Rhizomaticola guizhouensis gen. et sp. nov. and Five Rosellinia Like Species Isolated from Decaying Wood

: During the investigation of xylarialean taxa in China and Thailand, six rosellinia like taxa were collected. Rhizomaticola gen. nov. with type species of Rh. guizhouensis is established based on its morphology and multi-gene molecular data. Rhizomaticola owns no carbonaceous stromata and has black ascospores without a germ slit which are distinguished from those of Rosellinia , Dematophora , Stilbohypoxylon and Xylaria . Five r osellinia like species are introduced based on their morphology, inducing three new species ( Dematophora populi, Rosellinia thailandica , Ro. vitis ), one new record for China ( Ro. cainii ) and one known species ( D. necatrix ). Their descriptions and illustrations are detailed.

In the process of investigating xylarialean taxa in China and Thailand, six rosellinia like species were collected from the forests, which were regard as an undescribed xylariaceous genus, Rhizomaticola to accommodate Rh. guizhouensis, as well as three species of Rosellinia and two species of Dematophora.Their descriptions and illustrations are provided.

Collection and Isolation
Samples with black dots were collected from forests in China and Thailand in the rainy season.Samples were put into paper bags with some silica gel desiccant.Macroscopic characteristics of stromata were observed and photographed under an Olympus SZ61 stereomicroscope.Materials were mounted in water and Melzer's reagent for anatomical examination [31].Macroscopic photographs of asci and ascospores were taken under a Nikon digital camera (700 D) fitted to a light microscope (Nikon Ni, Nikon Corporation, Tokyo, Japan).At least 30 ascospores and 30 asci were measured using the Tarosoft ® image framework (v.0.9.0.7).Macroscopic photographs were made with the necessary changes and arranged for a plate.Single-spore isolation was used to obtain pure cultures [33].Herbarium materials were deposited in the Herbarium of Guizhou Agricultural College (GACP); the Herbarium of the Engineering and Research Center for Southwest Bio-Pharmaceutical Resources of National Education Ministry of China, Guizhou University (GZUH); the Herbarium of Mae Fah Luang University (MFLU); and the living cultures are deposited in Guizhou University Culture Collection (GZUHC) or Mae Fah Luang University Culture Collection (MFLUCC).

Sequence Alignment and Phylogenetic Analyses
All sequences for phylogenetic tree construction were chosen following published literature [40] and top hits of ITS blasted in the GenBank database (Table 1).ITS, LSU, tub2, rpb2 sequence data including all introns and exons were aligned separately using the MAFFT v.7.110 online programme (http://mafft.cbrc.jp/alignment/server/(accessed on 15 August 2022) [41]) with the default settings.Multiple sequence alignments were generated and adjusted using BioEdit v.7.0.5.3 [42].The MrModeltest 2.2 was used to perform the model of evolution [43].The final ML search was conducted using the GTRGAMMA + I model.The phylogenetic analyses were carried out for maximum likelihood in CIPRES web portal [44] using RAxML 7.4.2Black Box [45].
Bayesian analyses were performed in CIPRES web portal by using MrBayes on XSEDE [46].The model of evolution was calculated by using MrModeltest v. 2.2 [43].Markov Chain Monte Carlo sampling (MCMC) was used to determine posterior probabilities (PP) [47] in MrBayes on XSEDE.Six simultaneous Markov chains were run for 1,000,000 generations and trees were sampled every 1000th generation.The first 25% of trees were discarded during the burn-in phase of each analysis [48].Phylogenetic trees were visualized and arranged using FigTree v1.4.0.and were edited with Adobe Photoshop CS6 [38].The alignments were uploaded in TreeBASE (www.treebase.org/treebase-web/home.html(accessed on 15 August 2022) under ID 24609 for ITS-LSU-rpb2-tub2 alignment.

Taxonomy
Rhizomaticola Q.R. Li  Notes-Morphologically, this genus is similar to Dematophora, Rosellinia, Stilbohypoxylon and Xylaria, all of which have large stromata visible to the naked eye and unitunicate asci with a J+ apical ring bluing in Melzer's reagent, ascospores with germ slits [3,16,69,70].However, Rhizomaticola has no subiculum, non-carbonaceous stromata cracking on its surface, white external stromata and ascospores lacking germ slits which are different from those close genera.Rhizomaticola differs from Collodiscula and Astrocystis by its noncarbonaceous stromata.Moreover, Collodiscula has ascospores with one too many septa, most species of Astrocystis have the ascospores with germ slits [19,29].Molecular phylogenetic studies based on ITS, LSU, tub2 and rpb2 sequences in this study showed Rhizomaticola formed a distinct branch in Xylariaceae.Although the support values (48/0.91)are not high, its morphological characteristics are consistent with those of Xylariaceae.We would like to propose to temporarily place it in the Xylariaceae.Rhizomaticola guizhouensis Q.R. Li and J.C. Kang, sp.nov. Figure 2. Mycobank No.: 844446 Etymology: In reference to its collection location, Guizhou province, China.
Culture characteristics-no culture was obtained; DNA was extracted directly from asci and ascospores in stromata.
Notes-Rhizomaticola guizhouensis is designated as the type species of Rhizomaticola.Rh. guizhouensis was found from Guizhou, China.Rhizomaticola guizhouensis differs from the uniperithecial species of Xylaria by it non-carbonaceous stromata, the ascospores lacking germ slits and observation of no multiple perithecia on a stroma [3][4][5].Unfortunately, we could not obtain the pure culture of this species after many attempts.Ascospores did not germinate on PDA, OA (oatmeal agar) and MEA (malt extract agar) media.
Notes-Rosellinia cainii was introduced by Petrini [16] as a new species, since its broadly rounded ascospores without slimy sheaths and caps.No available description for asci in the original literature.The first collection was found on Corylus rostrata hort.ex Dippel from Canada.This is the first report for Ro.cainii collected in China.

Discussion
Xylariaceae is a worldwide distributed group which includes common characteristics such as ascomata visible to the naked eye, unitunicate asci with or without a J+, apical apparatus, brown to black, rarely hyaline, 1-2-celled ascospores mostly with a germ slit, geniculosporium-like or nodulisporium-like asexual morph [40].Here, we introduce a new genus, Rhizomaticola to accommodate the type species of Rh. guizhouensis isolated from China.Rhizomaticola have black ascospores without a germ slit and no hard carbonaceous stromata which can be distinguished from its similar genera.
Rosellinia and Dematophora are widely distributed in tropical and subtropical regions and mainly saprophytes on plant branches [27,32,74].In this paper, we introduced five species of Rosellinia and Dematophora which were collected from China and Thailand and identified them based on their morphology.We have attempted to isolate the pure cultures of these specimens, but only a part of the isolations has been obtained.We found that the larger the ascospores, the less likely it is to germinate in Rosellinia and Dematophora.Moreover, the culture is likely to die after being stored for a while at 4 °C.
Many taxonomic features are used for the identification of species within Rosellinia and Dematophora.The commonly used morphological characteristics mainly include: the size and shape of the stromata; the size and shape of the apical ring of ascus; the size and shape of the ascospores; the length of germ slits; the type of appendages; and the presence and type of sheaths covering the ascospores [16,[75][76][77].Anamorph is used for species identification as well, although only a few species of asexual stage have been observed [32,49,74].DNA sequences have also been carried out for the identification of species within those genera [30,73].However, there are only a few DNA sequences of Rosellinia available on Genbank.Secondary metabolites were attempted to be taken as a taxonomic feature to identify species of Rosellinia and Dematophora [32].Dematophora was resurrected as inferred from polythetic taxonomy, while the results of utility of secondary metabolites as genus-specific chemotaxonomic markers were inconclusive [32].

Figure 1 .Figure 1 .
Figure 1.Phylogeny of Xylariales obtained from a Maximum Likelihood analysis of the combined ITS, LSU, rpb2 and tub2 using RAxML-HPC BlackBox software online.Furfurella nigrescens (CE1) Figure 1.Phylogeny of Xylariales obtained from a Maximum Likelihood analysis of the combined ITS, LSU, rpb2 and tub2 using RAxML-HPC BlackBox software online.Furfurella nigrescens (CE1) andF.stromatica (CE4) were taken as outgroup taxa.Strains or specimen numbers were followed by their names.Type and authority strains are marked in bold.Bayesian posterior probabilities ≥0.95 and bootstrap support values for maximum likelihood (ML) higher than ≥70% are marked above the nodes; an en-dash ("-") indicates a value < 0.95 (PP) or <70% (BS).

Figure 4 .
Mycobank no.: 844442 Etymology: The name refers the name of host, Populus sp.

Table 1 .
List of taxa used for phylogenetic reconstruction.