Zygosporium cymodoceae Guerra, Baulin, Cano & Gené, sp. nov.

Index Fungorum number: IF 902401; Mycobank number: MB 902401; Facesoffungi number: FoF 14004; Fig. 1

Etymology – The name refers to the substrate where this fungus was isolated, Cymodocea nodosa.

Holotype – CBS H-25261

Saprobic in Cymodocea nodosa. Sexual morph not observed. Asexual morph on OA: Mycelium 1.5–2 µm wide, hyaline, smooth-walled to verrucose, branched, septate. Conidiophores (35–)40–50(–55) × 1.5–2.5 µm (x̅=46.9×1.9 µm, n=30), arising directly from the superficial mycelium, setiform, erect, 3(–4) septate, unilaterally branched near the base, brown and smooth-walled at the base, subhyaline to hyaline and often roughened towards the apex, lateral branch consisting in a short stalk, 0(–1)-septate, brown to pale brown, 3–5 ×2–3 µm (x̅=4×2.5 µm, n=30), with a terminal vesicular cell bearing two conidiogenous cells at the apex. Vesicular cells 6–7.5×4–5.5 µm (x̅=6.9×5 µm, n=30), in the broadest part, solitary, dark brown, smooth-walled, clavate, curved (hooked). Conidiogenous cells 4–4.5(–6)×2.5–3 µm (x̅=4.5×2.6 µm, n=30), holoblastic, hyaline, verruculose, ampulliform, curved. Conidia 4–6 µm diam (x̅=4.6 µm, n=30), solitary, pale brown, verrucose, globose.

Culture characteristics – after 2 weeks at 25 °C, colonies on PDA attaining 8 mm diam., raised, umbonate, grey (4F1, Kornerup & Wanscher 1978) at the centre, white (4A1) towards the periphery, margin entire; reverse yellowish white (4A2). On MEA, colonies reaching 8 mm diam., slightly raised, crateriform, radially sulcate, velvety, grey (4F1) at the centre, light orange (5A4) towards the periphery, margin entire and lobated; reverse grey (4F1) to light orange (5A4). On OA, colonies reaching 16–18 mm diam., flat, velvety, hyaline with black spots of sporulation at the centre; reverse colourless. Cardinal temperatures for growth: minimum at 10 °C, optimum at 25 °C, maximum at 30 °C.

Material examined – Spain, Catalonia, Tarragona, Platja del Miracle, N 41° 6′ 37″, E 1° 15′ 37″, on Cymodocea nodosa Asch. (Cymodoceae), at 6 m of depth, 14 October 2021, G. Quiroga-Jofre and D. Guerra-Mateo (CBS H-25261, holotype), ex-type CBS 149942, FMR 19851, MycoBank MB847540.

GenBank numbers – ITS=OQ589930, LSU=OQ589929.

Notes – Zygosporium cymodoceae is a novel species phylogenetically related to Z. oscheoides, the type species of the genus. Since DNA data of the species accepted in Zygosporium is very limited, morphological comparison is essential to distinguish species. Although the conidiogenous apparatus of Z. cymodoceae is similar to that of its closely related species, Z. minus and Z. oscheoides, they can
be distinguished mainly by their conidial morphology. The conidia in the new species are globose, conspicuously verrucose and measure 4–6 µm diam. (Fig. 1g), while in Z. minus, although globose, they are larger (5.5–10 µm diam.) and verruculose, and in Z. oscheoides are ellipsoid, smooth to verruculose and measure 7–12.5×4–9 µm diam. (Hughes 1951a; Whitton et al. 2002a). Other Zygosporium species that can be easily distinguished from Z. cymodoceae by the conidial features are Z. bioblitzi, Z. deightonii, Z. geminatum, Z. pacificum and Z. verruciferum, all with ellipsoid conidia (Whitton et al. 2002a; Mckenzie et al. 2007; Lucena and Fernández-Valencia 2017); Z. anupamvarmae shows falcate conidia with ridges (Monoharachary et al. 2006); the conidia of Z. majus, Z. tuberculatum and Z. pandanicola are globose but considerably larger (13–18, 12.5–16.5 and 11–14.5 µm diam., respectively); and Z. chartarum and Z. verticilatum show globose and ovoid conidia, respectively, but hyaline and smooth (Whitton et al. 2002a). Other features that distinguish Z. cymodoceae from the other species in the genus is the presence of a setiform conidiophore (Fig. 115a–f), which is absent in Z. cocos, Z. dilleniae, Z. gibbum, Z. masonii, Z. mycophilum, Z. tonellianum, Z. pseudogibbum and Z. pseudomasonii (Whitton et al. 2002a; Dubey 2014; Crous et al. 2018, 2019a), and the production of conidiophores unilaterally branched at the base bearing a single vesicular cell, in contrast to Z. verticillatum which produces 2–3 vesicular cells per conidiophore (Whitton et al. 2002a).

Phylogenetically, Z. cymodoceae represents an independent lineage with strong statistical support using the LSU region (99% MLBS, 0.99 BYPP) (Fig. 2). The ITS region was excluded from the phylogenetic tree due to the great phylogenetic distance between FMR 19851 and related taxa. The alignment of the ITS sequences revealed a 94% of similarity with Z. oscheoides (CBS 195.79, GenBank MH861194), and a 93% with Z. minus (HKAS99625, MF621586). BLAST searches on the NCBIs GenBank nucleotide database using the ITS region did not provide significant results. According to our results and previous works (Crous et al. 2020a), some Zygosporium species should be accommodated in other genera. Since Z. cymodoceae clusters with Z. oscheoides, we consider it a species of Zygosporium s. str.

Figure 1 – Zygosporium cymodoceae (FMR 19851). a, b Conidiophores. c, d Vesicular cells from superior view. e, f Vesicular cells from lateral view, bearing verruculose conidiogenous cells. g Conidia. h Colony on OA after 14 days at 25ºC. Scale bars: 10 µm

Figure 2 – Phylogram generated from a RaxML analysis based on LSU sequence data of Zygosporium isolates. Related sequences were obtained from NCBI´s GenBank. Fifteen taxa are included in the analysis, which represents 822 characters including gaps. The tree is rooted to Xylaria arbuscula CBS 126416 and Iodosphaeria tongrenensis MFLU 15-0393. Here is represented the best RaxML tree using the GTR model with a final likelihood of − 2334.079572. The matrix had 126 alignment patterns, with 1.82% of undetermined characters or gaps. Estimate base frequencies were as follows: A=0.250237, C=0.213354, G=0.301698, T=0.234711; substitution rates: AC=1.189322, AG=2.931397, AT=0.638000, CG=0.896786, CT=10.246864, GT=1.000000; gamma distribution shape parameter α=0.107590. RaxML bootstrap support values≥70% (MLBS) and Bayesian posterior probabilities≥0.95 (BYPP) are given at the nodes. The scale bar indicates 0.02 changes. The novel species proposed in this study is in blue and ex-types strains of different species are in bold