Cercophora dulciaquae M.S. Calabon, E.B.G. Jones & K.D. Hyde, sp.nov.

Index Fungorum number: IF 558645; MycoBank number: MB 558645; Facesoffungi number: FoF 09962; Fig. 1

Etymology – The specific epithet “dulciaquae” reflects the origin in a freshwater habitat.

Holotype – MFLU 21-0122

Saprobic on submerged decaying wood. Sexual morph Ascomata 390–700 μm high, 350–475 μm diam., perithecial, mostly solitary, superficial, with base immersed in the hyphae, subglobose to obpyriform with conical neck, black, centrally located ostiole, without periphyses. Peridium 30–40 μm thick, slightly thicker at apex, 2-layers, outer layer composed of dark brown, thick-walled, irregular, brown cells of textura angularis; inner layer thin-walled, composed of elongated, flattened, light brown to hyaline cells. Hamathecium 2–3 μm wide, filiform, septate, unbranched, abundant, hyaline paraphyses. Asci 110–180× 15–25 μm (x̅=135×16 µm, n=20), 8-spored, unitunicate, cylindrical with premature ascospores, cylindric-clavate at later stages, apex rounded, narrow, apical ring, thin-walled, with a short pedicel. Ascospores 35–60×6–8 μm (x̅=45×7 µm, n=20), 2-seriate, premature, hyaline, aseptate, cylindrical, slightly sigmoid to geniculate, numerous oil droplets, curved, rounded at both ends, with a lash-like appendage attached to both ends, 8–15 μm long; becoming differentiated into a swollen head and pedicel, 1-transversely septate, head 10–20×9–11 μm (x̅=17×10 µm, n=30), conical at the apex, truncate at the base, hyaline to dark brown, with several oil globules, pedicel 19–27×4–6 μm (x̅=23×5 µm, n=30), hyaline to pale brown, curved; appendages disappear with age. Asexual morph Undetermined.

Culture characters – Conidia germinated on MEA within 24 h. Colonies on MEA reaching 1–2 cm diam. after 4 weeks at room temperature, slow growing, colonies irregular, medium dense, flat with smooth and filiform margins; brown to dark brown in top view, reverse brown.

Material examined – THAILAND, Chiang Mai Province, Mushroom Research Center, on decaying wood submerged in a freshwater stream, 29 January 2019, S. Boonmee, SB17- 10 (MFLU 21-0122, holotype), ex-type living culture, MFLUCC 21-0104.

GenBank numbers – ITS=MZ497331, LSU=MZ497335.

Notes – Cercophora dulciaquae is the seventh species from Cercophora (C. appalachianensis, C. aquatica, C. caudata, C. costariensis, C. squamulosa, C. vinosa) observed from freshwater habitats (Luo et al. 2019; Calabon et al. 2020b). It closely resembles C. aquatica and C. costariensis. Still, it differs in ascomata (390–700 μm high vs. 400–600 μm high vs. 350–500 μm high), asci (110–180 × 15–25 μm vs. 180–250 × 13–16 μm vs. 135–186 × 13–16 μm) and ascospore size (35–60 × 6–8 μm vs. 36–51 × 4–6 μm vs. 40–49 × 4–5 μm) (Chaudhary et al. 2007; Catania et al. 2011). Furthermore, C. dulciaqaue (Fig. 1) has aseptate ascospores while C. aquatica has up to 5-septate ascospores after liberation from the ascus. Immature ascospores of C. dulciaquae have bipolar appendages, but these are absent in C. costariensis (Catania et al. 2011). Based on the phylogenetic analysis of combined LSU and ITS sequence data, C. dulciaquae clustered with Zopfiella marina (CBS 155.77) with low bootstrap support (Fig. 2). The two species are morphologically different and the novel species fits the generic description of Cercophora. Cercophora dulciaquae is phylogenetically close to C. tuberculata. The former species differs in ascomatal morphology (short neck vs. neck up to 150 μm high, with basal tubercles) and ascospore size (35–60×6–8 μm vs. 37.5–40×2.5–5 μm) (Chang and Wang 2005). Blast results of ITS sequence data revealed a 99.44% similarity to Apodus oryzae (E-219) while LSU sequence data showed a 99.88% similarity with Apodus oryzae (ATCC 38847), Triangularia mangenotii (CBS 419.67; ATCC 38847) and Zopfiella pilifera (CBS 413.73).

Figure 1 – Cercophora dulciaquae (MFLU 21-0122, holotype). a–c Appearance of ascomata on host substrate. d Vertical section through ascoma. e Section of pycnidial wall. f Paraphyses. g–l Asci. m–r Ascospores with bipolar appendages (arrow). Scale bars: a, b=1 mm, c, d=500 μm, e, i–r=50 μm, f=20 μm, g, h=100 μm

Figure 2 – Phylogenetic tree generated from maximum likelihood (ML) analysis based on combined LSU and ITS sequence data for the species from Lasiosphaeriaceae. Microthecium quadrangulatum (CBS 112763) and M. tenuissimum (CBS 112764) were used as outgroup taxa. The dataset comprised and 1243 characters after alignment including gaps (LSU=828 bp and ITS=415 bp). The RAxML analysis of the combined dataset yielded a best scoring tree with a final ML optimization likelihood value of − 4540.010304. The matrix had 520 distinct alignment patterns, with 17.16% undetermined characters or gaps. Estimated base frequencies were as follows: A=0.243204, C=0.239086, G=0.300179, T=0.217531; substitution rates AC=1.598327, AG=1.651124, AT=2.172105, CG=0.801233, CT=6.709684, GT=1.000000; gamma distribution shape parameter α=0.254987. Bootstrap support values for ML and MP equal to or greater than 60% and BYPP equal to or greater than 0.90 are given above the nodes. The newly generated sequence is in blue and bold