Neomonodictysaquatica sp. nov. (Pleurotheciaceae) from a plateau lake in Yunnan Province, China.

BACKGROUND: In this study, a new species Neomonodictysaquatica was collected from submerged decaying wood in Erhai Lake, Yunnan Province, China. NEW INFORMATION: Neomonodictysaquatica is characterised by acrogenous, solitary, oval, dictyospores (most are transverse septum, occasionally vertical septum, in immaturity the septum is clear, but when mature, the conidia becomes darker so the septum is not clear), smooth-walled conidia. The immature conidia are usually hyaline to olivaceous and mature conidia are usually darkened to black, sometimes with one pale basal cell. Phylogenetic analyses of combined ITS and LSU sequence data showed that the new collection is distinct from other Neomonodictys species. Description and illustration are provided as well. Si-Ping Huang, Dan-Feng Bao, Hong-Wei Shen, Hong-Yan Su, Zong-Long Luo.

Introduction

was introduced by Réblová et al. (2016), based on morphological characters and phylogenetic analyses. Members of are mostly saprobic on wood (Hyde et al. 2020) and some species have been identified as opportunistic human pathogens (Guarro et al. 2000, Chew et al. 2010, Réblová et al. 2020). Species of the order were collected on submerged decaying wood in lentic and lotic habitats in temperate, subtropical and tropical zones in Asia, Europe, Melanesia and North America (Matsushima 1971, Réblová et al. 2012, Réblová et al. 2016, Réblová et al. 2020, Hernández-Restrepo et al. 2017, Hyde et al. 2018, Hyde et al. 2020, Luo et al. 2018a).

is a single family of . It is typified by with as the type species (Morgan) Höhn (Réblová et al. 2016). Recently, Hyde et al. (2020) updated the phylogenetic tree for and introduced a new genus Y.Z. Lu, C.G. Lin & K.D. Hyde. Currently, ten genera are accepted in this family (Réblová et al. 2016, Maharachchikumbura et al. 2016, Hernández-Restrepo et al. 2017, Hyde et al. 2020, Goh and Kuo 2021).

The monophyletic asexual genus is established for a fungus () collected from a freshwater habitat in Thailand, which is morphologically similar to members of S. Hughes (Hyde et al. 2020). is characterised by holoblastic, monoblastic, integrated, terminal, determinate conidiogenous cells and muriform, subglobose to globose, smooth-walled, pale brown to darkened to black conidia (Hyde et al. 2020).

In this study, the fungus was isolated from submerged decaying wood in Erhai Lake, Yunnan Province in China. The morphology and phylogeny show that our collection is distinct from related species. We provide detailed descriptions, illustrations for and a synopsis table for the morphology comparison.

Materials and methods

Isolation and morphological examination

Submerged decaying wood was collected from Erhai Lake, Dali City, Yunnan, China. The coordinates of sampling sites are 25°44′29.65″N, 100°09′49.33″E and at an altitude of 1966 m. Samples were returned to the laboratory in plastic bags. The samples were incubated in aseptic plastic boxes, lined with moistened tissue paper at room temperature for one week. Specimen observations and morphological studies were conducted following the protocols provided by Luo et al. (2018b).

Morphological observations were made by using a SMZ760 series stereomicroscope and photographed using a Nikon-80i microscope. The fungal structures were measured with Tarosoft (R) Image Frame Work programme and images were processed using Adobe Photoshop CS6 extended version 13.0 (Adobe Systems, USA). Single spore isolation was carried out following the method described in Chomnunti et al. (2014). Germinating conidia were transferred aseptically to PDA plates with 0.5 mg/l of Amoxicillin and incubated at room temperature under dark conditions. The colonies were checked every three days. A herbarium was deposited in the herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (KUN-HKAS), Yunnan, China. Living cultures were deposited in Kunming Institute of Botany Culture Collection (KUNCC) and China General Microbiological Culture Collection Center (CGMCC). Facesoffungi numbers were registered as described in Jayasiri et al. (2015) and Index Fungorum numbers as in Index Fungorum(2021).

Molecular Phylogenetic Analyses

DNA Sequencing and Sequence Alignment

The appropriate fungal mycelium was scraped from the surface of colonies on Potato Dextrose Agar (PDA) plates with a scalpel into a 1.5 ml EP tube (Bao et al. 2018). Genomic DNA was extracted using the TreliefTM Plant Genomic DNA Kit (Beijing TsingKe Biological Technology and Services Co. Ltd, China) according to the manufacturer’s protocols.

The primers ITS4/ITS5 for Internal transcribed spacer (ITS) and LR0R/LR5 for Large subunit ribosomal ribonucleic acid (LSU rRNA) were selected for PCR amplification (Vilgalys and Hester 1990). Polymerase Chain Reaction (PCR) mixture was performed in a 25 μl system reaction containing 9.5 μl ddH2O, 12.5 μl of 2 × Power Taq PCR Master Mix, 1 μl of DNA template and 1 μl of each primer (10 μM) (Wang et al. 2019). The PCR thermal cycles for amplification of the ITS gene region were as per Su et al. (2015) and the LSU gene followed Sun et al. (2020). PCR amplifications were confirmed on 1% agarose electrophoresis gels stained with ethidium bromide.

Sequences were assembled with BioEdit. Sequences with high similarity indices were determined from a BLAST search to find the closest matches with taxa in and from recently published data (Ariyawansa et al. 2015, Wanasinghe et al. 2015, Hyde et al. 2019, Hyde et al. 2020). All consensus sequences and the reference sequences were aligned in MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html, Katoh and Standley 2013). Aligned sequences of each gene region (ITS and LSU) were combined and manually improved using BioEdit v. 7.0.5.2 (Hall 1999). Ambiguous regions were excluded from the analyses and gaps were treated as missing data.

Phylogenetic Analyses

Maximum Likelihood analysis was performed in the CIPRES Science Gateway v.3.3 (Miller et al. 2010) using RAxML v. 8.2.8 as part of the “RAxML-HPC2 on XSEDE” tool (Stamatakis 2006, Stamatakis et al. 2008). The final ML search was conducted using the GTRGAMMA + I model estimated using MrModeltest 2.2 (Nylander 2004), with ML bootstrap support being calculated from 1000 bootstrap replicates.

Bayesian analysis was performed using MrBayes v. 3.1.2. (Ronquist and Huelsenbeck 2003). The model of each gene was estimated using MrModeltest 2.2 (Nylander 2004), with GTR + I + G model being the best-fit model of ITS and LSU for Bayesian analysis. Posterior Probabilities (PP) (Rannala and Yang 1996) were performed by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v.3.1.2 (Liu et al. 2012). Six simultaneous Markov chains were run for 50 million generations and trees were sampled every 5000th generation (resulting in 10,000 trees). The first 2000 trees, representing the burn-in phase of the analyses, were discarded and the remaining 8000 (post burning) trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree (Cai et al. 2006, Liu et al. 2012).

Phylogenetic trees were visualised by FigTree v. 1.4.4 (Rambaut 2014) and edited in Microsoft Office PowerPoint 2016 (MicrosoftInc. United States). Newly-produced sequences in this study were submitted to GenBank (Table 1).

Table 1.

Isolates and sequences used in this study (newly-generated sequences are indicated in bold and with “*” after species name, the type strains are in bold).

Taxon	Strain	GenBank Accession No.
		ITS	LSU
Adelosphaeriacatenata	CBS 138679	NR_145396	MH877664
Anapleurotheciumbotulisporum	FMR 11490	NR_153582	KY853483
Ascotaiwaniamitriformis	HKUCC3706	−	AF132324
Ascotaiwaniasawadae	SS00051	HQ446340	HQ446363
Bactrodesmiastrumobovatum	FMR 6482	NR_152537	FR870266
Bactrodesmiastrumpyriforme	FMR 11931	HE646636	HE646637
Brachysporiellasetasa	HKUCC 3713	−	AF132334
Canalisporiumcaribense	SS03683	GQ390284	GQ390269
Canalisporiumelegans	SS00895	GQ390286	GQ390271
Canalisporiumexiguum	SS00809	GQ390296	GQ390281
Canalisporiumpulchrum	SS03982	GQ390292	GQ390277
Conioscyphajaponica	CBS 387.84	−	AY484514
Conioscyphalignicola	CBS 335.93	−	AY484513
Conioscyphaminutispora	CBS 137253	NR_137847	NG_066275
Conioscyphaperuviana	ILL 41202	−	NG_058867
Conioscyphavaria	CBS 113653	−	AY484512
Fuscosporellapyriformis	MFLUCC 16-0570	NR_152555	NG_059711
Helicoascotaiwaniafarinosa	DAOM 241947	JQ429145	JQ429230
Melanotrigonumovale	MR 3685	KT278726	KT278712
Melanotrigonumovale	CBS 138744	KT278725	KT278710
Melanotrigonumovale	CBS 138815	KT278722	KT278711
Melanotrigonumovale	CBS 138743	NR_145397	NG_058197
Melanotrigonumovale	CBS 138742	KT278723	KT278708
Mucisporaobscuriseptata	MFLUCC 15-0618	NR_152556	NG_059709
Neomondictysaquatica *	KUNCC21-10708	MZ686200	OK245417
Neomonodictysmuriformis	MFLUCC 16-1136	NR_168231	NG_068916
Parafuscosporellamoniliformis	MFLUCC 15-0626	NR_152557	NG_059710
Phaeoisariaaquatica	MFLUCC 16-1298	NR_160592	NG_066194
Phaeoisariaclematidis	MFLUCC 16-1273	MF399229	MF399246
Phaeoisariaclematidis	DAOM 226789	JQ429155	JQ429231
Phaeoisariaclematidis	MFLUCC 17-1968	MG837022	MG837017
Phaeoisariaclematidis	MFLUCC 17-1341	MF399230	MF399247
Phaeoisariafasciculata	DAOM 230055	KT278720	KT278706
Phaeoisariafasciculata	CBS 127885	NR_145395	NG_064241
Phaeoisariaguttulata	MFLUCC 17-1965	MG837021	MG837016
Phaeoisarialoranthacearum	CBS 140009	NR_56593	NG_064294
Phaeoisariapseudoclematidis	MFLUCC 11-0393	NR_155648	NG_059559
Phaeoisariasedimenticol	CGMCC 3.14949	MK878380	MK835851
Phaeoisariasparsa	FMR11939	HF677179	HF677185
Phaeoisariamicrospora	MFLUCC 16-0033	MF671987	−
Pleurotheciellaaquatica	MFLUCC 17-0464	NR_160591	NG_066193
Pleurotheciellacentenaria	DAOM 229631	NR_111709	NG_060098
Pleurotheciellalunata	MFLUCC 17-0111	NR_160593	NG_066195
Pleurotheciellarivularia	CBS 125238	NR_111711	NG_057950
Pleurotheciellarivularia	CBS 125237	JQ429161	JQ429233
Pleurotheciellafusiformis	KUMCC 15-0192	MF399234	MF399251
Pleurotheciellafusiformis	MFLUCC 17-0113	MF399233	MF399250
Pleurotheciellafusiformis	MFLUCC 17-0115	MF399232	MF399249
Pleurotheciellafusiformis	MFLUCC 16-1356	MF399235	MF399252
Pleurotheciellaguttulata	KUMCC 15-0442	MF399239	MF399256
Pleurotheciellaguttulata	KUMCC 15-0296	NR_160594	NG_066399
Pleurotheciellakrabiensis	MFLUCC 16-0852	MG837018	MG837013
Pleurotheciellakrabiensis	MFLUCC 16-0858	MG837019	MG837014
Pleurotheciellasaprophytica	MFLUCC 16-1251	NR_160595	NG_066196
Pleurotheciellasubmersa	MFLUCC 17-1709	NR_160596	MF399260
Pleurotheciellasubmersa	DLUCC 0739	MF399242	MF399259
Pleurotheciellasubmersa	MFLUCC 17-0456	MF399244	MF399261
Pleurotheciellatropica	MFLUCC 16-0867	MG837020	MG837015
Pleurotheciellauniseptata	KUMCC 15-0407	MF399231	MF399248
Pleurotheciumaquaticum	MFLUCC 17-1331	NR_160597	NG_066197
Pleurotheciumfloriforme	MFLUCC 15-0628	NR_156614	NG_059791
Pleurotheciumobovoideum	CBS 209.95	EU041784	EU041841
Pleurotheciumpulneyense	MFLUCC 16-1293	−	MF399262
Pleurotheciumrecurvatum	CBS 138686	KT278727	KT278715
Pleurotheciumrecurvatum	CBS 138747	KT278728	KT278714
Pleurotheciumrecurvatum	CBS 131646	JQ429150	JQ429236
Pleurotheciumrecurvatum	CBS 131272	JQ429149	JQ429237
Pleurotheciumrecurvatum	CBS 101581	JQ429148	−
Pleurotheciumsemifecundum	CBS 131482	JQ429158	JQ429239
Pleurotheciumsemifecundum	CBS 131271	NR_111710	NG_057951
Savoryellaaquatica	SS 03801	HQ446349	HQ446372
Savoryellalignicola	NF00204	HQ446357	HQ446378
Savoryellalongispora	SAT00322	HQ446359	HQ446380
Savoryellapaucispora	SAT00866	−	−
Savoryellaverrucosa	SS 00052	HQ446353	HQ446374
Sterigmatobotrysmacrocarpa	PRM 915682	JQ429153	_
Sterigmatobotrysmacrocarpa	DAOM 230059	−	GU017316
Sterigmatobotrysrudis	DAOM 229838	JQ429152	JQ429241
Triadelphiauniseptata	DAOMC 250376	−	KT278718

Taxon treatments

D.F. Bao, S.P. Huang & Z.L. Luo sp. nov.

A78A5B68-C181-5A92-892A-865AFBC15D3E

558842

Facesofungi number: FOF 10537

Type status: Holotype. Occurrence: recordedBy: Longli Li; Siping Huang; Taxon: scientificName: ; kingdom: ; phylum: ; class: ; order: ; family: ; genus: ; Location: waterBody: Erhai Lake; locality: Baitaiyi; verbatimElevation: 1966 m; locationRemarks: China, Yunnan Province, Dali, saprobic on submerged decaying wood in Erhai Lake; verbatimLatitude: 25 44 29.65N; verbatimLongitude: 100d 09' 49.33'' E; Event: year: 2020; habitat: freshwater, submerged decaying wood; Record Level: collectionID: 2EH 3-17-1 H; collectionCode: L127.

Description

Sexual morph Undetermined. Asexual morph Hyphomycetous (Fig. 1) sporodochia. Colonies on natural substratum superficial, scattered, black, glistening. Mycelium immersed in the substrate, composed of septate, smooth, thin-walled, light to dark brown, 2–3 μm wide hyphae. Conidiophores lacking. Conidiogenous cells short or occasionally missing, suborbicular, holoblastic, monoblastic, integrated, terminal, determinate, hyaline to pale brown. 3.7–6.4 × 2.9–4.7 μm (x̄ = 5.1 × 3.8 μm, n = 10). Conidia 23.1–29.5 × 8.5–11.5 μm (x̄ = 26 × 10 μm, n = 30), acrogenous, acrospore, oval, ellipsoidal to obovoid, muriform, smooth-walled, hyaline when young, becoming dark brown at maturity sometimes with one pale basal cell.

Figure 1.

(KUN-HKAS 115806, holotype). a Colonies on submerged wood; b-e Conidiophores with conidia; f-j Conidiogenous cells with conidia; k-p Conidia; q Germinating conidium; r, s Colony on PDA. Scale bars: b-c, e, k = 25 μm; d, f-i, l-p = 20 μm; j, q = 30 μm.

Culture characteristics: Conidia germinate on PDA in 36 h. Colonies growing on PDA, subglobose, with flat surface, edge jagged, reaching 3 cm long and 2.5 cm wide in 12 weeks at 28°C, dark grey in PDA medium. Mycelium superficial and partially immersed, branched, septate, hyaline to pale brown, smooth.

Material examined: China, Yunnan Province, Dali, sprobic on submerged decaying wood in Erhai Lake, September 2020, S. P. Huang, L-127 (KUN-HKAS 115806, holotype), ex-type living culture, KUNCC 21-10708 = CGMCC3.20681.

Etymology

Name reflects the aquatic habitat of this fungus

Notes

Morphologically, is easily distinguished from . has wider conidia than (15–25 vs. 8–12.2 μm). In addition, conidia of are oval or ellipsoidal to obovoid, while has subglobose to globose conidia. In the phylogenetic analysis, clustered with with strong support (99% ML and 1.00 PP) (Fig. 2). ITS comparison between our strain and MFLUCC 16-1136 revealed 57 bp difference in a total of 539 bp. LSU comparison between our strain and MFLUCC 16-1136 revealed 13 bp difference in total of 829 bp (Jeewon and Hyde 2016). Therefore, we introduce our new isolate as a new species.

Figure 2.

Phylogenetic tree based on RAxML, generated from a combined ITS and LSU dataset. Bootstrap support values for Maximum Likelihood (ML, black) higher than 75% and Bayesian posterior probabilities (BYPP, red) greater than 0.95 are indicated above the nodes as ML/PP. The tree is rooted to . The type-derived sequences are indicated in bold and new isolates are in red. Bootstrap values for Maximum Likelihood (ML) equal to or greater than 75% and clade credibility values greater than 0.90 from Bayesian-inference analysis labelled on the nodes. Ex-type strains are in bold and black, the new isolate is indicated in bold and red. (Fig. 2).

Analysis

Phylogenetic analyses

The phylogram generated from Maximum Likelihood analysis, based on combined ITS and LSU sequence data, represents and the closely related orders. Seventy-nine strains are included in the combined analyses, which comprise 2039 characters (ITS: 849 bp, LSU: 1190 bp) after aligning. (AFTOL-1) is the outgroup taxon in this phylogentic tree. The best RAxML tree with a final likelihood value of -12803.740107 is presented. The matrix had 698 distinct alignment patterns with 34.21% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.222096, C =0.295691, G = 0.272214, T = 0.209999; substitution rates AC = 1.588217, AG = 2.820721, AT = 2.535737, CG = 1.003016, CT = 5.905028, GT = 1.000000; gamma distribution shape parameter α = 0.570011.

In the phylogenetic analysis, our new isolate clustered as a sister taxon with with strong bootstrap support (99 ML/1.00 PP, Fig. 2).

Discussion

Up to now, two species are accepted in , including the newly-introduced species. Both of them are collected from submerged wood in freshwater habitats (Hyde et al. 2020) and only asexual morphs are reported. Morphologically, is similar to in having solitary, dictyospores conidia and monoblastic, hyaline to brown conidiogenous cells (Ellis 1971, Seifert et al. 2011). Compared with the diaphragms of them, have a mostly transverse septum, less of the vertical septum, but the transerve and vertical septa of are evenly distributed. The significant difference between and is conidiophores, which are shorter than in the former (Kukwa and Diederich 2005). Phylogenetically, they are distinct (Hyde et al. 2020). In the phylogenetic analysis, was placed in (Day et al. 2006, Seifert et al. 2011, Wijayawardene et al. 2020), while was placed in (Hyde et al. 2020).