683 MycoKeys MycoKeys 106: 303-325 (2024) DOI: 10.3897/mycokeys.106.127355 Research Article Phylogenomics, taxonomy and morphological characters of the Microdochiaceae (Xylariales, Sordariomycetes) Zhao-Xue Zhang™, Yu-Xin Shang', Meng-Yuan Zhang|, Jin-Jia Zhang', Yun Geng?, Ji-Wen Xia'™®, Xiu-Guo Zhang’ 1 Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests, College of Plant Protection, Shandong Agricultural University, Taian, 271018, China 2 Institute of Crop Germplasm Resources, Shandong Academy of Agricultural Sciences, Jinan, 250100, China Corresponding author: Xiu-Guo Zhang (sdau613@163.com) 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 OPEN Qaceess Academic editor: Danushka Sandaruwan Tennakoon Received: 12 May 2024 Accepted: 20 June 2024 Published: 3 July 2024 Citation: Zhang Z-X, Shang Y-X, Zhang M-Y, Zhang J-J, Geng Y, Xia J-W, Zhang X-G (2024) Phylogenomics, taxonomy and morphological characters of the Microdochiaceae (Xylariales, Sordariomycetes). Mycokeys 106: 303-325. https://doi. org/10.3897/mycokeys.106.127355 Copyright: © Zhao-Xue Zhang 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 Species of the family Microdochiaceae (Xylariales, Sordariomycetes) have been reported from worldwide, and collected from different plant hosts. The proposed new genus and two new species, viz., Macroidriella gen. nov., M. bambusae sp. nov. and Microdochium australe sp. nov., are based on multi-locus phylogenies from a combined dataset of ITS rDNA, LSU, RPB2 and TUB2 with morphological characteristics. Microdochium sinense has been collected from diseased leaves of Phragmites australis and this is the first report of the fungus on this host plant. Simultaneously, we annotated 10,372 to 11,863 genes, identified 4,909 single-copy orthologous genes, and conducted phylogenomic analysis based on genomic data. A gene family analysis was performed and it will expand the understanding of the evolutionary history and biodiversity of the Microdochiaceae. The detailed descriptions and illustrations of species are provided. Key words: Microdochiaceae, multigene phylogeny, new taxa, phylogenomics, taxonomy Introduction Microdochium Syd. & P. Syd., is the type genus of the family Microdochiaceae Hern.-Restr., Crous & J.Z. Groenew. This was first described by Syd. & P. Syd. (Sydow 1924). The holotype collection of the type species of Microdochium, M. phragmitis Syd. & P. Syd. was obtained in Germany from the leaves of Phragmites australis (Sydow 1924). Microdochium species were collected as endophytes, plant pathogens, and saprophytes, and were frequently isolated from different plant hosts (Von Arx 1987; Glynn et al. 2005; Jewell and Hsiang 2013; Mandyam et al. 2013; Hiruma et al. 2018; Liang et al. 2019; Lu et al. 2023; Zhang et al. 2023a). Prior research has indicated that the classification of Microdochium within the Amphisphaeriaceae is supported by its morphological similarities (Parkinson et al. 1981; Samuels and Hallett 1983; Von Arx 1984; Jaklitsch and Voglmayr 2012). Hernandez-Restrepo et al. (2016) was proposed that /driella and Microdochium may be closely related genera. Their phylogenetic analysis revealed that /driella, Microdochium, and Selenodriella formed a distinct 303 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae monophyletic group within the Xylariales. Therefore, Hernandez-Restrepo et al. (2016) established the new family Microdochiaceae to encompass this clade. Currently, there are approximately 68 species of Microdochium listed in the Index Fungorum (2024), with 45 species being accepted. Microdochium has a diverse range of hosts that are widely distributed worldwide (Zhang et al. 2017; Crous et al. 2018, 2019, 2021; Marin-Felix et al. 2019; Huang et al. 2020). However, only a few species of Microdochium have the capability to cause diseases, primarily impact- ing grasses and cereals. Zhang et al. (2015) identified Microdochium paspali Syd. & P. Syd., which was responsible for causing leaf blight on Paspalum vaginatum Sw. Liang et al. (2019) identified Mi. poae J.M. Liang & Lei Cai, which induced leaf blight disease in turf grasses like Poa pratensis and Agrostis stolonifera L. Stewart et al. (2019) identified Mi. sorghi U. Braun, which was responsible for the develop- ment of zonate leaf spots and decay on sorghum species. Mi. albescens (Thiim.) Hern.-Restr. & Crous was the causative agent of leaf scald and grain discoloration in rice, leading to a global decrease in rice yield (Dirchwolf et al. 2023). Mi. bolleyi (R. Sprague) de Hoog & Herm.-Nijh. was cited as the cause of root necrosis and basal rot in creeping bent grass (Hong et al. 2008). In addition to this, some species of Microdochium occur as endophytes or saprophytes. Liu et al. (2022) identified three species, Microdochium miscanthi S.B. Liu, X.Y. Liu, Z. Meng & X.G. Zhang, Mi. sinense S.B. Liu, X.Y. Liu, Z. Meng & X.G. Zhang, and Mi. hainanense S.B. Liu, X.Y. Liu, Z. Meng & X.G. Zhang, isolated from Miscanthus sinensis Anderss. and Phrag- mites australis (Cav.) Trin. ex Steud in Hainan, China. Zhang et al. (2023a) collected novel species (Mi. bambusae J. Zhang, Z.X. Zhang, & Z. Li, Mi. nannuoshanense J. Zhang, Z.X. Zhang, & Z. Li, and Mi. phyllosaprophyticum J. Zhang, Z.X. Zhang, & Z. Li) from leaves of Bambusaceae plant as a saprobe. With the advent of the sequencing era, genomics is increasingly being utilized for phylogenetic studies and can offer additional insights into pathogenic mecha- nisms (Manamgoda et al. 2011; Schoch et al. 2012; Jeewon et al. 2013; David et al. 2016; Mesny et al. 2021; Tsers et al. 2023). However, at present, only the genome information of three species of this taxon (Microdochium) can be retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 30 April 2024). In this study, we explored the species diversity of Microdochium and described one new species and one new host record based on the molecular phylogenet- ic analyses and morphological observations. In addition, we conducted genome and transcriptome sequencing of the new species, aiming to conduct phyloge- netic analysis, and gene structure annotation at the genomic level. By comparing and analyzing the obtained data with existing species genome information, we aim to reveal the genetic relationship and functional differences between the new species and other species. This will gain a more comprehensive understanding of the biological characteristics and evolutionary history of the new taxa. Materials and methods Morphological study During a series of field visits in 2023 in Hainan Province, China, plant spec- imens with necrotic spots were collected. Even though specimens harbor multiple fungi, we managed to obtain pure colonies through the single spore MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 304 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae isolation (Senanayake et al. 2020) and tissue isolation techniques (Zhang et al. 2023a). We retrieved small fragments (5 x 5 mm) from the damaged leaf edges, treated them by immersion in a 75% ethanol solution for 60 s, followed by rinsing in sterile distilled water for 45 s and a 10% sodium hypochlorite solution for 45 s. Subsequently, specimens were rinsed three times in ster- ile deionized water for 30 s. The processed fragments were then placed on Sterile filter paper to remove excess moisture before being transferred onto PDA for incubation at 24 °C for 3 days. The hyphal tips from growing colonies were transferred to fresh PDA plates. Images were captured using a Sony Al- pha 6400L digital camera (Sony Group Corporation, Tokyo, Japan) on days 7 and 14. Microscopic examination of the fungal structures was conducted us- ing an Olympus SZ61 stereo microscope and an Olympus BX43 microscope (Olympus Corporation, Tokyo, Japan), along with BioHD-A20c color digital camera (FluoCa Scientific, China, Shanghai) for recording. All fungal strains were preserved in 15% sterilized glycerol at 4 °C, with each strain stored in three 2.0 mL tubes for future studies. Structural measurements were carried out using Digimizer software (v5.6.0), with a minimum of 25 measurements taken for each characteristic such as conidiophores, conidiogenous cells, and conidia. Specimens were deposited in the HSAUP (Herbarium of Plant Pathol- ogy, Shandong Agricultural University) and HMAS (Herbarium Mycologicum Academiae Sinicae), while living cultures were stored in the SAUCC (Shan- dong Agricultural University Culture Collection) for preservation and further research purposes. Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org). DNA extraction, amplification and sequencing Fungal DNA was extracted from fresh mycelia grown on PDA using either the CTAB method or a kit method (OGPLF-400, GeneOnBio Corporation, Changchun, China) (Guo et al. 2000; Zhang et al. 2023a). Four gene regions, LSU, ITS, RPB2, and TUB2 were amplified using the primer pairs listed in Suppl. material 1 (Vilgalys et al. 1990; White et al. 1990; Liu et al. 1999; Sung et al. 2007; Jewell et al. 2013). The amplification reaction was con- ducted in a 25 uL reaction volume, consisting of 12.5 uL 2 x Hieff Canace® Plus PCR Master Mix (Shanghai, China) (with dye) (Yeasen Biotechnology, Cat No. 10154ES03), 0.5 uL each of forward and reverse primer, and 0.5 uL template genomic DNA, with the volume adjusted to 25 uL using distilled de- ionized water. PCR products were separated and purified using 1% agarose gel and GelRed (TsingKe, Qingdao, China), and UV light was used to visual- ize the fragments. Gel extraction was performed using a Gel Extraction Kit (Cat: AE0101-C) (Shandong Sparkjade Biotechnology Co., Ltd., Jinan, Chi- na). The purified PCR products were subjected to bidirectional sequencing by Biosune Company Limited (Shanghai, China). The raw data (trace data) were analyzed using MEGA v. 7.0 to obtain consistent sequences (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank under the accession numbers provided in Table 1. The abbreviations of the genera names used in our study are as follows: /. = Idriela; S. = Selenodriella; Ma. = Macroidriella; Mi. = Microdochium. MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 305 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae Table 1. GenBank accession number of the taxa used in phylogenetic reconstruction. Species Cryptostroma corticale Idriela lunata |. chlamydospora |. multiformispora Macroidriella bambusae Microdochium albescens Mi. australe Mi. bambusae Mi. bolleyi = i. chrysanthemoides = i. chrysopogonis = ij. chuxiongense = i, citrinidiscum = i, colombiense = i, dawsoniorum = i. fisheri S i, graminearum S i, hainanense S i, indocalami S i, insulare S ij. lycopodinum S i, maculosum S i, Majus S i, miscanthi = i; Musae = i; nannuoshanense Strain no. CBS 218.52 CBS 204.56* CBS 177.57 CGMCC 3.20778* GZUIFR 21.922 CGMCC 3.20779* GZUIFR 21.924 GZUIFR 21.925 SAUCC 6792-1* SAUCC 6792-2 SAUCC 6792-5 SAUCC 6113-1 SAUCC 6113-3 CBS 243.83 CBS 291.79 SAUCC 6322-5-1* SAUCC 6151-1 SAUCC 1862-1* SAUCC 1866-1 CBS 540.92 CPC 25994 CGMCC 3.17929* GDMCC 3.683 LNU-196 YFCC 8794* CBS 109067* CBS 624.94* BRIP 65649* CBS 242.90* CGMCC 3.23525* CGMCC 3.23524 SAUCC 210782 SAUCC 210781* SAUCC 1016* BRIP 75114a CBS 146.68 CBS 122885* COAD 3358* CBS 741.79 SAUCC 211092* SAUCC 211093 CBS 143499 CBS 143500* SAUCC 2450-1* SAUCC 2450-3 ITS HG934112 KP859044 KP859043 OL897016 OL897017 0L897018 OL897019 OL897020 PP716851 PP716852 PP716853 PP716854 PP716855 KP858994 KP858996 PP695312 PP695313 OR/02567 OR/02568 KP859010 KP859018 KU746690 MT988022 MT988020 OK586161 KP859003 KP858999 MK966337 KP859015 OP103966 OP103965 OM956296 OM956295 MT199884 0Q917075 KP858993 KP859016 0k966954 KP859001 OM956214 OM956215 MH107894 MH107895 OR702569 OR/02570 LSU MH868531 KP858981 KP858980 OL897058 OL897059 OL897060 OL897061 OL897062 PP716512 PP716513 PP716514 PP716515 PP716516 KP858930 KP858932 PP702043 PP702044 OR702576 OR/02577 KP858946 KP858954 KU746736 MT988024 MT988023 OK586160 KP858939 KP858935 KP858951 OP104016 OP104015 0M959324 0M959323 MT199878 0Q892168 KP858929 KP858952 0k966953 KP858937 OM957532 0M957533 MH107941 MH107942 OR/02578 OR7/02579 MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 GenBank accession number RPB2 HG934118 ON568988 ON568989 ON568990 PP729053 PP729054 PP729055 PP729056 PP729057 KP859103 KP859105 PP716780 PP716779 OR7/15785 OR/15786 KP859119 KP859127 MW002442 MW002445 OK584019 KP859112 KP859108 KP859124 OP236027 OP236026 OM981154 0M981153 MT510550 0Q889560 KP859102 KP859125 OL310501 KP859110 OM981148 OM981149 MH108003 OR/15787 OR/15788 TUB2 HG934104 ON569069 ON569070 ON569071 ON569072 | ON569073 PP729058 PP729059 PP729060 PP729061 PP729062 KP859057 KP859059 | PP716787 PP716788 PP445175 PP445176 KP859073 KP859074 KU746781 MW002441 MW002442 OK556901 KP859066 KP859062 KP859078 OM981147 OM981146 MT435653 KP859056 KP859080 KP859064 OM981141 OM981142 PP445177 PP445178 References Vu et al. (2019) Hernandez-Restrepo et al. (2016) Zhang et al. (2023b) This study Hernandez-Restrepo et al. (2016) This study Zhang et al. (2023a) Hernandez-Restrepo et al. (2016) Zhang et al. (2017) Lu et al. (2023) Tang et al. (2022) Hernandez-Restrepo et al. (2016) Crous et al. (2020) Hernandez-Restrepo et al. (2016) Gao et al. (2022) Liu et al. (2022) Huang et al. (2020) Hernandez-Restrepo et al. (2016) Crous et al. (2021) Hernandez-Restrepo et al. (2016) Liu et al. (2022) Crous et al. (2018) Zhang et al. (2023a) 306 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae Species Mi. ne Mi Mi. Mi Mi. Mi Mi oqueenslandicum . nivale . Nivale var. majus . Nivale var. nivales . Novae-zelandiae . paspali phyllosaprophyticum Mi. Mi SSS 8 = SES S Selenodriella cubensis S: . phragmitis . poae i, ratticaudae ij. rhopalostylidis i, salmonicolor i, seminicola i, shilinense i, sinense j. sorghi |, tainanense i. trichocladiopsis Mi . yunnanense fertilis Strain no. CBS 445.95 CBS 108926* CBS 116205* CBS 177.29 CBS 288.50 CPC 29376* CPC 29693 HK-ML-1371 CBS 138620* SAUCC 3583-1* SAUCC 3583-6 CBS 285.71* CBS 423.78 CGMCC 3.19170* LGA. LC 12116 BRIP 68298* CBS 145125* NC14-294 CBS 139951* CPC 26001 DAOM 250161 CGMCC 3.23531* SAUCC 211097* SAUCC 211098 SAUCC 3922-1 SAUCC 3922-3 CBS 691.96 CBS 269.76* CBS 270.76 CBS 623.77* SAUCC 1011* SAUCC 1012 CBS 683.96 CBS 772.83 ITS | KP858997 KP859002 KP859008 MH855031 LT990655 LT990656 KJ569509 KJ569513 OR/02571 OR7/02572 KP859013 KP859012 MH740898 MH740901 MH740902 MW481661 MK442592 MK836110 KP859038 KP859025 KP859034 OP103972 OM956289 OM956290 PP695314 PP695315 KP859000 KP859009 KP858995 KP858998 MT199881 MT199882 KP859053 KP859055 LSU KP858933 KP858938 KP858944 MH866500 MH868135 OR/02580 OR702581 KP858949 KP858948 MW481666 MK442532 MK836108 KP858974 KP858961 KP858970 OP104022 OM959225 OM959226 PP702045 PP702046 KP858936 KP858945 KP858931 KP858934 MT199875 MT199876 KP858990 KP858992 GenBank accession number RPB2 KP859106 KP859111 KP859117 LT990641 LT990642 OR715789 OR715790 KP859122 KP859121 MH740906 MH740909 MH740910 MW626890 MK442667 KP859147 KP859134 KP859143 OM981151 OM981152 PP716781 PP716782 KP859109 KP859118 KP859104 KP859107 MT510547 MT510548 TUB2 KP859060 | KP859065 KP859071 LT990608 LT990609 KJ569514 KJ569518 PP445179 PP445180 KP859077 KP859076 | MH740914 MH740917 MH740918 KP859101 KP859088 KP859097 | OP242834 OM981144 OM981145 PP716789 PP716790 KP859063 KP859072 KP859058 | KP859061 MT435650 MT435651 References Hernandez-Restrepo et al. (2016) Vu et al. (2019) Marin-Felix et al. (2019) Zhang et al. (2015) Zhang et al. (2023a) Hernandez-Restrepo et al. (2016) Liang et al. (2019) Crous et al. (2021) Crous et al. (2019) Das et al. (2020) Hernandez-Restrepo et al. (2016) Gao et al. (2022) Liu et al. (2022) This study Hernandez-Restrepo et al. (2016) Huang et al. (2020) Hernandez-Restrepo et al. (2016) Hernandez-Restrepo et al. (2016) Notes: Ex-type or ex-epitype strains are marked with “*” and the new species described in this study was marked in bold. Library construction, quality control and whole-genome sequencing Library construction and sequencing were carried out by Novogene Co., Ltd. (Beijing, China). Obtain FASTQ format data, which included sequence infor- mation and corresponding sequencing quality information (Cock et al. 2010). Preprocess the raw data that were obtained from the sequencing platform us- ing fastp (https://github.com/OpenGene/fastp) to obtain clean data for subse- quent analysis (Chen et al. 2018). Clean data were deposited in the National Center for Biotechnology Information (NCBI) under BioProject PRJNA1105317. MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 307 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae Genome assembly and annotation Genome data were assembled using the software SPAdes v 3.12.0 (Bankev- ich et al. 2012). Genome annotation mainly included three aspects: a. Masking of repetitive sequences (RepeatMasker version v4.1.4; RepeatModeler v2.0.3, https://www.repeatmasker.org/); b. Annotation of non-coding RNA (RNAmmer v1.2; tRNAscan-SE v2.0); c. Annotation of gene structure (RNA-seq prediction: Trinity v2.14.0, HISAT2 v2.2.1, StringTie v2.2.0; Ab inito prediction: BRAKER2; Homology protein prediction: GEMoMa v1.9) (Grabherr et al. 2011; Pertea et al. 2015; Keilwagen et al. 2016, 2018; Bruna et al. 2021). The final genome and annotation files were integrated using EVM and PASA (Haas et al. 2008, 2011). Phylogeny The generated consensus sequences were subjected to Megablast searches to identify closely related sequences in the NCBI's GenBank nucleotide database (Zhang et al. 2000). Newly generated sequences in this study were aligned with related sequences retrieved from GenBank (Table 1) using MAFFT 7 (Katoh et al. 2019; http://mafft.cbrc.jp/alignment/server/) online service with the default strategy and corrected manually used MEGA 7. For phylogenetic analyses, we operated following the methods by Zhang et al. (2023a), single and concatenated ITS rDNA, LSU, RPB2 and TUB2 sequence alignments were subjected to analysis by maximum likelihood (ML) and Bayesian Inference (BI) algorithms, respectively. ML and BI were run on the CIPRES Science Gateway portal (https://www.phylo. org/, accessed on 30 April 2023) or offline software (ML was operated in Rax- ML-HPC2 on XSEDE v8.2.12, and BI analysis was operated in MrBayes v3.2.7a with 64 threads on Linux). For ML analyses, the default parameters were used and 1,000 rapid bootstrap replicates were run with the GTR+G+I model of nucleotide evolution; BI analysis was performed using a fast bootstrap algorithm with an au- tomatic stop option (Zhang et al. 2023a). The GTR+I+G model was recommended for LSU, RPB2, and TUB2, while SYM+I+G was suggested for ITS. The Markov chain Monte Carlo (MCMC) analysis of the five concatenated genes was con- ducted over 1,130,000 generations, yielding 22,602 trees. Following the discard of the initial 5,650 trees generated during the burn-in phase, the remaining trees were used to compute posterior probabilities in the majority rule consensus trees. For phylogenomic analyses, the genome sequences were submitted to GenBank under the accession numbers in Table 2. The final annotated data were processed to retain the coding protein genes and the longest transcript. Extracted all coding protein genes to identify gene families and single copy orthologous genes using OrthoFinder v2.5.5 (https://github.com/davidemms/OrthoFinder), according to the method by Emms and Kelly (2015, 2019). Multiple sequence alignment was used ParaAT v1.0 (https://ngdc.cncb.ac.cn/tools/paraat) and merged into supergene us- ing seqkit v2.7.0 (https://github.com/shenwei356/segkit) (Zhang etal. 2012; Shenet al. 2016). Phylogenomic analysis was carried out following the methods by Stamat- akis et al. (2014), using RAXML-NG v1.2.1 (https://github.com/amkozlov/raxml-ng) with the LG+G8+F model and 100 bootstrap replications. All resulted trees were plotted using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) or ITOL: In- teractive Tree of Life (https://itol.embl.de/, accessed on 20 October 2023) (Letunic and Bork 2021) and the layout of the trees was edited in Adobe Illustrator CC 2019. MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 308 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae Table 2. BioSample and SRA NCBI number of the taxa used in phylogenomic reconstruction in this study. Species Asterophora parasitica Cryphonectria parasitica Diaporthe eres Macroidriella bambusae Microdochium australe Mi. bambusae Mi. bolleyi Mi. nannuoshanense Mi. nivale Mi. phyllosaprophyticum Mi. trichocladiopsis Pestalotiopsis fici Xylaria flabelliformis Strains BioSample SRA NCBI* References APO1 SAMN09737569 SRS3956156 EP155 SAMNO2744051 SRS6915724 Crouch et al. 2020 CBS 160.32 SAMN21449118 SRS10459569 Hilario et al. 2022 SAUCC 6792-1 SAMN41099213 SRR28834790 This study SAUCC 6322-5-1 SAMN41099214 SRR28834789 This study SAUCC 1862-1 SAMN41099215 SRR28834788 This study J235TASD1 SAMN04386150 SRS1667728 David et al. 2016 SAUCC 2450-1 SAMN41099216 SRR28834787 This study F00608 SAMN26062287 SRS14642463 Tsers et al. 2023 SAUCC 3583-1 SAMN41099217 SRR28834786 This study MPI-CAGE-CH-0230 SAMN06297163 SRS2394902 Mesny et al. 2021 W106-1 SAMN02369365 Wang et al. 2015 G536 SAMN11912834 SRS4852315 Species information described in this study is marked in bold. Results Phylogenetic and phylogenomic analyses A total of 80 isolates representing species within the Microdochiaceae fam- ily used for phylogenetic analysis. One strain of Cryptostroma corticale (CBS 218 52) was used as an outgroup taxon. The final alignment comprised 3,386 concatenated characters, spanning from positions 1 to 553 (ITS), 554 to 1,827 (LSU), 1,828 to 2,676 (RPB2), and 2,677 to 3,386 (TUB2). The maximum likeli- hood (ML) optimization likelihood was calculated to be -23041.844775. The matrix exhibited 1,071 distinct alignment patterns, with 25.57% of characters or gaps remaining undetermined. MrModelTest suggested that Dirichlet base fre- quencies be utilized for the ITS, LSU, RPB2, and TUB2 data partitions. The align- ment exhibited a total of 876 unique site patterns (ITS: 287, LSU: 186, RPB2: 386, TUB2: 213). The topology of the ML tree corroborated that of the tree obtained from Bayesian inference; therefore, only the ML tree is depicted (Fig. 1). Based on the four-gene phylogeny (Fig. 1), the 80 strains were classified into 47 spe- cies. To enhance the visual appeal and conciseness of the phylogenetic tree, 39 strains were collapsed within it (The complete ML phylogenetic tree is avail- able in the Suppl. material 5). Among them, five strains (SAUCC 6792-1, SAUCC 6792-2, SAUCC 6792-5, SAUCC 6113-1 and SAUCC 6113-3) identified a new ge- nus, Macroidriella gen. nov., with solid support (98% MLBV and 1.0 BIPP), and M. bambusae sp. nov. (SAUCC 6792-1) as the type species. Two strains (SAUCC 6322-5-1 and SAUCC 6151-1) identified as Microdochium australe sp. nov. We sequenced the genomes of six species in Microdochiaceae for phyloge- nomic analyses, and downloaded the published genomes of four species from in NCBI Datasets (https://www.ncbi.nlm.nih.gov/datasets/). Xylaria flabelliformis G536 was used as an outgroup taxon. Based on 4,909 clusters of orthologous proteins, the ML tree is depicted (Fig. 2). The phylogenomic tree was divided into two clades (excepted outgroup), viz, clade 1 (Microdochium nannuoshanense SAUCC 2450-1, Mi. phyllosaprophyticum SAUCC 3583-1, Mi. australe SAUCC 6322- 5-1 and Mi. bambusae SAUCC 1862-1) and clade 2 (Mi. nivale F00608, Mi. bolleyi J235TASD1, Mi. trichocladiopsis MPI-CAGE-CH-0230 and Macroidriella bambusae MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 309 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae 0.1). Due to limited genomic data, Macroidriella bambusae (SAUCC 6792-1) was not individually clustered, but the evolutionary distance of Macroidriella bambusae is relatively far compared to other species. Annotations and comparative analysis After structural annotation of the genomic data, we conducted a statistical summary, including, number of genes, total number of cds, total number of ex- ons, total number of introns, total cds length, total exon length and total intron length (Suppl. material 2). Due to the limited genomic data available for Micro- dochiaceae, we will conduct gene family analysis by comparing the self-tested data of the new genus (Macroidriella) with genomic data from the orders of Diaporthales (Cryphonectria parasitica EP155 and Diaporthe eres CBS 160.32), Xylariales (Pestalotiopsis fici W106-1 and Xylaria flabelliformis G536), and the Basidiomycota (Asterophora parasitica APO1). The intersections of gene fam- ily among the six representative strains (s 6) are 3431, the maximum number (508) of gene family intersections between Macroidriella bambusae and Micro- dochium trichocladiopsis, and the minimum number (4) of gene family inter- sections between Macroidriella bambusae and Asterophora parasitica (Fig. 3a). The intersections of gene family among the seven representative strains are 3,291, the unique number of genes in Asterophora parasitica was 513 (maxi- mum), the unique number of genes in Macroidriella bambusae was 42 (mini- mum) (Fig. 3b). We have presented the number of single-copy genes, multi-co- py genes and so on for the seven representative strains (Fig. 3c). Taxonomy Macroidriella Z.X. Zhang, J. W. Xia & X.G. Zhang, gen. nov. MycoBank No: 853699 Type species. Macroidriella bambusae Z. X. Zhang, J. W. Xia & X. G. Zhang. Etymology. Referring to the composed of “Macro-” and “-idriella” (Similar in morphology to /driella and bigger than /driella in conidia). Description. Genus of Microdochiaceae. Endogenic on diseased leaves of Bambusaceae sp. Sporodochia yellowish brown, slimy. Conidiophores are in- distinct and often reduced to conidiogenous cells. Conidiogenous cells are straight or slightly branched, smooth, curved, mono- or polyblastic, terminal, hy- aline, septate, cylindrical and ampulliform. Conidia are solitary, hyaline, lunate, curved, mooned, multi-guttulate, apex rounded, base usually flattened. Sexual morphs were not observed, chlamydospores were not observed. Notes. In the phylogenetic tree (Fig. 1), Macroidriella is allied to /driella, Microdochium and Selenodriella, but forms a separate lineage with good sta- tistical support (98% MLBV and 1.0 BIPP). In morphology, the conidia of Mac- roidriella are predominantly lunate and curved, unlike the elliptical conidia of Microdocium, suggesting a genus of its own, because it is similar to /driella in morphology (but the conidia of Macroidriella are longer than /driella), both being lunate conidia, it is named Macroidriella gen. nov. MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 311 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae Asterophora parasitica Cryphonectria parasitica Diaporthe eres Macroidriella bambusae Microdochium trichocladiopsis Pestalotiopsis fici Xylaria flabelliformis a 3431 aie 2774 L] [| [| fa] L gene family intersection size 7500. 5000 2500 is} gene family set number b C 15000 a Other orthologs || Unique paralogs a Multi-copy orthologs ay Single-copy orthologs Gene number Figure 3. Gene family analysis of Macroidriella a UpSet plot of six strains, showing the intersection counts between different strains in the form of a bar graph b petal plot of seven strains, the center of the petal represents the number of shared genes c bar chart of homologous genes for each strain. Macroidriella bambusae Z.X. Zhang & X.G. Zhang, sp. nov. MycoBank No: 853712 Fig. 4 Type. CHINA, Hainan Province, Danzhou City: Hainan tropical botanical garden, on diseased leaves of Bambusaceae sp., 15 October 2023, Z. X. Zhang (HMAS 352974, holotype), ex-holotype living culture SAUCC 6792-1. Etymology. Referring to the species name of the host plant Bambusaceae sp. Description. Endogenic on diseased leaves of Bambusaceae sp. Mycelia are superficial and immersed, 2—3.5 um wide, branched, membranous and hyaline. MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 312 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae Figure 4. Macroidriella bambusae (HMAS 352974, holotype) aa leaf of Bambusaceae sp. b, c colonies on PDA from above and below after 14 days d colony overview e, f conidiogenous cells and conidia g, h conidia. Scale bars: 10 um (eh). Sporodochia yellowish brown, slimy. Conidiophores are indistinct and often re- duced to conidiogenous cells. Conidiogenous cells are straight or slightly curved, 10.4-15 x 1.7-—2.8 um, mono- or polyblastic, terminal, hyaline, septate, cylindri- cal and smooth. Conidia are solitary, hyaline, lunate, curved, mooned, 16.5- 21.7 x 2-2.8 um, multi-guttulate, apex rounded, base usually flattened. Sexual morphs were not observed, chlamydospores were not observed, see Fig. 4. Culture characteristics. Cultures incubated on PDA at 25 °C in darkness, reaching 63-70 mm diam., had a growth rate of 4.5—5.0 mm/day after 14 days, with moderate aerial mycelia, the center and edges are milky white, with a yel- low-brown color in the middle, and sporodochia are observed. MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 313 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae Additional material studied. CHINA, Hainan Province, Danzhou City, Hainan trop- ical botanical garden, on diseased leaves of Bambusaceae sp., 15 October 2023, Z. X. Zhang (HSAUP 6792-2), living culture SAUCC 6792-2; ibid, (HSAUP 6792-5), living culture SAUCC 6792-5; on dead leaves, 15 October 2023, Z. X. Zhang (HSAUP 6113- 1), living culture SAUCC 6113-1; ibid., (HSAUP 6113-3), living culture SAUCC 6113-3. Notes. Phylogenetic analyses showed that Macroidriella bambusae formed an independent clade (Fig. 1), and closely related to /driella multiformispora (lu- nate, curved-shaped conidia) and Microdochium bolleyi. The Ma. bambusae was distinguished from |. multiformispora (CGMCC 3.20779) by 60/520, 22/1222, 74/848 and 57/710 base-pair differences, from Mi. bolleyi (CBS 540.92) by 40/514, 19/765, 138/850 and 51/710 base pairs in ITS, LSU, RPB2 and TUB2 se- quences, respectively. Morphologically, Ma. bambusae (16.5-21.7 x 2—2.8 pm) longer than /. multiformispora (8.5-13.5 x 1.0-2 um) and Mi. bolleyi (5-8.7 x 1.6-2.3 um) in conidia. Therefore, we describe this fungus as a novel species. Microdochium australe Z.X. Zhang, & X.G. Zhang, sp. nov. MycoBank No: 853695 Fig..5 Type. CHINA, Hainan Province, Jianfengling National Forest Park, on diseased leaves of Phragmites australis, 13 October 2023, Z. X. Zhang (HMAS 352973, holotype), ex-holotype culture SAUCC 6322-5-1. Etymology. Referring to the species name of the host plant Phragmites australis. Description. Endogenic on diseased leaves of Phragmites australis. Mycelia are superficial and immersed, 3-3.3 um wide, branched, membranous and hyaline. Sporodochia black, aggregative or solitary. Conidiophores are indistinct and often reduced to conidiogenous cells. Conidiogenous cells are straight or slightly curved, 15.4-23.5x2.8-—4 um, terminal, hyaline, septate, ampulliform or obpyriform, smooth. Conidia are solitary, hyaline, straight to slight curved, oblong to ellipsoid, 11.3-16.1 x 2.5-3.7 um, multi-guttulate, (2)3-septate, apex rounded, base usually flattened. Sexual morphs were not observed, chlamydospores were not observed, see Fig. 5. Culture characteristics. Cultures incubated on PDA at 25 °C in darkness, reaching 73-76 mm diam., had a growth rate of 5.2—5.4 mm/day after 14 days, with moderate aerial mycelia, milky white to grey-white, with regular margin, and sporodochia are observed, reverses black to brown in the centre, with grey- white and regular margin. Additional material studied. CHINA, Hainan Province, Jianfengling National Forest Park, on saprophytic leaves, 13 October 2023, Z. X. Zhang (HSAUP 6151- 1), living culture SAUCC 6151-1. Notes. Phylogenetic analyses showed that Microdochium australe sp. nov. formed an independent clade closely related to Microdochium bambusae and Mi- crodochium indocalami (Fig. 1). Mi. australe was distinguished from Mi. bambusae (SAUCC 1862-1) by 47/503, 2/836, 56/848 and 17/710 base pair differences, from Mi. bambusae and Mi. indocalami (SAUCC 1016) by 52/503, 2/848, 44/840 and 17/708 base pairs in ITS, LSU, RPB2 and TUB2 sequences, respectively. Morpho- logically, Mi. australe (11.3-16.1 x 2.5-3.7 um, (2)3-septate) differs from Mi. bam- busae (13.0-17 x 2.5-3.5 um, aseptate) and Mi. indocalami in conidia (13-15.5 x 3.5-5.5 um, 3-septate), and, therefore, we described this fungus as a novel species. MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 314 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae Figure 5. Microdochium australe (HMAS 352973, holotype) a a leaf of Phragmites australis b, c colonies on PDA from above and below after 14 days d colony overview e, f conidiogenous cells and conidia g, h conidia. Scale bars: 10 um (eh). Microdochium sinense S.B. Liu, X.Y. Liu, Z. Meng & X.G. Zhang, J. Fungi 2022, 8, 577. Fig. 6 Material examined. CHINA, Hainan Province, Jianfengling National Forest Park, on diseased leaves of Phragmites australis, 12 April 2023, Z. X. Zhang (HSAUP 3922-1), living culture SAUCC 3922-1; ibid., (HSAUP 3922-3), living culture SAUCC 3922-3. Description. Endogenic on diseased leaves of Phragmites australis. Mycelia are superficial and immersed, 2.1-2.9 um wide, branched, membra- nous and hyaline. Conidia are solitary, hyaline, straight, oblong to ellipsoid, MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 315 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae e a a fT Figure 6. Microdochium sinense a diseased symptoms on a leaf of Phragmites australis b, c colonies on PDA from above and below after 14 days d conidiomata on PDA e, f conidia. Scale bars: 10 um (e-f). 12.3-15 x 3.5-5.6 um, multi-guttulate, apex rounded, base usually flattened. Conidiophores were not observed, chlamydospores were not observed, sexual morphs were not observed, see Fig. 6. Culture characteristics. Cultures incubated on PDA at 25 °C in darkness, reach-ing 72-76 mm diam., had a growth rate of 5.1-5.4 mm/day after 14 days, with moder-ate aerial mycelia, milky white to grey-white, with irregular margin, reverses light brown in the centre, with grey-white and regular margin. Notes. Phylogenetic analyses of four combined genes (ITS, LSU, RPB2 and TUB2) showed that SAUCC 3922-1 and SAUCC 3922-3 clustered with the type collection of Microdochium sinense with strong support (Fig. 1). We, therefore, identified the isolated strains (SAUCC 3922-1 and SAUCC 3922-3) as Mi. sin- ense. Morphologically, the conidia of the both (newly isolated and type) were similar (12.3-15 x 3.5-5.6 vs. 11.5-19.34 x 2.8-5.4 um). Discussion The establishment of the family Microdochiaceae by Hernandez-Restrepo et al. (2016) to encompass the clade consisting of /driella, Microdochium, and Selenodriella within the Xylariales highlights the importance of phylogenetic analysis in understanding the evolutionary relationships among fungi. This new classification helps to better organize and categorize fungal species based on their genetic relatedness and morphological characteristics (Hernandez-Re- strepo et al. 2016; Liang et al. 2019; Huang et al. 2020; Liu et al. 2022; Lu et al. MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 316 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae 2023; Zhang et al. 2023a). In the recent study, nine strains isolated from two host plants, Phragmites australis and Bambusaceae sp., were introduced into a new genus, Macroidriella and two new species, Macroidriella bambusae and Microdochium australe. The Global Biodiversity Information Facility (GBIF) cur- rently hosts 1,594 georeferenced records of Microdochiaceae species world- wide (https://www.gbif.org/, accessed on April 30, 2024). The distribution of this family is predominantly in the United States, Europe, and Oceania, with fewer occurrences in Asia. In the recent study of the family, Microdochium emerged as a prominent re- search focus, with 12 species of this genus documented across five Provinces (Guizhou, Hainan, Henan, Shandong, and Yunnan) since the beginning of the 21st century in China (Zhang et al. 2015; Liang et al. 2019; Huang et al. 2020; Gao et al. 2022; Liu et al. 2022; Tang et al. 2022). Microdochium species have been identified on a variety of host families (10 families), with over half of the fungi associated with Poaceae plants. In contrast, /driella and Selenodriella have been less extensively studied, with /driella having only two reported spe- cies since the turn of the 21% century. Through the joint analysis of multiple gene fragments and genomes, the position of new taxa can be better deter- mined, especially through phylogenomic analyses, which was provided with more robust support values. Comparative analysis will help us determine the position of the Macroidriella genus on the evolutionary tree and its relationship with other fungi. By comparing the genomic data of different fungi, we can iden- tify common gene families and infer their evolutionary relationships. Through comparative genomic analysis, it can be observed that Macroidriella has 42 unique single-copy orthologous genes. Asterophora shares only 4 single-copy orthologous genes with Macroidriella, which also indicates that their relation- ship is very distant (belonging to different fungal phyla). This study represents a pioneering effort in Microdochiaceae as it integrates multi-gene fragments with genomic data to unveil the phylogenetic relation- ships within the family. By combining these diverse datasets, a comprehen- sive understanding of the evolutionary history of Microdochiaceae is achieved, shedding new light on its genetic landscape and evolutionary dynamics. Acknowledgements We would like to express our gratitude to Jie Zhang, a master’s student at Shan- dong Agricultural University, for her assistance in this study. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding This research was supported by National Natural Science Foundation of China (nos. 32100016, 32270024, U2002203 and 32370001). MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 317 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae Author contributions Sampling, molecular biology analysis: Zhao-Xue Zhang and Yu-Xin Shang; fungal isola- tion: Yu-Xin Shang and Jin-Jia Zhang; description and phylogenetic analysis: Meng-Yu- an Zhang; microscopy: Yun Geng; writing—original draft preparation: Zhao-Xue Zhang; writing—review and editing, Ji-Wen Xia and Xiu-Guo Zhang. All authors read and approved the final manuscript. Author ORCIDs Zhao-Xue Zhang ® https://orcid.org/0000-0002-4824-9716 Ji-Wen Xia © https://orcid.org/0000-0002-7436-7249 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. 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MycoKeys 98: 167-220. https://doi.org/10.3897/mycokeys.98.102816 MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 393 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae Supplementary material 1 The PCR primers, sequence and cycles used in this study Authors: Zhao-Xue Zhang, Yu-Xin Shang, Meng-Yuan Zhang, Yun Geng, Ji-Wen Xia, Xiu-Guo Zhang Data type: docx 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.106.127355.suppl1 Supplementary material 2 GenBank accession number of the taxa used in phylogenetic reconstruction Authors: Zhao-Xue Zhang, Yu-Xin Shang, Meng-Yuan Zhang, Yun Geng, Ji-Wen Xia, Xiu-Guo Zhang Data type: docx 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.106.127355.suppl2 Supplementary material 3 The sequence of phylogenetic analysis Authors: Zhao-Xue Zhang, Yu-Xin Shang, Meng-Yuan Zhang, Yun Geng, Ji-Wen Xia, Xiu-Guo Zhang Data type: txt 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.106.127355.suppl3 MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 394 Zhao-Xue Zhang et al.: Phylogenomics, taxonomy and morphological characters of the Microdochiaceae Supplementary material 4 The sequence of phylogenomic analysis Authors: Zhao-Xue Zhang, Yu-Xin Shang, Meng-Yuan Zhang, Yun Geng, Ji-Wen Xia, Xiu-Guo Zhang Data type: txt 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.106.127355.suppl4 Supplementary material 5 The complete ML phylogenetic tree Authors: Zhao-Xue Zhang, Yu-Xin Shang, Meng-Yuan Zhang, Yun Geng, Ji-Wen Xia, Xiu-Guo Zhang Data type: pdf 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.106.127355.suppl5 MycoKeys 106: 303-325 (2024), DOI: 10.3897/mycokeys.106.127355 395