Biodiversity Data Journal 9: e64505 doi: 10.3897/BDJ.9.e64505 Taxonomic Paper Veronaea aquatica sp. nov. (Herpotrichiellaceae, Chaetothyriales, Eurotiomycetes) from submerged bamboo in China Sajini K.U. Chandrasiri‡,§,|,¶, Yu-lin Liu§, Jun-En Huang‡, Milan C. Samarakoon|, Saranyaphat Boonmee|,¶, Mark S. Calabon|,¶, Dian-Ming Hu‡,§,# ‡ College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China § Jiangxi Environmental Engineering Vocational College, Ganzhou 341002, China | Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand ¶ School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand # Bioengineering and Technological Research Centre for Edible and Medicinal Fungi, Jiangxi Agricultural University, Nanchang 330045, China Corresponding author: Dian-Ming Hu (hudianming1@163.com) Academic editor: Danny Haelewaters Received: 16 Feb 2021 | Accepted: 26 Aug 2021 | Published: 23 Sep 2021 Citation: Chandrasiri SK.U, Liu Y-l, Huang J-E, Samarakoon MC, Boonmee S, Calabon MS, Hu D-M (2021) Veronaea aquatica sp. nov. (Herpotrichiellaceae, Chaetothyriales, Eurotiomycetes) from submerged bamboo in China. Biodiversity Data Journal 9: e64505. https://doi.org/10.3897/BDJ.9.e64505 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 brownspored 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. Veronaea aquatica sp. nov. differs from related taxa V. compacta and V. japonica in having longer © Chandrasiri S et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Chandrasiri S et al conidiophores and cylindrical to pyriform or subclavate conidia with 0–2 septa. Veronaea aquatica also has darker brown hyphae compared to V. japonica. A morphological description and detailed illustrations of V. aquatica are provided. Keywords one new taxon, hyphomycetes, molecular phylogeny, saprobe, taxonomy, freshwater fungi Introduction Freshwater fungi are those taxa that grow in freshwater bodies for the entirety or only part of their life cycle (Goh and Hyde 1996, Dhanasekaran et al. 2006). They are "recyclers" in that they decompose dead organic matter (Harms et al. 2011, Iskandar et al. 2011, Anastasi et al. 2013, Tsui et al. 2016, Grossart et al. 2019, Gulis et al. 2019). Freshwater fungi can be found in living (plants and animals) and non-living (decaying wood and leaves) substrates (Choi et al. 2019, Grossart et al. 2019, Tsui et al. 2000). They are accommodated in eight phyla: Aphelidiomycota, Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Monoblepharomycota, Mortierellomycota and Rozellomycota (Zhang et al. 2012, Bao et al. 2019, Calabon et al. 2020, Hyde et al. 2021). The majority of described freshwater fungi are members of Dothideomycetes and Sordariomycetes; however, several taxa have been recorded from Eurotiomycetes (Liu et al. 2015, Luo et al. 2016, Luo et al. 2018b, Luo et al. 2019, Dong et al. 2018, Dong et al. 2020a, Wang et al. 2019). Coreomyces chinensis and C. minor (Laboulbeniaceae, Laboulbeniales, Laboulbeniomycetes) were the first freshwater taxa reported from China (Thaxter 1931, Hu et al. 2013). During the past two decades, studies have used combined morphological and molecular data to describe freshwater taxa from China (Tsui et al. 2000, Li et al. 2017, Luo et al. 2017 , Luo et al. 2018a, Luo et al. 2018b, Luo et al. 2019, Su et al. 2018, Bao et al. 2020, Lu et al. 2020). Hu et al. (2013) reviewed the biodiversity of freshwater fungi in China and reported 782 species. Since Hu et al. (2013), this number has increased to more than 1,000 (Luo et al. 2019, Bao et al. 2020, Dong et al. 2020a, Dong et al. 2020b). Veronaea (Herpotrichiellaceae, Chaetothyriales) was introduced by Cifferi and Montemartini (1958) and is typified by V. botryosa, which was isolated from a decomposed rachis of a palm (Arecaceae) in Italy (Arzanlou et al. 2007). Twenty species have been described in Veronaea, but sequences are only available for four of them (V. botryosa, V. compacta, V. constricta and V. japonica) (Wijayawardene et al. 2020). Only the asexual morphs of Veronaea are presently known and they are related to black yeasts (Vicente et al. 2008, Badali et al. 2013, Bonifaz et al. 2013, Döğen et al. 2013). Species of Veronaea are characterised by polyblastic, terminally integrated, cylindrical, solitary, pale brown conidiogenous cells and smooth-walled, septate, cylindrical to pyriform pale brown to brown conidia (Arzanlou et al. 2007, Vicente et al. 2008, Badali et al. 2013, Veronaea aquatica sp. nov. (Herpotrichiellaceae, Chaetothyriales, Eurotiomycetes) ... 3 Döğen et al. 2013). 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. Materials and methods 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). 4 Chandrasiri S et al Sequencing and sequence alignment Consensus sequences were obtained using Lasergene SeqMan Pro v. 7. BLASTn searches were performed to identify highly similar sequences in NCBI GenBank. Other sequences, used in the phylogenetic analyses (Table 1), were downloaded from NCBI GenBank, based on recently-published data (Dong et al. 2018, Wijayawardene et al. 2020). Single-locus alignments were generated with MAFFT v. 7.036 (http://mafft.cbrc.jp/alignment/server; Katoh et al. 2019). Alignments were further improved manually when necessary in BioEdit v. 7.0.5.2 (Hall 1999). Ambiguous bases were removed using TrimAl v. 1.3 and the gappyout option (Capella-Gutiérrez et al. 2009). Table 1. Table of taxa used in this study and GenBank accession numbers of DNA sequences. The new strain is indicated in bold and type strains are indicated with an asterisk (*). Name Strain Number Gene bank accession number LSU ITS TUB2 - Aculeata aquatica * MFLUCC 11–0529 MG922575 MG922571 Brycekendrickomyces acaciae CBS 124104 NG_058633 NR_132828 - Byssochlamys lagunculariae * CBS 100.11 NG_058631 NR_144910 AY753354 Capronia pilosella AFTOL-ID 657 DQ823099 DQ826737 Cladophialophora carrionii* CBS 160.54 NG_055741 NR_121267 EU137201 Exophiala aquamarina FMR 3998 KU705846 KU705829 E. aquamarina* CBS 119918 - NR_111626 JN112434 E. brunnea CBS 587.66 MH870554 MH858890 JN112442 E. equina CBS 116009 KF928497 KF928433 KF928561 E. equina * CBS 119.23 - NR_111627 JN112462 E. jeanselmei* CBS 507.90 MH873915 MH862234 E. nigra* CBS 535.94 NG_059253 NR_154974 - E. pisciphila AFTOL-ID 669 DQ823101 - - E. psychrophila* CBS 191.87 MH873750 MH862061 JN112497 CBS 157.67 AY213702 NR_121270 JN112499 CBS 115831 FJ358246 - CBS 118157 - NR_111203 DQ182571 Fonsecaea monophora CBS 102243 FJ358247 EU938579 EU938542 F. pedrosoi BMU 07690 KJ930165 KJ701014 KM658155 E. salmonis * E. xenobiotica E. xenobiotica * - - EF551501 - Veronaea aquatica sp. nov. (Herpotrichiellaceae, Chaetothyriales, Eurotiomycetes) ... Name Strain Number 5 Gene bank accession number LSU ITS CBS 271.37 - NR_130652 - MFLUCC 16–1449 - NR_164246 - Melanoctona tectonae* MFLUCC 12–0389 KX258779 KX258778 - Metulocladosporiella musae CBS 113863 DQ008162 DQ008138 - Paecilomyces fulvus * CBS 146.48 NG_063990 NR_103603 FJ389986 Phialophora verrucosa BMU 07618 KJ930128 KJ700977 P. verrucosa* CBS 140325 - NR_146242 - Rhinocladiella atrovirens CBS 317.33 MH866906 MH855447 - Thysanorea lotorum* CBS 235.78 MH872892 MH861130 - T. papuana* CBS 212.96 EU041871 EU041814 - T. rousseliana CBS 126086 MH875246 MH863784 - Veronaea aquatica JAUCC2549 MW046893 MW046892 MW248394 V. botryosa CBS 102593 KF928493 KF928429 KF928557 V. botryosa CBS 122236 KF928491 KF928427 KF928555 V. botryosa MFLUCC 11–0072 MG922574 EU041817 - V. botryosa * CBS 254.57 EU041873 EU041816 JN112505 V. compacta * CBS 268.75 EU041876 EU041818 - V. constricta CBS 572.90 MH873920 EU041819 - V. japonica CBS 776.84 NG_057789 EU041821 - V. japonica * CBS 776.83 EU041875 EU041820 - Veronaea sp. DS253 - MK808629 - Veronaea sp. E6917h - HM992819 - Veronaea sp. HB - KR909168 - Veronaea sp. NWHC 24266–02–03–03 - KX148688 - Veronaea sp. NWHC 24266–02–04–01 - KX148689 - F. pedrosoi * Marinophialophora garethjonesii* TUB2 KM658080 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). For each single-locus sequence alignment, Chandrasiri S et al 6 GTRGAMMA + I was selected as the best-fit model in MrModeltest 2.3 (Nylander 2004). Bayesian (BYPP) analysis was performed using MrBayes v. 3.1.2. for the combined dataset (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). Phylograms were visualised using FigTree v. 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/). Modification of the final phylogenetic tree was done in Microsoft PowerPoint. Taxon treatment Veronaea aquatica Chandrasiri, J.E. Huang & D.M. Hu, sp. nov. • • IndexFungorum IF558295 Facesoffungi FoF 05435 Materials Holotype: a. kingdom: Fungi; phylum: Ascomycota; class: Eurotiomycetes; order: Chaetothyriales; family: Herpotrichiellaceae; taxonRank: species; genus: Veronaea; specificEpithet: aquatica; country: China; stateProvince: Jiangxi Province; locality: Lushan Mountains; verbatimElevation: 675; verbatimLatitude: 29°55'72''N; verbatimLongitude: 115°94'86''E; year: 2017; month: December; day: 31; habitat: stream in small forest, on submerged decaying bamboo culms; fieldNotes: Freshwater; recordedBy: J.E. Huang; identifiedBy: Sajini K. U. Chandrasiri; institutionID: HFJAU 0739; institutionCode: Herbarium of Fungi, Jiangxi Agricultural University; collectionCode: HJ054 Other material: a. type: ex-type living culture; collectionID: JAUCC2549; collectionCode: Jiangxi Agricultural University Culture Collection Description Saprobic on submerged decaying bamboo (Fig. 2). Sexual morph: Undetermined. Asexual morph: Hyphomycetous. Colonies effuse, spreading very widely, brown to dark brown, white hairy. Mycelium in the wood immersed or partly superficial, hyphae subhyaline to pale olivaceous, smooth, 1.5–3 μm wide. Conidiophores erect, the lower part is usually straight and the upper half is usually flexuose, usually loosely branched, macronematous, monomenatous, sometimes geniculate, smooth-walled, near the apex pale brown, dark brown at the middle and base, 2.5–4 μm wide and up to 280 μm long. Conidiogenous cells terminally integrated, polyblastic, occasionally intercalary, cylindrical, (3–)10–30 × 2–3.5 µm (x̄ = 16.5 × 2.5 μm, n = 30), variable in length, pale brown, later often becoming septate, fertile part subhyaline, wide at the basal part, rachis with crowded, flat to slightly prominent, faintly pigmented; scars flat, slightly pigmented, not thickened, about 0.65 μm diam. Conidia solitary, smooth, cylindrical to subpyriform and some subclavate, 6–11(–12) × 2.5–3.5(–4.0) µm (x̄ = 8.7 × 3.1 μm, n = Veronaea aquatica sp. nov. (Herpotrichiellaceae, Chaetothyriales, Eurotiomycetes) ... 7 50), pale brown, most medially 1-spetate, rarely 0 or 2-septate, often constricted at the septum and the colour septum middle brown and the conidia with a round apex and truncate base; with a faintly darkened, unthickened hilum, about 0.5–0.9 μm diam. Figure 1. Phylogenetic tree generated from RAxML analysis of a combined ITS, LSU and TUB2 dataset. ML bootstrap (BS) support values ≥ 60% and Bayesian PP ≥ 0.95 are indicated above branches as MLBS/PP. Paecilomyces fulvus (CBS 146.48) and Byssochlamys lagunculariae (CBS 100.11) serve as outgroup taxa. Type strains are highlighted in bold; the new species is shown in blue bold. 8 Chandrasiri S et al Figure 2. Veronaea aquatica (HFJAU0739, holotype) a Submerged bamboo; b Colonies on submerged bamboo culm; c, d Colony on PDA from above and below; e–h Conidiophores, conidiogenous cells and conidia; note the scars (arrowed in e, d); i–k Conidia. Scale bars: b = 100 μm, e–k = 10 μm. Culture characteristics: Conidia germinating on PDA within 24 hrs. Colonies growing on PDA, circular, reaching 10–20 mm diam. after 2–3 weeks at 28°C, from above flat, dense, olivaceous to medium brown, lightly raised at centre, surface rough; from below medium to dark brown. Etymology: Referring to the aquatic habitat. Notes Veronaea aquatica is morphologically most similar to V. japonica and V. botryosa. However, V. aquatica has 0–2-septate conidia, whereas those of V. japonica are 0–1septate. In addition, the conidiophores of both V. compacta (up to 50 μm) and V. japonica (up to 65 μm) are shorter compared to V. aquatica (280 μm). Veronaea aquatica has conidiogenous cells that are 10–30 μm in length, while those of V. botryosa are 100 μm long. In addition, the conidiophores of V. aquatica are 280 μm long; they are shorter in V. botryosa (73–124 μm) (Fig. 2). 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). Veronaea aquatica sp. nov. (Herpotrichiellaceae, Chaetothyriales, Eurotiomycetes) ... 9 Analysis Phylogenetic analyses The final aligned concatenated dataset (LSU, ITS, TUB2) was comprised of 44 strains including two outgroup taxa, Byssochlamys lagunculariae (CBS 100.11) and Paecilomyces fulvus (CBS 146.48) (Aspergillaceae) and 734 distinct alignment patterns, with 23.30% of undetermined characters or gaps. The best-scoring RAxML tree (-lnL = 12666.921) is shown in Fig. 1. Tree topologies from ML and Bayesian analyses were congruent; no significant differences were observed at the generic level. Veronaea aquatica (JAUCC2549) was retrieved as sister to V. japonica with high support (MLBS = 95%, PP = 1.00). Discussion The family Herpotrichiellaceae (Eurotiomycetes) was introduced by Munk (1953) and currently includes 16 genera (Wijayawardene et al. 2020). The anamorph–teleomorph relationships within Herpotrichiellaceae were described by Müller et al. (1987) and Untereiner et al. (1995). Most anamorphs are dematiaceous and opportunistic fungi (Capronia, Cladophialophora, Exophiala, Veronaea) ( Untereiner et al. 1995, Crous et al. 2007). 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, Bonifaz et al. 2013). 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 Table 2. Several unidentified Veronaea species have also been isolated, such as Veronaea sp. DS253 (from root of Bouteloua dactyloides), Veronaea sp. E6917h (from Socratea exorrhiza), Veronaea sp. HB (from grapevine), Veronaea sp. [NWHC 24266–02–03–03; NWHC 24266–02–04–01 (from snake)] (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 discoveries— including those from freshwater habitats. Exploring new fungal taxa, understanding their ecology and generating molecular phylogenetic data will promote fungal conservation (Cheek et al. 2020). 10 Chandrasiri S et al Table 2. Synopsis of related species Name Conidiophore Conidiogenous cell Conidia Conidial septation References Exophiala brunnea Branched 8-350 µm long Occasionally intercalary, variable in shape, flaskshaped, ovoid, oblong, symmetrical or curved, fimbriate Cylindrical to pyriform, aseptate proximally tapered and usually slightly stipitate 4.5-10 x 2-3 µm Veronaea aquatica Loosely branched, sometimes geniculate, up to 280 × 2.4–4 µm Occasionally intercalary, scars flat, rachis with crowded, flat to slightly Cylindrical to pyriform, some subclavate, rounded at the apex 6.3–11(–11.8) × 2.4– 3.7(–4.0) µm 0–1(–2) This study Veronaea botryosa Unbranched Integrated, occasionally 73–124.5 × 2–3 µm interspersed, flat to slightly prominent denticles, rachis with crowded Ellipsoidal or fusiform, rounded at the apex (3–)6.5–8.5(–12) × (1.5–)2–2.5(–3) µm 1(–2) Bonifaz et al. (2013), Dong et al. (2018) V. compacta Unbranched or branched at acute angles, rarely exceeding 50 µm Occasionally intercalary, integrated, hardly prominent denticles, scars flat 0–1(–2) Ellipsoidal to ovoid or oblong to subcylindrical, rounded at the apex, acropleurogenous (4–)6–7(–9) × 2–3 µm V. japonica Unbranched or occasionally branched 65 × 2–3 µm Occasionally intercalary, hardly prominent denticles, scars flat, slightly pigmented Ellipsoidal to ovoid, rounded at the apex (6–)7–8(–10) × 2– 2.5(–4) µm (0–)1 Arzanlou et al. (2007) V. oblongispora 320 × 3–5 µm Integrated, polyblastic, bearing thin, flat conidial scars Oblong, obtuse at the apex 7–8 ×4–5 µm aseptate Morgan-Jones 1982 Papendorf (1969) Papendrof (1976) Acknowledgements We would like to thank the National Natural Science Foundation of China (NSFC 32070023), the Key Project of Jiangxi Provincial Department of Science and Technology Youth Fund (20192ACBL21017), the Key Research and Development Plan of Jiangxi Province (20161BBF60078), the Natural Science Foundation of Education Department of Jiangxi Province (GJJ190168) and Thailand Research Fund (RDG6130001) for financial support. Saranyaphat Boonmee would like to thank the Mae Fah Luang University Fund (No. 631C15001). K.S.U. Chandrasiri would like to express her profound gratitude to S.M.B.C. Samarakoon, N. Wijesinghe and W.A.E. Yasanthika for their valuable support. Veronaea aquatica sp. nov. 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