Robillarda aquatica M.S. Calabon, E.B.G. Jones, K.D. Hyde, in Manawasinghe et al., Mycosphere 13(1): 494 (2022)

MycoBank number: MB 559337; Index Fungorum number: IF 559337; Facesoffungi number: FoF 10600;

Etymology – in reference to the habitat where the fungus was collected

Holotype – MFLU 21–0253

Saprobic on decaying wood submerged in freshwater. Sexual morph: Not observed. Asexual morph: Coelomycetous. Conidiomata 170–200 × 170–230 μm (x̅= 181.6 × 192.7 μm, n = 10), pycnidial, black, semi-immersed, mostly erumpent, uniloculate, solitary, scattered, irregular in shape, thick-walled, ostiolate. Conidiomatal wall 10–15 μm wide, thin-walled, slightly thick at the pycnidial upper wall, composed of two layers, dark brown to black, thick-walled cells in outer layer, arranged in textura angularis, becoming hyaline towards the inner layer. Conidiophores reduced to conidiogenous cells lining the cavity of the conidiomata. Conidiogenous cells 5–15 × 5–10 μm (x̅= 11.9 × 6.6 μm, n = 20), holoblastic, proliferating sympodially at apex, ampulliform to subcylindrical, hyaline, aseptate, smooth and thin-walled. Conidia 15–20 × 3–5 μm (x̅= 15.9 × 3.7 μm, n = 50), hyaline, oblong to ellipsoidal, narrow towards the basal cell, straight or slightly curved, (0–)1-septate, smooth and thin-walled, slightly constricted at the septum, apical cell modified into a branched appendage; appendages 30–45 × 1–2 μm (x̅= 37.3 × 1.4 μm, n = 50), dividing into 3–4 divergent branches, devoid of cell contents, flexuous, attenuated toward the apex, slightly swollen at the apex.

Culture characteristics – Conidia germinating on malt extract agar (MEA) within 24 h. Germ tubes produced from the basal and apical cell of conidia. Colonies on MEA reaching 30-35 mm diam. after 2 week at 25 °C. Mycelia superficial, circular, flat, surface slightly rough,  with entire margin, forming small, black pycnidia on colony after two weeks of incubation; from above, grey to light brown; reverse, light brown at the center and becoming dark brown towards margin; not producing pigmentation in agar.

Material examined – Thailand, Tak Province, Tha Sing Yang, Ban Mae Ja Wang, on decaying wood submerged in a river, 17 October 2019, N. Padaruth, CC20 (MFLU 21–0253, holotype), extype living culture, MFLUCC 21–0217.

GenBank submissions – LSU: OL477390, ITS: OL504777

NotesRobillarda aquatica is the third species recorded from freshwater habitats; the other two species, R. sohagensis and R. sessilis, were recorded from submerged decaying leaves of Phoenix dactylifera in a river (Egypt) and unidentified plant litter in a stream (Korea), respectively (Song et al. 2020, Goh et al. 2020). The phylogenetic analyses of a combined LSU and ITS sequence dataset show that R. aquatica clusters with other Robillarda species and is closely related to R. mangiferae (KUMCC 18-0180), R. sohangensis (CBS H-23861), and Robillarda sp. (MS9788) with 100% MP bootstrap and 1.00 BYPP support. Robillarda aquatica differs from R. mangiferae by having longer conidia (15–20 × 3–5 μm vs. (7.5–)10–11(–12) × (2.5–)3–4(–4.5) μm vs.), and longer apical appendages (30–45 × 1–2 μm vs. 25–35 × 1–2.5 μm) (Phookamsak et al. 2019). Robillarda sohagensis has larger conidiomata (765–2025 × 270–495 μm vs. 170–200 × 170–230 μm) compared to R. aquatica which has shorter appendages (26–41 × 1–1.5 μm vs. 30–45 × 1–2 μm) (Song et al. 2020). The morphological comparison with closely related taxa and two-locus phylogenetic analysis supports the introduction of a novel freshwater species, R. aquatica.

Figure 1 Robillarda aquatica (MFLU 21–0253, Holotype) a Host. b Appearance of conidiomata on natural substrate. c Section trough conidiomata, d Conidiomatal wall e–j Conidiogenous cells. k, l Conidia. m Germinated conidium. Colonies on MEA from above (n) and below (o). p, q Conidiomata on MEA. r Conidiogenous cells and conidia. s-w Conidia. Scale bars: a = 5 mm, b = 500 µm, c = 100 µm, d, k–m, r–w = 20 µm, e–j = 10 µm, p, q = 1000 µm.

Figure 2 – Phylogenetic tree generated by ML analysis of combined LSU and ITS sequence data of Sporocadaceae. The analyses included 63 strains, and the tree is rooted with Phlogicylindrium eucalyptorum (CBS 111689) and Phlogicylindrium uniforme (CBS 131312). The tree topology of the ML analysis was similar to the BYPP. The best-scoring RAxML tree with a final likelihood value of -8243.687976 is presented. The matrix had 594 distinct alignment patterns, with 33.45% of undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.261909, C = 0.211108, G = 0.259193, T = 0.267790; substitution rates AC = 0.998621, AG = 2.475880, AT = 1.324142, CG = 0.858286, CT = 4.291128, GT = 1.000000; gamma distribution shape parameter α = 0.182492. RAxML bootstrap support values ≥70% and ≥0.90 BYPP are shown near the nodes. New isolates recovered in this study are in red