Synnemellisia punensis K. S. Pawar, P. N. Singh, S. K. Singh sp. nov.

Index Fungorum number: IF 900322; Mycobank number: MB 900322; Facesoffungi number: FoF 10810; Figs. 1, 2

Etymology – specific epithet ‘punensis’ refers to name Pune (a district of Maharashtra state).

Holotype – NFCCI 5166.

Colour code follows – Methuen Handbook of Colour (Kornerup and Wanscher 1978).

Isolated in vitro from the leaf litter. Asexual morph: Synnemata produced in the form of aggregated mass of parallel bundles hyphae, hyaline. Vegetative hyphae aggregated in loose parallel bundles, rope like, branched to unbranched, anastomoses, smooth walled, thin and thick, hyaline, up to 3 μm wide. Setae and hyphopodia absent. Chlamydospores absent. Conidiophores arising from superficial hyphae, forming floccose sporodochia, unbranched to branched, branching unilateral to bilateral verticillate, hyaline, smooth walled, 9.68–127.7×1.97–2.77 μm. Conidiogenous cells polyblastic and polyphialidic, smooth walled, hyaline, 4.71–35.91 × 1.35–3.46 μm. Phialides solitary or in group of 2–4, sometimes directly produced from superficial hyphae, cylindrical, attenuated, smooth walled. Metulae 1–2, primary metulae solitary, spatulate, variable in size (8.59–10.27 × 1.05–1.83 μm), secondary metulae paired (6.98 × 2.12 μm), 8.29–25.36 × 1.35–3.58 μm. Conidia produced in long chain from polyblastic and polyphialidic conidiogenous cell, sometimes conidia accumulated in the form of chevron or zipper like, produced in loose mass on the tip of phialides, oval to broadly ovoid, allantoid to fusoid, base broader, sometimes base rarely tapered, base truncate, tip obtuse, aseptate, smooth walled, 1–2 gutulate, smooth walled, hilum unthickened, hyaline, 2.22–6.43×1.2–4 μm (x=3.84×2.17 μm, n=30). Sexual morph: Undetermined.

Culture characteristics – on semi-synthetic agar medium PDA white (6A1) to orange white (6A2), reaching 3.0 cm diam. in 5 days at 25 °C, forming synnemata, margin irregular, reverse wrinkled, pale orange (5A3); on PCA (Potato Carrot Agar) white (6A1), reverse yellowish white (4A2), reaching 3.7 cm diam. in 5 days at 25 °C, rarely forming dome like synnemata with pale orange (5A3) exudate. No sporulation on PDA and PCA but abundantly sporulating on grass leaf agar.

Material examined – India, Maharashtra, Pune District, from leaf litter, 1 October 2020, K.S. Pawar, NFCCI 5166 (holotype), ex-type living culture, National Fungal Culture Collection of India, WDCM 932.

GenBank numbers – ITS=ON059361, LSU=ON059433.

Notes – Synnemellisia punenesis is morphologically distinct from allied species of Synnemellisia. The conidia of S. hyalospora are solitary, slightly curved, 40–52 μm long, 4.2–5 μm wide at the middle, and 1–1.4 μm wide at the base (Rao et al. 1989). The conidia of type species S. aurantia (COAD 2070) are aggregated on a cushion-like head, navicular to fusiform, 23–30 long and 7–9 μm wide, apex subacute, base obtuse to subtruncate, aseptate, guttulate, subhyaline and smooth. (Crous et al. 2016). In present taxon S. punensis (NFCCI 5166), the conidia are produced from polyblastic, polyphialidic conidiogenous cells, ovoid, aseptate 2.22–6.43 μm long and 1.2–4 μm wide. Thus, S. punenesisis morphologically different in having much smaller shape and dimension of conidia as compared to the conidial dimension of S. hyalospora and S. aurantia. In addition, the conidia in new taxon produced in long chains and in loose aggregated mass, also forming zipper or chevron like pattern. Tan et al. (2021) described both the taxa based only on sequencing and phylogenetic analysis. Hence, morphological features of S. urenae (BRIP 71675) and S. acacia (BRIP 71652) are missing and hence cannot be compared.

Phylogenetic analysis of ITS and LSU sequence data specifies that S. punensis (NFCCI 5166), is a new species in Bionectriaceae, which is dissimilar from other known species of Synnemellisia (Fig. 3). Based on megablast analysis, ITS sequence of Synnemellisia punensis (NFCCI 5166) showed 94% (541/574) identity and 13 gaps (2%) with extype strain of Synnemellisia aurantia (COAD 2070), 96% (544/569) identity and 6 gaps (1%) with ex-type strain Synnemellisia urenae (BRIP 71675), and 95% (543/572) identity and 10 gaps (1%) with Synnemellisia acacia (BRIP 71652). Sequence data of Synnemellisia hyalospora is not available in the literature. Based on morphological and phylogenetic analysis, Synnemellisia punensis (NFCCI 5166) is proposed as novel species with strong ultrafast bootstrap (96%).

Figure 1 – Synnemellisia punensis (NFCCI 5166, holotype). a, b Colony morphology on PDA (front view). c colony morphology on grass leaf media. d Stereomicroscopic surface view of colony grown on PDA with hyphal bundles forming synnemata. e Stereomicroscopic surface view of colony grown on PCA with pale orange exudate. f Stereomicroscopic surface view of colony grown on grass leaf media with watery exudate. g, h Whitish puffy mass of sporodochia. i Anastomoses with phialides bearing long chains of conidia. j parallel hyphal bundles of hyphae forming synnemata. k anastomosed hyphae. l bipolar germination in spores (showing arrow). m Germinating conidia directly producing phialide with conidia (showing arrow). Scale bars: i–m=10 μm

Figure 2 – Synnemellisia punensis (NFCCI 5166, holotype). a, b Conidiophores with phialides bearing conidia. c Conidiophores with phialides bearing conidia (in higher magnification). d Conidiophores with phialides bearing chevron/zipper like arrangement of conidia (showing arrow). e Conidiophores, phialides with chains of conidia. f Numerous conidia. g SEM images of branched conidiophores, phialides and conidia. h Conidiophore with primary and secondary metulae bearing phialides and conidia. i Chain of conidia. Scale bars: a–f=10 μm, g, h=2 μm, i=1 μm

Figure 3 – Phylogenetic tree of Synnemellisia punensis (NFCCI 5166) inferred from Maximum Likelihood analysis based on combined sequence data of ITS, LSU. Verrucostoma freycinetiae MAFF 240100 was used as outgroup. The analysis involved 32 nucleotide sequences. Evolutionary analyses were conducted in IQ–TREE multicore version 1.6.11 (Nguyen et al. 2015) by the Maximum–Likelihood method using the best suitable model (TNe+I+G4 model). Ex-type strains are in bold and newly generated sequence is in blue. One–thousand bootstrap replicates were analyzed to get ultrafast bootstrap values, and the values above 50% were represented on nodes in the tree