Multigene phylogeny and taxonomy of Dendryphion hydei and Torula hydei spp. nov. from herbaceous litter in northern Thailand

During our studies on asexual fungi colonizing herbaceous litter in northern Thailand, we discovered two new fungal species, viz. Dendryphion hydei and Torula hydei spp. nov. The latter are examined, and their morphological characters are described as well as their DNA sequences from ribosomal and protein coding genes are analysed to infer their phylogenetic relationships with extant fungi. Torula hydei is different from other similar Torula species in having tiny and catenate conidia. Dendryphion hydei can be distinguished from other similar Dendryphion species in having large conidiophores and subhyaline to pale olivaceous brown, 2–4(–5)-septate conidia. Multigene phylogenetic analyses of a combined LSU, SSU, TEF1-α, RPB2 and ITS DNA sequence dataset generated from maximum likelihood and Bayesian inference analyses indicate that T. hydei forms a distinct lineage and basal to T. fici. Dendryphion hydei forms a distinct lineage and basal to D. europaeum, D. comosum, D. aquaticum and D. fluminicola within Torulaceae (Pleosporales, Dothideomycetes).

Dendryphion Wallr. was introduced by Wallroth [21] to accommodate hyphomycetous species, D. comosum Wallr. The genus is commonly known to be saprobic on dead stems of herbaceous plants and decaying wood, and is characterized by having erect, solitary, branched in upper part, polytretic conidiophores, forming septate, pigmented, thick-walled, finely roughened stipe and a distinct conidiogenous apparatus, with dark scars and catenate, in simple or branched chains of brown, septate (didymo-or cheiro) conidia [8,7]. Crous et al. [3] introduced D. europaeum Crous & R.K. Schumacher based on morphological characteristics and molecular data and later Crous et al. [8] accommodated the species in Torulaceae and further accepted Dendryphion in Torulaceae. Su et al. [6] circumscribed genera of Torulaceae from freshwater. Only seven Dendryphion species have DNA sequence data and their phylogenetic affinities to members of the Torulaceae have been investigated.
In this study, a novel Torula species was isolated from herbaceous litters collected from northern Thailand. Among collected samples, Dendryphion hydei is also recovered as another new species from northern Thailand. These species are described and illustrated. In addition, an updated phylogenetic tree with our new taxa for the family Torulaceae is provided in this study.

Isolation and identification
The specimens were collected from herbaceous litters (Chromolaena odorata Linn. and Bidens pilosa Linn.) in northern Thailand during the year 2015 to 2016. Samples were returned to the laboratory (Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand) for examination and description of morphological characteristics. The specimens were observed under a Motic SMZ 168 series dissecting stereomicroscope. The conidial structures were picked up by a sterilized surgical needle and transferred into 10% lacto-glycerol on a clean slide and examined under a Nikon Eclipse 80i compound microscope and photo-captured with a Canon 600D digital camera using DIC microscopy. Macro-morphological structures were photographed with a Discovery V.8 stereo microscope fitted with a CARL ZEISS Axio Cam ERc5S microscope camera. Tarosoft1 Image Frame Work program v.0.9.0.7 and Adobe Photoshop CS5 Extended version 10.0 software (Adobe Systems Inc., The United States) were used for measurements and drawing photographic plates.
Single conidium isolation was carried out to obtain pure cultures as described in Dai et al. [22]. Germinating conidia were transferred aseptically to potato dextrose agar (PDA) and malt extract agar (MEA) plates and grown at room temperature (16-30˚C) in alternating day and night light. Colony characters were observed and recorded after one week and at weekly intervals [23,24].
The type specimens were deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand and the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (KUN-HKAS), Yunnan, China. Ex-type living cultures were deposited in Mae Fah Luang University Culture Collection (MFLUCC 18-0250 and MFUCC 18-0236) and Kunming Institute of Botany Culture Collection (KUMCC 16-0037 and KUMCC 18-0009). Faces of Fungi and Index Fungorum numbers are registered as outlined in Jayasiri et al. [25] and Index Fungorum [26]. New species are established based on guidelines of Jeewon and Hyde [27].

DNA extraction, PCR amplification and sequencing
Fungal mycelium was scraped off and transferred to a 1.5 ml micro-centrifuge tube using a sterilized lancet for genomic DNA extraction. The Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux1, P.R. China) was used to extract fungal genomic DNA, following the protocols in the manufacturer's instructions.
DNA amplification was performed by polymerase chain reaction (PCR) using the following genes (ITS, LSU, SSU, RPB2 and TEF1-α). The primers ITS5 and ITS4 primer pairs were used to amplify the ITS and 5.8S regions of the rDNA gene [28]; The primers LR0R and LR5 were used to amplify the partial ribosomal RNA for the 28S nuclear large subunit (LSU) [29]; NS1 and NS4 were used to amplify the partial ribosomal RNA for the 18S nuclear small subunit (SSU) [28]; fRPB2-5F and fRPB2-7cR were used to amplify the partial RNA polymerase second largest subunit (RPB2) [30] and EF1-983F and EF1-2218R were used to amplify the translation elongation factor 1-alpha gene (TEF1-α) [31].
The final volume of the PCR reaction was 25 μl, containing 1 μl of DNA template, 1 μl of each forward and reward primer, 12.5 μl of 2×Easy Taq PCR SuperMix (mixture of Easy-TaqTM DNA Polymerase, dNTPs, and optimized buffer, Beijing TransGen Biotech Co., Ltd., Beijing, P.R. China) and 9.5 μl of ddH 2 O. The PCR thermal cycling conditions of ITS, LSU, SSU and TEF1-α were as follows: 94˚C for 3 minutes, followed by 35 cycles of denaturation at 94˚C for 30 seconds, annealing at 55˚C for 50 seconds, elongation at 72˚C for 1 minute, and a final extension at 72˚C for 10 minutes. The PCR thermal cycle program for RPB2 was as follows: initial denaturation at 95˚C for 5 minutes, followed by 40 cycles of denaturation at 95˚C for 1 minute, annealing at 52˚C for 2 minutes, elongation at 72˚C for 90 seconds, and final extension at 72˚C for 10 minutes. Purification and sequencing of PCR fragments with PCR primers mentioned above were carried out at Shanghai Majorbio Biopharm Technology Co., Ltd, China.

Sequence alignment and phylogenetic analyses
Phylogenetic analyses were performed from single gene (LSU dataset) as well as based on a combined LSU, SSU, TEF1-α, RPB2 and ITS sequence dataset. Sequences generated from this study were analyzed with other similar sequences obtained from GenBank and those derived from recent publications [32,10,19,9,5,6,7] (Table 1). The single gene alignment was performed by using MAFFT v. 7 [33] (http://mafft.cbrc.jp/alignment/server/) and manually aligned wherever necessary in MEGA version 7.0 [34]. Further analyses for the combined dataset were  [39,40] following the methodology in Li et al. [5].
The phylogram was represented in Treeview [57] and drawn in Microsoft PowerPoint and converted to jpeg file in Adobe Photoshop version CS5 (Adobe Systems Inc., the United States). The new sequences were submitted in GenBank ( Table 1). The alignment was deposited in TreeBASE [58] under the accession number 25462.

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies. In addition, new names contained in this work have been submitted to Index Fungorum from where they will be made available to the Global Names Index. The unique Index Fungorum number can be resolved and the associated information viewed through any standard web browser by appending the Index Fungorum number contained in this publication to the prefix www.indexfungorum.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central and LOCKSS.

Compliance with ethical standards
There is no conflict of interest (financial or non-financial) and all authors have agreed to submission of paper. The authors also declare that they have no conflict of interest and confirm that the field studies did not involve endangered or protected species.

Phylogenetic analyses
The combined LSU, SSU, TEF1-α, RPB2 and ITS sequence dataset comprises 71 taxa with Occultibambusa bambusae (MFLUCC 13-0855) and Neooccultibambusa chiangraiensis (MFLUCC 12-0559) as the outgroup taxa. Bayesian Inference (BI) and maximum likelihood (ML) analyses of the combined dataset were performed to determine the placement of our new taxa and infer relationships at the intrageneric level as well as resolving the phylogenetic relationships of the core families in Pleosporales. The phylogenetic trees obtained from BI and ML analyses resulted in trees with largely similar topologies and also similar to those generated from previous studies based on maximum likelihood analysis [18,5,7]. The best scoring RAxML tree is shown in Fig 1,  Most of the core genera of Torulaceae and other representative genera in Nigrogranaceae, Ohleriaceae, Roussoellaceae and Thyridariaceae are included in our phylogenetic analysis (Fig  1). Torulaceae formed a well-resolved clade (100% ML and 1.00 PP) with a close relationship to Roussoellaceae and Thyridariaceae. Species of different genera currently accommodated in Torulaceae formed well-resolved subclades except for Sporidesmioides which is recovered as basal to other genera with significant Bayesian support (1.00 PP) but with low support in ML analysis (56% ML). Torula is recovered as a strongly monophyletic genus in Torulaceae. Torula hydei is sister to T. fici with high support (100% ML and 1.00 PP). Notes-Dendryphion hydei is unique in having large conidiophores and subhyaline to pale olivaceous brown, 2-4(-5)-septate conidia to compare with other related species in Dendryphion. Dendryphion hydei resembles D. aquaticum and D. europaeum in morphology. However, these species can be distinguished based on the size of the conidiophores, conidiogenous cells and conidia, as well as conidial septation and habitats (see Table 2). Dendryphion hydei has 2-4(-5)-septate conidia and inhabit in a terrestrial environment, similar to D. europaeum. However, D. europaeum has smaller conidiophores and conidia, and the conidia of D. europaeum are (2-)3(-5)-septate while D. aquaticum inhabits in a freshwater environment and has 3-6-septate conidia [3,7]. In the phylogenetic tree, D. hydei forms a separate lineage and clustered with D. europaeum, D. comosum, D. aquaticum and D. fluminicola with significant support in Bayesian inference analysis (1.00 PP). A comparison of TEF1-α nucleotides shows that D. hydei differs from D. fluminicola in 20/852 bp (2.3% difference, no gap) and from D. submersum in 30/902 bp (3.3% difference, no gap). A comparison of ITS nucleotides shows that D. hydei differs from D. europaeum in 19/553 bp (3.4% difference, no gap) and differs from D. aquaticum in 6/398 bp (1.5% difference, no gap). Phylogenetic analyses support D. hydei as a new species in Dendryphion. These tally with recommendations outlined by Jeewon and Hyde [27] to establish our new species. In this study, we collected D. hydei from Bidens pilosa, which is a new host record for this species. A morphometric comparison of the new taxon with other similar taxa of Dendryphion provide in Table 2. Notes-Torula hydei resembles T. herbarum and T. fici in having 2-3-septate, catenated, brown, verruculose conidia, but differs in having smaller conidia [3]. Phylogenetic analyses showed that T. hydei constitutes an independent lineage basal to T. fici (100% ML and 1.00 BYPP). Morphologically T. hydei differs from T. fici in having smaller conidia (T. hydei, (7.5-) 8-14 × 2-4 μm versus (12-)13-17(-19) × 5(-6) μm, T. fici) and the conidia are also brown to dark brown, paler at the apex where branching occurs [8]. Whereas, T. fici has brown conidia, with a pale brown apex and the fertile cells in the conidial chain, where branching occurs, are darker brown than other cells [8]. The conidiogenous cells of T. fici are slightly larger than T. hydei and frequently clavate (T. fici, (5-)6(-8) × 5(-7) μm versus 3-5.5 × 4.3-5 μm, T. hydei), whereas, T. hydei has doliiform to ellipsoid conidiogenous cells [8]. We also note distinct nucleotide base pair differences between T. hydei and T. fici (CBS 595.96, type strain) across the ITS gene region (8/479 bp, 1.7% difference, no gap) and TEF1-α gene region analysed (43/ 760 bp, 5.7% difference, no gap). Based on distinct morphological characteristics and phylogenetic support, T. hydei is introduced as a new species in this study.

Discussion
Taxonomic characterizations of taxa in Torulaceae have been well-studied since Crous et al. [8] who re-classified Torula and Dendryphion in Torulaceae (Pleosporales, Dothideomycetes) based on phylogenetic analyses of LSU sequence data. Subsequent authors introduced new genera and species in this family based on multigene phylogenetic analyses coupled with morphological characteristics (see Table 3) [10,18,9,5,6,7,20]. Recently, there are more than 520 epithets in the genus Torula and 85 epithets in Dendryphion listed in Index Fungorum [26]. However, most of the described species lack DNA sequence data to verify their phylogenetic placement and affinities with other related fungi. Nevertheless, many species previously described as Torula and Dendryphion have also been synonymized to many genera in Sordariomycetes [26]. Taxa in these genera need to be clarified based on molecular data.  (HKAS 97478, holotype). a Colonies on dead branch of Chromolaena odorata. b-e Conidiophores with conidiogenous cell. f-j Budding on conidia. k, l Conidia in chain. m-t Conidia. Scale bars: a = 100 μm, b, k-l = 5 μm, c, f-j, q-t = 2 μm, d, e, m-p = 1 μm.
https://doi.org/10.1371/journal.pone.0228067.g003 Torula and Dendryphion have a wide host range in various habitats and are commonly found as saprobes in both terrestrial and aquatic habitats in temperate to tropical regions [10,3,59,18,9,5,6,7,20]. It is interesting to note that many Torula species have been found to be associated with the host family Asteraceae [59,5]. In this study, our new strains were collected from Asteraceae and Li et al. [5] also reported two novel Torula species, T. chromolaenae and T. mackenziei from Asteraceae, indicating that Asteraceae harbors a diversity of these taxa. Dendryphion hydei was also collected from Bidens pilosa (Asteraceae) and is the first record from northern Thailand.