Morphology and multi-gene phylogeny reveal a novel Torula (Pleosporales, Torulaceae) species from the plateau lakes in Yunnan, China

Abstract Background During an investigation into lignicolous freshwater fungi from the plateau lakes in Yunnan Province, China, two fresh collections of Torula taxa were collected and examined morpholgically. New information Torulaluguhuensis is characterised by: conidiophores which are semi-macronematous mononematous, erect, septate, smooth, slightly flexuous and pale brown; conidiogenous cells which are holoblastic, mono- to polyblastic, integrated, terminal, terminal or intercalary in conidial chains, doliiform and pale brown; conidia which are branched chains, acrogenous, straight or slightly curved, dark brown to blackish, pale brown or subhyaline at apex, 1–3 septate, strongly constricted at the septa, verruculose or finely echinulate and rounded at both ends. A new species was introduced, based on morphological and phylogenetic analysis of combined ITS, LSU, RPB2 and TEF sequence data. Detailed descriptions and illustrations are provided, with an updated phylogenetic tree depicting intergeneric relationships within the Torulaceae.


New information
Torula luguhuensis is characterised by: conidiophores which are semi-macronematous mononematous, erect, septate, smooth, slightly flexuous and pale brown; conidiogenous cells which are holoblastic, mono-to polyblastic, integrated, terminal, terminal or intercalary in conidial chains, doliiform and pale brown; conidia which are branched chains, acrogenous, straight or slightly curved, dark brown to blackish, pale brown or subhyaline at apex, 1-3 septate, strongly constricted at the septa, verruculose or finely echinulate and rounded at both ends. A new species was introduced, based on morphological and

Introduction
Torulaceae was introduced by Corda (Sturm 1829) with Torula as the type. The family is known only by the asexual morph which is characterised by: mostly immersed mycelium, erect, micro-or macronematous, straight or flexuous, subcylindrical conidiophores with or without apical branches and doliiform to ellipsoid or clavate, brown, smooth to verruculose and mono-to polyblastic conidiogenous cells and subcylindrical, phragmosporous, acrogenous, brown, dry and smooth to verrucose conidia that are characteristically produced in branched chains (Crous et al. 2015, Li et al. 2017. Currently, six genera, viz. Cylindrotorula, Dendryphion, Neopodoconis Neotorula, Rutola and Torula are accommodated in Torulaceae (Crous et al. 2015, Crous et al. 2020, Qiu et al. 2022. Torula was introduced by Persoon (1795) and is typified by T. herbarum. Members in this genus are hyphomycetes and characterised by superficial dark colonies, terminal or lateral, monoblastic or polyblastic conidiogenous cells with a basally thickened and heavily melanised wall, a thin-walled apex and medium to dark brown conidia in branched chains (Crane and Miller 2016). Torula has been investigated as an interesting source of secondary metabolites. For example, a new dechlorinated aromatic lactone produced by Torula sp. (YIM DT 10072) exhibited antibacterial activity against Staphylococcus aureus (Chunyu et al. 2018). Herbarin, dehydroherbarin and o-methylherbarin have been extracted from Torula herbarum (Narasimhachari and Gopalkrishnan 1974).
Yunnan is an inland province at a low latitude and high elevation, lying between 21°09'-29°15' N and 97°32'-106°12' E in south-western China, an area which is rich in freshwater resources. The nine major plateau lakes represented by Dianchi Lake, Erhai Lake and Fuxian Lake are major features of Yunnan. Abundant freshwater lake resources provide a favourable environment for the occurrence of lignicolous freshwater fungi (Shen et al. 2022 ). The studies of lignicolous freshwater fungi in Yunnan are mainly focused on lotic habitats , Luo et al. 2019. At present, only a limited number of early studies have explored the diversity of lignicolous freshwater fungi in Dianchi Lake and Fuxian Lake (Cai et al. 2002, Luo et al. 2004. Presently, we are conducting systematic research on lignicolous freshwater fungi from plateau lakes in Yunnan Province. In this study, two Torula species were collected from Luguhu Lake and their phylogenetic relationships were analysed, based on molecular sequence data.

Isolation and morphological study of strain
Submerged decaying woods were collected from Luguhu Lake, Yunnan Province and brought to the laboratory in zip-lock plastic bags. The samples were incubated in plastic boxes lined with moistened tissue paper at room temperature for one week and examined by methods following . Micromorphological characters were observed using an Optec SZ 760 compound stereomicroscope. Temporarily prepared microscope slides were placed under a Nikon ECLIPSE Ni-U compound stereomicroscope for observation and micro-morphological-photography. The morphology of colonies on native substrates were photographed with a Nikon SMZ 1000 stereo zoom microscope.
Single spore isolations were carried out following the methods described by Senanayake (2020). Germinating conidia were transferred aseptically to PDA plates supplemented with 0.5 mg/l of Amoxicillin and grown at room temperature.

DNA extraction, PCR and sequencing
Fungal mycelium was scraped from the surface of colonies grown on PDA at room temperature. The Trelief Plant Genomic DNA Kit (TSP101-50) was used to extract DNA from the ground mycelium according to the manufacturer's instructions. The primers used for PCR amplification were ITS = ITS5/ITS4 (White et al. 1990), LSU = LR0R/LR5 (Vilgalys and Hester 1990), TEF-α = 983F/2218R and RPB2 = fRPB2-5F/fRPB2-7cR (Liu et al. 1999). The final volume of the PCR reaction was 25 μl and contained 12.5 μl of 2× Power Taq PCR MasterMix, (20 mM Tris-HCL pH 8.3, 100 mM KCl, 3 mM MgCl , stabiliser and enhancer), 1 μl of each primer (10 μM), 1 μl genomic DNA extract and 9.5 μl deionised water. The PCR of ITS genes was processed as follows: 94℃ for 3 minutes, followed by 35 cycles of denaturation at 94℃ for 30 seconds, annealing at 56℃ for 50 seconds, elongation at 72℃ for 60 seconds and final extension at 72℃ for 10 minutes. The LSU and TEF genes were processed as follows: 94℃ for 3 minutes, followed by 35 cycles of denaturation at 94℃ for 30 seconds, annealing at 55℃ for 50 seconds, elongation at 72℃ for 60 seconds and final extension 72℃ for 10 minutes. The RPB2 gene region was amplified with an initial denaturation of 95℃ for 5 minutes, followed by 40 cycles of denaturation at 95℃ for 60 seconds, annealing at 52℃ for 2 minutes, elongation at 72℃ for 90 seconds and final extension at 72℃ for 10 minutes.
PCR amplification was confirmed on 1% agarose electrophoresis gels stained with ethidium bromide. Purification and sequencing of PCR products were sent for sequencing TM 2 at Tsingke Biological Engineering Technology and Services Company, Yunnan, China. The sequences were deposited in the GenBank database at the National Center for Biotechnology Information (NCBI) and the accession numbers are listed in Table 1

Sequencing and sequence alignment
Sequences were assembled with BioEdit (Hall 1999) and those with high similarity indices were determined from a BLAST search to find the closest matches with taxa in Torula and from recently-published data , Li et al. 2023, Tian et al. 2023. Aligned sequences of each loci (ITS, LSU, RPB2 and TEF) were combined and manually improved using BioEdit v.7.0.5.2 (Hall 1999

Etymology
Referring to Luguhu Lake, China, where the fungus was collected.
Culture characteristics: Conidia germinating on PDA within 10 hours and germ tubes produced from the apex. Colonies growing on PDA, reaching 10 cm in 15 days at 24℃. Colonies were raised in the middle, velvety on the surface and had a white centre fading to yellowish-brown, reverse, yellowish-brown in centre and white edges.

Notes
Torula submersa was introduced by Tian et al. (2023), collected on a submerged decaying branch from Sichuan Province. In this study, a fresh collection was made on submerged decaying wood in Luguhu Lake, Yunnan Province. Phylogenetic analysis showed that our collection (KUNCC 22-12426) sistered with T. submersa ( Fig. 3).
Morphological characteristics of our new collection are consistent with Torula submersa (Tian et al. 2023). Based on morphological characteristics and phylogenetic analysis, we, therefore, identified our new collection as Torula submersa, which was collected from a lentic freshwater habitat for the first time.

Discussion
Species of Torula are quite similar in morphology and most species lack DNA sequence data to support their phylogenetic relationships (Crous et al. 2015). This causes taxonomic confusion and some species may have been misidentified. Some Torula species may be the same or belong to other genera and their taxonomic statuses have to be further investigated (Crane and Miller 2016). In addition to the morphological examination, DNAbased phylogenetic analysis should be performed for more Torula species. Herein, we combined multi-loci phylogenetic analysis and morphological characterisation to introduce Phylogram generated from Maximum Likelihood analysis, based on combined ITS, LSU, RPB2 and TEF sequence data for species of Torulaceae. RAxML bootstrap support values equal to or greater than 75% are given before the forward slash. Branches with Bayesian posterior probabilities equal to or higher than 0.95 are given after the forward slash.
one new species which contributes to the taxonomy for the genus and addition of DNA sequence in databases.
Taxonomic research on Torulaceae in China is mainly concentrated in the south-western region and commonly found in freshwater habitats , Qiu et al. 2022, Tian et al. 2023). There are four genera of Torulaceae viz. Dendryphion, Neopodoconis, Neotorula and Torula which are reported from freshwater habitats in China. In this study, a checklist of Torulaceae species in China is provided. Torula is commonly found on submerged decaying wood in freshwater environments with most species having been isolated from lotic water (Table 2). In our study, we found a new species in a lake in Yunnan Province. Presumably there could be other new species in these habitats and it is necessary to investigate lignicolous freshwater fungi in other lakes in Yunnan. A checklist of Torulaceae species from freshwater habitats in China is shown in  Checklist of Torulaceae species from freshwater habitats in China.