Insights into the molecular phylogeny and morphology of three novel Dothiora species, along with a worldwide checklist of Dothiora

Most species of Dothiora are known from the dead parts of various host plants as saprobic fungi in terrestrial habitats occurring in tropical and temperate regions. In the present study, samples of Dothiora were collected from dead twigs and branches of Capparis spinosa, Rhaponticum repens, and an unknown angiosperm plant from the Tashkent and Jizzakh regions of Uzbekistan. Multi-gene phylogenetic analyses based on a combined ITS, LSU, SSU, TEF1, and TUB2 sequence data revealed their taxonomic positions within the Dothideaceae. Three new species of Dothiora, namely, Dothiora capparis, Dothiora rhapontici, and Dothiora uzbekistanica were proposed by molecular and morphological data. Likewise, the phylogenetic relationship and morphology of Dothiora are discussed. In addition, we provide a list of accepted Dothiora species, including host information, distribution, morphology descriptions, and availability of sequence data, to enhance the current knowledge of the diversity within Dothiora.

Most of the Dothiora species are reported from Europe and North America (Hyde et al., 2020).Targeting underexplored regions such as Central Asia, including Uzbekistan, might be helpful for the discovery of new fungi (Gafforov, 2017;Cheek et al., 2020).Recent studies have led to the discovery of several new genera and species of ascomycetous microfungi in Uzbekistan (Gafforov and Rakhimov, 2017;Pem et al., 2018;Pem et al., 2019a;Pem et al., 2019b;Gafforov et al., 2019;Abdurazakov et al., 2021;Appadoo et al., 2021;Htet et al., 2021;Lestari et al., 2021;Aluthmuhandiram et al., 2022;Dong et al., 2023).However, Dothiora is still poorly known in Asia including Central Asian regions.The aim of the present study was to clarify the taxonomic position of Dothiora and to identify new taxa through multi-gene phylogeny and morphological examination.Fresh specimes collected from Uzbekistan were examined and their DNA sequence data were obtained for use in multi-gene phylogenetic analyses.Moreover, an updated list of Dothiora species worldwide is also provided.

Sample collection and specimen examination
Specimens were collected from Capparis spinosa, Rhaponticum repens, and an unknown angiosperm plant from the Tashkent and Jizzakh regions of Uzbekistan.The collected specimens were brought to the laboratory in small plastic bags.Ascomata were sectioned by hand, examined, and captured under a Nikon SMZ800N stereomicroscope.The slides were prepared by mounting the materials in double-distilled water (ddH 2 O), lactophenol, and Indian ink stain.The micro-morphological characters were examined and captured using a Nikon DS-Ri2 camera connected with a Nikon ECLIPSE Ni (Tokyo, Japan) compound microscope.The measurement of structures was done by the Tarosoft ® Image Framework program (v.0.9.0.7).Adobe Photoshop Version: 22.4.2(Adobe Systems U.S.A.) was used to make the photographic plates.The specimens were deposited in the Herbarium of the Department of Biology (CMUB), Faculty of Science, Chiang Mai University, Thailand, and the Tashkent Mycological Herbarium (TASM) of the Institute of Botany, Uzbekistan Academy of Sciences, Uzbekistan.

DNA extraction, PCR amplification, and sequencing
To obtain pure cultures, single ascospore isolation was carried out following the methods of Senwanna et al. (2019) and Senanayake et al. (2020).However, no germinated ascospores were found on Petri dishes containing 2% malt extract agar (MEA; Gibco, Life Technologies Corporation, USA), 2% water agar (WA), and potato dextrose agar (PDA; BD Difco ™ , Becton, Dickinson and Company, USA) after incubation at 25°C to 30°C in the dark for 24-96 h.Fungal fruiting bodies, thus, were picked up and placed in a 1.5-mL sterilized tube.Genomic DNA was directly extracted, using E.Z.N.A. ® Genomic DNA Isolation Kits (OMEGA Bio-Tek, Georgia) following the manufacturer's protocol.Polymerase chain reaction (PCR) amplification was carried out using the primer pairs as follows: ITS5 and ITS4 (White et al., 1990) to amplify the partial gene regions of internal transcribed spacers (ITS); LR0R and LR5 (Vilgalys and Hester, 1990) to amplify the 28S large subunit (LSU); NS1 and NS4 (White et al., 1990) to amplify the 18S small subunit (SSU); and EF1-728F (Carbone and Kohn, 1999) and EF2 (O'Donnell et al., 1998) to amplify the protein coding region for the translation elongation factor 1-alpha gene (TEF1).The PCR mixture contained 6 µL of double-distilled water (ddH 2 O),10 mL of 2 × Quick TaqTM HS DyeMix (TOYOBO, Japan), 2 µL of genomic DNA, and 1 mL of each forward and reverse primer.The PCR thermal cycle programs for ITS, LSU, and SSU amplification were as follows: initial denaturing step of 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 45 s, elongation at 72°C for 1 min, and final extension at 72°C for 10 min.The PCR thermal cycle programs for TEF1 amplification were as follows: initial denaturing step of 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, elongation at 72°C for 1 min, and final extension at 72°C for 10 min.PCR products were examined on 1% agarose electrophoresis gels under UV light.PCR products were purified using the PCR clean-up Gel extraction NucleoSpin ® Gel and PCR Clean-up Kit (Macherey-Nagel, Germany) following the manufacturer's protocol.PCR fragments were performed and sequenced at 1st BASE Company (Kembangan, Malaysia).

Phylogenetic analyses
The generated sequence data were assembled using SeqMan 5.00, and the consensus sequences were subjected to BLASTn s e a r c h e s o f t h e N C B I n u c l e o t i d e d a t a b a s e ( h t t p : / / blast.ncbi.nlm.nih.gov/;accessed on 2 November 2023) to determine their most probable closely related taxa.The representative taxa used in the analyses were selected from GenBank based on the BLASTn searches and recently published data (Boonmee et al., 2021;Gao et al., 2021)     November 2023) and was improved manually where necessary.The phylogenetic tree was carried out using the maximum likelihood (ML).The single gene datasets were then combined using BioEdit v.7.0.9.1 (Hall, 1999).The final alignments of the combined ITS, LSU, SSU, TEF1, and beta-tubulin (TUB2) datasets were analyzed, and the phylogenetic trees were inferred based on ML and Bayesian inference (BI) analyses.
The ML tree was accomplished using the RAxML-HPC2 on XSEDE (v.8.2.12) (Stamatakis, 2014) under the GTRGAMMA substitution model of nucleotide substitution with 1,000 bootstrap (BS) iterations.For BI analyses, the best-fit model of the sequence evolution of each locus was estimated using the Akaike information criterion (AIC) in MrModeltest v. 2.3 (Nylander, 2008) implemented in PAUP v. 4.0b10 (Swofford, 2002).The GTR+G+I substitution model was the best-fit model for all loci.The BI tree was executed with MrBayes v. 3.2.6 (Ronquist et al., 2012) to evaluate posterior probabilities (PP) (Rannala and Yang, 1996;Zhaxybayeva and Gogarten, 2002) by Markov Chain Monte Carlo sampling (BMCMC).Four simultaneous Markov chains were run for 5,000,000 generations, with the trees sampled every 100th generation resulting in 50,000 trees.The run was stopped when the standard deviation of split frequencies reached below 0.01.The first 12,500 trees were discarded as the burn-in phase of the analyses, while the remaining 37,500 trees were calculated for PP in the majority rule consensus tree.The resulting phylogenetic trees were drawn using FigTree v1.4.0 (Rambaut, 2016) and edited using Adobe Illustrator Version 25.2.3 and Adobe Photoshop Version 22.4.2(Adobe Systems., U.S.A.).ML bootstrap values ≥50% and Bayesian PP ≥0.95 were placed above each node (Figure 1).The new nucleotide sequence data are deposited in GenBank (Table 1).The final alignment and tree were deposited in TreeBASE (http:// www.treebase.org/)under the accession number S31239 and URL

Phylogenetic analyses
The combined dataset of LSU, ITS, SSU, TEF1, and TUB2 sequence data comprises 74 taxa, which represent strains from Dothideaceae and two outgroup taxa in Saccotheciaaceae, Pseudoseptoria obscura Quaedvl., Verkley & Crous (CBS 135103) and Pseudosydowia eucalypti (Verwoerd & du Plessis) Thambug.& K.D. Hyde (CPC14028) (Table 1).The combined sequence alignment consisting of 4,055 characters was analyzed by ML and BI.A best scoring RAxML tree with a final likelihood value of −19,627.809539 is presented in Figure 1.The matrix of the combined dataset had 1,384 distinct alignment patterns and 53.70% of undetermined characters or gaps.Estimated base frequencies were A = 0.253918, C = 0.235773, G = 0.266686, T = 0.243623; substitution rates were AC = 1.637334,AG = 2.504185, AT = 1.806129,CG = 1.203612,CT = 7.247662, GT = 1.000000; and gamma distribution shape parameter a = 0.504007.Bayesian posterior probabilities (PP) from MCMC were evaluated with the final average standard deviation of split frequencies = 0.009894.The Bayesian analysis resulted in a tree with similar topology and clades as the ML tree.Phylogenetic analyses of a combined LSU, ITS, SSU, TEF1, and TUB2 sequence data (Figure 1  RAxML tree generated by maximum likelihood analysis of combined LSU, ITS, SSU, TEF1, and TUB2 sequence data representing Dothideaceae.Bootstrap support values for maximum likelihood (ML, left) ≥50% and Bayesian posterior probabilities (PP, right) ≥0.95 are indicated above the nodes.Hyphens (-) represent support values <50% ML/0.95PP.The tree is rooted to Pseudoseptoria obscura (CBS 135103) and Pseudosydowia eucalypti (CPC14028).The ex-type strains are in bold, and the newly generated sequences in this study are in blue.
MycoBank number: MB851612 Etymology: Name reflects the host genus Capparis from which it was isolated.
MycoBank number: MB851613 Etymology: The name reflects the host genus Rhaponticum from where it was isolated.
MycoBank number: MB851614 Etymology: The name refers to the country Uzbekistan, where it was collected.

Discussion
Historically, Dothiora has relied on morphological studies, and only a few sequences of species are available in GenBank.In this study, three novel species of Dothiora are introduced in the family Dothideaceae from the Central Asian region based on their morphological distinctiveness and phylogenetic analyses.Although a living culture from an isolated ascospore could not be obtained, the fungal DNA was extracted directly from the ascomata.The connection between sexual and asexual morphs is likewise unknown.The individual phylogenetic analyses of ITS or LSU separated Dothiora species from other genera in Dothideaceae, but their placement was otherwise unresolved (Crous and Groenewald, 2017;Crous et al., 2018a;Crous et al., 2018b).Therefore, the combination of LSU, ITS, and SSU sequence data was previously used to clarify the relationships among the species in Dothideaeceae, although there is no strong statistical support (Thambugala et al., 2014;Hyde et al., 2017;Crous et al., 2020, Crous et al., 2022;Hongsanan et al., 2020;Boonmee et al., 2021).Gao et al. (2021) recommended using a combination of the nuclear ribosomal region (ITS, LSU, and SSU) and the protein-coding gene regions (TEF1 and TUB2) to clarify the relationships of Dothideaceae.Our attempts to obtain TUB2 sequence data for our new strains were unsuccessful; however, the data from the combined sequence analyses of the ITS, LSU, SSU, and TEF1 loci are not well-resolved for most Dothiora species.Thus, a phylogenetic analysis based on a combination of five loci was generated for a better phylogenetic relationship within the family and genus.Our multigene phylogeny (Figure 1) revealed that the generic placement within Dothideaceae, comprising 14 genera and three Dothideales genera incertae sedis, viz., Coniozyma, Hormonema, and Rhizosphaera, was similar to those of Hongsanan et al. (2020) and Gao et al. (2021).Most Dothiora taxa clustered together in its own clade with 65% ML, 1 PP statistical support, excepting D. mahoniae (A.W. Ramaley) Crous (strain CBS 264.92) (Figure 1).
A checklist of 69 accepted Dothiora species, including details of each species based on recorded from Index Fungorum (2024), MycoBank (2024), and published articles, is provided in Table 2. Dothiora have a cosmopolitan distribution and are mainly saprobic, found in decaying wood and plant litter in terrestrial environments.Although the sexual morph of Dothiora species has been delimited according to morphological criteria by having one or more septate or muriform ascospores (Thambugala et al., 2014;Crous and Groenewald, 2017), many species with aseptate ascospores were also classified as Dothiora based on morphological characteristics and phylogenetic analyses (Hyde et al., 2016;Hyde et al., 2017;Boonmee et al., 2021).Most Dothiora species with hyaline to pale brown, one or more septate or muriform ascospores, form a separate clade without statistical support (Figure 1), while three new taxa, D. capparis, D. rhapontici, and D. uzbekistanica, cluster with D. buxi, D. coronillae, D. coronillicola, and D. spartii, which also have hyaline, aseptate, and fusoid to ovoid ascospores with 89% ML and 0.99 PP statistical support in different clades, agreeing with previous studies (Boonmee et al., 2021).Dothiora rhapontici and D. buxi differ     Asexual: Young hyphae were 3.5-8 mm diam, while older hyphae, having shorter, fatter, thicker-walled cells, were up to 13.5 mm diam.More or less cylindrical phialoconidia (8-20 × 3.3-5 mm) were produced on minute phialides.Several phialoconidia were often produced at one point on the hyphal cell (multiple phialoconidia).Secondary buds on the phialoconidia and chlamydospores were rarely seen.Arthroconidia and endospores were not observed.Yeast-like multiplication of the strains occurred by polar budding of single cells (4-12.5 × 1.5-4 mm) or by phialoconidial formation on centrally constricted septate cells (6.5-14 × 3-6 mm).Polar and lateral phialoconidia were also produced on cells with several septa.from related species in having polysporous asci, while other species have octosporous asci.These species can be distinguished from each other based on their asci and ascospores sizes; moreover, D. capparis can be distinguished from D. coronillae, D. coronillicola, D. spartii, and D. uzbekistanica by the presence of a mucilaginous sheath surrounding the ascospores.Dothiora cactacearum also clustered among these species; however, its morphological characteristics cannot be compared with others, as it is known only by its asexual morph.Dothiora buxi is phylogenetically close to D. cactacearum, and both species have similar asexual morphs to the generic, as described by Thambugala et al. (2014) and Crous and Groenewald (2017).The morphology of ascospores of Dothiora buxi, D. capparis, D. coronillae, D. coronillicola, D. rhapontici, D. spartii, and D. uzbekistanica, was clearly distinct from that of most Dothiora; however, only this evidence did not have enough support to accommodate a distinct lineage in Dothideaceae.Furthermore, an asexual morph of these species has not been cultured or reported to verify its morphological features, except for D. buxi.Due to the fact that most of the available sequences of Dothiora are only LSU and/or ITS, their taxonomic position remains uncertain.The taxonomic classification of Dothiora species is still incomplete; further investigations of freshly collected specimens in different regions and sequence data are needed to better understand their natural classification.Based on the Species Fungorum; the taxon was synonymized under other genera. 2 Based on the MycoBank database; the taxon was synonymized under other genera. 3 The species shows as Nom.inval., Art.36.1(c)(Melbourne) in Index Fungorum, but this became legitimate in MycoBank. 4 The species is regarded as an illegitimate name according to MycoBank (2024), but it was shown as valid in Index Fungorum (2024).Senwanna et al. 10.3389/fcimb.2024.1367673Frontiers in Cellular and Infection Microbiology frontiersin.orgCrous et al. (2018b) transferred Kabatina mahoniae A.W. Ramaley to Dothiora as D. mahoniae.According to the multigene analyses herein (Figure 1), Dothiora mahoniae (strain CBS 264.92) clustered with Neodothiora populina Crous, G.C. Adams & Winton (strain CBS 147087) with no statistical support for this relationship and separated from Dothiora species, which is consistent with Crous et al. (2020).Based on a comparison of morphology, Dothiora mahoniae fits well with the generic concepts of Kabatina rather than Dothiora and Neodothiora (Ramaley, 1992;Thambugala et al., 2014;Crous and Groenewald, 2017;Crous et al., 2020).In addition, only LSU and ITS are available for D. mahoniae in GenBank.Thus, the species is retained until more evidence of fresh collections with DNA sequence data is available to resolve its phylogenetic placement within the family.
In this study, three new species of hyaline-spored Dothiora (D. capparis, D. rhapontici, and D. uzbekistanica) are described and illustrated.It is interesting to note that Dothiora have morphological variability in their spores.Thus, it is inadequate to determine Dothiora spp.based solely on morphological data.It can be seen that phylogenetic analyses are necessary to confirm morphology-based identifications and detect species new to science.Many Dothiora on the list (Table 2) have not been verified yet based on the molecular data; however, these species have characteristics that match the generic description.Additionally, the classification of several species remains unclear due to the variability in some morphological characters, a lack of molecular information regarding protein-coding genes, and no sexual-asexual links.Hence, these species are not excluded from Dothiora until increased taxon samplings and sequence data are available.Further sampling is necessary to improve our knowledge of the diversity, ecology, and impacts of hyaline-spored Dothiora species on flowering plants in arid and semi-arid habitats.

TABLE 1
Taxa names, strain numbers, and GenBank accession numbers of sequences used in the phylogenetic analyses of this study.

TABLE 2
Morphology, host information, locality, sequence data, and related references Dothiora reported worldwide based on the record of Species Fungorum and MycoBank database 2024.

TABLE 2 Continued
Mycelium composed of hyaline, branched, strongly septate hyphae, smooth-and thin-walled, swollen at septa, 3.5-6 mm diam, becoming monilliform and dark brown with age due to the production of solitary to catenate chlamydospores of up to 20 mm diam, with some segments remaining hyaline or nearly so; Conidiogenous cells integrated on hyphae, intercalary or terminal, inconspicuously to conspicuously denticulate; Conidia holoblastic,