Phialolunulospora vermispora (Chaetosphaeriaceae, Sordariomycetes), a novel asexual genus and species from freshwater in southern China

Abstract

The asexual taxon Phialolunulospora vermisporagen. et sp. nov., collected from submerged dicotyledonous leaves in Hainan, China, is described and illustrated herein. Phialolunulosporagen. nov. is characterized by macronematous, semimacronematous, septate and pigmented conidiophores and acrogenous, long lunate, vermiform to sigmoid, hyaline conidia with an eccentric basal appendage. Complete sequences of internal transcribed spacer (ITS) and partial sequences of nuclear large subunits ribosomal DNA (LSU) genes are provided. Phylogenetic analyses of combined ITS and LSU sequences revealed its placement in the Chaetosphaeriaceae. The new fungus is compared with morphologically similar genera.

Introduction

China is considered an important Asian reservoir of biodiversity. The southern area of China ranks 34^th in biodiversity hotspots (Myers et al. 2000; Williams et al. 2001). Hainan Island, located in the south of China, harbors an incredibly high diversity of fungi. Its humid, subtropical climate, with an average annual temperature of 22 to 27 °C and an average annual precipitation of 1000–2600 mm, favors development of fungi. Our group has conducted investigations of freshwater fungi to increase knowledge of this important ecological group in China (Qiao et al. 2017a, b, 2018a, b, 2019a, b, 2020).

During our present investigation of freshwater fungi in Hainan Island, South China, an interesting species was collected on dead leaves of an unidentified dicotyledonous tree. This species is characterized by unbranched and septate conidiophores, phialidic conidiogenous cells and vermiform to sigmoid and aseptate conidia with an eccentric basal appendage. Based on preliminary analysis of morphological data, we place this unknown fungus in Chaetosphaeriaceae, but a literature search found that it did not belong to any known genus. To further confirm the position of the species, phylogenetic analyses with related taxa within Chaetosphaeriaceae were carried out based on complete sequences of internal transcribed spacer (ITS) and partial sequences of nuclear large subunits ribosomal DNA (LSU) genes.

Materials and methods

Isolation and morphological study

Submerged dicotyledonous leaves were collected from Limu Mountain Nature Reserve in Hainan Province. Samples were preserved in zip-lock plastic bags, labelled, and transported to the laboratory. The decomposed leaves were cut into several 2–4 × 2–4 cm sized fragments and then spread on to the surface of corn meal agar (CMA, 20 g cornmeal, 18 g agar, 40 mg streptomycin, 30 mg ampicillin, 1000 ml distilled water) medium for 10 days; single conidium was isolated with a sterilized needle and transferred to CMA plates while viewing with an Olympus BX51 microscope. The pure strain was further transferred to potato dextrose agar (PDA, 200 g potato, 20 g dextrose, 18 g agar, 40 mg streptomycin, 30 mg ampicillin, 1000 ml distilled water) medium. Colony morphology and microscopic characteristics were examined, and photographs were taken with an Olympus BX51 microscope connected to a DP controller digital camera. Measurement data were based on 30 random conidia and 10 conidiophores.

Pure cultures were deposited in the Herbarium of the Laboratory for Conservation and Utilization of Bio resources, Yunnan University, Kunming, Yunnan, China (YMF, formerly Key Laboratory of Industrial Microbiology and Fermentation Technology of Yunnan) and at the China General Microbiological Culture Collection Center (CGMCC).

DNA extraction, PCR amplification, and sequencing

Pure cultures were grown on PDA medium for 5 days at 25 °C. Actively growing mycelium was scraped off from the surface of the culture and transferred to 2 ml Eppendorf micro-centrifuge tubes. Total genomic DNA was extracted according to the procedures in Turner et al. (1997). Primers used for PCR amplification and sequencing of the nuclear large subunits ribosomal DNA (LSU) and the internal transcribed spacer (ITS) were LROR-LR7 and ITS1-ITS4, respectively (Vilgalys and Hester 1990; White et al. 1990). PCR products were purified and stored at -20 °C until sequencing. The same pairs of primers were used to obtain sequences, which was performed by Macrogen Europe (Macrogen Inc. Amsterdam, The Netherlands). Finally, the sequences were assembled and edited using SeqMan v. 7.0.0 (DNAStar Lasergene, Madison, WI, USA) to obtain the consensus sequences. The newly obtained sequences were submitted to GenBank nucleotide database (Table 1).

Table 1.

List of strains analyzed in this study, with GenBank accession numbers.

Species	Strain	ITS	LSU
Adautomilanezia caesalpiniae	LAMIC 010212	NR_153560	NG_058594
Anacacumisporium appendiculatum	HMAS 245593T	KT001555	KT001553
Anacacumisporium appendiculatum	HMAS 245602	KT001556	KT001554
Bahusutrabeeja dwaya	CBS 261.77T	MH861059	MH872829
Brunneodinemasporium brasiliense	CBS 112007T	JQ889272	JQ889288
Brunneodinemasporium jonesii	GZCC 16–0050T	KY026058	KY026055
Cacumisporium capitulatum	FMR 11339	HF677176	HF677190
Cacumisporium capitulatum	SMH 3766	–	AY017374
Calvolachnella guaviyunis	CBS 134695	NR_153892	NG_058879
Chaetosphaeria ciliata	CBS 122131T	MH863180	MH874726
Chaetosphaeria ciliata	ICMP 18253	–	GU180637
Chloridium chloroconium	FMR 11940	KY853435	KY853495
Chloridium sp.	HGUP 1806	MK372070	MK372068
Codinaea lambertiae	CBS 143419T	NR_156389	NG_059053
Codinaea pini	CBS 138866T	NR_137943	NG_058902
Conicomyces pseudotransvaalensis	HHUF 29956T	NR_138015	LC001708
Cryptophiale hamulata	MFLUCC 180098	–	MG386756
Cryptophiale udagawae	MFLUCC 180422	MH758198	MH758211
Cryptophialoidea fasciculata	MFLUCC 172119	MH758195	MH758208
Dendrophoma cytisporoides	CBS 223.95T	JQ889273	JQ889289
Dictyochaeta ellipsoidea	MFLUCC 181574T	MK828628	MK835828
Dictyochaeta lignicola	DLUCC 0899T	MK828630	MK835830
Dictyochaeta assamica	CBS 242.66	MH858788	MH870426
Dictyochaetopsis gonytrichoides	CBS 593.93	AF178556	AF178556
Dinemasporium morbidum	CBS 129.66T	JQ889280	JQ889296
Dinemasporium polygonum	CBS 516.95T	NR_137786	NG_059109
Echinosphaeria canescens	SMH 4791	–	AY436403
Eucalyptostroma eucalypti	CBS 142074T	NR_154027	NG_059257
Eucalyptostroma eucalyptorum	CPC 31800T	NR_159834	MH327838
Exserticlava vasiformis	TAMA 450	–	AB753846
Gelasinospora tetrasperma	CBS 178.33	NR_077163	DQ470980
Helminthosphaeria clavariarum	SMH 4609T	–	AY346283
Infundibulomyces cupulata	BCC 11929T	EF113976	EF113979
Infundibulomyces oblongisporus	BCC 13400T	EF113977	EF113980
Kionochaeta castaneae	GZCC 18–0025T	MN104610	MN104621
Kionochaeta microspora	GZCC 18–0036T	MN104607	MN104618
Lasiosphaeria ovina	SMH 4605	AY587923	AY436413
Lecythothecium duriligni	CBS 101317	–	AF261071
Leptosporella arengae	MFLUCC 150330T	MG272255	MG272246
Leptosporella gregaria	SMH 4290T	–	AY346290
Linocarpon arengae	MFLUCC 150331T	–	MG272247
Linocarpon cocois	MFLUCC 150812T	MG272257	MG272248
Menispora glauca	FMR 12089	HF678528	HF678538
Menispora tortuosa	DAOM 231154	KT225527	AY544682
Menispora tortuosa	CBS 214.56	AF178558	AF178558
Menisporopsis breviseta	GZCC 18–0071T	MN104612	MN104623
Menisporopsis dushanensis	GZCC 18–0084T	MN104615	MN104626
Morrisiella indica	HKUCC 10827	–	DQ408578
Multiguttulispora sympodialis	MFLUCC 180153T	MN104606	MN104617
Nawawia filiformis	MFLUCC 160853	–	MH758206
Nawawia filiformis	MFLUCC 172394	MH758196	MH758209
Neonawawia malaysiana	CBS 125544T	GU229886	GU229887
Paliphora intermedia	CBS 896.97T	NR_160203	NG_057766
Paliphora intermedia	CBS 199.95	–	EF204500
Phaeostalagmus cyclosporus	CBS 663.70	MH859892	MH871680
Phaeostalagmus cyclosporus	CBS 312.75	–	MH872661
Phialolunulospora vermispora	YMF 1.04260T	MK165444	MK165442
Phialosporostilbe scutiformis	MFLUCC 170227T	MH758194	MH758207
Phialosporostilbe scutiformis	MFLUCC 181288	MH758199	MH758212
Pseudodinemasporium fabiforme	MAFF 244361T	AB934068	AB934044
Pseudolachnea fraxini	CBS 113701T	JQ889287	JQ889301
Pseudolachnea hispidula	MAFF 244364	AB934071	AB934047
Pseudolachnella longiciliata	HHUF 29962	AB934081	AB934057
Pseudolachnella yakushimensis	HHUF 29683T	AB934087	AB934063
Pseudolachnella pachyderma	HHUF 29955	AB934085	AB934061
Pyrigemmula aurantiaca	CBS 126743T	HM241692	HM241692
Pyrigemmula aurantiaca	CBS 126744	HM241693	HM241693
Rattania setulifera	GUFCC 15501	GU191794	HM171322
Ruzenia spermoides	SMH 4606	–	AY436422
Sordaria fimicola	CBS 508.50	MH856730	MH868251
Sporoschisma hemipsilum	SMH 2125	–	AF466083
Sporoschisma hemipsilum	SMH 3251	–	AF466084
Stanjehughesia vermiculata	HKUCC 10840	–	DQ408570
Striatosphaeria codinaeaphora	MR 1230	AF178546	AF178546
Striatosphaeria codinaeaphora	SMH 1524	–	AF466088
Synaptospora plumbea	SMH 3962	–	KF765621
Tainosphaeria jonesii	GZCC 16–0053	KY026059	KY026056
Tainosphaeria jonesii	GZCC 16–0065	KY026060	KY026057
Tainosphaeria monophialidica	MFLUCC 180146T	–	MN104616
Thozetella pandanicola	MFLUCC 160253T	MH388366	MH376740
Thozetella tocklaiensis	CBS 378.58T	MH857817	MH869349
Verhulstia trisororum	CBS 143234T	MG022181	MG022160
Zanclospora iberica	CBS 130426T	KY853480	KY853544
Zanclospora iberica	FMR 12186	KY853481	KY853545

*Sequences generated in this study are emphasized in bold face. ^Tex-type cultures.

Sequence alignment and phylogenetic analysis

Preliminary BLAST searches with the ITS and LSU sequences of our strain against the GenBank nucleotide database determined the closely related species (Altschul et al. 1990). BLAST search showed that our strain has homology to species in Chaetosphaeriaceae. Based on this information, related sequences of the two marker loci, which include 72 representatives belonging to Chaetosphaeriaceae, 4 representatives of Helminthosphaeriaceae, 2 representatives of Linocarpaceae and 2 representatives of Leptosporellaceae, were downloaded according to recent studies (Yang et al. 2016, 2018; Wei et al. 2018; Lin et al. 2019). Sordaria fimicola (Roberge ex Desm.) Ces. & De Not, Gelasinospora tetrasperma Dowding and Lasiosphaeria ovina (Pers.) Ces. & De Not were used as the outgroup. These, together with the newly generated sequences, were aligned with ClustalX 1.83 (Thompson et al. 1997) with default parameters, and the consensus sequences were manually adjusted and linked through BioEdit v.7.0 (Hall 1999). Manual gap adjustments were done to improve the alignment and ambiguously aligned regions were excluded. Then, the combined alignment was converted to a NEXUS file using the program MEGA6 (Tamura et al. 2013) and a PHY files using the program ClustalX 1.83. The resulting combined sequence matrix included 1475 nucleotide positions (with alignment gaps) from two regions (607 from ITS, 868 from LSU). GenBank accession numbers of downloaded sequences are given in Table 1.

Maximum-likelihood (ML) analysis was computed with RAxML (Stamatakis 2006) with the PHY files generated with CLUSTAL_X version 1.83, using the GTR-GAMMA model. ML bootstrap proportions (MLBPs) were computed with 1000 replicates. Bayesian inference (BI) analysis was conducted with MrBayes version 3.2.2 (Ronquist and Huelsenbeck 2003). The Akaike information criterion (AIC) implemented in jModelTest version 2.0 was used to select the best fit models after likelihood score calculations were done (Posada 2008). The base tree for likelihood calculations was ML-optimized. HKY+I+G was estimated as the best-fit model under the output strategy of the AIC. Metropolis-coupled Markov chain Monte Carlo (MCMCMC) searches were run for 5 000 000 generations, sampling every 500^th generation. Two independent analyses with four chains each (one cold and three heated) were run until the average standard deviation of the split frequencies dropped below

0.01. The initial 25% of the generations of MCMC sampling were discarded as burn-in. The refinement of the phylogenetic tree was used for estimating BI posterior probability (BIPP) values. The tree was viewed in FigTree version 1.4 (Rambaut 2012).

Results

Phylogenetic analyses

The combined dataset comprised 71 taxa (including our strain) representing 52 genera, which include 60 species in the family Chaetosphaeriaceae, 4 species in Helminthosphaeriaceae, 2 species in Linocarpaceae and 2 species in Leptosporellaceae, with Gelasinospora tetrasperma CBS 178.33, Sordaria fimicola CBS 508.50 and Lasiosphaeria ovina SMH 4605 as the outgroup. The final alignment comprised a total of 1475 base pairs, containing the ITS and LSU sequences, and were analyzed by BI and ML method. The topology of the tree is shown in Fig. 1, with the Bayesian posterior probabilities above 95% and ML bootstrap support greater than 70% indicated for respective clades. In this tree, our strain occurred on an isolated clade within Chaetosphaeriaceae, and clustered together with Dictyochaetopsis Aramb. & Cabello, Bahusutrabeeja Subram. & Bhat and Dictyochaeta Speg. with good Bayesian posterior probabilities (100%) and ML bootstrap proportions (100%). Considering distinct morphological characters with these three genera, we propose to describe our unknown isolate as a new genus and species, Phialolunulospora vermispora.

Figure 1.

Phylogenetic tree derived from Bayesian analysis based on ITS and LSU sequences, depicting the relationships of the new taxon Phialolunulospora vermispora with closely related taxa. The numbers above branches represent BIPP (left) and MLBPs (right). BIPP over 95% and MLBPs greater than 70% are shown on the respective branches, and the bar represents the substitutions per nucleotide position. Gelasinospora tetrasperma CBS 178.33, Sordaria fimicola CBS 508.50 and Lasiosphaeria ovina SMH 4605 were used as outgroup.

Taxonomy

Phialolunulospora

Z. F. Yu & R. F. Castaneda gen. nov.

C8C22876-17B4-5984-B075-7703241266A8

828716

Type species.

Phialolunulospora vermispora Z. F, Yu & R. F. Castañeda

Etymology.

Phialo-Prefix, Phia. lis N.L fem. S. Phialide referring to the phialidic conidiogenous cells, and lunulospora, (lu.nu.la.tus N.L. adj. mean crescent-shaped + spo.ra N.L. fem. S. spora, referred to the conidia), referring to the genus Lunulospora.

Description.

Asexual fungus. Conidiophores macronematous, semimacronematous, mononematous, septate, prostrate or erect, straight or flexuous, pigmented. Conidiogenous cells integrated, terminal, cylindrical to subulate, pale brown to brown, monophialidic or polyphialidic, enteroblastic. Conidial secession schizolytic. Conidia solitary, acrogenous, long lunate, vermiform to sigmoid, unicellular, hyaline, truncate at the conspicuous or inconspicuous basal frill, with a cellular, unbranched, eccentric basal appendage.

Phialolunulospora vermispora

Z. F. Yu & R. F. Castaneda sp. nov.

6A1EB2AB-F23B-54E4-B808-E0AEF505B12D

828717

Figures 2 , 3 , 4

Figure 2.

Phialolunulospora vermispora (YMF 1.04260) A colony on PDA at day 10 B conidia C–F conidiophores, conidiogenous cells and conidia G conidiogenous cells H, I conidiophores and conidiogenous cells. Scale bars: 10 mm (A); 10 μm (B–I).

Figure 3.

Phialolunulospora vermispora conidiogenous cells and conidia.

Figure 4.

Phialolunulospora vermispora conidiogenous cells.

Type.

China, Hainan province, Limu Mountain, 19°29'40"N, 107°80'45"E, ca. 350 m alt., from leaves of an unidentified dicotyledonous plant submerged in a stream, Apr 2015, Zefen Yu, YMF 1.04260 – holotype; CGMCC 3.19632 – culture ex-type.

Etymology.

ver.mi- (from vermiformis), NL fem. adj mean worm-shaped + spo.ra N.L. fem. S. spora, referred to worm-shaped conidia.

Description.

Mycelium partly superficial and partly immersed, composed of septate, branched, smooth, hyaline, 1–2 μm wide hyphae. Conidiophores solitary, macronematous, semimacronematous, erect or prostrate, straight or flexuous, unbranched, up to 4-septate, cylindrical, up to 150 μm long, 3–4 μm wide, pale brown to brown, smooth, sometimes reduced to conidiogenous cells. Conidiogenous cells integrated, terminal, cylindrical to subulate, sometimes lageniform, determinate, smooth, pale brown to brown, mostly darker than conidiophores, phialidic, after secession leaving an inconspicuous basal frill, 12–47 × 2.6–3 μm. Conidia solitary, acrogenous, long lunate, vermiform to sigmoid, unicellular, guttulate, hyaline, smooth-walled, 31–55 × 2.5–3.5 μm, acute at the apex and narrow truncate at the base bearing minute marginal frills and a cellular, single, unbranched, somewhat attenuated or acuminate, eccentric basal appendage, 1.5–4.6 μm long.

Culture characteristics.

Colonies attain 2.4 cm diameter on PDA and 2.8 cm diameter on CMA after 10 days at 25 °C. On PDA, colonies flat to slightly raised, aerial mycelium abundant, margin entire to undulate, surface white initially, then become buff and grey with age, reverse same color. Colonies on CMA, center with aerial mycelium cottony, periphery with scarce aerial mycelium, olivaceous grey, dark green exudate and soluble pigment produced, reverse same color.

Distribution and ecology.

The species occurs on submerged leaves in stream. This species is currently known only from the type locality.

Discussion

The family Chaetosphaeriaceae was firstly introduced by Réblová et al. (1999) to accommodate Chaetosphaeria and its allies. Réblová et al. (1999) also suggested that Chaetosphaeriaceae should be placed in the Sordariales. However, based on the nuclear large subunit ribosomal RNA gene (LSU) sequence, Huhndorf et al. (2004) placed Chaetosphaeriaceae in order Chaetosphaeriales. In a recent review of the family Chaetosphaeriaceae based on morphology and phylogenetic analysis, Lin et al. (2019) accepted 49 genera (including three uncertain genera) within the family, among which 44 were asexual genera.

The asexual morph of the Chaetosphaeriaceae is hyphomycetous taxa. It is characterized by septate, branched or unbranched conidiophores with the conidiogenous cell monophialidic or polyphialidic, holoblastic or enteroblastic, smoothwalled (Réblová et al. 1999, 2011). Our new fungus, Phialolunulospora vermispora, fits the general description of asexual hyphomycetous Chaetosphaeriaceae well. Phialolunulospora is mainly distinguished from other species in the Chaetosphaeriaceae in having vermiform to sigmoid conidia. Conidia of typical members of the family, including Dictyochaeta and Codinaea Maire (Réblová 2000; Whitton et al. 2000; Cruz et al. 2008; Crous et al. 2014), are aseptate or 1-septate; they may be setulose or not. In this study, the phylogenetic analyses combining ITS and LSU sequences showed that P. vermispora is close to three asexual genera in Chaetosphaeriaceae (Fig. 1), Dictyochaetopsis, Bahusutrabeeja and Dictyochaeta. Morphologically, Bahusutrabeeja and Dictyochaeta are superficially similar to P. vermispora in septate and cylindrical conidiophores, but can be distinguished from the new genus in having globose conidia without appendages and long fusiform conidia with long appendage (Subramanian and Bhat 1977; Li et al. 2014; Liu et al. 2016; Lin et al. 2019), respectively. P. vermispora is clearly different from Dictyochaetopsis species in morphology, such as smooth and pale brown or brown conidiophores and long lunate, vermiform to sigmoid conidia (Arambarri and Cabello 1990; Whitton et al. 2000; Castañeda-Ruíz et al. 2008).

Phialolunulospora is morphologically similar to some other genera species of Chaetosphaeriaceae in hyaline conidia with basal eccentric cellular appendages, including Neopseudolachnella A. Hashim. & Kaz. Tanaka, Pseudolachnea Ranoj., Pseudolachnella Teng and Rattania Prabhug. & Bhat. Of these, species of Neopseudolachnella, Pseudolachnea and Pseudolachnella are different from Phialolunulospora in acervular, setose and stromatic conidiomata (Ranojevic 1910; Teng and Ling 1936; Hashimoto et al. 2015). The genus Rattania is distinguished from Phialolunulospora in having seta and smaller septate conidia (Prabhugaonkar and Bhat 2009). In addition, the type species of Lunulospora Ingold, L. curvula Ingold (Sordariomycetes, Sordariales incertae sedis), also has resemblance to Phialolunulospora in conidial shape (Ingold 1942; Seifert et al. 2011), but it has obviously bigger size of conidia, 70–90 × 4–5 μm vs. 12–47 × 2.6–3 μm, in Lunulospora.

Many freshwater species occur in the family Chaetosphaeriaceae. So far, approximately 16 genera in this family have been reported from fresh water, such as Codinaea (Luo et al. 2019). In this study, Phialolunulospora vermispora was also collected from freshwater habitats.

Citation

Zheng H, Wan Y, Li J, Castañeda-Ruiz RF, Yu Z (2020) Phialolunulospora vermispora (Chaetosphaeriaceae, Sordariomycetes), a novel asexual genus and species from freshwater in southern China. MycoKeys 76: 17–30. https://doi.org/10.3897/mycokeys.76.57410