﻿Three novel species of Aquapteridospora (Distoseptisporales, Aquapteridosporaceae) from freshwater habitats in Tibetan Plateau, China

﻿Abstract During an investigation of lignicolous freshwater fungi in the Tibetan Plateau, three Aquapteridospora taxa were collected from freshwater habitats in Xizang, China. The new species possess polyblastic, sympodial, denticles conidiogenous cells and fusiform, septate, with or without sheath conidial, that fit within the generic concept of Aquapteridospora, and multi-gene phylogeny placed these species within Aquapteridospora. Detailed morphological observations clearly demarcate three of these from extant species and are hence described as new taxa. The multi-gene phylogeny of the combined LSU, TEF1-α, and ITS sequence data to infer phylogenetic relationships and discuss phylogenetic affinities with morphologically similar species. Based on morphological characteristics and phylogenetic analyses, three new species viz. A.linzhiensis, A.yadongensis, and A.submersa are introduced. Details of asexual morphs are described, and justifications for establishing these new species are also provided in this study.


Introduction
Freshwater ascomycetes are the ecological groups that occur saprobically on submerged or partially submerged plant substrates in aquatic habitats (Shearer 1993).Lignicolous freshwater fungi represent a highly diverse taxonomic group with a substantial population.These fungi play a pivotal role in the transfer of nutrients and the flow of energy between trophic levels in the food chain.They achieve this by breaking down complex organic compounds into simpler inorganic materials derived from dead flora and fauna (Krauss et al. 2011;Sridhar et al. 2013;Wurzbacher et al. 2014;Tsui et al. 2016).Recent research showed that lignicolous freshwater fungi comprise a diverse taxonomic assemblage, MycoKeys 102: 183-200 (2024), DOI: 10.3897/mycokeys.102.112905 Rong-Ju Xu et al.: Three new species A. guttulata, A. yadongensis and A. submersa are introduced with more than 3,870 species listed (Calabon et al. 2022).Among them, most are in the classes Dothideomycetes and Sordariomycetes (Hyde et al. 2016;Maharachchikumbura et al. 2016;Luo et al. 2019;Dong et al. 2020;Calabon et al. 2022;Wijayawardene et al. 2022).Sordariomycetes is a prominent class within Ascomycota, encompassing a wide variety of fungi (Luo et al. 2019;Calabon et al. 2022;Yang et al. 2023).In freshwater environments, Sordariomycetes stands out as a significant fungal group, playing a pivotal role in ecosystems.This class is renowned for its production of bioactive compounds (e.g., penicillins, tetracyclines, macrolides, aminoglycosides, and cephalosporins) (Poch et al. 1992;Jones et al. 2014;Wright et al. 2014;Calabon et al. 2023).
Aquapteridospora was initially introduced and classified within the Diaporthomycetidae genera incertae sedis, based on morphological and phylogenetic analyses by Yang et al. (2015).Aquapteridospora, with A. lignicola as the type species, is characterized by polyblastic, sympodial, denticles conidiogenous cells and fusiform, with pale to dark brown central cells and subhyaline end cells, with or without sheath conidia.Furthermore, Hyde et al. (2021a) introduced the family Aquapteridosporaceae to accommodate Aquapteridospora and placed this family in order Distoseptisporales based on divergence estimates, morphological characters, and phylogenetic analyses.
Aquapteridospora is a hyphomycetous genus that are commonly found in freshwater habitats, but only a few terrestrial species, such as A. bambusinum (≡Pleurophragmium bambusinum) was collected from dead culms of bamboo (Yang et al. 2015;Dai et al. 2017;Luo et al. 2019;Bao et al. 2021;Dong et al. 2021;Ma et al. 2022;Peng et al. 2022).These fungi play an important role in the decomposition of organics and nutrient cycling in aquatic environments (Hyde et al. 2016;Luo et al. 2018).In recent years, an increasing number of species in Aquapteridospora have been described and documented, including A. aquatica, A. bambusinum, A. fusiformis, A. hyalina, A. jiangxiensis and A. lignicola (Yang et al. 2015;Luo et al. 2019;Bao et al. 2021;Dong et al. 2021;Ma et al. 2022;Peng et al. 2022).
During an investigation of freshwater fungal diversity on the Tibetan Plateau, six collections possessing morphological characteristics that fit within the genus Aquapteridospora were collected.In particular, their morphological characteristics revealed that these collections were morphologically different from the other species in Aquapteridospora.In addition, phylogenetic analyses of a combined LSU, TEF1-α and ITS sequence data show that our new collections belong to distinct clades, which are distinct from other species in Aquapteridospora.Therefore, three new species viz.Aquapteridospora linzhiensis, A. submersa and A. yadongensis are introduced, as well as details of asexual morphs being described, and justifications for establishing these new species are provided in this study.

Collection, morphological examination and isolation
Submerged decaying wood samples were collected from freshwater habitats in southeast Xizang, China.Fresh specimens were studied following the methods of Senanayake et al. (2020).Microscopic structures were examined by using a stereomicroscope (SteREO Discovery.V12, Carl Zeiss Microscopy GmBH, Germany), photographed by using a Nikon ECLIPSE 80i compound microscope fitted with a Nikon DS-Ri2 digital camera, and measured by using the Tarosoft (R) Image Frame Work program.Illustrated figures were processed by using Adobe Photoshop CS6 version 10.0 software (Adobe Systems, San Jose, CA, USA).
Single spore isolation was performed on potato dextrose agar (PDA) plates following the methods described in Senanayake et al. (2020).Fungal herbarium specimens and axenic living cultures were deposited in the Herbarium of Cryptogams of the Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS) and Kunming Institute of Botany Culture Collection (KUNCC), Kunming, China.Faceoffungi and Index Fungorum numbers of novel species were registered (Jayasiri et al. 2015, http://www.indexfungorum.org/Names/Names.asp).

DNA extraction, PCR amplification, and sequencing
Fresh mycelia were scraped off from colonies on PDA plates and transferred to a 1.5-ml microcentrifuge tube using a sterilized lancet for genomic DNA extraction.The TOLOBIO Plant Genomic DNA Extraction Kit, Shanghai Co. Ltd.P.R.China was used to extract fungal genomic DNA, following the protocols in the manufacturer's instructions.The DNA polymerase chain reaction (PCR) amplifications were performed by using primer pairs as follows: ITS5/ITS4 for internal transcribed spacer rDNA region and covered 5.8S ribosomal (ITS); LR0R/ LR5 for the nuclear ribosomal large subunit 28S rDNA gene (LSU), and TEF1-983F/TEF1-2218R for TEF1-α (Vilgalys and Hester 1990;White et al. 1990).DNA template was carried out in 25 μL reaction volume containing 21 μL of 1 × Power Taq PCR Master Mix, 1 μL of each primer (10 μL stock) and 2 μL of genomic DNA template.Amplifications were carried out by using the BioTeke GT9612 thermocycler (Beijing City, China).The PCR amplification conditions for ITS and LSU consisted of initial denaturation at 98 °C for 3 minutes, followed by 35 cycles of denaturation at 98 °C for 20 seconds, annealing at 53 °C for 10 seconds, extension at 72 °C for 20 seconds, final extension at 72 °C for 5 minutes; the PCR amplification conditions for TEF1-α consisted of initial denaturation at 98 °C for 3 minutes, followed by 35 cycles of denaturation at 98 °C for 20 seconds, annealing at 64 °C for 10 seconds, extension at 72 °C for 20 seconds, final extension at 72 °C for 5 minutes.PCR products were visualized by using 1% agarose gel electrophoresis stained with ethidium bromide and distinct bands were checked in Gel documentation system (Compact Desktop UV Transilluminator analyzer GL-3120).The PCR products were sequenced by Tsingke Company, Beijing, P.R. China.

Phylogenetic analyses
The sequences were uploaded in GenBank database (http://www.ncbi.nlm.nih.gov/blast/) to search for similar taxa.Sequences generated from the LSU, TEF1-α and ITS gene regions were carefully verified before further analyses.The new sequences were submitted to GenBank, and the strain information used in this paper was provided in Table 1.Multiple sequence alignments were aligned with MAFFT v.7 (Katoh and Standley 2016) http://mafft.cbrc.jp/alignment/server/index.html]and dataset was trimmed by TrimAlv.1.3using the gappyout option (http://phylemon.bioinfo.cipf.es/utilities.html)(Capella-Gutierrez et al. 2009).A combined sequence dataset was performed with the SquenceMatrix v.1.7.8 (Vaidya et al. 2011).Maximum likelihood (ML) analysis was performed by RAxML-HPC2 v.8.2.12 (Stamatakis 2014) in the CIPRES Science Gateway web server (http://www.phylo.org/portal2)by using 1,000 rapid bootstrap replicates and the GTRGAMMA+I model.Bootstrap support values for ML equal to or greater than 75% were given above the nodes in the phylogenetic tree (Fig. 1).The model of evolution for the Bayesian inference (BI) analysis was performed by using MrModeltest v2.3 (Nylander 2004).GTR+I+G was selected as the best-fitting model for LSU, TEF1-α and ITS dataset.The Markov chain Monte Carlo sampling (BMCMC) was carried out to assess posterior probabilities (PP) by using MrBayes v.3.2.7 (Ronquist et al. 2012).Six simultaneous Markov chains were run for random trees for 1,000,000 generations, and trees were sampled every 200 th generation.Bayesian posterior probabilities (PP) equal to or greater than 0.95 were given above the nodes in the phylogenetic tree (Fig. 1).Phylograms were visualized by using FigTree v1.4.0 (Rambaut 2012) and rearranged in Adobe Photoshop CS6 software (Adobe Systems, USA).The new sequences were deposited in GenBank (Table 1), and the final alignments and phylogenetic tree were registered in TreeBASE under the submission ID: 30133 (http://www.treebase.org/).
Culture characteristics.Conidia were germinated on PDA within 48 hours.Germ tubes produced from each end.Colonies grown on PDA, circular, flat, superficial, dark brown from above, reverse-side brown in the centre, with greyish white near the edge.
Culture characteristics.Conidia were germinated on PDA within 48 hours.Germ tubes produced from each end.Colonies grown on PDA, regular concentric circles, flat, superficial, with dense mycelium at around, grey brown from above, dark brown from below.
Culture characteristics.Conidia were germinated on PDA within 48 hours.Germ tubes produced from each end.Colonies grown on PDA, circular, flat, superficial, raised, with dense, pale mycelium in the centre.Grey brown from above, dark brown from below.

Discussion
Species of Aquapteridospora are morphologically unique in the taxonomic characteristics, especially in the features of the conidiophores and conidia (Table 2).In most species, the conidia are fusiform and pigmented, featuring brown to dark brown central cells and subhyaline end cells.However, some species exhibit conidia with a distinct sheath, such as A. aquatica, A. jiangxiensis and A. lignicola (Yang et al. 2015;Dong et al. 2021;Peng et al. 2022).Additionally, a few species are characterized by hyaline to sub-hyaline conidia, as observed in A. hyalina (Ma et al. 2022).In addition, the length of conidiophores in species of Aquapteridospora varies significantly.Most species have conidiophores ranging in length from 70 to 305 μm, as observed in species like A. aquatica, A. fusiformis, A. hyaline, A. jiangxiensis, A. lignicola and A. linzhiensis (Yang et al. 2015;Luo et al. 2019;Dong et al. 2021;Ma et al. 2022;Peng et al. 2022), a few species exhibit conidiophores exceeding 400 μm in length, with the longest reaching 856 μm.This is the case for species such as A. bambusinum, A. yadongensis and A. submersa (Bao et al. 2021, this study).
Molecular phylogenetic analyses play a crucial role in elucidating the classification of hyphomycetous fungi (Dhanasekaran et al. 2006;Tekpinar and Kalmer 2019).Pleurophragmium bambusinum was initially described by Dai et al. (2017), and was previously assigned to Sordariomycetes incertae sedis based on its morphological characteristics.According to the phylogenetic analysis conducted by Dong et al. (2021), P. bambusinum was found to cluster within the Aquapteridospora clade with (100% ML/1.00 PP) support.However, their studies did not synonymize P. bambusinum under Aquapteridospora due to the ellipsoidal and conidia without a sheath, which indicate that it does not fit within the characteristics of Aquapteridospora species.Subsequently, Bao et al. (2021) transferred P. bambusinum to Aquapteridospora and synonymized A. bambusinum instead of P. bambusinum, based on both phylogeny and morphology.
The Tibetan Plateau is renowned for its distinctive biological diversity and extensive array of aquatic habitats, encompassing lakes, rivers, and wetlands, which provide sustenance for various fungal communities (Yao et al. 2019).While freshwater fungi play a crucial role in the ecosystem, they have remained understudied in this region, primarily due to the limited number of researchers focusing on freshwater fungi in the Tibetan Plateau.During our investigation into freshwater fungal diversity on the Tibetan Plateau, we introduced three new species within the genus Aquapteridospora, supported by both phylogenetic analysis and morphology.The discovery of these new species revealed the abundant fungal diversity in Tibetan Plateau and more scientific studies in this region are expected in the future.

Conflict of interest
The authors have declared that no competing interests exist.

Ethical statement
No ethical statement was reported.

Figure 1 .
Figure1.Maximum likelihood (ML) tree is based on combined LSU, TEF1-α and ITS sequence data.ML bootstrap support values equal to or greater than 70% and Bayesian posterior probabilities (PP) equal to or greater than 0.95 given above the nodes, shown as "ML/PP".The tree is rooted with Pseudostanjehughesia aquitropica (MFLUCC 16-0569) and P. lignicla (MFLUCC 15-0352).New species are indicated in red and type strains are in bold.

Table 1 .
Strains used for phylogenetic analyses and their corresponding GenBank numbers.The newly generated sequences are in cells with light grey shading and the type strain are in bold font.

Table 2 .
Synopsis of known species in Aquapteridospora.