Morphology and Multi-Gene Phylogeny Reveal a New Species of Family Torulaceae from Yunnan Province, China

Abstract

The Family Torulaceae belongs to the Order Pleosporales (Class Dothideomycetes) and mainly comprises saprobes. The taxa are widely distributed in both terrestrial and aquatic habitats. In this study, we collected three dead leaf specimens of Carex baccans and two submerged wood specimens in Yunnan Province, China. A biphasic approach of morphological examination and multi-locus phylogenetic analyses conducted for internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), nuclear large subunit rDNA (28S), nuclear small subunit rDNA (18S), translation elongation factor 1-α (tef1) gene, and RNA polymerase II second-largest subunit (rpb2) revealed one new species Rutola kunmingensis and a new collection of Torula sundara. Rutola kunmingensis is characterized by black, powdery colonies, micronematous, creeping, reticular conidiophores bearing inconspicuous, monoblastic conidiogenous loci, and multi-septate, catenulate, verruculose, brown conidia. The conidiophores and conidia of each genus in Torulaceae are mapped onto the phylogenetic tree and the generic demarcations of this family are discussed and the significant divergence of ITS, 18S, 28S, rpb2, and tef1 sequences in Torulaceae is also discussed.

1. Introduction

The family Torulaceae was introduced by Sturm (1829) to accommodate Torula, which is typified by T. herbarum. This family is classified in Pleosporales, Dothideomycetes [1]. Most species of Torulaceae have been found as saprobes on submerged wood or dead branches of Asteraceae, Brassicaceae, Cyperaceae, Fabaceae, Iridaceae, and Ranunculaceae in Asia (China, India, Thailand), Europe (France, Germany, Italy), North America (Canada), and other regions [2,3,4,5,6,7]. Torula herbarum was reported as the causal agent of stem blight on Ziziphus mauritiana [8].

Torulaceae was demarcated only by asexual characters, and its concept has been revised in several articles [3,4,5,6,9]. Dark brown to black, effuse, or powdery colonies characterizes this family on natural substrates, with mycelia that can be immersed to superficial, ranging from hyaline to brown. The family features micro- or macronematous, brown, subcylindrical, erect conidiophores, which may or may not have apical branches, alongside mono-to polyblastic, doliiform to ellipsoid or clavate, and smooth to verruculose conidiogenous cells [10,11]. Within this family, subcylindrical, phragmosporous, acrogenous, brown, and dry conidia are prevalent, with a surface texture that can vary from smooth to verrucose, and these conidia adhere together in branched chains [3,4,5,6,12]. Qiu et al. [13] synonymized both Rostriconidium and Sporidesmioides under Neopodoconis [14]. Torulaceae includes Cylindrotorula, Dendryphion, Neotorula, Rostriconidium, Rotula, Sporidesmioides, and Torula [2,3,4,9,15,16]. Torula is the most speciose genus in Torulaceae with 542 epithets (Index Fungorum 2024); however, only 28 species have been confirmed with molecular data [12,17]. Torula was initially established and typified with T. monilis by Persoon (1795); however, the type species was invalid and replaced with T. herbarium (syn. Monilia herbarum) by Link (1809). Dendryphion was introduced by Wallroth (1833) with D. comosum as the type species and is the second largest genus in Torulaceae with 85 epithets (Index Fungorum 2024). Crous et al. [3] accepted Dendryphion and Torula in Torulaceae based on phylogenetic analysis of 28S sequences. Rotula was separated from Torula to accommodate Rotula graminis, a species lacking the diagnostic coronate conidiogenous cells [2]. Crous et al. [3] updated the morphological description of R. graminis without providing molecular data. Chen et al. [18] provided molecular data for R. graminis and confirmed its phylogenetic placement within Torulaceae. Neotorula was established to accommodate torula-like species by Su et al. [4], which is typified by N. aquatica. This genus contains two species, N. aquatica and N. submersa [19]. Sporidesmioides (=Neopodoconis) is a monotypic genus which is typified with N. thailandica, a species with sporidesmium-like asexual morph [15]. Su et al. [16] added another sporidesmium-like genus, Rostriconidium (=Neopodoconis), to Torulaceae based on phylogenetic inference of concatenated 28S, ITS, tef1, and rpb2 sequence. This genus contains three species, namely N. aquaticum, N. cangshanens, and N. pandanicola [6,16,20]. Cylindrotorula is a member of Torulaceae, comprising a single species, C. indica, which is distinguishable by its secondary conidia [9].

During a survey of microfungi in deciduous forests in Kunming City, Yunnan Province, China [21], we encountered five torula-like taxa with differences in the colors of colonies. This study aims to identify and describe new and known torula-like fungi on dead Carex baccans leaves and dead submerged wood by combining morphological characteristics and phylogeny based on ITS, 28S, 18S, rpb2, and tef1 sequence data.

2. Materials and Methods

2.1. Collection, Isolation, and Morphology

Samples were collected from submerged decaying wood in Xishan Park and dead leaves of Carex baccans (Cyperaceae) in Heilongtan Garden, Kunming city, Yunnan Province, China. The samples were collected into zip lock plastic bags and brought to the laboratory for further examination and were incubated in sterile plastic boxes lined with moistened tissue paper at room temperature for 10 days. The samples were examined following the methods described in Hyde and Jeewon [22] and Senanayake et al. [23]. Fungal characters were observed using NIKON MODEL C-PSN Series (Nikon Corporation, Tokyo, Japan) dissecting stereo microscope and the colonies on the substrate surface were transferred to a clean glass slide with a needle. The conidiophore, conidiogenous cells, and conidia of these asexual taxa were recorded by a NIKON ECLIPSE Ni-U compound microscope equipped with DS-Ri2 camera (Nikon Corporation, Tokyo, Japan). The images used for the figures were processed using Adobe Photoshop CC 2019 and microstructures were measured using Image Frame Work software (Version 0.9.7). Pure cultures were obtained via single spore isolation on Potato dextrose agar (PDA) and the germinating conidia were photographed using the compound microscope. The specimens were deposited at the Herbarium of Cryptogams Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China (HKAS). Pure cultures were deposited at the Kunming Institute of Botany Culture Collection (KUNCC). The novel species was linked with Facesoffungi, Index Fungorum, and Fungalpedia [24,25].

2.2. DNA Extraction and PCR Amplification

Fresh mycelia were scraped from cultures grown on PDA for 20 days at 25 °C for DNA extraction. The Biological Technology Trelief Plant Genomic DNA Extraction Kit was used to extract genomic DNA following the manufacturer’s instructions. The primer pairs ITS5/ITS4 [26], LR0R/LR5 [27], NS1/NS4 [26], EF1-983F/EF1-2218R [28], and fRPB2-5F/fRPB2-7cR [29] were used for amplification of the ITS, 28S, 18S, tef1, and rpb2, respectively. The PCR reaction was performed in a 25 μL reaction volume, comprising 21 μL Taq GoldenStar T6 Super PCR Mix (Vazyme 2 × Rapid Taq Master Mix, Vazyme Biotech Co., Ltd., Nanjing, China), 1 μL of each primer, and 2 μL DNA template. The PCR conditions of ITS, 28S, and 18S were as follows: initialization at 98 °C for 5 min, followed by 40 cycles of denaturation at 98 °C for 30 s, annealing at 53 °C for 40 s and extension at 72 °C for 30 s, and a final extension at 72 °C for 10 min. PCR reaction condition of tef1 was as follows: 5 min at 98 °C, followed by 35 cycles of 45 s at 98 °C, 45 s at 52 °C, and 70 s at 72 °C, and a final extension of 10 min at 72 °C. PCR condition of rpb2 was as follows: 5 min at 98 °C, followed by 10 cycles of 45 s at 98 °C, 120 s at 55 °C, and 50 s at 72 °C; 10 cycles of 45 s at 98 °C, 120 s at 50 °C, and 50 s at 72 °C; and a final extension of 10 min at 72 °C. The PCR products were visualized through agarose gel electrophoresis stained by 1% ethidium bromide-stained agarose gel (TS-GelRed Ver. 2, Tsingke Biotechnology Co., Ltd., Beijing, China). Those products were sent to Tsingke Biotech Co., Ltd., Kunming, China for sequencing.

2.3. Sequence Alignment and Phylogenetic Analyses

The quality of raw sequences was checked using BioEdit 7.0.9 [30]. The forward and reverse sequences were assembled using Sequencing Project Management (SeqMan) [31]. Assembled sequences were subjected to BLAST (https://www.ncbi.nlm.nih.gov/, accessed on 28 July 2024) search to reveal the close matches. Reference sequences were selected following recent publications (Table 1). The newly generated sequences and the reference sequences were aligned using MAFFT v 6.8 [32]. Uninformative gaps and ambiguous regions were removed using Trimal on the Phylemon 2.0 online platform [33]. Individual datasets were combined using SequenceMatrix 1.7.8 [34]. The combined alignment was used for Bayesian inference (BI) and maximum likelihood (ML) analyses.

The best-fit evolutionary models for each marker were estimated using MrModeltestv.2.2 with Akaike Information Criterion (AIC). Bayesian inference analyses were conducted using MrBayes v. 3.2.6 [35]. Six simultaneous Markov Chain Monte Carlo (MCMC) chains were run for 1,000,000 generations and trees were sampled every 1000th generation until the standard deviation of the split frequencies fell below 0.01. The phylogenetic trees were summarized and posterior probabilities (PP) were calculated by discarding the first 25% of generations as the burn-in phase. The ML analysis was performed using RAxML-HPC2 on XSEDE (8.2.10) in CIPRES Science Gateway V. 3.3 [36] by employing default parameters but with the following adjustments: bootstrap iterations were set to 1000 and substitution model was set to GTR + GAMMA + I. The phylogenetic tree was visualized using FigTree v.1.4.0. and edited with Adobe Illustrator 2020 and Adobe Photoshop CC 2019. The final alignments and phylogenies can be accessed in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S31037, accessed on 15 December 2023).

Table 1. GenBank accession numbers of the taxa used in the phylogenetic analyses in this study.

Taxa	Culture Collection/Voucher No.	GenBank Accession Numbers					Reference
		ITS	28S	18S	rpb2	tef1
Cylindrotorula indica	NFCCI:4837	MT339445	MT339443	N/A	MT321491	MT321493	Boonmee et al. [9]
C. indica	NFCCI:4836	MT339444	MT339442	N/A	MT321490	MT321492	Boonmee et al. [9]
Dendryphion aquaticum	MFLUCC 15-0257	KU500566	KU500573	KU500580	N/A	N/A	Su et al. [4]
D. comosum	CBS 208.69	MH859293	MH871026	N/A	N/A	N/A	Li et al. [37]
D. europaeum	CPC 23231	KJ869145	KJ869202	N/A	N/A	N/A	Crous et al. [38]
D. fluminicola	KUMCC 15-0321	MG208160	MG208139	N/A	MG207971	MG207990	Su et al. [16]
D. hydei	HKAS 112706	MW723060	MW879527	MW774583	MW729781	MW729786	Boonmee et al. [9]
D. submersum	DLUCC 0698	MG208158	MG208137	N/A	MG207969	MG207988	Su et al. [16]
Neopodoconis aquaticum	KUMCC 15-0297	MG208165	MG208144	N/A	MG207975	MG207995	Su et al. [16]
N. aquaticum	MFLUCC 16-1113	MG208164	MG208143	N/A	MG207974	MG207994	Su et al. [16]
N. pandanicola	KUMCC 17-0176	MH275084	MH260318	MH260358	MH412759	MH412781	Su et al. [16]
N. thailandica	MFLUCC 13-0840	MN061347	KX437757	KX437759	KX437761	KX437766	Li et al. [15]
N. thailandica	KUMCC 16-0012	MN061348	KX437758	KX437760	KX437762	KX437767	Li et al. [15]
Neotorula aquatica	MFLUCC 15-0342	KU500569	KU500576	KU500583	N/A	N/A	Su et al. [4]
Ne. submersa	KUMCC 15-0280	KX789214	KX789217	N/A	N/A	N/A	Hyde et al. [19]
Rutola graminis	CPC 33695	MN313815	MN317296	N/A	N/A	N/A	Chen et al. [18]
R. graminis	CPC 33267	MN313814	MN317295	N/A	N/A	N/A	Chen et al. [18]
R. kunmingensis	HKAS 124483	OR470710	OR470705	OR470700	OR753783	OR753778	Present study
R. kunmingensis	HKAS 124484	OR470711	OR470706	OR470701	OR753784	OR753779	Present study
R. kunmingensis	HKAS 124485	OR470712	OR470707	OR470702	OR753785	OR753780	Present study
Torula acaciae	CPC 29737	KY173471	KY173560	N/A	KY173594	N/A	Crous et al. [39]
Torula aquatica	DLUCC 0550	MG208166	MG208145	N/A	MG207976	MG207996	Su et al. [16]
T. aquatica	MFLUCC 16-1115	MG208167	MG208146	N/A	MG207977	N/A	Su et al. [16]
T. breviconidiophora	KUMCC 18-0130	MK071670	MK071672	MK071697	N/A	MK077673	Li et al. [37]
T. calceiformis	HKAS 125551	OP751054	OP751052	OP751050	OQ630510	OQ630512	Hyde et al. [26]
T. calceiformis	HKAS 125552	OP751055	OP751053	OP751051	OQ630511	OQ630513	Hyde et al. [26]
T. camporesii	KUMCC 19-0112	MN507400	MN507402	MN507401	MN507404	MN507403	Hyde et al. [40]
T. canangae	MFLUCC 21-0169	OL966950	OL830816	N/A	N/A	ON032379	de Silva et al. [41]
T. chiangmaiensis	KUMCC 16-0039	MN061342	KY197856	KY197863	N/A	KY197876	Li et al. [5]
T. chinensis	UESTCC 22.0085	OQ127986	OQ128004	OQ127995	N/A	N/A	Tian et al. [17]
T. chromolaenae	KUMCC 16-0036	MN061345	KY197860	KY197867	KY197873	KY197880	Li et al. [5]
T. fici	CBS 595.96	KF443408	KF443385	KF443387	KF443395	KF443402	Crous et al. [3]
T. fici	KUMCC 16-0038	MN061341	KY197859	KY197866	KY197872	KY197879	Li et al. [37]
T. gaodangensis	MFLUCC 17-0234	MF034135	NG_059827	NG_063641	N/A	N/A	Hyde et al. [42]
T. goaensis	MTCC 12620	NR_159045	NG_060016	N/A	N/A	N/A	Pratibha et al. [43]
T. herbarum	CPC 24414	KR873260	KR873288	N/A	N/A	N/A	Crous et al. [3]
T. hollandica	CBS 220.69	NR_132893	NG_064274	KF443389	KF443393	KF443401	Crous et al. [3]
T. hydei	KUMCC 16-0037	MN061346	MH253926	MH253928	N/A	MH253930	Li et al. [37]
T. lancangjiangensis	HKAS 112709	NR_175706	NG_081516	NG_078759	MW729780	MW729785	Boonmee et al. [9]
T. longiconidiophora	UESTCC 22.0088	OQ127983	OQ128001	OQ127992	OQ158967	OQ158977	Tian et al. [17]
T. longiconidiophora	UESTCC 22.0125	OQ127984	OQ128002	OQ127993	OQ158972	OQ158976	Tian et al. [17]
T. mackenziei	MFLUCC 13-0839	MN061344	KY197861	KY197868	KY197874	KY197881	Li et al. [5]
T. mackenziei	HKAS 112705	MW723058	MW879525	MW774581	N/A	N/A	Li et al. [5]
T. masonii	CBS 245.57	NR_145193	NG_058185	N/A	N/A	N/A	Crous et al. [3]
T. masonii	MFLUCC 20-0239	MW412523	MW412517	MW412514	MW422160	MW422157	Samarakoon et al. [44]
T. phytolaccae	ZHKUCC 22-0107	ON611796	ON611800	ON611798	ON660879	ON660881	Li et al. [45]
T. pluriseptata	MFLUCC 14-0437	MN061338	KY197855	KY197862	KY197869	KY197875	Li et al. [5]
T. polyseptata	MFLUCC 17-1495	MT214382	MT214476	MT214427	MT235830	MT235791	Li et al. [5]
T. sichuanensis	UESTCC 22.0087	OQ127981	OQ127999	OQ127990	N/A	N/A	Tian et al. [17]
T. submersa	UESTCC 22.0086	OQ127985	OQ128003	OQ127994	OQ158968	OQ158972	Tian et al. [17]
T. sundara	MFLU 21-0089	OM276824	OM287866	N/A	N/A	N/A	Jayawardena et al. [12]
T. sundara	HKAS 124486	OR470713	OR470706	OR470703	OR753786	OR753781	Present study
T. sundara	HKAS 124487	OR470714	OR470706	OR470704	OR753787	OR753782	Present study
T. thailandica	GZCC20-0011	MN907426	MN907428	MN907427	N/A	N/A	Hongsanan et al. [6]
Ohleria modesta	Wu 36870	KX650562	KX650562	N/A	KX650582	KX650533	Jaklitsch et al. [46]
O. modesta	CBS 141480	KX650563	KX650563	KX650513	KX650583	KX650534	Jaklitsch et al. [46]
Paradictyoarthrinium hydei	MFLUCC 17-2512	MG747498	MG747497	MH454349	MG780232	N/A	Liu et al. [47]
P. diffractum	MFLUCC 13-0466	N/A	N/A	KP753960	KX437764	N/A	Liu et al. [47]

Notes: The newly generated sequences are shown in blue and the type strains are in bold. N/A means the data are not available in GenBank.

Table 1. GenBank accession numbers of the taxa used in the phylogenetic analyses in this study.

Taxa	Culture Collection/Voucher No.	GenBank Accession Numbers					Reference
		ITS	28S	18S	rpb2	tef1
Cylindrotorula indica	NFCCI:4837	MT339445	MT339443	N/A	MT321491	MT321493	Boonmee et al. [9]
C. indica	NFCCI:4836	MT339444	MT339442	N/A	MT321490	MT321492	Boonmee et al. [9]
Dendryphion aquaticum	MFLUCC 15-0257	KU500566	KU500573	KU500580	N/A	N/A	Su et al. [4]
D. comosum	CBS 208.69	MH859293	MH871026	N/A	N/A	N/A	Li et al. [37]
D. europaeum	CPC 23231	KJ869145	KJ869202	N/A	N/A	N/A	Crous et al. [38]
D. fluminicola	KUMCC 15-0321	MG208160	MG208139	N/A	MG207971	MG207990	Su et al. [16]
D. hydei	HKAS 112706	MW723060	MW879527	MW774583	MW729781	MW729786	Boonmee et al. [9]
D. submersum	DLUCC 0698	MG208158	MG208137	N/A	MG207969	MG207988	Su et al. [16]
Neopodoconis aquaticum	KUMCC 15-0297	MG208165	MG208144	N/A	MG207975	MG207995	Su et al. [16]
N. aquaticum	MFLUCC 16-1113	MG208164	MG208143	N/A	MG207974	MG207994	Su et al. [16]
N. pandanicola	KUMCC 17-0176	MH275084	MH260318	MH260358	MH412759	MH412781	Su et al. [16]
N. thailandica	MFLUCC 13-0840	MN061347	KX437757	KX437759	KX437761	KX437766	Li et al. [15]
N. thailandica	KUMCC 16-0012	MN061348	KX437758	KX437760	KX437762	KX437767	Li et al. [15]
Neotorula aquatica	MFLUCC 15-0342	KU500569	KU500576	KU500583	N/A	N/A	Su et al. [4]
Ne. submersa	KUMCC 15-0280	KX789214	KX789217	N/A	N/A	N/A	Hyde et al. [19]
Rutola graminis	CPC 33695	MN313815	MN317296	N/A	N/A	N/A	Chen et al. [18]
R. graminis	CPC 33267	MN313814	MN317295	N/A	N/A	N/A	Chen et al. [18]
R. kunmingensis	HKAS 124483	OR470710	OR470705	OR470700	OR753783	OR753778	Present study
R. kunmingensis	HKAS 124484	OR470711	OR470706	OR470701	OR753784	OR753779	Present study
R. kunmingensis	HKAS 124485	OR470712	OR470707	OR470702	OR753785	OR753780	Present study
Torula acaciae	CPC 29737	KY173471	KY173560	N/A	KY173594	N/A	Crous et al. [39]
Torula aquatica	DLUCC 0550	MG208166	MG208145	N/A	MG207976	MG207996	Su et al. [16]
T. aquatica	MFLUCC 16-1115	MG208167	MG208146	N/A	MG207977	N/A	Su et al. [16]
T. breviconidiophora	KUMCC 18-0130	MK071670	MK071672	MK071697	N/A	MK077673	Li et al. [37]
T. calceiformis	HKAS 125551	OP751054	OP751052	OP751050	OQ630510	OQ630512	Hyde et al. [26]
T. calceiformis	HKAS 125552	OP751055	OP751053	OP751051	OQ630511	OQ630513	Hyde et al. [26]
T. camporesii	KUMCC 19-0112	MN507400	MN507402	MN507401	MN507404	MN507403	Hyde et al. [40]
T. canangae	MFLUCC 21-0169	OL966950	OL830816	N/A	N/A	ON032379	de Silva et al. [41]
T. chiangmaiensis	KUMCC 16-0039	MN061342	KY197856	KY197863	N/A	KY197876	Li et al. [5]
T. chinensis	UESTCC 22.0085	OQ127986	OQ128004	OQ127995	N/A	N/A	Tian et al. [17]
T. chromolaenae	KUMCC 16-0036	MN061345	KY197860	KY197867	KY197873	KY197880	Li et al. [5]
T. fici	CBS 595.96	KF443408	KF443385	KF443387	KF443395	KF443402	Crous et al. [3]
T. fici	KUMCC 16-0038	MN061341	KY197859	KY197866	KY197872	KY197879	Li et al. [37]
T. gaodangensis	MFLUCC 17-0234	MF034135	NG_059827	NG_063641	N/A	N/A	Hyde et al. [42]
T. goaensis	MTCC 12620	NR_159045	NG_060016	N/A	N/A	N/A	Pratibha et al. [43]
T. herbarum	CPC 24414	KR873260	KR873288	N/A	N/A	N/A	Crous et al. [3]
T. hollandica	CBS 220.69	NR_132893	NG_064274	KF443389	KF443393	KF443401	Crous et al. [3]
T. hydei	KUMCC 16-0037	MN061346	MH253926	MH253928	N/A	MH253930	Li et al. [37]
T. lancangjiangensis	HKAS 112709	NR_175706	NG_081516	NG_078759	MW729780	MW729785	Boonmee et al. [9]
T. longiconidiophora	UESTCC 22.0088	OQ127983	OQ128001	OQ127992	OQ158967	OQ158977	Tian et al. [17]
T. longiconidiophora	UESTCC 22.0125	OQ127984	OQ128002	OQ127993	OQ158972	OQ158976	Tian et al. [17]
T. mackenziei	MFLUCC 13-0839	MN061344	KY197861	KY197868	KY197874	KY197881	Li et al. [5]
T. mackenziei	HKAS 112705	MW723058	MW879525	MW774581	N/A	N/A	Li et al. [5]
T. masonii	CBS 245.57	NR_145193	NG_058185	N/A	N/A	N/A	Crous et al. [3]
T. masonii	MFLUCC 20-0239	MW412523	MW412517	MW412514	MW422160	MW422157	Samarakoon et al. [44]
T. phytolaccae	ZHKUCC 22-0107	ON611796	ON611800	ON611798	ON660879	ON660881	Li et al. [45]
T. pluriseptata	MFLUCC 14-0437	MN061338	KY197855	KY197862	KY197869	KY197875	Li et al. [5]
T. polyseptata	MFLUCC 17-1495	MT214382	MT214476	MT214427	MT235830	MT235791	Li et al. [5]
T. sichuanensis	UESTCC 22.0087	OQ127981	OQ127999	OQ127990	N/A	N/A	Tian et al. [17]
T. submersa	UESTCC 22.0086	OQ127985	OQ128003	OQ127994	OQ158968	OQ158972	Tian et al. [17]
T. sundara	MFLU 21-0089	OM276824	OM287866	N/A	N/A	N/A	Jayawardena et al. [12]
T. sundara	HKAS 124486	OR470713	OR470706	OR470703	OR753786	OR753781	Present study
T. sundara	HKAS 124487	OR470714	OR470706	OR470704	OR753787	OR753782	Present study
T. thailandica	GZCC20-0011	MN907426	MN907428	MN907427	N/A	N/A	Hongsanan et al. [6]
Ohleria modesta	Wu 36870	KX650562	KX650562	N/A	KX650582	KX650533	Jaklitsch et al. [46]
O. modesta	CBS 141480	KX650563	KX650563	KX650513	KX650583	KX650534	Jaklitsch et al. [46]
Paradictyoarthrinium hydei	MFLUCC 17-2512	MG747498	MG747497	MH454349	MG780232	N/A	Liu et al. [47]
P. diffractum	MFLUCC 13-0466	N/A	N/A	KP753960	KX437764	N/A	Liu et al. [47]

Notes: The newly generated sequences are shown in blue and the type strains are in bold. N/A means the data are not available in GenBank.

3. Results

3.1. Phylogenetic Analyses

The combined ITS, 28S, 18S, rpb2, and tef1 dataset comprised 57 taxa, with Paradictyoarthrinium hydei and P. diffractum as the outgroup. The ingroup taxa were sampled from seven genera of Torulaceae. The dataset consisted of 3740 characters including gaps (28S: 1–798 bp, 18S: 799–1655 bp, tef1: 1656–2473 bp, rpb2: 2474–2990 bp, ITS: 2991–3740 bp). The matrix had 1147 distinct alignment patterns, with 30.63% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.243127, C = 0.260508, G = 0.270495, T = 0.225871; substitution rates: AC = 1.746274, AG = 3.241931, AT = 1.507101, CG = 0.851080, CT = 7.791880, GT = 1.000000; gamma distribution shape parameter α = 0.382044. The best-scoring ML tree with the final optimization likelihood value of −20021.477139 is shown in Figure 1 and Figure 2.

Furthermore, we discuss the significant differences in ITS, 18S, 28S, rpb2, and tef1 sequences within Torulaceae using single-gene phylogeny.

(1)

The ITS dataset for Torulaceae contains 54 taxa; the final alignment contained 517 characters, of which 284 were parsimony informative. Substitution model: GTR + I + G

(2)

The 28S dataset for Torulaceae contains 54 taxa; the final alignment contained 798 characters, of which 130 were parsimony informative. Substitution model: GTR + I + G

(3)

The 18S dataset for Torulaceae contains 36 taxa; the final alignment contained 845 characters, of which 105 were parsimony informative. Substitution model: GTR + I

(4)

The rpb2 dataset for Torulaceae contains 35 taxa; the alignment contained 744 characters, of which 347 were parsimony informative. Substitution model: SYM + I + G

(5)

The tef1 dataset for Torulaceae contains 36 taxa; the final alignment contained 818 characters, of which 248 were parsimony informative. Substitution model: GTR + I + G

The two specimens HKAS 124486 and HKAS 124487 formed a monophyletic clade with Torula longiconidiophora and T. sundara with support value of 66% MLBS and 1.00 BPP (Figure 2), indicating they are closely related. The three specimens HKAS 124483, HKAS 124484, and HKAS 124485 formed a sister clade to Rutola with high support (100 ML/1.00 PP). This clade was proposed as a new species of Rutola.

3.2. Taxonomy

Torulaceae Corda, in Sturm, Deutschl. Fl., 3 Abt. (Pilze Deutschl.) 2: 71 (1829)

Index Fungorum number: IF 81478; Facesoffungi number: FoF 01740

Saprobic on freshwater and terrestrial habitats. Sexual morph: Not observed. Asexual morph: Hyphomycetous. Colonies effuse, dark brown to black, powdery to hairy. Conidiophores macronematous, semi-macronematous or micronematous, erect or reduced to conidiogenous cells, septate, verrucose, straight or slightly flexuous, with or without apical branches, dark brown to black. Conidiogenous cells terminal, mono- to polyblastic, monotretic to polytretic, discrete or integrated, smooth to verruculose, doliiform, pale brown. Conidia phragmosporous, solitary or catenate, in simple or branched chains, acrogenous, brown to dark brown, smooth to verrucose, multiseptate, subcylindrical, ampulliform with sheath at the tip and scars at base, sometime secondary conidia form from intercalary of primary conidia by monotretic conidiogensis.

Type genus. Torula Pers., Ann. Bot. (Usteri) 15: 25 (1795)

Host and distribution. This fungus can be found on submerged wood or dead branches within Asteraceae, Brassicaceae, Cyperaceae, Fabaceae, Iridaceae, and Ranunculaceae in various regions, including Asia (China, India, Thailand), Europe (France, Germany, Italy), and North America (Canada), as well as other locations [2,3,4,5,6,7].

Notes. Torulaceae formed a well-supported clade in Pleosporales based on multi-gene phylogenetic analyses [3,16]. Molecular data have revealed that Torulaceae contain species with a richer diversity of morphologies compared to traditional concept provided by previous studies wherein the general concept of Torulaceae fit well with Neotorula, Torula, Rotula, and Dendryphion but not with Cylindrotorula, Rostriconidium, and Sporidesmioides [3,4,9]. Herein, we revise the concept of Torulaceae by inserting mono-to polytretic conidiogenous cells in Neotorula [4], solitary ampulliform conidia in Rostriconidium and Sporidesmioides [15], as well as secondary conidia in Cylindrotorula [9].

Rutola J.L. Crane & Schokn., Can. J. Bot. 55(24): 3015 (1978) [1977]

Index Fungorum number: IF9768; Facesoffungi number: FoF 06656

Type species. Rutola graminis (Desm. ex Fr.) J.L. Crane & Schokn., Can. J. Bot. 55(24): 3015 (1978) [1977]

Saprobic on terrestrial habitats. Sexual morph: Not observed. Asexual morph: Hyphomycetous. Colonies circular to oval, dark brown, powdery. Conidiophores on the substrate, micronematous, branched, septate, subhyaline to brown, reduced to conidiogenous cells, appressed to substrate. Conidiogenous cells terminal or intercalary, monoblastic, subhyaline, brown. Conidia phragmosporous, in branched chains which break into many segments, conidia segments various in number of septa, constricted, verruculose, dark brown.

Host and distribution. On dead stem of Typha sp. dead and still attached leaves, as well as Scirpus sylvaticus, including dead and still attached leaves, with a distribution in Germany and Norway [3].

Notes. Rutola is a monotypic genus that was separated from Torula to accommodate R. graminis [2]. The main characters are the presence of conidiogenous loci on the mesh-like conidiophores that are appressed to the substrate [2]. Rutola is distributed in France, Germany, and Norway and are found on dead leaves of Scirpus sylvaticus, Typha sp. (Typhaceae) [2,18].

Rutola kunmingensis S.C. He, K.D. Hyde & D.P. Wei, sp. nov. (Figure 3).

Index Fungorum number: IF901355; Facesoffungi number: FoF 14741

Etymology. The species epithet “kunmingensis” refers to the collecting site, Kunming City, Yunnan, China where the holotype was collected.

Typification. China. Yunnan province: Kunming city, Pan long District, Heilongtan Garden, on Carex baccans (Cyperaceae), 24 October 2021, Shucheng He, HSC86 (holotype HKAS 124483). Ex-type culture KUNCC22-12396.

Saprobic on the dead leaves of Carex baccans (Cyperaceae). Sexual morph: Not observed. Asexual morph: Hyphomycetous. Colonies effuse on natural substrate, powdery, scattered, black. Mycelium immersed to superficial, composed of pale brown to brown, septate, branched hyphae. Conidiophores appressed to the substrate, reticular, pale brown, smooth-walled. Conidiogenous cells monoblastic, pale brown, smooth-walled, creeping, integrated on micronematous conidiophores, 1.9–2.2 μm ($\overline{\mathrm{x}}$ = 2.05 μm, n = 30) wide. Conidia monilioid, pleuroacrogenous, verruculose, brown, guttulate, thick-walled, 1–2-septate, adhering in branched chains, constricted at septa, 10.3–11.2 × 5.0–5.8 μm ($\overline{\mathrm{x}}$ = 10.75 × 5.4 μm, n = 30).

Culture characteristics. Conidia germinating within 20 h on PDA media at 25 °C, reaching 4.5–4.7 cm after 20 days incubation, colony surface pale olivaceous-grey, reverse pale black, dense, circular, slightly raised, smooth, entire, hairy, wrinkled folded, pigmentation not produced on PDA.

Additional material examined. China, Yunnan province, Kunming city, Pan long District, Songhuaba Reservoir, on Carex baccans (Cyperaceae), 27 November 2021, Shu-Cheng He, HSC250, (HKAS 124484, paratype); living cultures, KUNCC22-12397; HSC251, (HKAS 124485, paratype); living cultures, KUNCC22-12398.

Notes. Chen et al. [18] obtained the culture of R. graminis and provided molecular data (ITS, 28S). In the single-phylogenetic analysis, for ITS and 28S, there is a clear genetic distance between R. kunmingensis and R. graminis with 100% MLBS/1.00 BPP, support (Figure 1), For 18S, rpb2, and tef1, R. kunmingensis formed a distinct clade. In the single and multi-phylogenetic analyses, Rutola forms a distinct clade from other genera, Rutola kunmingensis clustered as a clade sister to Rutola graminis with 100% MLBS/1.00 BPP support (Figure 1 and Figure 2). Rutola kunmingensis shares similar characteristics with R. graminis in that the conidiophores that are appressed to the substrate, branched, septate, and the conidiogenous cells are unconspicious, monoblastic, intergrated, intercalary, or terminal, hyaline to pale brown [2]. However, R. kunmingensis differs from R. graminis in having brown to dark brown, guttulate conidia, that are yellow brown and eguttulate in the latter species [2,3]. Furthermore, a nucleotide comparison showed that R. kunmingensis was distinct from R. graminis by 2% in 28S, 10% in ITS, and 5% in rpb2, suggesting significant differences between these two species. We follow the guidelines of Maharachchikumbura et al. [48] and based on a polyphasic approach, we introduce a new species.

Torula Pers., Ann. Bot. (Usteri) 15: 25 (1795)

Index Fungorum number: IF10248; Facesoffungi number: FoF 01740

Type species. Torula herbarum (Pers.) Link, Mag. Gesell. naturf. Freunde, Berlin 3(1–2): 19 (1809)

Saprobic on freshwater and terrestrial habitats. Sexual morph: Not observed. Asexual morph: Hyphomycetous. Colonies on natural substrate, effuse, scattered, powdery, brown or black. Mycelia immersed to superficial, pale brown to dark brown. Conidiophores macronematous, mononematous, reduced to conidiogenous cells or with one pale brown to brown supporting hypha. Conidiogenous cells holoblastic, mono-to polyblastic, solitary on supporting hypha, erect, doliiform, clavate to ellipsoidal, pale brown to brown. Conidia catenated, dry, acrogenous, phragmosporous, verrucose, brown to dark brown, fertile cells often are melanized in the basal region and becoming paler at apical cells.

Host and distribution. On dead stem of decaying wood and dead herbaceous plants, the distribution of Asteraceae and Iridaceae varies across China, India, Italy, Thailand, and other regions [3,4,6,9].

Notes. Torula is abound in terrestrial and freshwater habitats and visible due to formation of black, powdery colonies on dead vegetation. Species of this genus are characterized by mono-to poloblastic, intact or cupulate conidiogenous cells that produce chains of rough-walled conidia. The distal conidial cells are usually thin-walled and paler in pigmentation [49].

Torula sundara (Subram.) Y.R. Sun, Yong Wang bis & K.D. Hyde, in Jayawardena et al. Fungal Diversity 117: 65 (2023) [2022]

=Torula longiconidiophora W.H. Tian, Y.P. Chen & Maharachch., in Tian, Su, Chen & Maharachchikumbura, Journal of Fungi 9 (2, no. 150): 6 (2023)

Index Fungorum number: IF559464; Facesoffungi number: FoF 09933

Saprobic on decaying submerged wood in freshwater. Sexual morph: Not observed. Asexual morph: Hyphomycetous. Colonies effuse on natural substrate, scattered, hairy, brown-yellow when young, becoming black with aging. Mycelium mostly immersed, composed of branched, septate, smooth-walled hyphae. Conidiophores macronematous, erect, simple, straight, branched, smooth-walled. Conidiogenous cells monoblastic to polyblastic, arise terminally or laterally from a subtending cell or a fertile conidium which is melanized in the basal region. Terminal cells of conidia frequently are conidiogenous and enteroblastic. Conidia acrogenous, pleuroacrogenous, in simple or branched chain, frequently disarticulate into segments which are thick-walled, 1–5-septate, pale brown to dark brown, slightly fusiform to cylindrical with round ends, verruculose, constricted at septa, 9.7–11.8 × 3.3–2.8 μm ($\overline{\mathrm{x}}$ = 10.75 × 3.05, n = 30).

Culture characteristics. Conidia germinating within 15 h on PDA media at 25 °C, reaching 5.8–6.0 cm after 30 days incubation, colony white, medium dense, circular, depressed in the center, smooth, entire, hairy, pigmentation not produced on PDA.

Material examined: China, Yunnan Province, Kunming City, Pan long District, Songhuaba reservoir, on decaying wood submerged in freshwater, 10 December 2021, Shu-Cheng He, HSC332 (HKAS 124486); living culture, KUNCC22-12399. HSC383, (HKAS 124487); living culture, KUNCC22-12400.

Host and distribution. On the decaying branch of an unidentified host, bamboo culms, and the dead leaf rachis of Phoenix canariensis (Yunnan Province, Sichuan Province), India and New Zealand [12,17].

Notes. In the single phylogenetic tree, the ITS phylogenetic analysis indicates a notable genetic distance for T. sundara from other Torula species. However, the genetic distance for certain species, including T. chinensis, T. hollandica, T. phytolaccae, T. pluriseptata, and T. snbmersa, is less apparent. The use of 18S and 28S is not conducive to distinguishing Torula species. Conversely, rpb2 and tef1 effectively differentiate Torula species, highlighting a significant genetic gap between T. sundara and other members. Through a single-gene phylogenetic analysis, we re-evaluated the contribution of five genes to the identification of Torulaceae species (rpb2 ≈ tef1 > ITS > 28S > 18S).

In the multi phylogenetic tree, our specimens (HKAS 124486 and HKAS 124487) grouped with T. sundara and T. longiconidiophora, forming a monophyletic clade in Torula. The taxa in this clade show low genetic difference, suggesting that they might be conspecific; however, they were considered separate species (Figure 4). The phylogenetic relationship between T. longiconidiophora and T. sundara has not been investigated in previous studies. Torula sundara was introduced by Jayawardena et al. [12] from decaying bamboo culms in a terrestrial habitat of Thailand and T. longiconidiophora was described by Tian et al. [17] from decaying wood in a damp environment. These two species are similar in having slightly fusiform, dark brown to black conidia. Our specimens present minute differences between the two species in having cylindrical and smaller conidia as well as being isolated from a freshwater habitat (see

Table 2. Difference between T. sundara and T. longiconidiophora.

Species	Specimen’s No.	Habitat	Country	Conidia (μm)	References
T. sundara	HKAS 124486	freshwater	China: Yunnan	9.7–11.8 × 3.3–2.8, cylindrical to slightly fusiform, 1—pale brown to dark brown, 5—septate,	This study
T. sundara	MFLU21-0089	terrestrial environment	Thailand: Chiang Mai	41–60 × 9–15, broadly fusiform, yellow brown to dark brown, 5–10—septate.	Jayawardena et al. [12]
T. longiconidiophora	HKAS 126512	damp environment	China: Sichuan	12–46 × 6–11, broadly fusiform, dark brown to black, 1–7—septate.	Tian et al. [17]

), indicating that the habitat or substrate could shape the morphology of Torula species. We identify our specimens as T. sundara as this species name was published prior to T. longiconidiophora. Therefore, we propose Torula longiconidiophora as a synonym of Torula sundara.

4. Discussion

With the application of molecular data, Torulaceae was expanded to comprise seven morphologically contrasting genera. Multigene phylogenetic analysis showed that each genus formed well-supported monophyletic lineage. The type species of each genus were drawn from the original literature and mapped onto the phylogenetic tree (Figure 2) to demonstrate that the micromorphologies are phylogenetically informative. The key trait of Torula is the presence of conidiogenous cells with melanized basal regions. Cylindrotorula is unique in producing sporodochial conidiomata and cylindrical elongated primary conidia which give rise to secondary conidia [9]. Dendryphion has distinctly differentiated, septate conidiophores terminating in a group of clavate, cylindrical or doliiform, cicatrized polytretic conidiogenous cells [4]. Neotorula has similar morphology with some species of Dendryphion, such as Dendryphion nanum in the stalked, multiseptate conidiophores. However, Neotorula has monotretic conidiogenous cells while the conidiogenous cells of Dendryphion are polytretic [4]. Rostriconidium (=Neopodoconis I) has erect, septate conidiophores, integrated, terminal, monotretic or polytretic conidiogenous cells and fusiform to pyriform, rostrate conidia with a black truncate scar at the base and pale pigment cell above the scar [16]. Sporidesmioides (=Neopodoconis II) has similar conidiophores and conidiogenous cells to Rostriconidium. However, it can be distinguished from the latter in producing flap-like, hyaline, thick sheath at the tip the of conidia [15]. Rutola is the most recognizable genus by producing the reticular, septate, creeping conidiophores that are appressed to the substrate. In summary, Neotorula, Rutola, and Torula are similar in forming black, powdery colonies on the substrate with undifferentiated or almost reduced conidiogenous cells, while they are distinguishable in the morphology of conidiogenous cells [1,3,4,5,6,7]. Neopodoconis I and Neopodoconis II have similar conidiophores and conidia, while they are distinct genera based on phylogenetic analysis, suggesting molecular data is essential for the natural classification of these two genera.

It is interesting that Rostriconidium (=Neopodoconis I) and Sporidesmioides (=Neopodoconis II) are the basal clades in Torulaceae, and both have determinate, stipitate conidiophores with mono- to polytretic conidiogenous cells and solitary, pyriform, rostrate, smooth-walled conidia [15,16]. The distinct phylogenetic nature of these genera (Rostriconidium and Sporidesmioides) was also found in the study of Wang et al. [14]. Rotula constitutes a distinct clade that nests between “Neopodoconis I + Neopodoconis II” and the rest of the genera. Conidiogenous cells of Rutola arise from abundant creeping conidiophores, giving rise to catenulate conidia that readily break into numerous segments. Toward the terminal lineages, the genera Neotorula, Dendryphion and Torula also reproduce by producing abundant, catenulate, verruculose conidia [4,19]. There is a general tendency that species of Torulaceae produce plenty of conidia which makes it easier to spread and attach to the substrate with a rough wall [4,19]. However, this hypothesis needs to be confirmed by further sampling and evolutionary analysis. This study utilized five genetic markers (ITS, 28S, 18S, rpb2, and tef1) to construct individual phylogenetic trees for each gene (Figure 1). The results indicate that single-gene phylogenies based on ITS, 28S, and 18S exhibit low identification for Torulaceae species, particularly for Torula and Dendryphion. In contrast, the utilization of rpb2 and tef1 demonstrates a high divergence among Torulaceae species and reveals a robust topology. Furthermore, our strains (HKAS 124483, HKAS 124484, and HKAS 124485) form monophyletic clades with strong support (100% MLBS/1.00 BPP) in separate phylogenetic trees for each gene (Figure 1). Although species recognition is higher for rpb2 and tef1 than for ITS, 28S, and 18S, not all recognized species possess the rpb2 and tef1 genes [3,4,6,9]. Consequently, we recommend combining all five markers (ITS, 28S, 18S, rpb2, and tef1) to identify species within Torulaceae.

The phylogenetic tree showed that our strains (HKAS 12486 and HKAS 124487) clustered with Torula longiconidiophora and T. sundara. Torula longiconidiophora was introduced as a new species by Tian et al. [17] and T. sundara was introduced as a new combination [12]. Subramanian (1958) introduced Dwayabeeja sundara as the type species of Dwayabeeja (Subramanian 1958), which is characterized by its dark blackish-brown colonies and the presence of two types of conidia. Subsequently, based on the observation of catenate phragmoconidia, Crane and Miller [2] transferred D. sundara to Pseudotorula [11]. Jayawardena et al. [12] provided sequences for D. sundara and proposed Pseudotorula sundara as a synonym of Torula sundara. We suspect that Torula longiconidiophora and T. sundara are conspecific based on the phylogenetic result, even though they have different conidia sizes (Table 2), and T. longiconidiophora was collected from Yunnan Province, China, while T. sundara was collected from Chiang Mai Province, Thailand. Different ecological environments may result in differences within the same species. This case indicates that the morphology of Torula species is overlapping, and thus, molecular data are needed for interspecific classification. However, Torula contains many morphologically designated species which need to be clarified by re-examination of the type specimens or ex-living cultures. New collections of the morpho species are also needed for molecular data.