Phylogenomics, taxonomy and morphological characters of the Microdochiaceae (Xylariales, Sordariomycetes)

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.

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. 2018Crous et al. , 2019Crous et al. , 2021;;Marin-Felix et al. 2019;Huang et al. 2020).However, only a few species of Microdochium have the capability to cause diseases, primarily impacting 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 development of zonate leaf spots and decay on sorghum species.Mi. albescens (Thüm.)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)  With the advent of the sequencing era, genomics is increasingly being utilized for phylogenetic studies and can offer additional insights into pathogenic mechanisms (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 phylogenetic analyses and morphological observations.In addition, we conducted genome and transcriptome sequencing of the new species, aiming to conduct phylogenetic 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.

Morphological study
During a series of field visits in 2023 in Hainan Province, China, plant specimens with necrotic spots were collected.Even though specimens harbor multiple fungi, we managed to obtain pure colonies through the single spore isolation (Senanayake et al. 2020) and tissue isolation techniques (Zhang et al. 2023a).We retrieved small fragments (5 × 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 sterile 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 Alpha 6400L digital camera (Sony Group Corporation, Tokyo, Japan) on days 7 and 14.Microscopic examination of the fungal structures was conducted using 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 Pathology, Shandong Agricultural University) and HMAS (Herbarium Mycologicum Academiae Sinicae), while living cultures were stored in the SAUCC (Shandong 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 conducted in a 25 μL reaction volume, consisting of 12.5 μL 2 × Hieff Canace® Plus PCR Master Mix (Shanghai, China) (with dye) (Yeasen Biotechnology, Cat No. 10154ES03), 0.5 μL each of forward and reverse primer, and 0.5 μL template genomic DNA, with the volume adjusted to 25 μL using distilled deionized water.PCR products were separated and purified using 1% agarose gel and GelRed (TsingKe, Qingdao, China), and UV light was used to visualize the fragments.Gel extraction was performed using a Gel Extraction Kit (Cat: AE0101-C) (Shandong Sparkjade Biotechnology Co., Ltd., Jinan, China).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: I. = Idriela; S. = Selenodriella; Ma. = Macroidriella; Mi. = Microdochium.

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 information and corresponding sequencing quality information (Cock et al. 2010).
Preprocess the raw data that were obtained from the sequencing platform using fastp (https://github.com/OpenGene/fastp) to obtain clean data for subsequent analysis (Chen et al. 2018).Clean data were deposited in the National Center for Biotechnology Information (NCBI) under BioProject PRJNA1105317.Notes: Ex-type or ex-epitype strains are marked with "*" and the new species described in this study was marked in bold.

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 automatic 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 conducted 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.

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 exons, 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 Microdochiaceae, 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 AP01).The intersections of gene family among the six representative strains (≤ 6) are 3431, the maximum number (508) of gene family intersections between Macroidriella bambusae and Microdochium trichocladiopsis, and the minimum number (4) of gene family intersections 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 (maximum), the unique number of genes in Macroidriella bambusae was 42 (minimum) (Fig. 3b).We have presented the number of single-copy genes, multi-copy genes and so on for the seven representative strains (Fig. 3c).
Etymology.Referring to the composed of "Macro-" and "-idriella" (Similar in morphology to Idriella and bigger than Idriella in conidia).
Culture characteristics.Cultures incubated on PDA at 25 °C in darkness, reaching 73-76 mm diam., had a growth rate of 5.2-5.4mm/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 greywhite and regular margin.
Culture characteristics.Cultures incubated on PDA at 25 °C in darkness, reach-ing 72-76 mm diam., had a growth rate of 5.1-5.4mm/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.

Discussion
The establishment of the family Microdochiaceae by Hernández-Restrepo et al. (2016) to encompass the clade consisting of Idriella, 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 (Hernández-Restrepo et al. 2016;Liang et al. 2019;Huang et al. 2020;Liu et al. 2022;Lu et   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) currently hosts 1,594 georeferenced records of Microdochiaceae species worldwide (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 research focus, with 12 species of this genus documented across five Provinces (Guizhou, Hainan, Henan, Shandong, and Yunnan) since the beginning of the 21 st 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, Idriella and Selenodriella have been less extensively studied, with Idriella having only two reported species since the turn of the 21 st century.Through the joint analysis of multiple gene fragments and genomes, the position of new taxa can be better determined, 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 identify 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 relationship 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 relationships within the family.By combining these diverse datasets, a comprehensive understanding of the evolutionary history of Microdochiaceae is achieved, shedding new light on its genetic landscape and evolutionary dynamics.

Figure 1 .
Figure 1.A maximum likelihood tree was constructed using a combined dataset of ITS, LSU, RPB2, and TUB2 sequence data.Branch support values, shown as ML/BIPP, are indicated above the nodes: MLBV ≥ 70% on the left and BIPP ≥ 0.90 on the right.Ex-type cultures are denoted in bold and marked with an asterisk (*).Strains from the current study are highlighted in red.The tree was rooted with Cryptostroma corticale (CBS 218.52).The scale bar at the bottom center represents 0.05 substitutions per site.

Figure 2 .
Figure 2. A Maximum Likelihood phylogenomic tree was constructed using a combined 4,909 clusters of orthologous proteins.Maximum Likelihood bootstrap values (≥ 70%) are indicated along branches.Genera are highlighted in different colors.The scale bar at the bottom represents 0.1 substitutions per site.

Figure 3 .
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.

Figure 4 .
Figure 4. Macroidriella bambusae (HMAS 352974, holotype) a a 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 μm (e-h).

Figure 5 .
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 μm (e-h). al.

Figure 6 .
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 μm (e-f).

Table 1 .
GenBank accession number of the taxa used in phylogenetic reconstruction.

Table 2 .
BioSample and SRA NCBI number of the taxa used in phylogenomic reconstruction in this study.