Four new species of Dothideomycetes (Ascomycota) from Para Rubber (Hevea brasiliensis) in Yunnan Province, China Author Xu, Rui-Fang https://orcid.org/0000-0003-1207-8254 Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering, Qujing Normal University, Qujing, Yunnan 655011, China & Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand Author Karunarathna, Samantha C. https://orcid.org/0000-0001-7080-0781 Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering, Qujing Normal University, Qujing, Yunnan 655011, China & School of Science, Mae Fah Luang University, Chiang Rai, 57100, Thailand Author Phukhamsakda, Chayanard https://orcid.org/0000-0002-1033-937X Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand Author Dai, Dong-Qin https://orcid.org/0000-0001-8935-8807 Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering, Qujing Normal University, Qujing, Yunnan 655011, China Author Elgorban, Abdallah M. https://orcid.org/0000-0003-3664-7853 National Institute of Fundamental Studies (NIFS), Kandy, Sri Lanka Author Suwannarach, Nakarin https://orcid.org/0000-0002-2653-1913 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia Author Kumla, Jaturong https://orcid.org/0000-0002-3673-6541 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia Author Wang, Xiao-Yan https://orcid.org/0009-0009-6430-3637 Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand & Center of Excellence in Microbial Diversity and Sustainable Utilization, Chiang Mai University, Chiang Mai, Thailand 527010142@qq.com Author Tibpromma, Saowaluck https://orcid.org/0000-0002-4706-6547 Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering, Qujing Normal University, Qujing, Yunnan 655011, China saowaluckfai@gmail.com text MycoKeys 2024 2024-03-22 103 71 95 http://dx.doi.org/10.3897/mycokeys.103.117580 journal article http://dx.doi.org/10.3897/mycokeys.103.117580 1314-4049-103-71 0C28E032647A557A9563F8DDF53D2D47 Nigrograna lincangensis R.F. Xu & Tibpromma sp. nov. Fig. 4 Etymology. The name refers to the location "Lincang, Yunnan, China", where the holotype was collected. Holotype. ZHKU 23-0104. Description. Saprobic on a dead branch of Hevea brasiliensis . Sexual morph : Ascomata 285-360 μm high, 230-307 μm diam. (x‾ = 337 x 272 μm , n = 5), immersed, under the clypeus, sometimes inconspicuous on host surface and small bumps can be seen, solitary, dark brown, globose or ellipsoid, with papilla. Ostioles 117-217 x 68-124 μm (x‾ = 152 x 99 μm , n = 10), central, brown, papillate. Peridium 16-45 μm wide, comprising several layers with dark-brown to dark cells of textura angularis. Hamathecium comprises 1.5-3 μm wide, unbranched, septate, hyaline, pseudoparaphyses. Asci 45-70 x 9-12 μm (x‾ = 57 x 10 μm , n = 10), 8-spored, bitunicate, pedicellate, club shape, cylindrical to clavate, straight or slightly curved, apically rounded, thick-walled. Ascospores 10-15 x 4-6 μm (x‾ = 13 x 4.8 μm , n = 30), 1-2-seriate, initially 1-septate, becoming 3-septate at the maturity, fusoid to narrowly ellipsoid, upper part or second cell slightly wider and tapering towards narrow ends, constricted at the septa, hyaline to yellow-brown to brown with age, guttulate, think-walled. Asexual morph : Undetermined. Culture characteristics. Spores germinated within 12 hours, colonies grow on PDA at 28 °C, circular, floppy, entire edge, raised, grey to taupe, reverse dark brown. Material examined. China , Yunnan Province , Lincang , on a dead branch of Hevea brasiliensis , 28 July 2022 , Rui-Fang Xu, LCR06, (ZHKU 23-0104, holotype ); ex-type ZHKUCC 23-0798, ZHKUCC 23-0799 . GenBank numbers. ZHKUCC 23-0798 = ITS: OR853099, LSU: OR922323, SSU: OR941079, tef 1-α: OR966282, rpb 2: OR966280; ZHKUCC 23-0799 = ITS: OR853100, LSU: OR922324, SSU: OR941080, tef1-α : OR966283, rpb 2: OR966281. Notes. In the phylogenetic analyses, Nigrograna lincangensis (ZHKUCC 23-0798) forms a closely-related clade to N. asexualis (ZHKUCC 22-0214), N. aquilariae (ZHKUCC 23-0070) and N. verniciae with 100% ML and 1.00 PP support (Fig. 3 ). However, we could not compare the morphological characteristics of N. lincangensis and N. asexualis , because N. lincangensis was described only from its sexual morph in nature, while N. asexualis was described by its asexual morph in nature from coffee in China. A comparison of the ITS region of N. lincangensis and N. asexualis revealed 16 base pair differences (3.46%) across 462 nucleotides, 40 base pair differences (4.21%) across 949 nucleotides in tef 1-α gene, 124 base pair differences (12%) across 1033 nucleotides in rpb 2 gene. Nigrograna aquilariae and N. verniciae have very similar morphological characteristics, but they can be differentiated by having wider ascomata (285-360 μm vs. 180-270 μm ), larger asci (45-70 x 9-12 μm vs. 49-57 x 7-9 μm ) and larger ascospores (10-15 x 4-6 μm vs. 10-13 x 3.5-4.5 µm ) in N. lincangensis ( Du et al. 2024 ); while N. verniciae has larger ascomata (340-360 x 350-370 μm vs. 85-360 μm x 230-307 μm ) and asci with knob-like to furcate pedicels ( Li et al. 2023 ). Figure 3. Phylogram generated from Maximum Likelihood analysis based on combined LSU, ITS, SSU, tef 1-α and rpb 2 sequence data of 119 taxa, which comprised 4399 base pairs (LSU = 908 bp, ITS = 512 bp, SSU = 1000 bp, tef 1-α = 925 bp, rpb 2 = 1054 bp). The best scoring RAxML tree with a final likelihood value of -38918.764563 is presented. The matrix had 2023 distinct alignment patterns, with 39.00% of undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.245191, C = 0.247520, G = 0.268228, T = 0.239061; substitution rates: AC = 1.533778, AG = 3.877174, AT = 1.672983, CG = 1.254032, CT = 8.838860, GT = 1.000000; gamma distribution shape parameter α = 0.208600. Bootstrap support values for ML equal to or greater than 60% and Bayesian Inference analysis values equal to or greater than 0.90 PP are labelled at each node. The tree is rooted with Seriascoma didymospora (MFLUCC 11-0179) and S. didymospora (MFLUCC 11-0194). Related sequences were obtained from De Silva et al. (2022) , Lu et al. (2022) and Li et al. (2023) . The new isolates are indicated in red and the ex-type strains are in bold. Nigrograna lincangensis has similar ascomata, asci and ascospore characteristics similar to other Nigrograna species ( Jaklitsch and Voglmayr 2016 ; Hyde et al. 2017 ; Tibpromma et al. 2017 ; Dayarathne et al. 2020 ; Mapook et al. 2020 ; Lu et al. 2022 ). However, N. lincangensis differs from N. cangshanensis by having larger ascomata (285-360 x 230-307 μm vs. 120-135 x 135-155 μm ) ( Tibpromma et al. 2017 ). Nigrograna chromolaenae can be distinguished from N. lincangensis in having smaller ascomata (160-280 x 115-130 μm vs. 285-360 x 230-307 μm ), smaller asci (40-55 x 7-10 μm vs. 45-70 x 9-12 μm ), and greyish-brown to dark brown ascospores ( Mapook et al. 2020 ). Nigrograna coffeae differs from N. cangshanensis by having smaller ascomata (90-140 x 140-200 μm vs. 285-360 x 230-307 μm ), 1-septate ascospores ( Lu et al. 2022 ). Nigrograna novergica differs from N. lincangensis as it occurs on pseudostromata from the host of Diaporthe sp. ( Jaklitsch and Voglmayr 2016 ). Nigrograna mycophila and N. obliqua are distinct from N. lincangensis by having dark brown ascospores ( Jaklitsch and Voglmayr 2016 ). Nigrograna puerensis differs from N. lincangensis by having acute apical and basal cells and the apical cells are wider than the basal cells ( Lu et al. 2022 ). Nigrograna samueliana differs from N. lincangensis by the absence of ostiole ( Dayarathne et al. 2020 ). Nigrograna thymi can be easily distinguished from N. lincangensis in having 4-5 septate ( Hyde et al. 2017 ). Therefore, N. lincangensis is described here as a new species, based on phylogeny and morphology. Figure 4. Nigrograna lincangensis (ZHKU 23-0104, holotype ) a-c appearance of ascomata on the host surface d vertical section of an ascoma e vertical section of ostiole f hamathecium and asci g section of peridium h-k asci l ascospores m a germinated ascospore n, o colonies on PDA (n-front and o-reverse views). Scale bars: 100 µm ( d ); 50 µm ( e ); 30 µm ( f ); 200 µm ( g ); 10 µm ( h-k ); 5 µm ( l ); 20 µm ( m ).