Phylogeny and a new species of Polylobatispora

Abstract

During surveys conducted on freshwater fungi from Vietnam, a new aquatic anamorphic fungus was isolated from decaying leaves in Vietnam. The fungus produced phialidic, swollen conidiophore and conidiogenous cells with one-celled conidia with four very tiny lobes. Based on its conidial development and other morphological characteristics, we confirmed that this is a novel fungus and described it as P. ambigua sp. nov. Phylogenetic analyses of P. ambigua based on the LSU nrDNA sequences showed that this fungus clusters in a single clade with the two known Polylobatispora spp. with 97% bootstrap value which is sister with Pseudoprobiscisporaceae. The Polylobatispora clade is nested in the Attracrosporales, Sordariomycetes. Furthermore, in this study two known species of Polylobatispora (P. deltoidea and P. quinquecornuta) were also isolated and illustrated.

1. Introduction

Members of the genus Polylobatispora Matsush. are rare aquatic fungi. They were characterised based on reduced or sparingly branched conidiophores, phialidic, hyaline conidiogenous cells which produce one-celled, triangular or lobed, pale brown conidia. The genus currently includes three species: P. deltoidea Matsush., P. quinquecornuta Matsush. (Matsushima, 1996) and P. setulosa L.B. Moro (Moro, Delgado, & Schoenlein-Crusius, 2015). The type species, P. deltoidea has triangular conidia with three lobes; P. quinquecornuta has quinquangular, 5-lobed conidia; in P. setulosathey are also triangular, 3-lobed, but have a hyaline setula at each tip. The members of Polylobatispora were isolated from aquatic habitats in Malaysia (Matsushima, 1996), Brazil (Moro et al., 2015) and Norway (Magyar, Vass, & Oros, 2017). Since no teleomorph has been known, nor DNA sequences were available, the relationship of this genus within the Ascomycota was completely unknown.

During the survey of micro-fungi in Vietnam, four hyphomycete strains were isolated from fallen leaves collected near Thac Bac waterfall in Bach Ma National forest and Suoi Tranh stream in Phu Quoc National forest, Vietnam. Since three of them were similar to the known Polylobatispora species (P. deltoidea and P. quinquecornuta), but the remaining one differed by its shape and size of the conidium, one aim of the study was to characterize the undescribed species.

Another aim was to investigate the systematic position of Polylobatispora with phylogenetic analyses of the large subunit ribosomal RNA gene (LSU rDNA) sequences from new isolates of Polylobatispora and related ascomycetes.

2. Materials and methods

2.1. Sampling, isolation and preservation

Fallen leaves of broad-leaved trees were collected near Thac Bac waterfall in Bach Ma National Forest, Hue prov., Vietnam, in May 2005 and Phu Quoc National Forest, Kien Giang, Vietnam, in Nov 2011. Samples were put in clean plastic bags, sealed and transported to the laboratory. The leaves were rinsed in tap water and incubated in moist chambers. After 3 to 7 d, each leaf was stamped on low carbon agar (LCA) (Miura & Kudo, 1970). A single spore on the LCA was isolated by a Skerman’s micromanipulator under a light microscope to obtain a pure culture. For inducing sporulation 10–20 d after inoculation, the fungal colonies were submerged in water for 1–2 d. Cultures were deposited in the Vietnam Type Culture Collection (VTCC), Institute of Microbiology and Biotechnology, Vietnam National University, Ha Noi, Vietnam, and the National Biological Resources Center (NBRC), National Institute of Technology and Evaluation (NITE), Japan (Table 1).

Table 1 List of the fungi used for phylogeny analysis

No	Family	Taxon/ Scientific name	Fungal strains number	GenBank number of 28S rDNA sequence	GenBank number of ITS rDNA sequence
1	Polylobatispora cluster	Polylobatispora deltoidea	NBRC 106820	LC495605	LC495612
2		Polylobatispora deltoidea	NBRC 106821	LC495606	LC495613
3		Polylobatispora deltoidea	NBRC 106822	LC495607	LC495614
4		Polylobatispora deltoidea	VTCC 31228	LC495601
5		Polylobatispora ambigua	VTCC 31218 = NBRC 111196	LC495600	LC495609
6		Polylobatispora quinquecornuta	VTCC 31229	LC495602	LC495610
7		Polylobatispora quinquecornuta	VTCC 31230	LC495603	LC495611
9		Polylobatispora quinquecornuta	NBRC 106823	LC495608	LC495615
10	Pseudoprobiscisporaceae	Cataractispora receptaculorum	HKUCC3710	NG_058725
11	Pseudoprobiscisporaceae	Pseudoproboscispora thailandensis	MFLUCC 15-0989	MF374369
12	Sordariomycetes genera incertae sedis	Phialemoniopsis endophytica	ACCC:38978	KT799558
13	Annulatascaceae	Vertexicola sp. confuse		AF177151
14	Sordariomycetidae genera incertae sedis	Cancellidium applanatum	CBS 337.76	MH872755
15	Attactosporacea	Rubellisphaeria abscondita	CBS 132078	KT991666
16	Attactosporacea	Atractospora aquatica	MFLU:18-2322	MK849848
17	Attactosporacea	Atractospora reticulata	CBS 127884	KT991660
18	Attactosporacea	Atractospora verruculosa	CBS 132040	KT991659
19	Annulatascaceae	Annulatascus saprophyticus	MFLUCC 14-0035	KR868947
20	Annulatascaceae	Longicollum biappendiculatum	INPA-A.C.2-1a	KU975075
21	Annulatascaceae	Submersisphaeria aquatica		KU975067
22	Annulatascaceae	Annulusmagnus triseptatus	CBS 127688	MH864680
23	Annulatascaceae	Ascitendus austriacus		KU975065
24	Annulatascaceae	Ascitendus austriacus	CBS 102665	NG_056942
25	Conlariaceae	Riomyces rotundus		JF775589
26	Conlariaceae	Conlarium duplumascospora	CGMCC 3.14938	JN936991
27	Conlariaceae	Conlarium aquaticum	MFLUCC 15-0992	MF374363
28	Conlariaceae	Conlarium thailandense	MFLUCC:17-2349	MH624127
29	genus incertae sedis	Clohiesia corticola		AF132329
30	Cordanaceae	Cordana pauciseptata	CBS:121804	HE672149
31	Cordanaceae	Cordana pauciseptata	CBS 160.56	MH869099
32	Cordanaceae	Cordana bisbyi	CBS 213.65	NG_067376
33	Cordanaceae	Cordana verruculosa	CBS 127868	NG_067464
34	Cordanaceae	Cordana inaequalis	CBS 508.83	NG_067424
35	Cordanaceae	Cordana mercadiana	FMR:11828	HE672165
36	Coniochaetaceae	Coniochaeta hoffmannii	IFM4922	AB100627
37	Coniochaetaceae	Coniochaeta ostrea	CBS 507.70	MH859821

2.2. Morphological study

The isolates were cultured at 25 °C on potato carrot agar (PCA), LCA and potato dextrose agar (PDA) for morphological observation. Observations were made under a differential interference contrast microscope (DIC; Axioplan 2, Zeiss, Jena, Germany) and a scanning electron microscope (SEM; JSM-6060, JEOL, Tokyo, Japan). For SEM observation, a small piece (ca, 5 × 5 mm) of the colony was cut and fixed with 1% OsO₄ aq. sol. at room temperature for 2 h, dehydrated in an ethanol series and finally substituted with isoamyl acetate. After critical point drying (HCP-2; Hitachi, Tokyo, Japan) and coating with platinum-palladium (JUC-5000, JEOL), the specimens were observed by SEM at 15 kV.

2.3. Molecular phylogeny

2.3.1. DNA extraction, PCR amplification and sequencing

Small pieces of a colony (3 × 3 mm) grown on malt extract agar (MEA) medium at 25 °C for 10 d were put into 2-mL Cryo tubes. DNA was extracted using the PrepMan™ Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA). PCR was performed using a KOD-Plus Kit (Toyobo, Osaka, Japan) following the manufacturer’s protocol. The rDNA large subunit region (LSU D1/D2) was amplified with the primer pair NL1/NL4 (O’donnell, 1993). To amplify the ITS region, the primers ITS1 and ITS4 were used (White, Bruns, Lee, & Taylor, 1990). Amplification of the DNA fragments was performed using the GeneAmp PCR System 9700 (Applied Biosystems). PCR products were checked by agarose gel electrophoresis and purified using an AMPureKit (Agencourt Biosciences, Beverly, MA, USA). Sequencing reactions were performed by using the Big Dye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems) and the same PCR primers. The newly generated sequence data were deposited in the DNA Data Bank of Japan (DDBJ) under the accession numbers provided in Table 1.

2.3.2. Phylogenetic analysis

All the sequences were assembled and edited manually using BioEdit ver. 7.09 (Ibis Biosciences, Carlsbad, CA, USA). They were aligned with GenBank sequences retrieved from the BLAST searches in the NCBI database (http://www.ncbi.nlm.nih.gov/) by using MEGA X (Kumar, Stecher, Li, Knyaz, & Tamura, 2018).

To determine the relationship of Polylobatispora with other ascomycetous fungi, phylogenetic analysis of the LSU rDNA partial sequence data was performed (Fig. 1). This analysis involved 36 nucleotide sequences obtained from 36 taxa belonging to six families: Pseudoprobiscisporaceae, Attactosporacea, Annulatascaceae, Conlariaceae, Cordanaceae, and Coniochaetaceae of the subclass Diaporthomycetidae of Sordariomycetes. Coniochaetaceae was used as out-group. The evolutionary history was inferred by using the maximum likelihood method and general time reversible model (Nei & Kumar, 2000). The alternative model was tested by the program installed in MEGA. The tree with the highest log likelihood (-3215.96) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach. A discrete Gamma distribution was used to model evolutionary rate differences among sites [(5 categories (+G, parameter = 0.4436)]. The rate variation model allowed for some sites to be evolutionarily invariable [(+I), 29.95% sites)]. All positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There were a total of 494 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018).

Fig. 1 - Phylogram generated from maximum likelihood analysis based on LSU rDNA partial sequences of selected species of Sordariomycetes. RAxML bootstrap support values equal to or greater than 50% are given above the nodes.

3. Results

3.1. Taxonomy

Polylobatispora ambigua L.T.H. Yen, K. Yamaguchi et K. Ando, sp. nov. Fig. 2.

MycoBank no.: MB 832137.

Diagnosis: Polylobatispora ambigua is similar to other known species of Polylobatispora by having hyaline phialides and lobed ameroconidia. It differs from other species of Polylobatispora by conidial lobes being so minute that they can be characterized better with SEM than with light microscopy.

Type: VIETNAM, Bach Ma National Park, Hue province, on fallen leaves of an unidentified deciduous broad-leaved tree, May 2005, leg. K. Ando (holotype, VTCCH 31218; isotype; ex-type culture, VTCC 31218 = NBRC 111196.

Gene sequence: ex-holotype: LC495600 (LSU) and LC495609 (ITS).

Etymology: ambigua = uncertain shape of the conidia when observed under light microscopy.

Colonies slow-growing on LCA and PCA, 5–10 mm diam after 10 d at 25 °C, light grey, partially immersed in agar. Colony on PDA growing moderately, 8–12 mm diam after 10 d at 25 °C, hyaline to light cream, mycelium thick, mostly immersed in agar, composed of branched, septate, hyaline to olivaceous, smooth, 1.0–2.5 µm wide hyphae. Sexual morph undetermined. Conidiophores absent or present. Conidiophores of P. ambigua budding from the hyphae as outgrowths, then elongating, finally composed of 1–2 cylindrical or swollen stalk cells or with 1–2 terminal conidiogenous cells (white circle in Fig. 2D), 2–5 × 2–2.5 µm, or reduced to conidiogenous cell (arrow in Fig. 2B). Conidiogenous cells swollen, 1.2–2.5 µm, constricted at the base, 0.5–1.0 µm wide at the apex, after conidium dehiscence with inconspicuous flaring collarette (arrow in Fig. 2B). Conidia hyaline to light brown, at first globose, 0.5–1.0 µm diam (arrow in Fig. 2A, D), when becoming 1.5–2.5 µm diam, four small lobes appearing at the top (arrow in Fig. 2E), when mature subglobose with four tiny lobes, 4.5–5.5 µm diam including lobes (Fig. 2C–F), lobes 0.2–0.5 × 0.2–0.5 µm (Fig. 2C). Conidium dehiscence schizolytic.

Fig. 2 - Morphological features of Polylobatispora ambigua . A–C: Conidiophores and conidia observed by light microscopy. D–F: Conidiophores and conidia observed by SEM; Bars : 5 µm.

Habitat and distribution: On fallen leaves of unidentified deciduous broad-leaved tree near waterfall, Vietnam.

Polylobatispora deltoidea Matsush. Mycol. Mem. 9: 21 (1996) ……Fig. 3A, B.

Material examined: VTCC 31228; dried culture on PDA (Nov 2011) of an isolate from fallen leaves collected in Phu Quoc National forest, Vietnam; living culture deposited at Vietnam Type Culture Collection, Institute of Microbiology and Biotechnology, Vietnam National University, Ha Noi, Vietnam, as VTCC 31228.

DNA sequence: LC495601 (LSU)

Colonies slow growing on LCA and PCA 5–10 mm diam after 10 d at 25 °C, hyaline to light grey, partially immersed in agar. Colony on PDA medium grew moderately slow-growing, 7–15 mm diam after 10 d at 25 °C, hyaline to light cream, mycelium thick, mostly immersed in agar. Mycelium composed of branched, septate, and hyaline to olivaceous, smooth, 1.0–2.5 µm wide hyphae. Sexual morph undetermined. Conidiophores absent or present, hyaline and doliiform-shaped, 2–5.5 × 2– 3 µm. Conidiogenous cells solitary, sometimes in clusters, cylindrical or bottle-shaped, producing hyaline to light brown stauroconidia with a central body 6–8 µm diam and three elliptical to oblong lobes, 3–3.5 µm wide. There was a small scar formed after conidium detachment from conidiogenous cell.

Polylobatispora quinquecornuta Matsush., Matsush. Mycol. Mem. 9: 21 (1996) ……Fig. 3C, D

VTCC 31229 and VTCC 31330; dried culture on PCA of an isolate from broad leaf collected from Bach Ma National Park- Vietnam (May 2005), living strains deposited at Vietnam Type Culture Collection, Institute of Microbiology and Biotechnology, Vietnam National University, Ha Noi, Vietnam, as VTCC 31229 and VTCC 31230.

DNA sequences: LC495602 (LSU), LC495610 (ITS) for VTCC 31229 and LC495603 (LSU), LC495611 (ITS) for VTCC 31230.

Colony slow growing on LCA and PCA, 0.5–10 mm diam after 10 d at 25 °C, hyaline to light grey, partially immersed in agar. Colony on PDA moderately slow-growing, 7–15 mm diam after 10 d at 25 °C, hyaline to light cream, mycelium thick, mostly immersed in agar. Mycelium composed of branched, septate, and hyaline to olivaceous, smooth, 1.0–2.0 µm wide hyphae. Sexual morph undetermined. Conidiophores absent or present, hyaline, 3.5–10.0 × 3–4 µm. Conidiogenous cells solitary, sometimes in clusters cylindrical or doliiform, producing 3–4 conidia at the same point. Stauroconidia hyaline to light brown, composed of a central body 11–15 µm diam and five to six (generally five) elliptical to oblong lobes, 5–6 µm wide. There was a small scar formed after conidium detachment from conidiogenous cell.

Fig. 3 - Mature conidia of Polylobatispora deltoidea and P. quinquecornuta. A: P. deltoidea VTCC 31228 observed by light microscopy. B: P. deltoidea VTCC 31228 under SEM. C, D: P. quinquecornuta VTCC 31229 observed by light microscopy; Bars : 10 µm.

3.2. Molecular phylogeny

The phylogenetic analysis revealed that Polylobatispora spp. clustered in one separated clade with high bootstrap support (97%), and this clade was sister to Pseudoproboscisporaceae, Attracrosporales, Diaporthomycetidae, Sordariomycetes, but with low support. Four strains of P. deltoidea (NBRC106820, NBRC106821, NBRC106822 and VTCC 31228) clustered in a separate clade with 100% bootstrap support. Polylobatispora quinquecornuta (NBRC106823, VTCC 31229 and VTCC 31230) clustered in one single clade with 100% bootstrap value. Polylobatispora ambigua sat alone in a single clade that separated it from known species and clustered with the P. quinquecornuta clade with a bootstrap value of 69% (Fig. 1). Based on a MegaBLAST search at NCBI GenBank, the closest hits using the ITS rDNA sequence were an uncultured fungus from a soil sample of a tea plantation in China, GenBank KT957779 (490/491 bp, 99.8% identity), P. deltoidea NBRC 106821 (446/512 bp, 87.1% identity) and Cordana bisbyi CBS 213.65 (433/507 bp, 85.4% identity). The closest hits using the LSU partial sequence were an uncultured fungus, GenBank JQ311650, (490/491 bp, 97.54% identity), P. quinquecornuta VTCC 31230 (446/512 bp, 94.51% identity) and C. abramovii PE 0063-1a (433/507 bp, 91.93% identity).

4. Discussion

4.1. Ecology of Polylobatispora

Members of the genus Polylobatispora are aquatic hyphomycetes because all the species were isolated from submerged or partially submerged substrates in aquatic habitats: P. deltoidea and P. quinquecornuta were isolated from fallen leaves collected in a stream in Malaysia (Matsushima, 1996) and P. setulosa from fallen leaves in a stream in Brazil. According to Shearer (1993) these fungi are absolutely aquatic fungi. Polylobatispora spp. have been recorded from Malaysia (Matsushima, 1996), Brazil (Moro et al., 2015) and Hungary (Magyar et al., 2017). This indicates that the genus Polylobatispora is probably widespread, but rarely encountered because aquatic hyphomycetes usually produce big, tetraridiate and branched spores which could help them easily to disperse and colonize on litter (Dang, Gessner, & Chauvet, 2007). However, they are difficult to germinate or to produce conidia on artificial cultural media (Descals & Moralejo, 2001). Even in Moro et al. (2015), P. setulosa did not form cultures on artificial media.

However, in our study, P. deltoidea, P. quinquecornuta and P. ambigua were successfully isolated from fallen leaves collected near streams or a water-fall in Vietnam. But they need water in their life cycle to induce their spore forming on the natural substrate and on artificial medium. Before isolation, the samples had to be submerged in water and put in moist chamber for several days for spore forming. Then spores were observed and transferred into new medium. After 10–20 d inoculation, there were no spores formed until the fungal colonies were submerged in water for 1–2 d. Furthermore, the 99.8% ITS sequence identity with only a single differing base pair of our strain of P. ambigua with an unpublished environmental sample from tea orchard soil in China (GenBank KT957779), however, indicates that the fungus also occurs in terrestrial habitats. Since the habitats of Polylobatispora species are not strictly aquatic, they must be terrestrial-aquatic fungi.

4.2. Taxonomy and phylogeny of Polylobatispora

Polylobatispora spp. are distinct to other hyphomycetes because they have swollen conidiophore or conidiogenuos cells. From conidiogenous cells, one-celled, triangular or lobed, light grey and conidia are produced. This combination of characteristics is somewhat similar to Anthopsis or Spegazzinia. However, the brown conidiogenous cells with brown stauro- or dictyoconidia of Spegazzinia spp. (Seifert, Kendrick, Morgan-Jones, & Gams, 2011) distinguish them from Polylobatispora spp. Anthopsis species are similar to Polylobatispora by having one-celled, triangular, light grey conidia. However, the clusters of inversed phialides (Seifert et al., 2011) make Anthopsis different from Polylobatispora.

The differences at species level of the Polylobatispora species are based on the shape and the size of conidia. Polylobatispora deltoidea has three-lobed conidia (Fig. 3A, B); P. setulosa also has three-lobed conidia, but by their size and the appendages at the tips of the conidia is differs from P. deltoidea (Moro et al., 2015); P. quinquecornuta has five-lobed conidia (Fig. 3C, D); P. ambigua has four-lobed conidia, however, the lobes of P. ambigua are so small that it was hard to visualize them under the light microscope (Fig. 2A–C). But under SEM, four lobes were clearly observed (Fig. 2D–F).

DNA barcoding of aquatic fungi is crucial because most publications on Ingoldian fungi have been based on morphological observations. Recently, to solve this problem, some authors performed phylogenetic analyses of these fungi (Shearer et al., 2009; Duarte, Batista, Bärlocher, Cássio, & Pascoal, 2015; Zhang et al., 2017; Luo et al., 2019). Based on morphological and phylogenetic analysis, Luo et al. (2019) introduced a new order Distoseptisporales, two new families, viz, Ceratosphaeriaceae and Triadelphiaceae, three new genera, viz, Aquafiliformis, Dematiosporium and Neospadicoides, 47 new species which belonging to the Sordariomycetes. However, the molecular phylogenetic position of Polylobatispora has not yet been studied.

In this study, based on phylogenetic analyses of the ITS and the partial LSU rDNA sequences, Polylobatispora formed a separate clade in Attractosporales, Sordariomycetes. This clade is sister to Pseudoproboscisporaceae, a family comprising aquatic hyphomycetes with presently the four genera Aquaticola, Cateractispora, Diluvicola, and Pseudoproboscispora, accordingly to Luo et al. (2019). In our analysis, however, the support for the sister relationship was low. Further study may show whether proposing a new family for including Polylobatispora or accommodating this genus in a known family will be the more appropriate taxonomical choice.

Disclosure

The authors declare no conflicts of interest. All the experiments undertaken in this study complied with the current laws of the country where they were performed.

Acknowledgments

This work was conducted under the Joint Research Project “Taxonomic and ecological studies of microorganisms in Vietnam and the utilization” between the Biological Resource Center, National Institute of Technology and Evaluation, Japan & the Institute of Microbiology and Biotechnology, Vietnam National University. We thank Mr. Kamijo, NBRC, for kindly performing the sequencing described in this study.

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