New species in Dictyosporium, new combinations in Dictyocheirospora and an updated backbone tree for Dictyosporiaceae

Abstract A survey of freshwater fungi on submerged wood in China and Thailand resulted in the collection of three species in Dictyocheirospora and four species in Dictyosporium including two new species in the latter genus. Morphological characters and phylogenetic analyses based on ITS, LSU and TEF1α sequence data support their placement in Dictyocheirospora and Dictyosporium (Dictyosporiaceae). An updated backbone tree is provided for the family Dictyosporiaceae. Descriptions and illustrations of the new taxa and re-collections are provided. Four new combinations are proposed for Dictyocheirospora.


Introduction
The family Dictyosporiaceae was introduced by Boonmee et al. (2016) to accommodate mostly aquatic lignicolous species with cheiroid, digitate, palmate and/or dictyosporous conidia and their sexual morphs that form a monophyletic clade in the class Dothideomycetes.
Dictyosporium, the type genus of the family, has been reported worldwide from dead wood and plant litter in terrestrial and aquatic habitats (Hyde and Goh 1998, Ho et al. 2002, Pinnoi et al. 2006, Pinruan et al. 2007). Corda (1836) established the genus with D. elegans Corda as the type species. The holomorph genus is characterised by dark brown, subglobose superficial ascomata, bitunicate cylindrical asci and hyaline, fusiform uniseptate ascospores with or without a sheath; sporodochial colonies, micronematous to macronematous conidiophores and cheiroid, digitate complanate conidia with several parallel rows of cells. Goh et al. (1999) reviewed the genus accepting 22 species and the remaining 16 species were doubtful or excluded. Tsui et al. (2006) first considered that the genus is closely related to Massarinaceae (Pleosporales) based on phylogenetic analysis using SSU and LSU sequence data. Tanaka et al. (2015) and Boonmee et al. (2016) confirmed the phylogenetic placement of Dictyosporium in Dictyosporiaceae (Massarineae, Pleosporales). Recent comparisons of Dictyosporium species were provided by Whitton et al. (2012), Prasher and Verma (2015) and Silva et al. (2015) with up to 48 accepted species. Since Silva et al. (2015),  (Prasher and Verma 2015, Tanaka et al. 2015, Abdel-Aziz 2016, Boonmee et al. 2016, da Silva et al. 2016, Alves-Barbosa et al. 2017, Zhang et al. 2017) and nine species were re-assigned to Dictyocheirospora, Jalapriya and Vikalpa (Boonmee et al. 2016). Wijayawardene et al. (2017a) provided information on the availability of cultures and references to accessible sequence data.
Dictyocheirospora was introduced by Boonmee et al. (2016) with Di. rotunda D'souza, Bhat & K.D. Hyde as the type species. Dictyocheirospora is morphologically similar to Dictyosporium except in having cheiroid, non-complanate or cylindrical conidia, mostly with conidial arms closely gathered together at the apex. Ten species are accepted in the genus including four species transferred from Dictyosporium (Boonmee et al. 2016, Wang et al. 2016, Li et al. 2017.
During a survey of freshwater fungi on submerged wood along a north/south gradient in the Asian/Australasian region , two new freshwater species and five previously described species were collected and identified based on phylogenetic analyses and morphological characters. We therefore introduce Dictyosporium tubulatum and Dictyosporium tratense as new species, with an illustrated account and phylogenetic evidence for the new taxa. An updated backbone tree based on the combined ITS, LSU and TEF1α sequence data is provided for Dictyosporiaceae. Four new combinations are proposed in Dictyocheirospora.

Collection and examination of specimens
Specimens of submerged, decaying wood were collected from streams in Chiang Rai, Prachuap Khiri Khan, Phang Nga and Trat Provinces, Thailand, in December 2014, April 2016and Guizhou Province, China, in October 2016. Specimens were brought to the laboratory in plastic bags and incubated in plastic boxes lined with moistened tissue paper at room temperature for one week. Morphological observations were made using a Motic SMZ 168 Series dissecting microscope for fungal structures on natural substrate. The fungal structures were collected using a syringe needle and transferred to a small drop of distilled water on a clean slide and covered with a cover glass. The fungi were examined using a Nikon ECLIPSE 80i compound microscope and photographed with a Canon 550D, 600D or 70D digital camera fitted to the microscope. Measurements were made with the TAROSOFT (R) IMAGE FRAME WORK programme and images used for figures were processed with ADOBE PHOTOSHOP CS6 software. Single spore isolations were made on to potato dextrose agar (PDA) or water agar (WA) and later transferred on to malt extract agar (MEA) or PDA following the method of Chomnunti et al. (2014). Specimens (dry wood with fungal material) are deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand and Kunming Institute of Botany, Academia Sinica (HKAS), China. Axenic cultures are deposited in Mae Fah Luang University Culture Collection (MFLUCC). Facesoffungi and Index Fungorum numbers are registered as outlined in Jayasiri et al. (2015) and Index Fungorum (2018).

DNA extraction, PCR amplification and sequencing
Isolates were grown on PDA and/or MEA medium at 25 °C for one month. Fungal mycelium was scraped off and transferred to a 1.5-ml microcentrifuge tube using a sterilised lancet for genomic DNA extraction. Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, China) was used to extract DNA following the manufacturer's instructions. ITS, LSU and TEF1α gene regions were amplified using the primer pairs ITS5 or ITS1 with ITS4 (Vilgalys and Hester 1990), LROR with LR5 or LR7 (White et al. 1990) and EF1-983F with EF1-2218R (Rehner 2001). The amplifications were performed in a 25 μl reaction volume containing 9.5 μl ddH 2 O, 12.5 μl 2 × Taq PCR Master Mix with blue dye (Sangon Biotech, China), 1 μl of DNA template and 1 μl of each primer (10 μM). The amplification condition for ITS, LSU and TEF1α consisted of initial denaturation at 94 °C for 3 min; followed by 40 cycles of 45 s at 94 °C, 50 s at 56 °C and 1 min at 72 °C and a final extension period of 10 min at 72 °C. Purification and sequencing of PCR products were carried out using the above-mentioned PCR primers at Sangon Biotech (Shanghai) Co. Ltd. in China.

Phylogenetic analyses
The taxa included in the phylogenetic analyses were selected and obtained from previous studies and GenBank (Boonmee et al. 2016, Wang et al. 2016, Li et al. 2017. Three gene regions (ITS, LSU and TEF1α) were used for the combined sequence data analyses. SEQMAN v. 7.0.0 (DNASTAR, Madison, WI) was used to assemble consensus sequences. The sequences were aligned using the online multiple alignment programme MAFFT v.7 (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013). The alignments were checked visually and improved manually where necessary.
Phylogenetic analysis of the sequence data consisted of maximum likelihood (ML) using RAxML-HPC v.8 (Stamatakis 2006, Stamatakis et al. 2008) on the XSEDE Teragrid of the CIPRES science Gateway (https://www.phylo.org) (Miller et al. 2010) with rapid bootstrap analysis, followed by 1000 bootstrap replicates. The final tree was selected amongst suboptimal trees from each run by comparing likelihood scores under the GTRGAMMA substitution model.
Maximum parsimony (MP) analyses were performed with PAUP v. 4.0b10 (Swofford 2003) using the heuristic search option with 1000 random taxa addition and tree bisection and reconnection (TBR) as the branch swapping algorithm. All characters were unordered and of equal weight and gaps were treated as missing data. Maxtrees were unlimited, branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. Clade stability was assessed using a bootstrap (BT) analysis with 1000 replicates, each with 10 replicates of random stepwise addition of taxa (Hillis and Bull 1993).
The programme MRMODELTEST2 v. 2.3 (Nylander 2008) was used to infer the appropriate substitution model that would best fit the model of DNA evolution for the combined datasets for Bayesian inference analysis with GTR+G+I substitution model selected. Posterior probabilities (PP) (Rannala andYang 1996, Zhaxybayeva andGogarten 2002) were determined by Markov Chain Monte Carlo sampling (MCMC) in MRBAYES v. 3.0b4 (Huelsenbeck and Ronquist 2001). Six simultaneous Markov chains were run for 1 million generations, with trees sampled every 100 generations (resulting in 10000 trees). The first 2000 trees, representing the burn-in phase of the analyses were discarded and the remaining 8000 trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree (Larget and Simon 1999).
The resulting trees were printed with FIGTREE v. 1.4.0 (http://tree.bio.ed.ac.uk/ software/figtree/) and the layout was created in MICROSOFT POWERPOINT for Mac v. 15.19.1. The alignment of phylogenetic analyses and resultant tree were deposited in TreeBASE (www.treebase.org, submission number 22802). Sequences generated in this study were submitted to GenBank (Table 1).
Cultural characteristics. Conidia germinating on PDA within 24 h and germ tubes produced from the basal cell. Colonies on MEA reaching 5-10 mm diam. in a week at 25 °C, in natural light, circular, with fluffy, dense, white mycelium on the surface with entire margin; in reverse yellow in the middle and white at the margin. Hyde in conidial ontogeny and conidial shape, colour and appendages. Dictyosporium tubulatum differs from the three species in the number of conidial cell rows. There are mostly four conidial columns in D. tubulatum while mostly five columns in the others. Dictyosporium tubulatum has smaller conidia (25-38 × 14-22 μm) than those in D. canisporum (32.5-47.5 × 20-25 μm) but has similar conidial size with D. alatum (26-32 × 15-24 μm) and D. thailandicum (15.4-34.5 × 14.5-20.6 μm) (Cai et al. 2003, Liu et al. 2015. Based on the molecular phylogeny, D. tubulatum is distinct from D. thailandicum and D. alatum. Unfortunately, molecular data are unavailable for D. canisporum. Etymology. Referring to the collecting site in Trat province, Thailand. Description. Saprobic on decaying plant substrates. Asexual morph: Colonies punctiform, sporodochial, scattered, black, glistening. Mycelium mostly immersed, composed of smooth, septate, branched, hyaline to pale brown hyphae. Conidiophores micronematous, mononematous, septate, cylindrical, hyaline to pale brown, smooth-walled, sometimes reduced to conidiogenous cells. Conidiogenous cells monoblastic, integrated, terminal, determinate, hyaline to pale brown. Conidia (40-)43-54(-57) × (20-)23-32(-36) μm (x = 49.5 × 26 μm, n = 40), acrogenous, solitary, cheiroid, smooth-walled, complanate, yellowish-brown to light brown, consisting of 39-68 cells arranged in 4-6 (mostly 5) closely compact columns, 9-11-euseptate in each column, guttulate; the inner columns nested within the outer columns, the outer columns derived from the basal cell of the conidium; the intermediate columns are derived from the first or second cell of the outer columns; the inner columns derived from the first or second cell of the intermediate columns; usually with 2-3 central columns longest and of equal length, 2-3 peripheral columns shorter and of equal length; sometimes with hyaline globose appendages at the apical cells of outer columns with hyaline cloud-shaped mucilaginous sheath. Sexual morph: Undetermined.
Cultural characteristics. Conidia germinating on PDA within 24 h and germ tubes produced from basal cell. Colonies on MEA reaching 5-10 mm diam. in a week at 25 °C, in natural light, circular, with fluffy, dense, pale yellow mycelium in the middle and sparse mycelium in the outer ring on the surface with irregular margin; in reverse, dark yellow to brown in the middle and pale yellow at the margin.
Notes. Phylogenetic analyses indicated Dictyosporium tratense nested within Dictyosporium and close to D. wuyiense. It is distinguished from the other species in the genus in having a mucilaginous sheath. Morphologically, D. tratense is most comparable to D. elegans in conidial colour and shape, but conidia of the new taxon (40-57 × 20-36 μm) are smaller than those of D. elegans (40-80 × 24-36 μm) (Goh et al. 1999 (Chen et al. 1991, Liu et al. 2015. Dictyosporium stellatum dif- fers from D. digitatum and D. aquaticum in lacking conidial appendages (Crous et al. 2011). In this case, it is difficult to identify our collection based on the recommendations advocated by Jeewon and Hyde (2016) for differentiating species or establishing new species. Thus, we recommend designating this collection as unknown species until enough evidence is available for its identification. Notes. Conidia in Dictyosporium nigroapice are characterised by conspicuously darker apical cells of the two inner arms, rarely darker at the apex of the outer arms. Morphological characters of this collection well agree with the original diagnosis of the holotype of D. nigroapice (Goh et al. 1999).

Discussion
Dictyosporiaceae accommodates a holomorphic group of Dothideomycetes, including 12 genera with nine being dictyosporous (Wijayawardene et al. 2017b, Wijayawardene et al. 2018. Dictyocheirospora and Dictyosporium are the two largest genera in the family. Dictyosporium has cheiroid, digitate and complanate conidia without separating arms, while Dictyocheirospora is characterised by non-complanate conidia with arms arising from the basal cell and closely gathered at the apex and compact. Thus, Dictyosporium hydei, D. indicum, D. musae and D. tetraploides are transferred to Dictyocheirospora based on the clear morphological characters. Phylogenetic analyses revealed the placement of Dictyocheirospora indica (MFLUCC 15-0056 reference specimen) within Dictyocheirospora. We believe that the other three species belong to Dictyocheirospora in having similar conidia and appendages to Dictyocheirospora indica, although molecular data are unavailable for them. specialist fungi associated with ants, Rhododendron species and Dracaena species" (grant no: DBG6080013) and "Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion" (grant no: RDG6130001) for supporting this study. Jing Yang thanks Shaun Pennycook for the corrections to the Latin names. Zong-Long Luo is acknowledged for the help with phylogenetic analyses.