Veronaeaaquatica sp. nov. (Herpotrichiellaceae, Chaetothyriales, Eurotiomycetes) from submerged bamboo in China

Abstract Background Freshwater fungi are highly diverse and ecologically important in freshwater systems. In China, more than 1000 species of freshwater fungi are known. Here, we present a brown-spored hyphomycetes that was collected on a submerged decaying bamboo culm in a forest stream in China. New information Phylogenetic analyses of combined LSU, ITS and TUB2 sequences confirm the placement of our new strain in Veronaea (Herpotrichiellaceae), sister to V.japonica. Veronaeaaquatica sp. nov. differs from related taxa V.compacta and V.japonica in having longer conidiophores and cylindrical to pyriform or subclavate conidia with 0–2 septa. Veronaeaaquatica also has darker brown hyphae compared to V.japonica. A morphological description and detailed illustrations of V.aquatica are provided.

). There are three records of Veronaea species from freshwater habitats, all on submerged wood. These are V. botryosa from Thailand (Dong et al. 2018), V. coprophila from the Republic of Seychelles (Hyde and Goh 1998) and V. oblongispora from Hong Kong (Tsui 1999). Since most Veronaea species lack molecular data, recollecting and sequencing are essential to investigate the phylogenetic relationships amongst species.
In the present study, we introduce Veronaea aquatica sp. nov., a freshwater species from submerged decaying bamboo culms collected in a stream in Jiangxi Province, China. A morphological description, illustrations and a multi-loci phylogeny are presented. The new species is compared with related taxa.

Sample collection and morphological examination
Submerged decaying bamboo culms were collected from a small forest stream in Lushan Mountains, Jiangxi Province, China in December 2017. Samples were incubated at room temperature for two weeks. Microscopic observation was conducted following Hu et al. (2010) and fungal characters were documented using a microscope. The holotype and extype living culture were deposited in the Herbarium of Fungi, Jiangxi Agricultural University (HFJAU), Nanchang-China and Jiangxi Agricultural University Culture Collection (JAUCC), respectively.

Fungal isolation
Single conidia were isolated in the potato dextrose agar (PDA) plate, following the method of Zhang et al. (2013). Germinated conidia were transferred to PDA plates and incubated at 16°C. Colonial characteristics were described after obtaining the pure cultures.

DNA extraction and PCR amplification
Mycelia were scraped off from six week-old colonies grown on PDA and transferred into a 1.5 ml centrifuge tube, followed by grinding in liquid nitrogen. DNA was extracted from the ground mycelium using the EZ gene TM fungal gDNA kit (GD2416) according to the manufacturer's instructions. The partial large subunit rDNA (LSU), internal transcribed spacer (ITS) and partial beta-tubulin (TUB2) were amplified using primer pairs LR0R/LR5, ITS1/ITS4 and T1/Bt2b, respectively (Vilgalys and Hester 1990, White et al. 1990, Hopple 1994, Glass and Donaldson 1995, Rehner and Samuels 1995, O'Donnell and Cigelnik 1997. The amplifications were performed according to Hu et al. (2012) as follows: initial denaturation at 94°C for 3 minutes; followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 50 seconds, elongation at 72°C for 1 minute; and a final extension at 72°C for 10 minutes. Purification of PCR products and sequencing, using the same primers, were outsourced to Tsingke Biological Technology Company (Beijing, China).

Phylogenetic analysis
Phylogenetic analyses were performed for both individual (LSU, ITS, TUB2) and combined (LSU-ITS-TUB2) datasets. Maximum Likelihood (ML) analyses were performed in the CIPRES Science Gateway v. 3.3 using the RAxML-HPC2 on XSEDE tool (Stamatakis et al. 2008, Miller et al. 2010, Stamatakis 2014 (Ronquist and Huelsenbeck 2003). Six simultaneous Markov Chains were run for 2,000,000 generations and trees were sampled every 100th generation. The first 2,000 trees were discarded as burn-in; the remaining 18,000 trees were used for calculating posterior probabilities (PP) (Cai et al. 2006

Etymology:
Referring to the aquatic habitat.
Veronaea aquatica shares the highest identity with V. japonica (CBS 776.83) in its LSU (99.65%) and ITS (98.08%). In its TUB2, it shares 89.61% identity with Exophiala brunnea (CBS 587.66). However, not enough TUB2 data are available to make conclusions about relationships, based on this gene region. Our tree topology (Fig. 1) is similar to Wang et al. (2019), although these authors did not include E. brunnea (CBS 587.66) in their analysis. In our study, E. brunnea (CBS 587.66) is clustered with V. compacta (CBS 268.75) with poor support (Fig. 1).
Species of Veronaea can be found on wood submerged in freshwater, in soil and on different terrestrial hosts. Fungi in the genus are saprobes (V. coprophila, V. japonica) or pathogens of plants (V. ficina, V. filicina) (Kharwar and Singh 2004, Arzanlou et al. 2007, Dong et al. 2018. Veronaea botryosa is a human pathogen, which causes phaeohyphomycosis disease (Kondo et al. 2007, Sang et al. 2011.
Veronaea is widely distributed across Australia, Brazil, China, Egypt, India, New Zealand, North America, South Africa and the UK (Dingley 1972, Papendrof 1976, Morgan-Jones 1982, Moustafa and Abdul-Wahid 1990, Kharwar and Singh 2004, Soares and Barreto 2008, Pan et al. 2009, Pan et al. 2012, Pan and Zhang 2010. This paper introduces a new species of Veronaea, bringing the number of species to twenty-one, based on morphology and molecular phylogenetic analyses. We compared the new species to the most related species in  (Fig. 1). These taxa are needed to be studied and identified in future research. The Kingdom of Fungi is an incredibly diverse group, with many taxa awaiting discoveriesincluding those from freshwater habitats. Exploring new fungal taxa, understanding their ecology and generating molecular phylogenetic data will promote fungal conservation (Cheek et al. 2020).