﻿Three novel species of Helminthosporium (Massarinaceae, Pleosporales) from China

﻿Abstract Three new species of Helminthosporium, H.nabanhensis, H.sinensis and H.yunnanensis collected on dead branches of unidentified plants in Xishuangbanna, China, were proposed by morphological and molecular phylogenetic analysis. Phylogenetic analysis of the combined data of ITS-SSU-LSU-TEF1-RPB2 sequences was performed using Maximum-Likelihood and Bayesian Inference, although H.nabanhensis and H.sinensis lack the RPB2 sequences. Both molecular analyses and morphological data supported H.nabanhensis, H.sinensis and H.yunnanensis as three independent taxa within the Massarinaceae.


Introduction
Helminthosporium Link was originally erected by Link (1809) with H. velutinum as the type species, and was mainly characterized by macronematous, cylindrical, septate conidiophores with polytretic conidiogenous cells that producing solitary (rarely in short chains), acropleurogenous, clavate or obclavate, distoseptate conidia with a flat, ringed pore at the base (Ellis 1961(Ellis , 1971Luttrell 1964;Seifert et al. 2011). The genus became a repository for a large amount of species due to a lack of understanding of the generic concepts. To date, about 770 epithets for Helminthosporium are listed in Index Fungorum (2022), but most of these were not congeneric with the generic type in development of conidia and conidiophores. Ellis (1961) provided a review on Helminthosporium, and accepted ten species. Luttrell (1963Luttrell ( , 1964 examined the type species and defined the generic concept, and Sivanesan (1987) transferred several unrelated pathogens of the Poaceae from Helminthosporium to the genera Cochliobolus (anamorph Bipolaris), Setosphaeria (anamorph Exserohilum) and Pyrenophora (anamorph Drechslera). Siboe et al. (1999) subsequently provided a synoptic table of the main morphological features that distinguish 27 accepted Helminthosporium species. Since then, 27 additional species have been described in the genus (Zhang et al. 2004(Zhang et al. , 2007Shirouzu and Harada 2008;Zhang and Zhang 2009;Zhang and Sun 2010;Zhao and Zhao 2012;Wang et al. 2014;Tanaka et al. 2015;Zhu et al. 2016;Alves-Barbosa et al. 2017;Tian et al. 2017;Crous et al. 2018Crous et al. , 2019Zhao et al. 2018;Boonmee et al. 2021;Chen et al. 2022). Voglmayr and Jaklitsch (2017) revealed the phylogenetic relationships of Corynespora, Exosporium and Helminthosporium species, synonymized Exosporium with Helminthosporium, and confirmed 17 species in Helminthosporium by morphological and molecular systematic analysis, but the generic concept has been widened by adding four Corynespora species that produce terminal, monotretic conidiogenous cells. So it is challenging to classify Corynespora and Helminthosporium species based on morphology alone because the distinction between monotretic vs. polytretic conidiogenous cells is the only character for separating Corynespora and Helminthosporium. Based on the records of Species Fungorum 2021, Konta et al. (2021) summarized the morphology, host information, locality, sequence data and related references of 216 Helminthosporium species reported worldwide. Unfortunately, sequence data for most species are unavailable, and only 27 species are represented by the DNA sequence in GenBank (Chen et al. 2022).
Xishuangbanna lies on the northern edge of tropical Southeast Asia. It is located in the southwestern part of Yunnan Province, China. It covers 19,125 km 2 and has a mountainous topography and humid tropical monsoon climate, with an av-erage annual temperature of 19.3-23.9 °C, and an average annual precipitation of 1200-1800 mm. The primary forest vegetation types are tropical seasonal rain forest, tropical montane rain forest, evergreen broad-leaved forest, monsoon forest over limestone, and monsoon forest on river banks (Cao and Zhang 1997). Such conditions create a very wide range of habitats favoring the growth of various microbial species. During our continuing mycological surveys of saprobic microfungi from plant debris in this region, three interesting hyphomycetes with morphological features typical of Helminthosporium were collected on dead branches. Based on morphological data and multi-locus phylogenetic analysis, they were described as new to science in the present study.

Sample collection, isolation and morphological studies
Samples of dead branches were collected from humid environments and river banks in the forest ecosystems of Xishuangbanna, Yunnan Province, China, and returned to the laboratory in Ziploc bags. Samples were processed and examined following the methods described in Ma et al. (2011). Fungi were mounted in a drop of lactic acid on microscope slides, and examined and photographed with an Olympus microscope (model BX 53), with a 100 × (oil immersion) objective at the same background color and scale. Adobe Photoshop 7.0 was used for image processing to assemble photographs into images. Single-spore isolations were made on potato dextrose agar (PDA) following Goh (1999). Colony colors were assessed according to the charts of Rayner (1970). All fungal strains were stored in 10% sterilized glycerin at 4 °C for further studies. The studied specimens and cultures were deposited in the Herbarium of Jiangxi Agricultural University, Plant Pathology, Nanchang, China (HJAUP).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from fungal mycelia grown on PDA, using the Solarbio Fungi Genomic DNA Extraction Kit following the manufacturer's protocol (Solarbio, China). The DNA amplification was performed by polymerase chain reaction (PCR) using the respective loci (ITS, SSU, LSU, TEF1, RPB2). Primer sets used for these genes were as follows: ITS: ITS5/ITS4 (White et al. 1990), SSU: 18S-F/18S-R, LSU: 28S1-F/28S3-R (Xia et al. 2017), TEF1: EF1-983F/EF1-2218R (Rehner 2001;Zhao et al. 2018) and RPB2: dRPB2-5f/dRPB2-7r (Voglmayr et al. 2016). The final volume of the PCR reaction was 25 μl, containing 1 μl of DNA template, 1 μl each of the forward and reverse primer, 12.5 μl of 2 × Power Taq PCR Master-Mix and 9.5 μl of double-distilled water (ddH 2 O). The PCR thermal cycling conditions of ITS, SSU and LSU were initialized at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 50 s, elongation at 72 °C for 1 min, a final extension at 72 °C for 10 min, and finally kept at 4 °C, the TEF1 and RPB2 were initialized at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at a suitable temperature for 30 s, elongation at 72 °C for 1 min, a final extension at 72 °C for 10 min, and finally kept at 4 °C. Annealing temperature was 60 °C for TEF1, 56 °C for RPB2. The PCR products were checked on 1% agarose gel electrophoresis stained with ethidium bromide. Purification and DNA sequencing were carried out at Beijing Tsingke Biotechnology Co., Ltd. China.

Sequence alignment and phylogenetic analysis
The newly generated sequences together with other sequences obtained from GenBank (Table 1) were initially aligned using MAFFTv.7 (Katoh and Standley 2013) on the online server (http://maffTh.cbrc.jp/alignment/server/), and optimized manually when needed. To establish the identity of the isolates at species level, phylogenetic analyses were conducted first individually for each locus and then as combined analyses of five gene loci (ITS, LSU, SSU, TEF1 and RPB2). Five aligned data sets of ITS, LSU, SSU, TEF1 and RPB2 are concatenated using the concatenated sequence function of Phylosuite software v1.2.1 (Zhang et al. 2020a), and absent sequence data (i.e., ITS, LSU, SSU, TEF1 and RPB2 sequence data) in the alignments were treated with the question mark as missing data. Phylosuite software v1.2.1 (Zhang et al. 2020a) was used to construct the phylogenetic tree based on ITS, SSU, LSU, TEF1 and RPB2 sequence data. The concatenated aligned dataset was analyzed separately using Maximum likelihood (ML) and Bayesian inference (BI). Maximum likelihood phylogenies were inferred using IQ-TREE (Nguyen et al. 2015) under Edge-linked partition model for 10000 ultrafast bootstraps (Hoang et al. 2017). The final tree was selected among suboptimal trees from each run by comparing the likelihood scores using the TIM2e+I+G4 for ITS+RBP2, TVMe+I+G4 for LSU+SSU, and TNe+R2 for TEF1 substitution model. Bayesian Inference phylogenies were inferred using MrBayes 3.2.6 (Ronquist et al. 2012) under partition model (2 parallel runs, 2000000 generations), in which the initial 25% of sampled data were discarded as burn-in. The best-fit model was SYM+I+G4 for ITS+RBP2, LSU+SSU; SYM+G4 for TEF1. ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the best-fit partition model (Edge-linked) using BIC criterion. The trees were viewed in FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and further edited in Adobe Illustrator 2021.
Cultural characteristics. Colony on PDA reaching 50-55 mm diam. after 2 weeks in an incubator under dark conditions at 25 °C, irregular circular, surface velvety, with white and denser mycelium at the center, becoming olivaceous and sparser towards the edge; reverse pale brown at the center, dark brown at the periphery.

Helminthosporium sinensis Jing W. Liu & Jian Ma, sp. nov.
IndexFungorum No: 559981 Fig. 3 Etymology. Referring to the country in which the fungus was collected.
Cultural characteristics. Colony on PDA reaching 75-82 mm diam. after 2 weeks in an incubator under dark conditions at 25 °C, irregular circular, surface velvety, with brown and denser mycelium at the center, becoming white and sparser towards the edge; reverse pale brown at the center, with little black dots.

Discussion
The taxonomic history of the genus Helminthosporium is complex. To date, about 770 epithets for Helminthosporium are listed in Index Fungorum (2022), but most of these were not congeneric with the generic type. Konta et al. (2021) listed 216 Helminthosporium species based on records from Species Fungorum, but most species are identified based on morphological studies, and so far only 27 species are represented by a DNA sequence in GenBank (Voglmayr and Jaklitsch 2017;Boonmee et al. 2021;Chen et al. 2022). Morphological comparison is important for fungal identification, but species identification only based on morphological studies is not comprehensive. With the availability of supplementary sequence data for Helminthosporium species, the molecular phylogenetic analysis is being used to evaluate previously described Helminthosporium-like species by molecular methods. The introduction of a phylogenetic analysis of Helminthosporium led to a better improvement of the heterogeneity of the genus and further clarified the taxonomic status of Helminthosporium. Voglmayr and Jaklitsch (2017) revisited Corynespora, Exosporium and Helminthosporium, with phylogenetic and morphological analyses. Zhang et al. (2020b) (Crous et al. 2018(Crous et al. , 2019Zhao et al. 2018;Boonmee et al. 2021;Chen et al. 2022). Based on previous studies, we proposed three new species by morphological and molecular phylogenetic analysis. Chen et al. (2022) described two new species, H. chengduense and H. chinense, based on combined ITS, LSU, SSU, TEF1 and RPB2 sequence data and morphological characters. Accordingly, we also used ITS, LSU, SSU, TEF1 and RPB2 for phylogenetic analysis and obtained high phylogenetic support, although our two species, H. nabanhensis and H. sinensis, lack the RPB2 sequences. They are considerably distinct from all other described Helminthosporium species by morphological characters and multi-locus phylogenetic analysis, so we are convinced that the newly introduced species are new to science.