Two new species of Jalapriya and a new record, Dictyocheirosporavinaya from freshwater habitats in China

Abstract Background Pleosporales is the largest order of Dothideomycetes. In recent years, systematics of Pleosporales have undergone considerable revisions. Dictyosporiaceae is one of the newly established families within this order proposed to accommodate holomorphic saprobic Dothideomycetes. Currently 18 genera are recognised in Dictyosporiaceae. New information The new species, Jalapriyaaquaticum sp. nov. and J.apicalivaginatum sp. nov. were collected from freshwater habitats in Gansu and Yunnan Provinces, China, respectively and are introduced, based on morphology and molecular analysis of combined ITS, LSU, SSU and TEF1-α sequence data. We also recovered one fresh collection of Dictyocheirosporavinaya D’souza, Bhat & K.D. Hyde, which is a new record for China. Jalapriyaaquaticum differs from extant species of Jalapriya in rows converging at the apex and apical cells with spherical-like appendages. Jalapriyaapicalivaginatum differs from extant species of Jalapriya in having the rows of conidia mostly arranged in a plane. The phylogenetic analysis place the new collections within Dictyosporiaceae (Pleosporales). Descriptions and illustrations of Jalapriyaaquaticum, J.apicalivaginatum and Dictyocheirosporavinaya are provided. A synopsis of characters of species of Jalapriya is also provided.


Introduction
Pleosporales is the largest order of Dothideomycetes. In recent years, various families and genera in the Pleosporales have undergone considerable revisions (Goh and Hyde 1999, Cai et al. 2008, Zhang et al. 2009, Zhang et al. 2012, Hyde et al. 2013.  accepted eleven genera in the family Dictyosporiaceae (Pleosporales) to accommodate most cheirosporous hyphomycetous genera that are saprobes on decaying wood and plant debris in terrestrial and freshwater habitats. One of the diagnostic characteristics of Dictyosporiaceae is their multicellular cheiroid conidia and this morphological feature distinguishes it from other families in the suborder Massarineae .  and Yang et al. (2018) updated the phylogenetic tree for Dictyosporiaceae and introduced two new genera Aquadictyospora and Dendryphiella in the family. Subsequently, three additional genera, Neodendryphiella, Pseudoconiothyrium and Paradictyocheirospora were added (Iturrieta-González et al. 2018, Crous et al. 2019, Rajeshkumar et al. 2021. Currently, 18 genera are accepted in Dictyosporiaceae (Boonmee et al. 2016, Iturrieta-González et al. 2018, Yang et al. 2018, Crous et al. 2019, Rajeshkumar et al. 2021).
The genus Jalapriya was introduced by Boonmee et al. (2016) with Jalapriya pulchra D'souza, Su, Luo & K.D. Hyde as type species; It is characterised by dark brown to black colonies, acrogenous, solitary and cheiroid conidia ). Presently, three species are accepted in the genus, Jalapriya inflata, J. pulchra and J. toruloides. Dictyocheirospora was established by  to accommodate three new species, Dictyocheirospora bannica, D. rotunda and D. vinaya and four new combinations, D. gigantica, D. heptaspora, D. pseudomusae and D. subramanianii. Dictyocheirospora is characterised by non-complanate conidia with arms arising from the basal cell and closely gathered at the apex and compact . The species of Dictyocheirospora have been reported from freshwater and terrestrial habitats in China, Japan and Thailand , Yang et al. 2018, Tibpromma et al. 2018. Currently, 23 species are accepted in the genus , Yang et al. 2018.
In this study, two new species Jalapriya aquaticum and J. apicalivaginatum and a new geographic record, Dictyocheirospora vinaya are introduced, based on morphology and phylogenetic analyses. Detailed descriptions and illustrations are provided.

Isolation and morphological examination
Submerged woody substrates were collected from dynamic waters, Gansu and Yunnan Provinces and taken back to the laboratory in Zip-lock plastic bags. The samples were incubated in plastic boxes lined with moistened tissue paper at room temperature for one week. Methods of morphological observation and isolation follow Luo et al. (2018) and .
The pure cultures were developed by single spore isolation following the method provided by Chomnunti et al. (2014). The cultures are deposited in Kunming Institute of Botany, Chinese Academy of Sciences (KUMCC) and China General Microbiological Culture Collection Center (CGMCC). Herbarium specimens are deposited at the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (Herb. HKAS). Facesoffungi and Index Fungorum numbers were obtained as in  and Index Fungorum -Search Page.

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from fresh mycelia grown on PDA at room temperature. The EZ geneTM Fungal gDNA kit (GD2416) was used to extract DNA according to the manufacturer's instructions. ITS, LSU, TEF1-α, SSU gene regions were amplified using the primer pairs ITS5/ITS4, LROR/LR5, EF1-983F/EF1-2218R and NS1/NS4. The final volume of the PCR reaction was 25 µl and contained 12.5 µl of 2 × Power Taq PCR MasterMix (a premix and ready-to-use solution, including 0.1 Units/µl Taq DNA Polymerase, 500 µM dNTP Mixture each (dATP, dCTP, dGTP, dTTP), 20 mM Tris-HCl pH 8.3, 100 mM KCl, 3 mM MgCl , stabiliser and enhancer), 1 μl of each primer (10 μM), 1 µl genomic DNA extract and 9.5 µl deionised water. The PCR thermal cycle programme for ITS, LSU, TEF1α and SSU amplification was as follows: initial denaturation of 94°C for 3 minutes, followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 56°C for 50 seconds, elongation at 72°C for 1 minute and the final extension at 72°C for 10 minutes. PCR products were purified using minicolumns, purification resin and buffer according to the manufacturer's protocols (Amershamproduct code: 27-9602-01). The sequencing works were carried by Tsingke Biological Engineering Technology and Services Co. Ltd (Yunnan, P.R. China).

Phylogenetic analysis
Sequence data for relevant strains were downloaded from GenBank following recent publications . The consensus sequences were initially aligned using MAFFT v.7 (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013) and optimised manually when needed. The aligned dataset was analysed by Maximum Likelihood (ML) and Bayesian Inference (BI).
Maximum Likelihood analysis was performed using RAxMLGUI v.1.3 (Silvestro and Michalak 2011). The optimal ML tree search was conducted with 1,000 separate runs using RAxML tree generated from combined LSU, ITS, TEF1-α and SSU sequence data. Bootstrap support values for Maximum Likelihood (the first value) ≥ 75% and Bayesian posterior probabilities (the second value) ≥ 0.95 are placed near the branches as ML/BYPP. The tree is rooted to Periconia igniaria (CBS 379.86 and CBS 845.96). Newly-generated sequences are indicated in red and strains isolated from the holotype and reference specimens are indicated with a red superscript T. the default algorithm of the programme from a random starting tree for each run. The final tree was selected amongst suboptimal trees from each run by comparing the likelihood scores using the GTR+GAMMA substitution model. Maximum Likelihood bootstrap values equal to or greater than 75% were given as the first set of numbers above the nodes in the resulting ML tree (Fig. 1).
Bayesian analysis was conducted with MrBayes v.3.1.2 (Ronquist and Huelsenbeck 2003) to evaluate posterior probabilities (Rannala and Yang 1996) by Markov Chain Monte Carlo sampling (MCMC). The best-fit models of evolution were estimated by MrModeltest V.2.2 (Nylander and Uppsala University 2004). ITS, LSU and TEF selected the GTR+I+G model with inverse gamma-distributed rate in Bayesian analyses. SSU selected the GTR+G model with inverse gamma-distributed rate in Bayesian analyses. The ML analyses were conducted with RAxML v.7.2.6 (Stamatakis and Alachiotis 2010) using a GTRGAMMA substitution model with 1000 bootstrap replicates. The robustness of the analyses was evaluated by bootstrap support (MLBS). Six simultaneous Markov chains were run for 10 million generations and trees were sampled every 100 generation and 100,000 trees were obtained. The first 20,000 trees, representing the burn-in phase of the analyses, were discarded, while the remaining 80,000 trees were used to calculate posterior probabilities in the majority rule consensus tree (the critical value for the topological convergence diagnostic was 0.01). Through the posterior probabilities (PP) to reflect visually the reliability of each branch without the test for bootstrap method.
The phylogenetic trees were viewed and optimised in FigTree v.1.2.2 (Rambaut and Drummond 2008) and edited further using Microsoft Office PowerPoint. Newly-generated sequences in this study were deposited in GenBank (Table 1).  T   T   T   T   T   T   T   T   T   Table 1.
Isolates and sequences used in this study (newly-generated sequences are indicated in bold, strains isolated from the holotype and reference specimens are indicated in with a T, without GenBank accession numbers are indicated in "_") .
Culture characteristics: Conidia germinating on PDA within 24 h, germ tubes arising from the outermost cells of the conidium. Colonies on MEA covering 9 cm diam., in 4 weeks at 28°C. On the obverse, the edges are white and the middle is greyish-white. On the reverse, colonies appear pale yellow. Sporulation not observed in culture.

Etymology
Referring to the conidia with an apical mucilaginous sheath.
Culture characteristics: Conidia germinating on PDA within 24 h, germ tubes arising from the outermost cells of the conidium. Colonies on MEA covering 9 cm diam., in 4 weeks, at 28°C, white to cream. Sporulation not observed in culture.

Etymology
Referring to the species collected from aquatic habitats.

Notes
In the phylogenetic analysis, J. aquaticum nested in Jalapriya and sister to J. toruloides. Morphologically, J. aquaticum is similar to J. inflata in having 3-4 rows of conidia, but differs from J. inflat in the shape of the conidia, the cells of J. inflata are fuller and more three-dimensional. J. inflata arranged more loosely in the rows of conidia and J. aquaticum packed more tightly. J. aquaticum has larger conidia than those of J. inflata (22-53 × 16-24 vs. 28.5-38 × 14.5-21.5 μm). Jalapriya quaticum similar to J. pulchra in having appendages on the apical cells of the conidia, but differs in the rows of J. aquaticum not being separable without manual force.   Two newly-collected Jalapriya aquaticum isolates grouped with species of Jalapriya and basal to the genus with highly-supported value (100 ML/1.00 PP). Jalapriya apicalivaginatum formed a distinct lineage between J. toruloides and Jalapriya sp. (19VA07) with high bootstrap (97 ML/1.00 PP). Dictyocheirospora vinaya (HKAS 115802) clustered with its ex-type strains with high support (93 ML/1.00 PP).

Discussion
Dictyosporiaceae accommodates a holomorphic group of Dothideomycetes, including 18 genera , Rajeshkumar et al. 2021. Dictyocheirospora is the second largest genus of Dictyosporiaceae, followed by Dictyosporium. Dictyocheirospora is morphologically similar to Dictyosporium in having cheiroid, cylindrical conidia; However, Dictyocheirospora differs from Dictyosporium in having non-complanate conidia with arms arising from the basal cell and closely gathered at the apex and compact, while Dictyosporium has complanate conidia without separating arms. Thus, eight species were transferred from Dictyosporium to Dictyocheirospora, based on themorphological characters and phylogenetic analyses. , Yang et al. 2018. Dictyocheirospora is cosmopolitan in distribution and commonly reported from freshwater habitats in China, India, Japan and Thailand. Nine species of Dictyocheirospora were found on submerged decaying wood, others were found in terrestrial habitats. Currently, nine species have been discovered in China including Dictyocheirospora vinaya, which is mentioned in this article. , Yang et al. 2018, Tibpromma et al. 2018, Rajeshkumar et al. 2021).
Currently, three species are accepted in Jalapriya, of which, J. toruloides (Corda) is a terrestrial species discovered by Henningsson (1974) in Sweden; Afterwards it has been found in subtropical to temperate areas of both hemispheres, seemingly more often reported from coastal localities, considered an euryhaline species (Tibell et al. 2020), but our fresh collections are all from submerged wood in freshwater lotic habitats. In addition, both J. pulchra and J. acuaticum were all found in Yunnan Province, China on decaying wood submerged in a stream ( Table 2). The morphological differences between J. apicalivaginatum and J. pulchra are not significant, but they are phylogenetically distinct. Morphology of J. toruloides is not available; However, the new species J. aquaticum forms a distinct clade from J. toruloides. Jalapriya aquaticum is different from other species in Jalapriya and forms a separate branch with high support value (100% ML and 1.00 BYPP).