Freshwater ascomycetes from southern Australia: Melanascomaceae fam. nov., Melanascoma panespora gen. et. sp. nov., and Pleurothecium brunius sp. nov.

Abstract

During a survey of freshwater fungi in temperate southern Australia, two new taxa were found, Melanascoma panespora and Pleurothecium brunius. Morphological and molecular data place Melanascoma panespora in the Diaporthomycetidae representing a new genus. Melanascoma, along with Proliferophorum and Paraproliferophorum, form a new lineage and the family Melanascomaceae is introduced. Phylogenetic analyses using ITS, 28S, and 18S nrRNA gene sequences,, along with morphological examination revealed Pleurothecium brunius to be a new species of Pleurothecium, sister to P. aquaticum.

Citation: Fryar SC, Catcheside DEA (2023). Freshwater ascomycetes from southern Australia: Melanascomaceae fam. nov., Melanascoma panespora gen. et. sp. nov., and Pleurothecium brunius sp. nov. Fungal Systematics and Evolution 11: 85–93. doi: 10.3114/fuse.2023.11.07

INTRODUCTION

Freshwater fungi are abundant and diverse in rivers, streams and lakes around the world (Jones et al. 2014). They include saprophytes, plant parasites, animal parsites, endophytes and some form mycorrhizae (Jones et al. 2014). Fungi form a rich tapestry of interactions with other organisms within freshwater ecosystems, playing critical roles such as decomposition of organic matter (Calabon et al. 2020).

A major impediment to full understanding of fungal ecology and conservation is the lack of taxonomic information (May et al. 2019). With only 3–8 % of fungal species described (Hawksworth & Lücking 2017), it is imperative to accurately describe and delimit many more fungi to allow ecologists, conservation, and evolutionary biologists to accurately study them and their interactions. During a survey of freshwater fungi on wood in streams in southern Australia, two distinctive and novel ascomycetes were collected.

The first ascomycete species found during our survey was morphologically similar to many species within the Diaporthomycetidae by having wide, septate paraphyses, unitunicate, cylindrical asci with a distinctive non-amyloid apical apparatus (Maharachchikumbura et al. 2015), but we were unable to assign it any known taxa. Maharachchikumbura et al. (2015) described the subclass Diaporthomycetidae (Sordariomycetes) and outlined the orders and families included in this new subclass. Hyde et al. (2021) further refined the phylogeny of this subclass including 21 orders.

The second species had morphological characters consistent with the genus Pleurothecium but did not fit the description of any of the previously described species. Höhnel (1919) established Pleurothecium for P. recurvatum, a species that is commonly found in freshwater habitats. Réblová et al. (2016) introduced Pleurotheciales and Pleurotheciaceae to accommodate Pleurothecium within the Sordariomycetes. There are currently eight accepted species of Pleurothecium, six of which occur in fresh water (Réblová et al. 2012, Hyde et al. 2017, Luo et al. 2018, 2019, Shi et al. 2021).

In this study, we provide morphological descriptions and phylogenies for these two novel taxa based on multigene analyses.

MATERIALS AND METHODS

Collection details and examination

Samples of submerged wood less than 5 cm diam were collected in Scott Creek Conservation Park, South Australia from two streams, approximately 50 cm deep with a muddy base, that flow only during winter. Samples were sealed into plastic bags for transport to the laboratory. The riparian vegetation is a mixture of native vegetation and invasive weeds.

Samples were incubated in sterile plastic containers and regularly examined for fungi over 6 months using a Leica MZ7s dissecting microscope. Any fungi observed were photographed, described, and transferred to a microscope slide with a drop of distilled water. A cover slip was added and the slide was examined using a Nikon Eclipse Ni with differential interference contrast. Photographs were taken using either a Sony RX-100 or Canon D6 camera.

Single spore isolation

Potato dextrose agar (PDA, BD micro) was autoclaved, cooled to 60 °C, 100 mg/L streptomycin and 70 mg/L of penicillin added as filter-sterilised stock solutions and 10 mL poured into each 60 mm diam plate.

Ascospores or conidia were transferred to a sterile 1.5 mL microtube with 20 μL of sterile water using a sterile micro needle. The mixture was agitated for several seconds, then transferred to a PDA plate using a pipette. Plates were incubated at room temperature and checked over 5 d for germinating spores using a Leica MZ7s dissecting microscope. Germinated spores were picked off the agar surface using a sterile needle and transferred to individual PDA plates which were incubated at room temperature.

DNA extraction, amplification, and sequencing

Standard method

Approximately 50 mg of fungal mycelium was scraped from the surface of agar cultures with a sterile scalpel and the genomic DNA was isolated using a Qiagen DNeasy Plant Mini kit following the manufacturer’s protocols. The final DNA extracts were eluted into 100 μL of buffer.

Primers LROR/LR5 (Vilgalys & Hester 1990, Rehner & Samuels 1994) were used to amplify the sequences from the 28S nrRNA gene (28S), while ITS1/ITS 4 (White et al. 1990) were used to amplify the internal transcribed spacer regions and the intervening 5.8S nr RNA gene (ITS), and NS1/NS4 (White et al. 1990) were used to amplify the 18S nrRNA gene. Sequences from protein-coding gene, translation elongation factor 1-alpha (TEF1), were amplified with primers were EF1-983F and EF1-2218R (Rehner & Buckley 2005).

Reaction mixtures contained 5 μL buffer, 1 μL (10 mM each) dNTPs, 1 μL (10 μM) of each primer, 0.25 μL hotStart Taq DNA polymerase (New England Biolabs), 1 μL DNA template, and 16.75 μL sterile milliQ water.

PCR amplification was performed in an Applied Biosystems 2720 Thermo Cycler. Cycling conditions for PCR were initial denaturation at 95 °C for 3 min; 35 cycles of denaturation at 95 °C for 1 min, annealing at 52 °C (ITS) or 54 °C (28S) for 50 s, and extension at 72 °C for 1 min; and a final extension at 72 °C for 10 min. Cycling conditions for TEF1 was 95 °C for 1 min; 35 cycles of 95 °C for 30 s, 57 °C for 50 s, and 68 °C for 1 min; followed by 68 °C for 5 min. PCR products were observed on a 1.5 % agarose electrophoresis gel stained with Gel Red (Gene Target Solutions).

The resulting amplicons were purified using a Qiagen QIAquick PCR Purifcation Kit and sequenced in both directions using the respective primers by the Australian Genome Research Facility. Raw sequence reads were assembled, examined, and edited using Sequencher v. 5.3 (Gene Codes Corporation). Newly generated sequences were submitted to NCBI GenBank under the accession numbers listed in Tables 1 and 2.

Table 1.

GenBank accession numbers of selected taxa from Diaporthomycetidae used for phylogenetic analyses. Newly generated sequences are shown in bold.

Species	Strain	ITS	28S	TEF1
Acrodictys bambusicola	HSAUP myr9510	KU999973	KX033564	—
Acrodictys elaeidicola	HSAUP mj5528	KU999978	KX033569	—
	HSAUP mj5536	KU999977	KX033568	—
Acrodictys fluminicola	KUMCC 15-0240	MK828642	MK849786	—
Acrodictys globulosa	HSAUP myr4696	KU999970	KX033562	—
Acrodictys liputii	HSAUP myr7561	KU999974	KX033565	—
Acrodictys malabarica	HSAUP myr9509	KU999968	KX033560	—
Acrodictys peruamazonensis	HSAUP myr4694	KU999969	KX033561	—
Acrodictys porosiseptata	HSAUP myr4698	KU999967	KX033559	—
Ascobrunneispora aquatica	HKUCC 3708	AF177154	AF132326	—
Cancellidium cinereum	MFLUCC 18-0424	MT370353	MT370363	MT370488
Cancellidium atrobrunneum	MFLUCC 20-0100	MT422724	MT422740	MT436438
Fluminicola aquatica	MFLUCC 15-0962	MF374357	MF374366	MF370960
Fluminicola saprophytica	MFLUCC 15-0976	NR_153493	NG_069504	MF370956
	MFLUCC 15-0984	MF374359	MF374368	—
	MFLUCC 18-1244	MW286504	MW287778	MW396649
Fluminicola striata	MFLUCC 18-0990	MW286496	MW287770	—
Fluminicola thailandensis	MFLUCC 14-0037	MK828644	MK849789	MN194049
	MFLUCC 15-0984	MF374359	NG_069505	—
Melanascoma panespora	AD291710	—	OQ789909	—
	AD219607	—	OQ799385	OQ870569
Obliquiminima hyalina	MFLUCC 18-1401	MW286507	MW287781	MW396652
Papulosa amerospora	AFTOL-ID 748	—	DQ470950	DQ471069
Paraproliferophorum hyphaenes	CPC 40103	ON603770	ON603790	ON605632
Platytrachelon abietis	CBS 125235	—	NG_057957	—
Proliferophorum thailandicum	MFLUCC 17-0293	MK028344	MK028343	—
Pseudostanjehughesia aquitropica	MFLU 17-0857	MF077548	MF077559	MF135655
Pseudostanjehughesia lignicola	MFLUCC 15-0352	NR_168808	MK849787	MN194047
Wongia aquatica	MFLUCC 18-1607	MK828645	MK849788	MN194048
Wongia fusiformis	DLUCC:1767	MZ420746	MZ420761	—
Wongia griffinii	BRIP 60377	KU850472	KU850470	KU850466

Table 2.

GenBank accession numbers of selected taxa from Pleurothecium used for phylogenetic analyses. Newly generated sequences are shown in bold.

Species	Strain	ITS	28S	18S
Phaeoisaria clematidis	MFLUCC 18-1017	MW131990	MW132065	MW132063
Pleurothecium aquaticum	GZCC19-0546	MW133897	—	MW134679
	MFLU 21-0148	OM654775	OM654772	OM654807
	B-27	—	MK835854	MK834786
	MFLU 17-0922	NR_160597	NG_066197	—
Pleurothecium brunius	AD291640	OQ799373	OQ799347	OQ799346
	AD291629	OQ799378	OQ799377	OQ799376
Pleurothecium floriforme	MFLU 15-1163	NR_156614	NG_059791	—
Pleurothecium guttulatum	IFRD 9203	NR_176728	MT559115	NG_081395
Pleurothecium obovoideum	CBS 209.95	EU041784	EU041841	—
Pleurothecium pulneyense	MFLUCC 16-1293	—	MF399262	MF399228
Pleurothecium recurvatum	GZCC19-0441	MW133898	—	MW134680
	CBS 138686	KT278727	KT278715	KT278702
	CBS 138747	KT278728	KT278714	KT278703
Pleurothecium semifecundum	CBS 131271	NR_111710	NG_057951	NG_062854
	CBS 131482	JQ429158	JQ429239	JQ429253

Direct PCR method

Numerous attempts were made to culture specimens. When cultures were not obtained, conidiophores or ascomata were used for direct PCR. In these cases, Phire Plant Direct PCR Master Mix (Thermo Fisher Scientific) was used. Ascomata or conidiophores were placed into 20 μL of Phire dilution buffer at 4 °C, heated in a dry block to 98 °C for 10 min, then on ice for 5 min, vortexed briefly, then centrifuged for 1 min at 11 000 rcf.

Reaction mixtures for the Phire kit contained 7 μL sterile milliQ water, 10 μL master mix, 1 μL of each primer, 1 μL DMSO, and 1 μL DNA template. PCR cycling conditions for Phire kit extracts was 98 °C for 5 min; 35 cycles 98 °C for 5 s, 60 °C (ITS), 56.8 °C (28S) or 55.5 °C (18S) for 5 s, 72 °C for 20 s; followed by a final extension at 72 °C for 1 min.

For some samples using the Phire kit, a second band was persistent on electrophoresis gels, despite optimisation of PCR conditions. In these cases, the band-stab and re-amplification technique of Bjourson & Cooper (1992) was used. Re-amplification used the standard PCR protocol and primers described above. PCR primers, product clean up and sequencing were as described above for the standard method.

Phylogenetic analyses

The generated sequences for each gene were used in megablast searches (Zhang et al. 2000) to identify closely related sequences in NCBI’s GenBank nucleotide database. Other sequences used in this study were derived from GenBank. Sequences were aligned in Geneious Prime v. 2023.0.4 (https://www.geneious.com) using MUSCLE. Ambiguously aligned regions were removed from the alignments using GBlocks v. 0.91b (Castresana 2000, Talavera & Castresana 2007) on the Phylogeny.fr platform (Dereeper et al. 2008). Alignments were imported into Mega X v. 10.2.6 (Stecher et al. 2020) to find the best substitution models for phylogenetic analyses. The best substitution models were K2+G (ITS) and TN93+G (28S and TEF1) for the first species and K2+G (ITS and 28S), K2 (18S) for the second species. However, RAxML and MrBayes do not use these models, so GTR+G+I was used for all analyses as recommended by Abadi et al. (2019). Genes were then concatenated and maximum-likelihood phylogenetic trees were constructed with partitions for each gene region using RAxML v. 8.2.11 (Stamatakis 2014) within Geneious using the GTR + I + G substitution model and branch support values were calculated with 1 000 rapid bootstrap inferences. The same alignment was analysed using Bayesian analysis with MrBayes using the GTR GAMMA I substitution model (v. 3.2.6; Huelsenbeck & Ronquist 2001) with partitions for each gene region within Geneious. All resulting trees were formatted in Geneious, then further edited in Adobe Illustrator v. 27.0.

RESULTS

Phylogenetic analyses

Phylogenetic trees based on multi-locus analyses (Figs 1, 2) show the relationships between the new species and other related taxa. Branch supports of Maximum Likelihood bootstrap ≥ 70 % and Bayesian PP value ≥ 0.90 are indicated above the branches.

Fig. 1.

Phylogram generated from maximum likelihood analysis based on combined ITS, 28S and TEF1 sequence data of Melanascoma panespora and closely related taxa. Bootstrap values equal to or great that 70 % and Bayesian posterior probabilities equal to or greater than 0.90 are given above the nodes. Ex-type strains are shown in bold and newly generated sequences shown in blue.

Fig. 2.

Phylogram generated from maximum likelihood analysis based on combined ITS, 28S and 18S sequence data of Pleurothecium species. Bootstrap values equal to or great than 70 % and Bayesian posterior probabilities equal to or greater than 0.90 are given above the nodes. Ex-type strains are shown in bold and newly generated sequences shown in blue.

Despite numerous attempts, we were unable to recover high-quality sequence data from the ITS region of either of the specimens of the first species. To place this undescribed perithecial species (AD291710 and AD219607) from our survey, the 28S and TEF1 sequences were analysed with ITS, 28S and TEF1 sequences of 30 species in five families in the Sordariomycetes (Fig. 1). The dataset comprised 1 670 characters: 321 bp for ITS, 506 bp for 28S, 843 bp for TEF1. The 18S sequences were mostly uninformative and were not included. The tree is rooted to Cancellidium applanatum, C. cinereum and Obliquiminima hyalina. The undescribed ascomycete nested within the Diaporthomycetidae and formed a well supported clade (100/1) with Proliferophorum thailandicum and Paraproliferophorum hyphaene, which together form a new family (Fig. 1).

For the undescribed Pleurothecium (AD291629 and AD291640) the combined ITS, 18S and 28S sequences were assessed with the eight previously recognised species in Pleurothecium (Fig. 2). The dataset comprised 2 161 characters: 511 bp for ITS, 814 bp for 28S, 836 bp for 18S. The tree is rooted to Phaeoisaria clematidis. The two new specimens form a sister clade to Pleurothecium aquaticum and Pleurothecium guttulatum, representing a new species, Pleurothecium brunius (Fig. 2).

Taxonomy

Melanascomaceae Fryar & D.E.A. Catches, fam. nov. Index Fungorum IF 900400.

Asexual morph: Hyphomycetous. Conidiophores macronematous, mononematous, sub-cylindrical to cylindrical, unbranched or branched, erect, olivaceous brown to dark brown, light brown at the apex, septate, smooth or ornamented. Conidiogenous cells holoblastic, polyblastic, terminal, subhyaline to brown. Conidia fusiform to cylindrical, subhyaline to brown, 0–3-septate when mature, guttulate, smooth.

Sexual morph: Ascomata perithecial, non-stromatic, subglobose with a straight neck. Ascomatal wall textura angularis, dark brown. Paraphyses persistent, cylindrical, septate, unbranched. Asci unitunicate, eight-spored, apex with a non-amyloid apical ring. Ascospores ellipsoid, septate, ornamented, without appendages or sheath.

Type genus: Melanascoma Fryar & D.E.A. Catches

Melanascoma Fryar & D.E.A. Catches, gen. nov., Index Fungorum IF 900396.

Etymology: The prefix “Melan-” means darkly coloured. Melanascoma refers to the darkly coloured ascomata.

Asexual morph: undetermined. Sexual morph: Ascomata perithecial, non-stromatic, immersed, subglobose with a straight neck, dark brown. Ascomatal wall textura angularis, dark brown. Paraphyses persistent, cylindrical, septate, unbranched. Asci unitunicate, pedicellate, eight-spored, apex with a non-amyloid apical ring. Ascospores ellipsoid, hyaline, septate, ornamented, without appendages or sheath.

Type: Melanascoma panespora Fryar & D.E.A. Catches

Melanascoma panespora Fryar & D.E.A. Catches, sp. nov. Fig. 3. Index Fungorum IF 900395.

Fig. 3.

Melanascoma panespora (holotype AD291710). A. Necks on wood from fresh water. B. Sectioned perithecium in wood. C. Paraphyses. D. Ascus tip. E, F. Asci. G. Peridium. H–L. Ascospores. Scale bars: A = 400 μm; B = 200 μm; C–L = 5 μm.

Typus: Australia, South Australia, Scott Creek Conservation Park (S35°5’45.90”, E138°40’59.16), on submerged decaying wood in an ephemeral stream, 30 Aug. 2020, S.C. Fryar (holotype AD219607; GenBank sequences: 28S - OQ799385; 18S - OQ799375).

Etymology: The specific epithet refers to the appearance of the ascospores, like Vienna bread loaves.

Sexual morph: Perithecia immersed, subglobose, solitary or gregarious, 300–432 × 216–312 μm. Ascomatal wall dark brown, textura angularis (35–50 μm thick). Neck long, cylindrical, black, apex hyaline, (469–)536–771 × (67–)80–87 μm. Paraphyses hyaline, septate, unbranched, constricted at septa up to 4 μm wide, tapering to rounded ends 2 μm wide. Asci cylindrical, uniseriate, inamyloid apical ring, 4–5 μm wide, (1.5–)2 μm high, 8-spored, pedicellate, slowly dissolving in water, (155–) 160–165(–178) × (12–)15–18 μm. Ascospores hyaline, ellipsoid, 1–3-septate, very slightly constricted at the septa, finely verrucose 18–22(–24) × 7–9 μm. Asexual morph undetermined.

Additional material examined: Australia, South Australia, Scott Creek Conservation Park (S35°5’45.90”, E138°40’59.16), 30 Aug. 2020, on submerged decaying wood in an ephemeral stream, S. Fryar (paratype AD219607; GenBank sequences: 28S - OQ789909; 18S - OQ789908; TEF1 - OQ870569).

Notes: Melanascoma panespora forms a clade with Proliferophorum thailandicum and Paraproliferophorum hyphaenes (Fig. 1) both of which form monotypic genera. They are described from asexual morphs, with sexual morphs unknown, therefore morphological comparisons with these genera are not possible at this stage. This clade is sister to Papulosaceae and forms a larger clade with Pseudostanjehughesiaceae and Acrodictyaceae. Further analysis and data will be required to decide which of these clades represents a new order within the Diaporthomycetidae.

Morphologically M. panespora resembles other species within the subclass Diaporthomycetidae in having globose to subglobose brown to black ascomata, wide, septate paraphyses, unitunicate, cylindrical asci with a distinctive non-amyloid apical apparatus (Maharachchikumbura et al. 2015). Rhamphoria is similar to Melanascoma but has dictyosporous ascospores (Réblová et al. 2018). Atractospora is also similar but has fusiform ascospores and lateral necks (Réblová et al. 2016).

Melanascoma panespora shares some characteristics with Rivulicola species (Hyde et al. 1997, Raja et al. 2009, Ranghoo et al. 2000). The asci of M. panespora resemble those of Rivulicola species having uniseriate asci with large inamyloid apical rings and a pedicel. The ascospores of Rivulicola are ellipsoid, hyaline, and septate as in M. panespora but Rivulicola has ascospores with a mucilaginous sheath and without constriction at the septa. In addition, the ascomata and necks of Rivulicola are hyaline to pale brown compared with the dark brown ascomata and necks of Melanascoma. There are currently no available sequences for Rivulicola in GenBank for phylogenetic comparison.

Hyde et al. (2021) noted that, in their analyses, Proliferophorum diverged from Platyrachelon around 76 MYA, which falls within the family range (50–130 MYA). In our analysis the clade including Melanascoma, Proliferophorum and Paraproliferophorum is sister to Platyrachelon, forming a distinct lineage. We therefore introduce the new family Melanascomaceae to accommodate these three genera.

Pleurothecium brunius Fryar & D.E.A. Catches, sp. nov., Index Fungorum IF 900397. Fig. 4.

Fig. 4.

Pleurothecium brunius (holotype AD291640). A. Conidiophores on wood from fresh water. B–D. Conidiophores and conidiogenous cells. E–J. Conidia. Scale bars: A = 100 μm; all others = 5 μm.

Etymology: The specific epithet refers to the brown colour of the conidia.

Typus: Australia, South Australia, Scott Creek Conservation Park (S35°5’45.90”, E138°40’59.16) on submerged decaying wood in an ephemeral stream, S.C. Fryar (holotype AD291640; GenBank sequences: ITS - OQ799373; 28S - OQ799347; 18S - OQ799346).

Asexual morph: Conidiophores macronematous, mononematous, straight or slightly flexuous, septate, smooth, unbranched, dark brown, paler towards the apex, apex hyaline, (60–)80–100(–115) × 3–3.5 μm. Conidiogenous cells integrated, terminal, polyblastic, denticulate, hyaline, swollen, with 6–8 cylindrical denticles, swollen part 3–7 × 3–5 μm, denticles (2–)2.5–4 μm long, 1 μm wide. Conidia hyaline to dark brown, ellipsoid, 1–3-septate with conspicuous septa, not constricted at the septa, straight to slightly curved, smooth-walled (14–)16–19 × 5–6 μm. Sexual morph undetermined.

Distribution: Found in South Australia, Australia.

Additional material examined: Australia, South Australia, Scott Creek Conservation Park (S35°5’45.90”, E138°40’59.16), on submerged decaying wood in an ephemeral stream, S.C. Fryar (paratype AD291629; GenBank sequences: ITS - OQ799378; 28S - OQ799377; 18S - OQ799376; TEF1 - OQ784578).

Notes: Pleurothecium brunius forms a clade with P. aquaticum and P. guttulatum (Fig. 2). Morphologically it is different to P. aquaticum in having dark brown, shorter conidia, and longer conidiophores. Pleurothecium brunius is different to P. guttulatum by having dark brown, septate conidia, and a bulbous apex.