Molecular phylogeny of Aplosporella abexaminans: a novel species revealing the second report of sexual-asexual connection in Aplosporellaceae (Botryosphaeriales) from India Author Verma, Sanjeet Kumar 0000-0002-2579-5917 Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, U. P., India 221005 & sanjeetmycology @ gmail. com; https: // orcid. org / 0000 - 0002 - 2579 - 5917 sanjeetmycology@gmail.com Author Yadav, Sanjay 0000-0003-1850-7566 Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, U. P., India 221005 & sanjaymycology @ gmail. com https: // orcid. org / 0000 - 0003 - 1850 - 7566 Author Singh, Raghvendra 0000-0002-8672-6149 Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, U. P., India 221005 & singhr. bot @ bhu. ac. in; https: // orcid. org / 0000 - 0002 - 8672 - 6149 singhr.bot@bhu.ac.in text Phytotaxa 2021 2021-11-19 525 3 205 222 journal article 3399 10.11646/phytotaxa.525.3.3 151a5f33-096d-4052-8877-4d327f3874ea 1179-3163 5712372 Aplosporella abexaminans Sanjeet, Sanjay & Raghv. Singh , sp. nov. ( Fig 1–4 ) MycoBank number: MB 837539 Diagnosis:— Aplosporella abexaminans is characterized by its very larger ascostromata, locules without ostioles, peridium of thinner locules, two to multi-layered, larger asci, surrounded by an additional hyaline sac like structure, 8- spored, larger ascospores, hyaline to light olivaceous, asexual state without conidiomata formation, conidiogenous cells brown, absence of paraphyses, conidia brown without granular content, smooth and presence of chlamydospores. Etymology:— “ ab -” (Latin) means “away” or “separation” implied its similarity but difference to Bagnisiella examinans . Type:— India , Madhya Pradesh , Panchmarhi , on the stem of Murraya koenigii L. ( Rutaceae ) , 6 March 2018 , coll. Sanjeet Kumar Verma , AMH 10212 ( holotype ), MH-BHU 09 (isotype), NFCCI 5010 (ex-type living culture). Sexual morph: Ascostromata 0.5–2.5 mm wide, 0.25–0.45 mm height, stiff, black, leathery, gregarious, partially under the host surface and erumpent, circular to subcircular in outline, solitary to gregarious, coriaceous, flattened at the upper surface with many polygonal areas, multiloculate, with up to70 locules (each polygonal areas on the upper surface represents each locule), each locule contains single ascus, cells of ascostromata brown to dark brown-walled, textura angularis. Locules (70)80–100(115) × (16)20–25(30) μm, without ostioles. Peridium of locules 4–25 µm thick at the sides, two to multi-layered, dark brown, thick-walled cells, textura angularis, pseudoparenchymatous. Paraphysis absent. Asci (70)75–110(115) × (17)20–25(30) µm, cylindrical to cylindro-clavate, bitunicate, fissitunicate, surrounded by an additional hyaline sac like structure, 8-spored, apex rounded and thick-walled with a narrow inversely funnel-shaped ocular chamber, very short pedicellate (upto 30 µm). Ascospores (17)23–29(30) × 10–14 µm, over-lapping, 1 to 2-seriate, hyaline to light olivaceous, aseptate, ellipsoid to oval, smooth. Asexual morph: Conidiomata lack on PDA . Hyphae 2–6 µm wide, branched, septate, smooth to verruculose, subhyaline to light brown. Conidiophores develop as a lateral branch of creeping hyphae. Conidiogenous cells 15–19 × 3.5–6.5 μm, usually not distinct, sometimes cylindrical to phialidic, light brown to brown, smooth to slightly roughened. Conidia (10)16–18(20) × (6)9–10(12) μm, aseptate, mid brown to dark brown, smooth-walled, ellipsoid to oval, apex rounded, sometimes hilum with peg like projection. Chlamydospores 7–14 × 5–13 μm, mid brown to dark brown, spherical to oval. FIGURE 1. Stem of Murraya koenigii . a, b. Habit of ascostromata on the bark of host stem. Scale bars a, b = 5 mm. Culture characteristics Ascospores germinated on PDA within 24 h. Colonies on PDA attained a diameter of 9 cm after 21 days at 25˚ C , upper surface whitish to brownish with rounded brownish to blackish raised spots, reverse brown to black with round blackish spots, irregular, velvety and margin scattered. Phylogeny Based on a megablast search of NCBI GenBank nucleotide database, the closest hits using ITS and LSU sequences were Bagnisiella examinans strain CBS 551.66 (GenBank: EU 167562 , Identities = 575/575 (100%), gap = 0/575 (0%)) and Bagnisiella examinans strain CBS 551.66 (GenBank: KF 766316 , Identities = 788/789 (99%), gap = 1/789 (0%)) respectively. FIGURE 2. Sexual morph of Aplosporella abexaminans (AMH 10212, holotype). a–e Vertical sections through ascostromata. f. Released asci from ascostromata. g, h. Ascus released from an additional sac like structure (blue arrows). Scale bars a, b = 200 µm, c = 100 µm, d–h = 50 µm. FIGURE 3. Asci of Aplosporella abexaminans (AMH 10212, holotype). a–f. Asci. g–i. Apical operculum. j–l. ascospores. Scale bars a = 20 µm, b–d = 30 µm, e–l = 20 µm. FIGURE 4. Asexual morph of Aplosporella abexaminans on PDA (NFCCI 5010, ex-type culture). a. Colony on PDA from above. b. Colony on PDA from below. c, d. Initial stage of germinated ascospores on PDA. e, f. Development of hyphae from germinated ascospores on PDA. g. Verruculose hyphae. h. Smooth hyphae. i–m. Conidia. n, o. Chlamydospores (yellow arrows). Scale bars a, b = 20 mm, c–o = 10 µm. FIGURE 5. Phylogram generated from Maximum likelihood analysis based on ITS region and LSU gene of strains belong to Aplosporellaceae . Kellermania dasylirionicola (CBS 131720) was chosen as outgroup. Bootstrap values for ML and MP above 50% and BPP support above 0.50 are indicated. Red names indicate Aplosporella abexaminans . Phylogenetic analysis based on ITS-LSU concatenated sequences ( Fig. 6 ), Aplosporella abexaminans clustered closer ( ML / MP /BPP: 98/98/0.99) to Bagnisiella examinans (Mont. & Berk.) Arx & E. Müll. (1975) among the members of Aplosporella in Aplosporellaceae . A best scoring RAxML tree is presented in Fig. 6 , with the Likelihood value of −6121.803898. The most parsimonious tree showed length = 889 steps, consistency index = 0.414, retention index = 0.851 and the composite index is 0.413861 (0.353318) for all sites and parsimony-informative sites (in parentheses).