Four New Species of Torula (Torulaceae, Pleosporales) from Sichuan, China

Torula is an asexual and hyphomycetous genus in the family Torulaceae. Torula species are generally saprophytic. They have a worldwide distribution and abound in humid or freshwater habitats. In order to better understand this genus, we carried out several field collections from Sichuan, China. As a result, we obtained nine Torula isolates from dead woody substrates in terrestrial and freshwater habitats. Based on a biphasic approach of morphological examination and multi-locus phylogenetic analyses (ITS, SSU, LSU, TEF, RPB2), these collections were identified as belonging to seven Torula species. Four of them were new species (Torula chinensis, T. longiconidiophora, T. sichuanensis and T. submersa), and the other three belonged to existing species, though one was found for the first time in China (T. masonii). Morphological and updated phylogenetic delamination of the new discoveries is also discussed. This study provides further insights into our understanding of wood-based Torula species in China.

The genus Torula was initially described by Persoon [9]. Conidiogenesis in Torula was observed by Crane and Schoknecht [10] using light and transmission electron microscopy. The phylogenetic relationships of the Torulaceae (Pleosporales) were studied by Crous et al. [1], and Torula was accepted within the family. Additionally, they identified three new species based on the number of septa per conidium and designated a neotype (CBS H-22275) for the generic type, Torula herbarum [1]. Torula has an ancient origin and has 541 species epithets in Index Fungorum (http://www.indexfungorum.org/; accessed on 20 January 2023). However, most species of Torula were introduced before the arrival of Sanger sequencing; therefore, there are a few molecular data available. Torula species are commonly collected from dead branches and submerged wood in terrestrial or freshwater habitats [4,5]. The genus is characterized by terminal or lateral, monoblastic or polyblastic conidiogenous cells with a basally thickened and heavily melanized wall and a thin-walled apex, frequently collapsing and becoming coronate [1,4,11]. This genus is a mold that lacks a known sexual state. The different species of Torula can be distinguished by conidial size and the number of septa and chains [1,4]. Recently, some species were introduced based on both molecular data and morphology [5,[12][13][14].
We are studying the diversity of fungi in southwestern China, Sichuan, along the Yangtze River, and surveying the taxonomy of hyphomycetes [15][16][17][18][19]. During the survey of torula-like species in Chengdu Province (July to September 2021), nine isolates were obtained from wood-based substrates. Based on the in-depth phylogenetic analysis of combined (ITS, LSU, SSU, RPB2 and TEF) sequence data and morphological examination, these isolates were identified as three known Torula species, including a new geographic record (T. masonii) and four new species, viz., T. chinensis sp. nov., T. submersa sp. nov., T. sichuanensis sp. nov. and T. longiconidiophora sp. nov., which are introduced herein.

Sample Collection, Isolation and Morphological Examination
A survey on the diversity of hyphomycetes in Sichuan, China, was conducted between July and September of 2021. Dead branches of unknown hosts were collected from 5 locations (Yunqiao Wetland, Chengdu City; Baiyungou, Chongzhou City; Huilonggou, Pengzhou City; Hongfengling, Deyang City; Longchang, Neijiang City) in Sichuan. The specimens were taken into the laboratory in paper envelopes for examination. Microscopic characteristics were observed and recorded using a Nikon SMZ800N stereo microscope equipped with a Nikon DS-Fi3 microscope camera and a Nikon ECLIPSE Ni-U microscope fitted with a Nikon DS-Ri2 microscope camera, respectively. Measurements were conducted using the Nikon NIS-Elements Documentation Imaging Software Version 5.21.00. All photos were processed using Adobe Photoshop software version 22.0. Single conidium isolation was performed following the method described by Senanayake et al. [20]. Germinated conidia were individually transferred to potato dextrose agar (PDA) media plates and incubated in the dark at 25 • C. Culture characteristics were examined and recorded after one week and at regular intervals.
Herbarium specimens were deposited in the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China; and the Herbarium of the University of Electronic Science and Technology (HUEST), Chengdu, China. The living cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, and the University of Electronic Science and Technology Culture Collection (UESTCC) in Chengdu, China.

DNA Extraction, PCR Amplification and Sequencing
Fungal genomic DNA was extracted from mycelia using Trelief TM Plant Genomic DNA Kit (TSINGKE Biotech, Shanghai, P.R. China) according to the manufacturer's protocol. Five partial loci, including the nuclear ribosomal internal transcribed spacer (ITS: ITS1-5.8S-ITS2), the nuclear ribosomal small subunit rRNA gene (SSU), the nuclear ribosomal large subunit rRNA gene (LSU), the translation elongation factor 1-alpha gene (TEF) and the second largest subunit of RNA polymerase II gene (RPB2) were amplified by polymerase chain reaction (PCR). The corresponding primer pairs and PCR conditions are listed in Table 1. The final reaction volume of the PCR reagent was 25 µL containing 2 µL of DNA template, 1 µL each of the forward and reverse primer, 8.5 µL of double-distilled water (ddH 2 O) and 12.5 µL of 2 × lash PCR MasterMix (mixture of DNA Polymerase, dNTPs, Mg 2+ and optimized buffer; CoWin Biosciences, Jiangsu, China). The PCR products were visualized by 1% agarose gel electrophoresis. Sanger sequencing was conducted by Tsingke Biological Technology (Beijing, China).

Phylogenetic Analyses
According to the corresponding Sanger sequencing chromatograms, misleading data from the ends of raw sequencing fragments were manually trimmed and assembled into consensus sequences using SeqMan Pro version 7.1.0 (DNASTAR, Inc. Madison, WI, USA). Barcode sequences of all Torula species for which the sequence data are available and the outgroup taxon Sporidesmioides thailandica (MFLUCC 13-0840) were downloaded from the NCBI nucleotide database (Table 2) using the function read. GenBank data were integrated within the R package Analysis of Phylogenetics and Evolution (APE) [29]. The multiple sequence alignments were conducted using MAFFT [30] version 7.310 with options "-adjustdirectionaccurately -auto", and the alignment files were further trimmed using trimAl version 1.4 [31] with the option "-gapthreshold 0.5", which only allows 50% of taxa with a gap in each site. The best-fit nucleotide substitution models for each locus were selected using PartitionFinder version 2.1.1 [32] under the corrected Akaike information criterion (AICC). All sequence alignments were combined using an in-house Python script.
Maximum likelihood (ML) and Bayesian analysis were conducted based on individual and combined datasets. Five alignment datasets of SSU, ITS, LSU, TEF, and RPB2 were concatenated for multi-locus phylogenetic analysis. ML phylogenetic tree was obtained using the IQ-TREE version 2.0.3 [33], and the topology was evaluated using 1,000 ultrafast bootstrap replicates. The Bayesian analysis was conducted using parallel MrBayes version 3.2.7a [34]. Two different runs with 20 million generations and four chains were executed, and the initial 25% of sample trees were treated as burn-in. Tracer version 1.7.1 [35] was used to confirm that the MCMC runs reached convergence, and all ESS values were above 200. Then, the ML tree was annotated by TreeAnnotator version 2.6.4 implemented in BEAST [36] based on Mrbayes MCMC trees with no discard of burn-in and no posterior probability limit. The ML tree was visualized using ggtree [37] and further edited in Adobe Illustrator software version 16.0.0.
The best-scoring ML tree (lnL = −14437.4141) with ultrafast bootstrap values from ML analyses and posterior probabilities from MrBayes analysis at the node is shown in Figure 1. Nine newly obtained Torula isolates represent seven different species.
Material examined: CHINA: Sichuan, Chongzhou City, Baiyungou, on a submerged decaying branch of an unknown host, N  Culture characteristics: Conidia germinated on PDA within 24 h at 25 • C. Colonies reached 32 mm after 10 days in an incubator under dark conditions at 25 • C. Colonies were raised in the middle; they were irregular circles; surface velvety; they had a white center and were yellowish-brown at the edges, with clear margins; reverse, yellowish brown with pale edges.
Culture characteristics: Conidia germinating on PDA within 24 h at 25 • C. Colonies reaching 34 mm after 10 days in an incubator in the dark at 25 • C. Colonies raised in the middle, irregularly circular, surface velvety, with white centers and becoming light brown, with a white and clear margin; reverse, yellow to light brown in the center and white at the margin.   (Figure 4).
Culture characteristics: Conidia germinated on PDA within 24 h at 25 • C. The colony reached 21 mm after 10 days in an incubator in the dark at 25 • C. Colonies were white and irregularly circular with a velvety surface, with denser mycelium in the center and becoming sparser towards the edge, with clear margins; in reverse, pale green in the center and becoming white towards the edge.
Notes: Torula masonii was introduced by Crous et al. [1] based on the fungus sporulating in culture, which was collected from Brassica sp. in the UK. In this study, the phylogenetic tree shows that our isolate (UESTCC 22.0089) from decaying wood in a damp environment is clustered with the strain T. masonii (KUMCC 16-0033). In addition, isolate HUEST 22.0089 displays similar morphological characteristics with the type species of T. masonii (CBS 245.57) in colony shape, conidiophores, conidiogenous cells and conidia. We identified the isolate UESTCC 22.0089 as T. masonii, a new geographic record from a humid habitat in China.
Notes: The phylogenetic tree shows that the isolate UESTCC 22.0086 is clustered with the ex-type strain of Torula chinensis (UESTCC 22.0085) (Figure 1). Multigene phylogenetic analysis of a combined dataset of the LSU, ITS, SSU, TEF, and RPB2 showed that T. chinensis and T. submersa are close relatives with 95% ML support ( Figure 1). Morphologically, T. submersa differs from other Torula species by having black conidia composed of subglobose cells and 1-4 septa (see notes under T. chinensis). Therefore, we introduce the isolate UESTCC 22.0086 as a new species.

Discussion
Even though over 541 epithets of Torula have been reported, previously, many Torula species were identified based on morphology alone, and there were very little data relating to the phylogenetic relationships until the study of Crous et al. [1]. Therefore, it is possible that a number of the species in Torula are conspecific or belong to different genera. Re-examining type specimens of Torula-like species described prior to the advent of molecular technology is necessary to address this issue. Additionally, fresh specimens Scale bar of (d) applies to (d-g). Scale bar of (h) applies to (h,i). Scale bar of (j) applies to (j-q).

Discussion
Even though over 541 epithets of Torula have been reported, previously, many Torula species were identified based on morphology alone, and there were very little data relating to the phylogenetic relationships until the study of Crous et al. [1]. Therefore, it is possible that a number of the species in Torula are conspecific or belong to different genera. Reexamining type specimens of Torula-like species described prior to the advent of molecular technology is necessary to address this issue. Additionally, fresh specimens should be collected, sequenced and combined with multi-locus phylogenetic analysis and morphological examination, and designation of epi-types is essential. For instance, Crane et al. [11] re-examined several hyphomycetous species previously placed in the Torula and found that T. rhombica and T. terrestris do not seem to be congenerous with T. herbarum, and transferred to them Bahusandhika. Recently, new species have been identified based on molecular data and morphology, and to date, only 23 species have sequence data [1,[3][4][5]8,[11][12][13][14]20,[38][39][40].
The genus Torula contains many diverse species, frequently isolated from submerged decaying wood, living leaves, dead wood and twigs of various terrestrial plants, soil and earthworm casts [41]. They are commonly isolated as saprobes in temperate and tropical climate regions [42]. A few species are plant pathogens, such as T. herbarum, causing stem blight in Indian jujube (Ziziphus mauritiana) [43]. They are also isolated from the air of natural environments, parks and industrial zones and are reported to cause seasonal fungal allergies in humans [44][45][46][47][48]. The genus has been shown to produce a wide range of chemically novel diverse secondary metabolites. For example, T. herbarum displays antibacterial, antifungal, antiamoebic and potentially anti-cancer properties [49][50][51]. Therefore, more taxonomic, phylogenic and biochemical studies of this bioprospecting genus should be performed.