Paracremonium aquaticum M.S. Calabon, E.B.G. Jones & K.D. Hyde, sp. nov.

Index Fungorum number: IF 559840;  Mycobank number: MB 559840; Facesoffungi number: FoF 05445; Fig. 1

Etymology – “aquaticum” in reference to the aquatic habitat.

Holotype – MFLU 22-0120.

Sexual morph: Undetermined. Asexual morph: Hyphomycetous. Saprobic on submerged decaying wood from a freshwater habitat. Colonies on natural substrate effuse, greyish white, velvety. Mycelium immersed, composed of hyaline to pale brown, branched, septate hyphae. Conidiophores 18–30×1.4–2.4 μm (x̄=23.8×1.8 μm, n=15), erect, subcylindrical, unbranched, hyaline, smooth. Conidiogenous cells terminal, elongate-ampulliform, tapering towards apex, with prominent periclinal thickening and inconspicuous collarette, hyaline, smooth. Conidia 4.5–8.5×2.0–3.0 μm (x̄=6.3×2.5 μm, n=50), formed in heads at apex of conidiogenous cells, aseptate, ellipsoidal to fusiform, acute at both ends, smooth, slightly to strongly curved, with two large guttules. Chlamydospores not observed.

Culture characteristics – Conidia germinating on malt extract agar (MEA) and producing germ tubes at both ends within 24 h. Colonies growing on MEA, circular, with flat surface, margin entire, reaching 40–45 mm in 4 weeks at 25 °C, powdery, from above pale yellow to white from edge to center, from white to pale yellow from edge to center. Hyphae 2–5.5 μm wide, septate, hyaline, mostly smooth, thick-walled, moniliform, abundant. Conidiophores erect, simple or mostly branched, septate, bearing whorls of 2–4 conidiogenous cells. Conidiogenous cells 8–15×1.5–6.0 μm (x̄=11.2×3.6 μm, n=20), terminal or lateral, straight, flask-shaped, tapering towards apex, hyaline, with prominent periclinal thickening and inconspicuous collarette, 1.5–2.0 µm in diam. Conidia 2.5–8.5×1.5–3 μm (x̄=5.8×2.4 μm, n=50), unicellular, hyaline, ellipsoidal to fusiform, smooth-walled, with slightly apiculate base, sightly curved, guttulate. Chlamydospores hyaline, formed intercalary in chains or solitary, smooth, thinwalled, ellipsoidal to mostly cylindrical, guttulate.

Habitat and distribution – Paracremonium aquaticum was observed from submerged decaying wood in stream, and is currently only found in Thailand.

Material examined – Thailand, Chiang Mai Province, Mushroom Research Center, on submerged wood in an artificial lake, 13 September 2019, M.S. Calabon, MRC67 (MFLU 22-0120, holotype), ex-type living culture, MFLUCC 22-0077.

GenBank numbers – ITS=OP216410, LSU=OP216405, RPB2 = OP251199, TEF1-α = OP251195, TUB2=OP251200.

Notes – Multi-locus phylogenetic analysis showed that Paracremonium aquaticum MFLUCC22-0077 shared the same subclade with the type species P. inflatum CBS 485.77 (Fig. 2). Paracremonium aquaticum MFLUCC 22-0077 differs from P. inflatum in having flask-shaped conidiogenous cells with larger conidia (2.5–8.5×1.5–3 μm vs. 5–6×1–2 μm). In pairwise nucleotide comparisons of P. aquaticum with the type strain of P. inflatum CBS 124513, there is a nucleotide difference of 2.51% (14/558 bp) in ITS, 4.70% (25/532 bp) in TEF1-α, 8.37% (72/860 bp) in RPB2 and 3.15% (11/349 bp) in TUB2 genes. Another strain of P. inflatum CBS 482.78, is a sister taxon to P. aquaticum (Fig. 2), but pairwise nucleotide comparison showed 0.89% (5 bp) in ITS (of 564 nucleotides altogether), 1.16% (10 bp) in RPB2 (of 862 nucleotides altogether) and 0.37% (2 bp) in TEF1-α (of 533 nucleotides altogether). Morphological comparison is not possible between P. inflatum CBS 482.78 and P. aquaticum because the former does not have morphological data. As the strain P. inflatum CBS 482.78 forms a sister taxon to P. aquaticum, we rename this strain as P. aquaticum CBS 482.78. Paracremonium contagium also clustered with strains of P. pembeum but with inclusions of three more additional loci (calmodulin, histone H3-like and actin) in a separate phylogenetic analysis of Paracremonium species (data not shown), P. contagium has a distinct lineage basal to P. pembeum strains. Phylogenetic analysis also shows that two strains of P. lepidopterorum DY10351 and DY10352, a species isolated from an insect pupa in China (Ming et al. 2021), did not cluster within the Paracremonium clade, but forms a strongly supported clade with members of Cordycipitaceae (Fig. 2). A wider taxon sampling of taxa under Cordycipitaceae will help establish the phylogenetic placement of P. lepidopterorum within this family. Paracremonium aquaticum from freshwater habitats in Thailand is the third freshwater species of Paracremonium, with P. binnewijzendii recorded from submerged wood and P. variiforme from cave water, both from China (Zhang et al. 2017a; Luo et al. 2019).

Figure 1 – Paracremonium aquaticum (MFLU 22-0120, holotype). a, c Conidiophores and conidia. d Conidia. e Germinated conidium. f Colonies on MEA from surface and in reverse. g–ab Sporulation on MEA. g–t Various moniliform hyphal elements. m–t Chlamydospores. w–aa Conidiophores, conidiogenous cells, and conidia. ab Conidia Scale bars: a–c=50 µm, d, e, i–k, m–t=20 µm, g, h, l=100 µm, w–ab=10 µm

Figure 2 – Phylogram generated from maximum likelihood analysis based on combined ITS, LSU, RPB2, TEF1-α, and TUB2 sequence data representing Nectriaceae (Hypocreales) and closely related families. Seventy-eight strains are included in the combined analyses which comprised 3663 characters (ITS: 499, LSU: 817, RPB2: 1037, TEF1-α: 714, TUB2: 596) after alignment. Seven Phaeoisaria species (P. annesophieae MFLU 19-0531; P. aquatica MFLUCC 16–1298; P. clematidis MFLUCC 18–1017; P. fasciculata CBS 127885, DAOM 230055; P. pseudoclematidis MFLUCC 11-0393; P. sedimenticola CGMCC 3.14949) in Pleurotheciaceae (Pleurotheciales) were used as the outgroup taxa. The best scoring RAxML tree with a final likelihood value of−30,486.223016 is presented. The matrix had 1831 distinct alignment patterns, with 38.42% of undetermined characters or gaps. Estimated base frequencies were as follows: A=0.230840, C=0.279253, G=0.276292, T=0.213616; substitution rates: AC=1.237490, AG=2.611546, AT=1.138497, CG=0.936411, CT=6.064431, GT=1.000000; gamma distribution shape parameter α=0.282744. Bootstrap support values for ML equal to or greater than 75% are given above the nodes (left side). Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are given above the nodes (right side). Ex-type strains are in bold and newly generated sequences are in blue