Phylogenetic analysis shows that Pyrenula (Pyrenulaceae) diversity is larger than expected: three new species and one new record discovered in China Author Dou, Mingzhu 0000-0001-5160-1517 College of Agriculture and Life Sciences, Liaocheng University, Liaocheng 252059, China Author Li, Jiechen https://orcid.org/0009-0001-8819-8407 College of Agriculture and Life Sciences, Liaocheng University, Liaocheng 252059, China Author Hu, Yongshun https://orcid.org/0009-0002-6764-4364 College of Agriculture and Life Sciences, Liaocheng University, Liaocheng 252059, China Author Aptroot, André 0000-0001-7949-2594 Laboratório de Botânica, Liquenologia, Instituto de Biociências, Universidade Federal de Mato Grosso do Sul, Avenida Costa e Silva s / n, Bairro Universitário, CEP 79070 - 900, Campo Grande, Mato Grosso do Sul, Brazil Author Jia, Zefeng College of Agriculture and Life Sciences, Liaocheng University, Liaocheng 252059, China text MycoKeys 2024 2024-11-13 110 159 183 journal article 305174 10.3897/mycokeys.110.131741 ce58e353-081f-47ed-ba34-9ed539493661 Pyrenula macularis (Zahlbr.) R. C. Harris Fig. 6 Basionym. Anthracothecium maculare Zahlbr. , Mycologia 22: 70 (1930) . Holotype . Yauco , Porto Rico [ Puerto Rico ], 30 Dec. 1915 . Description. Thallus corticolous, crustose, olive-green in the field and khaki after drying, surface dull, corticate with abundant pseudocyphellae, UV-. Apothecia perithecioid, conical, dispersed, aggregated occasionally when crowded, with crystals, immersed in the thallus, small, to 0.3 mm wide at the early developmental stage, then the sides partly covered by the thallus. Excipulum carbonized when mature and falls apart when over-mature. Ostioles apical, white. Hamathecium not inspersed, IKI + red. Ascospores 8 per ascus, subbiserial, fusiform, with pointed or blunt ends, (33 –) 37–50 × (13 –) 14–16 μm, hyaline to brown, muriform, with c. 8 × 1–3 locules, lumina rounded, old spores containing globules of red oily substance. Figure 6. Pyrenula macularis ( LCUF HNX 18017) A, B thallus with ascomata and pseudocyphellae C, D ascospores at different development stages E ascospores with gelatinous halo shown by red arrows F, G over-mature ascospores with orange oil. Scale bars: 1 mm ( A, B ); 10 µm ( C ); 20 µm ( D, E ); 10 µm ( F, G ). Chemistry. Thallus UV-. TLC with solvent C showed unidentified black spots at Rf two, four and five under 254 nm ultraviolet light on fresh plate; unidentified green spot at the dividing line of Rf four and five on charred plate under daylight; unidentified red spots at Rf three, four and red spots at Rf five on charred plate under 365 nm ultraviolet light (Suppl. material 4 ). Additional specimens examined. China • Hunan Province , Wugang City , Yun Shan , Shuanghua Pavilion , 26°39'28"N , 110°36'37"E , alt. 730 m , on bark of Zelkova serrata , 28 April 2018 , Z. F. Jia ( LCUF HNX 18016 ; GenBank PP 692368 for ITS) . China • Hunan Province , Wugang City , Yun Shan , Shuanghua Pavilion , 26°39'28"N , 110°36'37"E , alt. 730 m , on bark of Zelkova serrata , 28 April 2018 , Z. F. Jia ( LCUF HNX 18017 ; GenBank PP 692369 for ITS) . China • Hunan Province , Wugang City , Yun Shan , Shuanghua Pavilion , 26°39'28"N , 110°36'37"E , alt. 730 m , on bark of Zelkova serrata , 28 April 2018 , Z. F. Jia ( LCUF HNX 18018 ; GenBank PP 659691 for mtSSU, PP 692370 for ITS and PP 692473 for nuLSU) . Habitat and distribution. Growing on exposed tree in pantropical forest. Previously reported from Porto Rico ( Zahlbruckner 1930 ), U. S. A. ( Harris 1989 ), Australia ( Aptroot 2009 ), Puntarenas ( Aptroot et al. 2008 ), Muri Lagoon ( Mccarthy 2000 ), HongKong ( Aptroot and Seaward 1999 ) and Taiwan ( Aptroot 2003 ) of China . Notes. The morphology and anatomy characteristics of the Chinese specimens correspond to Pyrenula macularis (Zahlbr.) R. C. Harris described from Yauco , Porto Rico . P. breutelii is distinguished by different lichen substances (Suppl. material 4 ), smaller ascomata ( 0.3–0.5 mm ), smaller ascospores (( 23 –) 25–37 (– 41) × (10 –) 12–15 (– 18 )) μm and more locules (1–4). P. macularis (no. nr. 5 and 6) has two more black spots at Rf two under 254 nm ultraviolet light on fresh plate than P. breutelii (no. nr. 2 and 3) (Suppl. material 4 ). In the protolog, TLC and KI result was not mentioned and molecular sequences were not provided. Here, we provide TLC, KI result and ITS, nuLSU sequences. This species has previously been reported in China only in Hong Kong ( Aptroot and Seaward 1999 ) and Taiwan ( Aptroot 2003 ). Because the difference in ascospores between P. macularis and P. breutelii is not very significant, P. macularis was synonymous with P. breutelii ( Aptroot 2012 ; Aptroot et al. 2013 ). But the phylogenetic result and TLC results prove they are two different species.