DOI: https://doi.org/10.21203/rs.3.rs-2900241/v1
During a survey of freshwater fungi in temperate southern Australia, two previously unknown anamorphic ascomycetes were found. The coelomycetous ascomycete was placed in the family Morosphaeriaceae (Pleosporales) as a new genus based on molecular data. We introduce the new genus and species Microvesuvius unicellularis with morphological and molecular data. The Dictyochaeta-like hyphomycete was placed in Achrochaetabased on both morphological characters and phylogenetic analyses using ITS, 28S, and tef1 sequences. Achrochaeta rivulata is the second species described within this genus.
Freshwater fungi are an ecologically important group within freshwater ecosystems around the world. They are found in lakes, rivers, streams, and waterholes as decomposers, endophytes, plant and animal pathogens, and some form mycorrhizae. There are 3,870 species described, mostly within the Sordariomycetes and Dothideomycetes, with many more being found every year (Calabon et al., 2022). It is estimated that there are 20,000 freshwater fungal species worldwide (Gessner & Van Ryckegem, 2003).
Freshwater fungi are any species which, for the whole or part of their life cycle, rely on free freshwater, or which uses any resource of a predominantly aquatic or semi-aquatic nature as a substratum (Thomas, 1996). This excludes spores or fungal DNA from a terrestrial origin found in a freshwater habitat.
During a survey of freshwater fungi on submerged wood in southern Australia, a coelomycete resembling Hongkongmyces and a Dictyochaeta-like hyphomycete species were found. Coelomycetous fungi are often found in freshwater habitats and occur throughout the ascomycota (Wijayawardene et al., 2020). Dictyohaeta and similar genera are found in the Chaetosphaeriaceae.
Réblová (1999) introduced Chaetosphaericaceae to accommodate Chaetosphaeria and it is now a genus- and species-rich family, most species of which are only known by their asexual morph (Réblová et al., 2021; Lin et al., 2019). They are commonly found in both terrestrial and freshwater habitats.
In this study, we provide morphological descriptions and phylogenies of these two novel taxa based on multigene analyses.
Submerged wood samples less than 5 cm in diameter were collected from Scott Creek Conservation Park, South Australia from two streams that flow only during winter and are approximately 50cm deep with a muddy base. Samples were sealed in plastic bags for transport to the laboratory. The riparian vegetation is a mixture of native vegetation and invasive weeds.
The wood samples were incubated in sterile plastic containers and regularly examined for fungi using a Leica MZ7s dissecting microscope over 6 months. All fungi observed were photographed, described, then examined using a Nikon Eclipse Ni with differential interference contrast. Imaged were captured using either a Canon 6D or Sony RX-100 camera.
Potato dextrose agar (PDA, BD micro) was autoclaved, cooled to 60°C, 100mg/L streptomycin and 70mg/L of penicillin added as filter-sterilised stock solutions and 10 mL poured into each 60mm diameter plate.
Using a sterile needle, spores were transferred to 20 µL of sterile water in an Eppendorf tube and agitated for several seconds to suspend them. The suspension was then pipetted onto a PDA plate. Plates were incubated at room temperature and checked over 5 days 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.
Approximately 50 mg of fungal mycelium was scraped from the surface of agar cultures with a sterile scalpel and genomic DNA isolated using a Qiagen DNeasy Plant Mini kit following the manufacturer's protocols. The final DNA extracts were eluted into 100 µL of buffer.
Sequences from translation elongation factor 1-alpha (TEF1) were amplified with primers were EF1-983F and EF1-2218R (Rehner & Buckley 2005). For nuclear ribosomal genes, primers ITS1/ITS 4 (White et al., 1990) were used to amplify ITS1, 5.8S and ITS2 and LROR/LR5 (Vilgalys & Hester, 1990; Rehner & Samuels, 1994) to amplify the sequences from the 28S nrRNA gene
Reaction mixtures contained 1 µL (10 mM each) dNTPs, 1 µL (10µM) of each primer, 0.25 µL hotStart Taq DNA polymerase (New England Biolabs), 1 µL DNA template, 5 µL buffer, 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 30s, 57°C for 50 s, and 68°C for 1 min; followed by 68°C for 5 min.
PCR amplicons were visualised on 1.5% agarose electrophoresis gels stained with Gel Red (Gene Target Solutions).
PCR products 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.
| Species | Strain | ITS | 28S |
|---|---|---|---|
| Aliquandostipite khaoyaiensis | ISAN100 | MT864350 | MT860428 |
| Aquilomyces metrosideri | CBS 146782 | NR_173036 | NG_076729 |
| Aquilomyces patris | REF101 | JN859321 | JN859476 |
| Aquilomyces patris | CBS 135760 | KP184004 | KP184042 |
| Aquliomyces patris | REF099 | JN859319 | JN859475 |
| Aquliomyces patris | CBS 135662 | KP184003 | KP184043 |
| Aquliomyces patris | CBS 135661 | NR_137961 | NG 057057 |
| Aquliomyces rebunensis | KT 732 2 | AB809630 | AB807542 |
| Aquliomyces rebunensis | HHUF 27556 | NR_154664 | NG_056937 |
| Clypeoloculus hirosakiensis | HHUF 30144 | NR_153866 | NG_068961 |
| Clypeoloculus microsporus | HHUF 30143 | NR_153867 | NG_068960 |
| Clypeoloculus towadaensis | HHUF 30145 | NR_153865 | NG_058722 |
| Helicascus alatus | MFLUCC 17–0147 | MG356480 | MG356478 |
| Helicascus mangrovei | MCR355 | KX957960 | KX639747 |
| Helminthosporium chengduense | CGMCC 3.23575 | ON557751 | ON557745 |
| Jahnula dianchia | MFLUCC 16-1353 | MH793538 | MH793544 |
| Leptosphaeria etheridgei | CBS 125980 | NR_111620 | JF740291 |
| Leptosphaeria irregularis | MFLUCC 15-1118 | NR_171725 | KX856055 |
| Leptospora macarangae | MFLUCC 18–0553 | NR_174831 | MW063232 |
| Massarina pandanicola | MFLUCC 17–0596 | MG646958 | MG646947 |
| Microvesuvius unicellularis | AD291626 | OQ799384 | OQ799383 |
| Microvesuvius unicellularis | AD291633 | OQ799382 | OQ799391 |
| Morosphaeria muthuputensis | PUFD87 | MF614795 | MF614796 |
| Morosphaeria velatispora | PUFD25 | MK026766 | MK026764 |
| Neohelicascus aquaticus | KUMCC 19–0107 | MT627719 | MT627662 |
| Neohelicascus submersus | MFLU 20–0436 | NR_172425 | MT627656 |
| Periconia cyperacearum | CPC 32138 | NR_160357 | NG_064549 |
| Periconia neobritannica | CPC 37903 | NR_166344 | NG_068342 |
| Phaeosphaeria sinensis | MFLUCC 18-1552 | NR_163350 | NG_070076 |
| Species | Strain | ITS | 28S | TEF1 |
|---|---|---|---|---|
| Achrochaeta rivulata | AD291612 | OQ799381 | OQ799389 | OQ866587 |
| Achrochaeta rivulata | AD291619 | OQ799348 | OQ799390 | OQ866588 |
| Achrochaeta talbotii | ICMP 15161 | MT454480 | MT454495 | OL653988 |
| Chaetosphaeria dilabens | CBS 734.83 | MH861683 | MH873395 | - |
| Chaetosphaeria guttulata | MFLU 18-1617 | MK828636 | MK835837 | MN194087 |
| Chaetosphaeria hebetiseta | MR 938 | AF178549 | AF178549 | - |
| Chaetosphaeria hebetiseta | SMH 2729 | AY906955 | AF466069 | - |
| Chaetosphaeria innumera | M.R. 1175 | AF178551 | AF178551 | - |
| Chaetosphaeria mangrovei | MCD 069 | MG813821 | MG813820 | - |
| Chaetosphaeria myriocarpa | CBS 143389 | MH107883 | MH107931 | - |
| Dictyochaeta callimorpha | ICMP 15130 | MT454483 | MT454498 | MT454673 |
| Dictyochaeta detriticola | ICMP 14948 | MT454486 | MT454501 | MT454676 |
| Dictyochaeta fuegiana | ICMP 15153 | MT454487 | EF063574 | MT454677 |
| Dictyochaeta montana | CBS 145342 | MT454488 | MT454502 | MT454678 |
| Dictyochaeta querna | CBS 145503 | MT454490 | MT454504 | MT454680 |
| Dictyochaeta stratosa | CBS 138739 | MT454492 | MT454506 | MT454682 |
| Fuscocatenula submersa | MFLU 18-1616 | NR_168802.1 | NG_068637.1 | MN194085.1 |
| Gongromeriza pygmaea | M.R. 1365 | AF178545 | AF178545 | - |
| Sporoschisma hemipsilum | S-877 | MK828617.1 | MK835817.1 | MN194070.1 |
| Sporoschisma hemipsilum | S-627 | MK828618.1 | MK835818.1 | MN194071.1 |
| Sporoschisma juvenile | MFLU 18-1608 | MK828619.1 | MK835819.1 | MN194072.1 |
The generated sequences for each gene were subjected to 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 using MUSCLE. Alignments were imported into Mega X 10.2.6 (Stecher et al., 2020) to find the best substitution models for phylogenetic analyses.
Genes were then concatenated and maximum-likelihood phylogenetic trees were constructed using RAxML v. 8.2.11 (Stamatakis, 2014) within Geneious and branch support values were calculated with 1000 rapid bootstrap inferences. The same alignment was analysed with MrBayes (v 3.2.6, Huelsenbeck & Ronquist, 2001) within Geneious. All resulting trees were formatted in Geneious, then further edited in Adobe Illustrator v. 27.0.
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 p value ≥ 0.90 are indicated near the branches.
BLAST searches showed that the undescribed coelomycetous species from our survey was closest to species in the Morosphaeriaceae. In order to classify this species, ITS and 28S sequences were aligned with 24 species within the Pleosporales, including representatives of all accepted genera in the Morosphaeriaceae. The dataset including alignment gaps, comprised 1884 characters: 890 for ITS and 994 for 28S. The tree is rooted to Aliquandostipite khaoyaiensis (ISAN100) and Jahnula dianchia (MFLUCC 16-1353). The undescribed fungus formed a new clade within the Morosphaeriaceae, separate to previously described genera, therefore representing a new genus (Fig. 1).
BLAST searches of ITS sequences from the Dictyochaeta-like species, showed the closest match to be Achrochaeta talbotti at 92% similarity. Phylogenetic analyses of 21 of the closest species within Chaetosphaeriaceae included 2433 characters: 573 for ITS, 875 for 28S, and 985 for tef1. The tree is rooted to 3 Sporoschisma strains. The undescribed species formed a well-supported clade sister to Achrochaeta tablotti (Fig. 2).
Microvesuvius Fryar & D.E.A. Catches. gen. nov. MB848293
Conidiomata pycnidial, immersed in substrate, semi-immersed, or superficial, globose to ellipsoid, black, ostiolate. Wall made of indistinct dark brown cells, irregular thickness and an inner layer of hyaline, ellipsoid, thick-walled cells. Conidiogenous cells hyaline, cylindrical, percurrent proliferation. Conidia hyaline, appendages absent, sheath present.
Etymology: This genus is named after Mount Vesuvius due to the resemblance of the some of the pycnidia to small volcanoes.
Microvesuvius unicellularis Fryar & D.E.A. Catches. sp. nov. MB848294, Figs. 3 & 4
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. Fryar (holotype AD291633). GenBank numbers: ITS - OQ799382; 28S - OQ799391; TEF1 - OQ866585.
Etymology: The epithet refers to the aseptate conidia.
Saprobic on decaying wood in an ephemeral freshwater stream. Sexual morph undetermined. Asexual morph: On natural substrate. Conidiomata pycnidial, immersed in substrate, semi-immersed or rarely superficial, globose to ellipsoid, lying horizontal to substrate, scattered, black, ostiolate, (134)335–840 ⋅ (135)194–480 µm. Conidiomatal wall made of indistinct dark brown cells, irregular thickness up to 80 µm and an inner layer of hyaline, ellipsoid, thick-walled cells, irregular thickness up to 50 µm. Conidiogenous cells hyaline, cylindrical, percurrent proliferation, (12)22–29(35) ⋅ 3.5–4 µm. Conidia hyaline, aseptate, 1 large oil globule and multiple smaller oil globules, appendages absent, thin irregular sheath surround most conidia, 11–18 ⋅ 10–15 µm.
Culture characteristics: On PDA after 13 days 18 x 14 mm, irregular shape, 1 main colony, several satellite colonies, grey tinged with brown, margin white, 1 mm, slimy appearance, centre fluffy. Agar not discoloured. Reverse grey interior, white margin. Hyphae hyaline, twisted and intertwined, branched, septate, 2.5–3 µm wide. Aerial hyphae hyaline to dark grey, breaking easily, verrucose, 2–4 µm wide.
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. Fryar, AD291626, AD291608, AD291689. GenBank numbers (AD291626): ITS - OQ799384; 28S - OQ799383; TEF1 - OQ866586.
Notes:
Phylogenetic analyses of ITS and 28S sequences place our specimens in a clade representing Morosphaericaeae (Pleosporales) alongside Aquilomyces and Clypeoloculus as a separate lineage (Fig. 1). There were very few comparison TEF1 Morosphaeriaceae sequences in GenBank, so those data were not used in our analyses.
Most of the species described in the Morosphaericaeae only have descriptions of the sexual morph, so morphological comparisons are mostly not possible. However, the asexual morph of Neohelicascus aquaticus, also within the Morosphaeriaceae, is described and is coelomycetous (Zhang et al., 2013). Microvesuvius unicellularis is different to N. aquaticus in many ways. The conidia of M. unicellularis are globose to subglobose compared with the ellipsoid to obovoid conidia of N. aquaticus, the conidiogenous cells of M. unicellularis are larger, and percurrent, and the pycnidia are immersed and black, rather than superficial and brown.
Morphologically, M. unicellularis shares characters with Hongkongmyces aquaticus. They both have globose to subglobose, hyaline, aseptate conidia with large oil globules. However, the conidia of H. aquaticus do not have a sheath and the conidiogenous cells of H. aquaticus are shorter and not described as percurrent. Hongkongmyces is a member of the family Lindgomycetacae, also within the Pleosporales (Tsang et al., 2014).
Achrochaeta rivulata Fryar & D.E.A. Catches. sp. nov. MB848295, Figs. 5 & 6
Etymology: The epithet refers to the habitat where this species was found, a small stream.
Typus: AUSTRALIA, Scott Creek Conservation Park (S35° 5' 46", E138° 40' 59"), on decaying wood submerged in a stream, 26 August 2020, AD291612. GenBank numbers: ITS - OQ799381; OQ799389; TEF1 - OQ866587
Saprobic on decaying wood in an ephemeral freshwater stream. Sexual morph undetermined. Asexual morph: On natural substrate. Conidiophores, mononematous, macronematous, 77–170 ⋅ 2.5–3.6 µm, simple, dark brown, hyaline or subhyaline at the apex, septate, single or branched at the base into 3, straight or slightly flexuous, cylindrical. Conidiogenous cells 20–37 ⋅ 3 µm, monophialidic, tapering towards the apex, hyaline to subhyaline. Collarettes funnel shaped, 1.5–2 ⋅ 1.5–2 µm. Setae 70–99 ⋅ 3–3.5 µm, rounded, slightly bulbous apex, dark brown at base graduating through to subhyaline at the apex, septate. Conidia 6–10 ⋅ 2.5 µm, hyaline, aseptate, without appendages, straight or curved, ornamentation or sheath, smooth, ellipsoid, one end rounded, one end pointed.
Colonies on PDA after 4 weeks 15 ⋅ 12 mm, margin undulate, finely furrowed, velvety, dark grey in patches, white in patches, white outer zone, slightly convex (indented), reverse dark grey, with white margin. Hyphae hyaline, septate, branching, 2.5–4 µm wide. Sheath 8–18 ⋅ 7–10 µm, on some of the darker hyphae, often near the base of the conidiophores. Conidiophores monophialidic, tapering towards the apex, 12–54 ⋅ 2.5–4 µm, mononematous, subhyaline to brown, collarette funnel-shaped 1–2 x 1–2 µm. Conidia 5–13 ⋅ 2–3 µm, hyaline, mostly straight, some curved, aseptate, without sheaths or appendages, ellipsoid, one end pointed, other end rounded.
Additional material examined: AUSTRALIA, Scott Creek Conservation Park (S35° 5' 46", E138° 40' 59"), on decaying wood submerged in a stream, 26 August 2020, (AD291619, AD291630, AD291604, AD291618, AD219605. GenBank numbers (AD291619): ITS - OQ799348, 28S - OQ799390, TEF1 - OQ866588
Notes:
The conidiophores of Achrochaeta rivulata on PDA appear different to those on the natural substrate, wood. They are shorter and lighter in colour. There is also a sheath present on some hyphae which is not visible on the natural substrate. Also, no setae were observed in culture.
The phylogram of ITS, 28S and TEF1 sequences (Fig. 2) shows our new species to be in a clade with Achrochaeta talbotti, Chaetosphaeria hebetiseta and C. dilabens, sister to A. talbotti. In a megaBLAST search of ITS sequences, Achrochaeta rivulata was most similar to A. talbotii (MT454480) with 92% similarity. The two specimens share many characters such as unbranched, brown, septate conidiophores with hyaline phialidic conidiogenous cells, and funnel-shaped collarettes. However, A. talbotti (ICMP 15161) does not have setae, and the conidia are cylindrical-clavate rather than the ellipsoid. In addition, A. rivulata only has an apical opening, whereas A. talbotti (ICMP 15161) has either a single apical opening on the conidiogenous cells or also has lateral openings.
Achrocheata talbotti was originally described as Chaetosphaeria talbotii S. Hughes, W.B. Kendr. & Shoemaker (Hughes & Kendrick, 1968). The original collection of C. talbottti by Hughes and Kendrick was in Kuitpo Forest, South Australia, which is 20 km from the collection site of A. rivulata. There are close similarities between the original description of C. talbotti and A rivulata, but as with the comparison with A. tablotti (ICMP 15161), A. rivulata is monophialidic, not polyphialidic, has setae, and has ellipsoid conidia rather than cylindrical-clavate conidia.
Microvesuvius forms a well-supported clade within the Morosphaeriaceae based on phylogenetic analyses of combined rDNA sequences. Suetrong et al. (2009) introduced the Morosphaeriaceae based on molecular data and sexual morphs, placing Morosphaeria and Helicascus within this family. The family now has 4 addition genera, Aquilhelicascus, Aquilomyces, Clypeoloculus, and Neohelicascus, plus our newly described genus, Microvesuvius. The ascomata in the family are subglobose, conical, lenticular, immersed or superficial and are dark coloured. They have clavate to cylindrical, thick-walled, fissitunicate asci, with an ocular chamber and a non-amyloid apical ring. Ascospores are hyaline to brown, septate and can be with or without a sheath or cap (Suetrong et al., 2009).
Microvesuvius is not unusual within Morosphaeriaceae in being from a freshwater habitat. A number of species from Morosphaeriaceae are found in fresh water including Aquihelicascus thalassiodieus, A. yunnanensis, A. songkhlaensis (Dong et al., 2020), Clypeoloculus akitaensis, C. hirosakiensis, C. microsporus, C. towadaensis (Tanaka et al., 2015), Helicascus alatus (Zeng et al., 2018), Neohelicascus aquaticus, N. aegyptiacus, N. chiangraiensis, N. elaterascus, N. gallicus, N. submersus (Dong et al., 2020), N. unilocularis (Zhang et al., 2015), and N. uniseptatus (Luo et al., 2016).
While coelomycetes are rare within Morosphaeriaceae, Pleosporales commonly have both coelomycetous and hyphomycetous asexual morphs (Yu et al., 2022) and are mostly coelomycetous (Zhang et al., 2012). Phoma or Phoma-like anamorphs are the most common anamorphs of Pleosporales (Zhang et al., 2012) and Microvesuvius unicellularis has many similarities to these anamorphs such as pycnidia that are immersed to erumpent, ostiolate, dark-coloured, with annellidic conidiogenous cells and globose to obovoid, unicellular conidia (Kohlmeyer & Kohlmeyer 1979).
Based on morphological differences and multi-gene phylogenies, we introduce the new genus Microvesuvius and the two new species Microvesuvius unicellularis and Achrochaeta rivulata.
Acknowledgements
Financial support from the Australian Biological Resources Survey (ABRS) is gratefully acknowledged.
The authors declare that there are no competing interests.