Additions to Rhytidhysteron (Hysteriales, Dothideomycetes) in China

In this study, twelve terrestrial hysteriaceous saprobic fungi growing on different pieces of dead wood were collected from Yunnan Province, China. All hysteriaceous strains isolated in this study tallied with the general characteristics associated with Rhytidhysteron. Detailed morphological characteristics and combined multigene phylogeny of LSU, ITS, SSU, and TEF showed that the twelve hysteriaceous fungi strains represent four distinct new species, and seven new host or geographical records of Rhytidhysteron. Based on morphological and phylogenetic evidence, the four new species (Rhytidhysteron bannaense sp. nov., R. coffeae sp. nov., R. mengziense sp. nov., and R. yunnanense sp. nov.) expand the number of species of Rhytidhysteron from thirty-three to thirty-seven, while seven new geographical records expand the records of Rhytidhysteron in China from six to thirteen. In addition, 10 new Rhytidhysteron host records are reported for the first time, thus expanding the known hosts for Rhytidhysteron from 52 to 62. Full descriptions, images of the morphology, and phylogenetic analyses to show the position of the Rhytidhysteron taxa are provided. In addition, the present study summarizes the main morphological characteristics, host associations, and locations of this genus.


DNA Extraction, PCR Amplification, and Sequencing
Dissanayake et al. [30] was followed for molecular studies. Fresh mycelia which grew on PDA plates for two weeks were scraped off the plates, and then DNA was extracted using DNA Extraction Kit-BSC14S1 (BioFlux, Hangzhou, P.R. China), following the manufacturer's protocol. Polymerase chain reaction (PCR) was used to amplify four gene regions and the primers and protocols were used for the amplification following Wanasinghe et al. [25]. The LSU gene was amplified by using the primers LR0R and LR5 [31], the ITS gene was amplified by using the primers ITS5 and ITS4 [32], the SSU gene was amplified using the primers NS1 and NS4 [32], and the TEF gene was amplified using the primers EF1-983F and EF1-2218R [33]. The total volume of the PCR mixture for amplifications was 25 µL, which consisted of 12.5 µL 2xMaster Mix (mixture of Easy Taq TM DNA Polymerase, dNTPs, and optimized buffer (Beijing Trans Gen Biotech Co., Chaoyang District, Beijing, China)), 8.5 µL ddH 2 O, 2 µL of DNA template, and 1 µL of each forward and reverse primer (10 pM) [34]. Purification and sequencing of PCR products were carried out by Qinke Biotech Co., Kunming, China.
In phylogenetic analyses, Randomized Accelerated Maximum Likelihood (RAxML) and Bayesian inference analyses (BI) were carried out in the CIPRES Science Gateway (https://www.phylo.org/portal2/login!input.action (accessed on 10 November 2022)) [39]. The RAxML trees are performed using RAxML-HPC2 on XSEDE (8.2.12) [40,41] with the GTR + I + G model of evolution. Additionally, Bayesian analyses were conducted using the Markov Chain Monte Carlo (MCMC) method in MrBayes on XSEDE (3.2.7a) [42] to evaluate posterior probabilities [43,44]: the best model of LSU and ITS is GTR + I+G, the best model of SSU is HKY + I, and the best model of TEF is GTR + G. Six simultaneous Markov chains were run for 1,000,000 generations, and trees were sampled at every 100th generation. Maxtrees was set to 5000 and clade robustness was assessed using a bootstrap (BT) analysis of 1000 replicates. Phylogenetic trees were visualized with FigTree v.1.4.2 [45], bootstrap values showing at the nodes, and edited by Microsoft Office PowerPoint 2010. The newly obtained alignments and phylogenetic trees were deposited in TreeBASE, submission ID: 30049 (https://treebase.org/treebase-web/user/submissionList.html, accessed on 28 November 2022).

Phylogenetic Analyses
The phylogenetic trees obtained from RAxML and BI analyses provided essentially similar topologies. The RAxML analyses of the combined dataset yielded the best scoring tree (Figure 1), with a final ML optimization likelihood value of −12081.382479. The matrix had 761 distinct alignment patterns, with 23.87% being undetermined characters or gaps. Parameters for the GTR + I + G model of the combined LSU, ITS, SSU, and TEF were as follows: estimated base frequencies A = 0.239683, C = 0.247793, G = 0.276186, T = 0.236338; substitution rates AC = 1.225201, AG = 2.493063, AT = 1.186723, CG = 0.732384, CT = 4.977839, GT = 1.0; proportion of invariable sites I = 0.675821; and gamma distribution shape parameter α = 0.554013. The final RAxML tree is shown in Figure 1.

Taxonomy
Culture characteristics: Ascospores germinated on PDA within 24 h and germ tubes produced from one or both ends. Colonies on PDA reached a 6 cm diameter after two weeks at 28 • C. The colony is soft, circular, irregularly raised, with an undulated edge, white to gray on the forward, and grayish yellow in reverse.

Rhytidhysteron camporesii
Culture characteristics: Ascospores germinated on PDA within 24 h and germ tubes produced from one or both ends. Colonies on PDA reached a 6 cm diameter after two weeks at 28 • C. The colony is flossy, velvety, circular, slightly raised, with an entire edge, reddish brown on the forward and in reverse, with a green circle in the middle. Notes: In the phylogenetic analyses, Rhytidhysteron coffeae clearly separated from R. mangrovei with good statistical support (99% ML/1.00 PP). With respect to morphology, R. coffeae is distinct from R. mangrovei in having branched pseudoparaphyses, 8-spored asci, and 3-septate ascospores when mature, while R. mangrovei has unbranched pseudoparaphyses, (2-6-)8-spored asci, and (1-)3-septate ascospores [12]. In addition, the ascomata, asci, and ascospore size of R. coffeae are larger than those of R. mangrovei (ascomata:1520 × 1120 × 450 µm vs. 940 × 800 × 500 µm, asci: 179.5 × 13 µm vs. 146 × 9.5 µm, ascospores: 26 × 10 µm vs. 23 × 8.3 µm) [12]. Moreover, according to the comparison results of different gene fragments, R. coffeae is different from R. mangrovei in ITS (77/651 bp, 11.83%, without gaps) and TEF (21/960 bp, 2.19%, without gaps) genes (≥1.5%). Therefore, in this study, R. coffeae is introduced as a new species.  ses, forming a reddish brown to brown epithecium above asci when mounted in water, becoming purple epithecium above the asci when mounted in 10% KOH, and turns hyaline after 30 s, while appendages turn dark brown. Notes: In phylogenetic analyses, Rhytidhysteron mengziense was well separated from R. camporesii with low statistical support. However, R. mengziense is distinct from R. camporesii in having exciple cells of textura angularis to prismatica, and rough-walled ascospores, while R. camporesii has exciple cells of textura globulosa to angularis, and smooth-walled of ascospores [49]. In addition, the ascomata and ascospore size of R. mengziense are larger than those of R.  [16,18]. This species was previously only recorded in Thailand and Mexico. Therefore, we report our collection as new geographical and host record of R. neorufulum from the decaying wood of Elaeagnus sarmentosa (Elaeagnaceae) in the Yunnan Province of China. This is the first record of R. neorufulum on Elaeagnus sarmentosa.   Host: Betula sp., an unidentified member of the Fabaceae [18], Magnolia delavayi (this study), and Tectona grandis [53]. Notes: Rhytidhysteron tectonae was introduced by Doilom et al. [53] based on both morphology and phylogenetic analyses. According to phylogenetic analyses based on combined multi-gene (LSU, ITS, SSU, and TEF), our collection grouped together with R. tectonae (MFLUCC 13-0710, MFLUCC 21-0034, and MFLUCC 21-0037). In addition, our collection shows similar morphological characteristics to R. tectonae, having solitary to aggregated, semi-immersed to superficial, non-striated, yellow at the center hysterothecia, septate, branched pseudoparaphyses, 8-spored, cylindrical, short pedicellate asci, and uniseriate, slightly overlapping, 1-3-septate ascospores, dark brown when mature [18,53]. This species was previously only recorded in Thailand. Therefore, we report our collection as a new geographical and host record of R. tectonae from decaying wood of Magnolia delavayi (Magnoliaceae) in the Yunnan Province of China. This is the first record of R. tectonae on Magnolia delavayi. Notes: Rhytidhysteron thailandicum was introduced by Thambugala et al. [16] based on both morphology and phylogenetic analyses. According to phylogenetic analyses based on combined multi-gene (LSU, ITS, SSU, and TEF), our collection grouped together with R. thailandicum (MFLUCC 14-0503, MFLUCC 12-0530, MFLU17-0788, and MFLUCC 13-0051).
Culture characteristics: Ascospores germinated on PDA within 24 h and germ tubes produced from one or both ends. Colonies on PDA reached a 6 cm diameter after two weeks at 28 • C. The colony is velvety, circular, slightly raised, with a filiform edge, white on the forward and white in reverse.
Material Notes: In phylogenetic analyses, Rhytidhysteron yunnanense was well separated from R. mesophilum with good statistical support (90% ML/0.92 PP). In morphology, R. yunnanense is distinct from R. mesophilum, having navicular to irregular ascomata, each hysterothecia has two parallel striae parallel to the longitudinal slit, and slight perpendicular striae, and a longitudinal slit with no opening, while R. mesophilum has boat-shaped ascomata, with perpendicular striae with a perpendicular to longitudinal slit, and a longitudinal slit opening [24]. In addition, the asci and ascospore size of R. yunnanense are smaller than those of R. mesophilum (asci: 230 × 14 µm vs. 267-282 × 15.5-16 µm, ascospores: 32.5 × 13 µm vs. 44.2 × 13.6 µm) [24]. Moreover, according to the comparison results of different gene fragments, R. yunnanense is different from R. mesophilum in the ITS (28/651 bp, 4.30%, without gaps) gene. Therefore, in this study, R. yunnanense is introduced as a new species.
Moreover, in the previous study of the genus Rhytidhysteron, ascomata have a transversestriae, perpendicular to the longitudinal slit, and this study is the first to find parallel striae that are parallel to the longitudinal slit.

Discussion
Based on the morphological study and phylogenetic analyses, four new species and seven new records of Rhytidhysteron are introduced in this paper. Rhytidhysteron bannaense sp. nov., R. coffeae sp. nov., R. mengziense sp. nov., and R. yunnanense sp. nov. are proposed as new to science based on their unique morphological characteristics and moderate to good statistical support. Seven collections of R. bruguierae, R. camporesii, R. hongheense, R. magnoliae, R. neorufulum, R. tectonae, and R. thailandicum are identified as new records because of their identical morphological characteristics with the type species of the same species and high statistical support.
In this study, we found that the pseudoparaphyses of all 11 species are branched and septate. Interestingly, the epithecia of ten species all turn purple in 10% KOH, but the purple color of the epithecium fades and becomes hyaline in a short period of time (5-30 s). On the contrary, the epithecium of R. yunnanense sp. nov. (HKAS 122696) turns hyaline in 10% KOH, and the appendages become dark. In addition, R. yunnanense is unique in Rhytidhysteron because it has both parallel and perpendicular striae, relative to the longitudinal slit. This is also the first discovery of parallel striae in this genus, while other species of Rhytidhysteron have perpendicular striae or are non-striated ( Table 3). The presence or absence of striae on the margin of ascomata is one of the important characteristics of this genus and is used to identify different species [16].
The results of the phylogenetic tree generated in this study are consistent with those reported by Ren et al. [18], and R. erioi Ekanayaka & K.D. Hyde is grouped as a sister to R. bruguierae. Therefore, to find out the correct taxonomic placement of R. erioi, it is necessary to recollect more samples and confirm their placement. Boehm et al. [13] suggested that R. opuntiae (J.G. Br.) M.E. Barr should be removed from Rhytidhysteron based on morphological and molecular data. Subsequently, Almeida et al. [15] suggested that R. opuntiae should be accommodated by a new genus in future studies. The main reason is that R. opuntiae grouped with Hysterodifractum partisporum D.A.C. Almeida, Gusmão & A.N. Mill. [15,56], but morpho-molecular differences exist between Hysterodifractum D.A.C. Almeida, Gusmão & A.N. Mill. and R. opuntiae [12]. This study agrees with Boehm et al. [13] and Almeida et al. [15] in that R. opuntiae should be included in a new genus, due to the fact that morphomolecular data of R. opuntiae are different from those of Rhytidhysteron and Hysterodifractum. Therefore, more studies on R. opuntiae are needed.
Rhytidhysteron neorufulum and R. rufulum are the most common and most reported species in this genus [51]. Rhytidhysteron rufulum is considered a complex species based on studies on molecular and chemical data [16,22,27,51,57]. Thambugala et al. [16] indicated that some fungal specimens were incorrectly classified as R. rufulum, which needs to be reviewed exhaustively, as they might represent new species [51]. This action is meaningful, and can clarify the taxonomic placement of unclear species and enrich the diversity of Rhytidhysteron. Unfortunately, in this study, no strains with very similar morpho-molecular data to R. rufulum were found.
In previous studies, six species were reported in China. These are R. camporesii [49], R. hongheense [25], R. magnoliae [6], R. rufulum [15], R. thailandicum [6], and R. xiaokongense [18]. This study provides seven additional species from China-R. bannaense sp. nov., R. bruguierae, R. coffeae sp. nov., R. mengziense sp. nov., R. neorufulum, R. tectonae, and R. yunnanense sp. nov. With these additions, the number of species of Rhytidhysteron in China increases from six to thirteen. At the same time, the four new species added in this study expand the species of Rhytidhysteron from thirty-three to thirty-seven, and the known hosts for Rhytidhysteron expand from 52 to 62 records. However, only 22 species of Rhytidhysteron have sequence data (including this study), so more research needs to be carried out, and more samples need to be collected, isolated, and sequenced. This study also summarizes the morphological characteristics, hosts, and countries of the species of this genus for the first time (Table 3), which provides references for future research on Rhytidhysteron.         Remarks: the symbol "-" denotes no information available.