16-1104 Kirschsteiniothelia atra 3 783 Kirschsteiniothelia atra MFLUCC 15-0424 Kirschsteiniothelia sp. KUNCC 10431 Kirschsteiniothelia lignicola MFLUCC 10-0036T Sequences Number: 40 taxa — ASAP Group: 31 Kirschsteiniothelia lignicola MFLUCC 10-0105 Kirschsteiniathelia zizyphifolii MFLUCC 23-0270T Kirschsteiniathelia rostrata MFLUCC 15-0619T Kirschsteiniathelia rostrata MFLU 15-1154T. Kirschsteiniathelia submersa MFLUCC 15-0427T Kirschsteiniothelia xishuiensis GZCC 24-0052 I Kirschsteiniothelia xishuangbannaensis ZHKUCC 22-0221 Kirschsteiniothelia xishuanghannaensis ZHKUCC 22-0220T Kirschsteiniothelia xishuangbannaensis MFLUCC 23-0274 Kirschsteiniothelia xishuangbannaensis MFLUCC 23-0273 Kirschsteiniothelia inthanonensis MFLUCC 23-0277T Kirschsteiniothelia nabanheensis HJAUP C2006 Kirschsteiniothelia septemseptatum MFLU 21-0126T Kirschsteiniothelia nabanheensis HJAUP C2004T Kirschsteiniothelia thujina IF 13210 Kirschsteiniothelia dujuanhuensis KUNCC 22-12671 Kirschsteiniothelia tectonae MFLUCC 23-0271 Kirschsteiniothelia tectonae MFLUCC 23-0272 Kirschsteiniothelia thailandica MFLUCC 20-0116T. Kirschsteiniothelia thailandica MFLU 20-0263 Kirschsteiniathelia thailandica MFLUCC 22-0020 Kirschsteiniothelia chiangmaiensis MFLU 23-0358T Kirschsteiniothelia puerensis ZHKUCC 22-0272 Kirschsteiniothelia puerensis ZHKUCC 22-0271T Kirschsteiniathelia guizhouensis GZCC 24-0034 Kirschsteiniothelia dushanensis GZCC 19-0415 Kirschsteiniothelia phoenicis MFLUCC 18-0216T —> hy ss —=_ + =a | % C Rank Nb subsets/asap score [31/10 5] (2516.5) [2/9.8] [2077.0] [18/75] [188.5] [179.5] [18"9.0} [18/8.0) [1179.5] ii om o B o] (i m 6 4) fa Kirschsteiniothelia acutisporum MFLU 21-0127T e eo oe o 12] Legend: <0.001 <0.05 <0.1 20.1 NIA Kirschsteiniothelia chiangmaiensis MFLU 23-0358T Kirschsteiniothelia thailandica MFLUCC 20-0116T Kirschsteiniathelia thailandica MFLU 20-0263 Kirschsteiniathelia thailandica MFLUCC 22-0020 LSU Sequences Number: 40 taxa ASAP Group: 31 Kirschsteiniothelia thujina JF 13210 Kirschsteiniothelia dujuanhuensis KUNCC 22-12671 Kirschsteiniathelia submersa MFLUCC 15-0427T Kirschsteiniathelia xishuiensis GZCC 24-0052 Kirschsteiniothelia guizhouensis GZCC 24-0034 Kirschsteiniothelia aquatica MFLUCC 16-1685T Kirschsteiniothelia sp. KUNCC 23-13756 Kirschsteiniothelia sp. KUNCC 23-13755 Kirschsteiniothelia pini UESTCC 24-0131 Kirschsteiniothelia weiningensis GZCC 24-0072 Kirschsteiniothelia dushanensis GZCC 19-0415 Kirschsteiniothelia phoenicis MFLUCC 18-0216T Kirschsteiniothelia arasbaranica IRAN 2509C Kirschsteiniathelia arasharanica IRAN 2508CT Kirschsteiniothelia atra MFLUCC 16-1104 Kirschsteiniothelia atra 8 783 Kirschsteiniothelia sp. KUNCC 10431 Kirschsteiniothelia atra MFLUCC 15-0424 Kirschsteiniathelia lignicola MFLUCC 10-0036T Kirschsteiniothelia lignicola MFLUCC 10-0105 0) c Kirschsteiniothelia xishuangbannaensis ZHKUCC 22-0221 Kirschsteiniothelia xishuangbannaensis ZHKUCC 22-0220T Kirschsteiniathelia xishuangbannaensis MFLUCC 23-0274 Kirschsteiniothelia xishuanghannaensis MFLUCC 23-0273 Kirschsteiniothelia puerensis ZHKUCC 22-0272 Kirschsteiniothelia puerensis ZHKUCC 22-0271T Kirschsteiniathelia inthanonensis MFLUCC 23-0277T Kirschsteiniothelia septemsepratum MFLU 21-0126T Kirschsteiniothelia nabanheensis HJAUP C2006 Kirschsteiniothelia nabanheensis HJAUP C2004T Kirschsteiniothelia rostrata MFLUCC 15-0619T E | Kirschsteiniathelia rostrata MFLU 15-1154T Kirsehsteiniathelia tectonae MFLUCC 23-0271 Kirschsteiniothelia tectonae MFLUCC 23-0272 Kirschsteiniothelia zizyphifolii MFLUCC 23-0270T P| ———> Rank Nb subsets/asap score Legend: (26/8.0) [258.5] [22/6.5) [20/7.5] [196.5] [18/25] [1773.5] [1576.0] [14/3.5] [2/70] @ 10) 4 ul & ty) cit it Liat 64 Kirschsteiniothelia acutisporum MFLU 21-0127T Kirschsteiniathelia aquatica MFLUCC 16-1685T Kirschsteiniothelia sp. KUNCC 23-13756. Kirschsteiniothelia sp. KUNCC 23-13755 Kirschsteiniothelia pini UESTCC 24-0131 Kirschsteiniothelia weiningensis GZCC 24-0072 Kirschsteiniothelia dushanensis GZCC 19-0415 Kirschsteiniothelia phoenicis MFLUCC 18-0216T Kirschsteiniothelia atra MFLUCC 16-1104 Kirschsteiniothelia atra MFLUCC 15-0424 Kirschsteiniothelia atra $ 783 Kirschsteiniothelia zizyphifolii MFLUCC 23-0270T Kirschsteiniothelia lignicola MFLUCC 10-0036T Kirschsteiniothelia lignicola MFLUCC 10-0105 Kirschsteiniothelia submersa MFLUCC 15-0427T e e e Oo ° <0.001 <0.05 <01 >01 NIA SSU Kirschsteiniothelia xishuiensis GZCC 24-0052 Kirsehsteiniothelia inthanonensis MFLUCC 23-0277T Kirschsteiniothelia septemseptatum MFLU 21-0126T Kirschsteiniathelia rostrata MFLUCC 15-0619T Sequences Number: 40 taxa SAP Group: 26 Kirschsteiniothelia rostrata MFLU 15-1154T Kirschsteiniothelia tectonae MFLUCC 23-0271 Kirschsteiniathelia tectonae MFLUCC 23-0272 Kirschsteiniothelia chiangmaiensis MFLU 23-0358T Kirschsteiniathelia thailandica MFLUCC 20-0116T Kirschsteiniothelia thailandica MFLUCC 22-0020 Kirschsteiniathelia thailandica MFLU 20-0263 Kirschsteiniothelia xishuangbannaensis ZHKUCC 22-0221 Kirschsteiniathelia xishuangbannaensis ZHKUCC 22-0220T Kirschsteiniothelia xishuangbannaensis MFLUCC 23-0273 Kirschsteiniothelia xishuangbannaensis MFLUCC 23-0274 Kirschsteiniothelia puerensis ZHKUCC 22-0272 Kirschsteiniothelia puerensis ZHKUCC 22-0271T Kirschsteiniothelia dujuanhuensis KUNCC 22-12671 Kirschsteiniothelia nabanheensis HJAUP C2006 Kirschsteiniothelia nabanheensis HJAUP C2004T Kirschsteiniothelia arasbaranica [RAN 2509C Kirschsteiniathelia arasharanica [RAN 2508CT Kirschsteiniothelia thujina IF13210 Kirschsteiniothelia guizhouensis GECC 24-0034 Kirschsteiniathelia sp. KUNCC 10431 > ° [a P| © > Rank Nb subsets/asap score [262.5] [258.0] [2315.5] [22/12.0] (2012.5) [19/100] (1879.0) [17/9.5] [169.5] [14/11.5] B wa mt ce en. I Bl Legend: e e e ° <0.001 <0.05