Plant and Fungal Systematics 65(1): 167–175, 2020 ISSN 2544-7459 (print) DOI: https://doi.org/10.35535/pfsyst-2020-0011 ISSN 2657-5000 (online) Three new lichen species from Macaronesia belonging in Ramalinaceae, with the description of a new genus Pieter P. G. van den Boom1,2* & Nicolas Magain2 Abstract. Tylocliostomum is described as a new genus and T. viridifarinosum as a new Article info species. Two Bacidina species, B. pallidocarpa and B. violacea, are also described as new Received: 30 Sept. 2019 to science. They all occur in Macaronesia (Azores, Madeira). Revision received: 6 Mar. 2020 Accepted: 11 Mar. 2020 Key words: Ascomycota, Lecanorales, Tylothallia, type species, molecular phylogenetics Published: 2 Jun. 2020 Associate Editor Bernard Goffinet Introduction An extensive study of lichens and lichenicolous fungi Ascospores and other anatomical details were studied and in the Azores and Madeira by the first author resulted in measured in water or in 10% potassium hydroxide (K) if several works already published from Sao Miguel and features were otherwise unseparated. In each collection Terceira (van den Boom 2015b, 2016; Guzow-Krzemińska (~5 well-developed ascospores representing the observed et al. 2019) and from Madeira (van den Boom 2015a, b; variation of size and shape), conidia and paraphyses were van den Boom & Ertz 2014). Several Bacidia or Bacidina measured to 0.1 µm accuracy. species are recorded in van den Boom (2015a) and van Chemical spot tests were performed under a compound den Boom & Alvarado (2019). Ten species of these genera microscope using sodium hypochlorite (C) and K (Orange are recorded in the checklist of Madeira, including the et al. 2001). Pigments were determined following the endemic species Bacidia albonigrans, B. endoleucoides system of Meyer & Printzen (2000). The crystals from and B. fritzei (Carvalho et al. 2008). Fifteen species of all studied specimens were investigated in tissue sections these genera are recorded in the checklist of the Azores, by using a compound microscope with polarization filters. with none of them being endemic (Aptroot et al. 2010). For a detailed comparison of the main diagnostic features In this study we describe two new Bacidina species: of the two new Bacidina species and the most similar B. violacea, known from the Azores and Madeira, and Bacidina species, see Table 1. B. pallidocarpa, endemic to the Azores. Tylocliostomum is described as a new genus and T. viridifarinosum as a new Molecular data species, known only from the type locality in Madeira. DNA was extracted from two specimens of Bacidina pallidocarpa and two specimens of B. violacea, and two Materials and methods DNA extractions were performed from the same material of Tylocliostomum viridifarinosum (Table S1). Extraction Material acquisition of DNA followed the protocol of Cubero et al. (1999). This study is mainly based on extensive collections of We sequenced the ribosomal nuclear loci ITS using prim- Bacidia s.l. made by the first author on several Mac- ers ITS1F (Gardes & Bruns 1993) and ITS4 (White et al. aronesian islands in 2017 and 2019 and hosted in LG 1990), the mitochondrial ribosomal locus mtSSU with and the private herbarium of the first author. Hand-cut primers SSU1 and SSU3R (Zoller et al. 1999), and part apothecial sections and squashed thallus preparations were of the protein-coding gene RPB1 with RPB1AF (Stiller examined with a compound microscope (Olympus BX2). & Hall 1997) and VH6R (Hofstetter et al. 2007). For the three loci, PCR conditions were as follows: ini- 1 Arafura 16, 5691JA, Son, The Netherlands tial denaturation at 95°C for 1 min, followed by 35 cycles 2 Evolution and Conservation Biology, InBios Research Center, Institut of denaturation at 95°C for 45 sec, a different annealing de Botanique B22, Université de Liège, Chemin de la vallée 4, 4000 Liège, Belgium phase for each locus, then an elongation phase of 1 min * Corresponding author e-mail: [email protected] at 72°C, followed by a final elongation phase of 10 min at © 2020 W. Szafer Institute of Botany, Polish Academy of Sciences. This is an Open Access article distributed under the terms of the Creative Commons Attribution License CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/) 168 Plant and Fungal Systematics 65(1): 167–175, 2020 Table 1. Main diagnostic features of Bacidina species similar to B. pallidocarpa and B. violacea. Species Thallus Colour Apothecia Margin Disc Ascospores Pycnidia smooth to fine greenish with somewhat paler than white to pale 0.15– 25–38 white, B. pallidocarpa warted, granular ± pale brown disc, soon becoming cream with 0.55 mm × 1–1.2 µm one type to fine isidiate tinge immarginate yellowish tinge yellowish paler than disc, with 25–50 B. violacea areolate, farinose brown to very 0.2–0.7 mm dark brown unknown violet tinge × 1–1.5 µm pale brown concolorous with sorediate to pale pink to 40–50 white, B. adastra green 0.4–0.7 mm or slightly paler thickly leprose dark blue-black × 0.9–1.2 µm one type than disc concolorous with concolorous rimose to areolate, pale grey to or slightly paler pale pink to 23–41 with B. brittoniana 0.2–0.4 mm areoles convex grey-green than disc, finally purple-brown × 1.7–2.4 µm apo thecia, excluded one type wrinkled or concolorous with or pale pink to warted, ± rimose, pale pink to 31–53 70–100 µm, B. californica 0.4–0.7 mm paler or darker than purple-brown to or granular orange brown × 1.9–3.3 µm two types disc blackish areolate dull or grey- whitish or 20–38 white, B. chloroticula scurfy granular green to pale 0.1–0.2 mm paler than disc pink-orange to × 1–1.8 µm one type brownish pale grey-brown pale beige, rugulose to olivaceous 0.2– concolorous with waxy white or 32–41 B. contexta unknown microsquamulose green 0.45 mm disc pale pinkish × 1.2–1.6 µm orange pale green to white, beige or 24–48 white, B. delicata fine granular 0.3–0.6 mm paler than disc fawn orange-pink × 1–1.5 µm one type pale orange to hyaline, concolorous with or 25–34 B. egenuloidea fine granular grey-green 0.3–0.4 mm dark purple- 75–200 µm, darker than disc × 1.1–1.9 µm brown three types paler than disc, white or dark granular warted, grey-green to pale to dark 24–43 B. inundata 0.2–0.6 mm often becoming brown, cracked green-fawn brown × 2–2.5 µm excluded two types scurfy, uneven concolorous with pale, straw-coloured from whitish to 30–38 B. mendax crust or small 0.2–0.7 mm or paler than disc, 150–300 µm, to bright green fuscous-brown × 1.2–1.5 µm warts ± finally excluded one type colourless to pink-buff to olive-grey, green-grey grey to dark brown, flesh-coloured 40–55 B. neosquamulosa squamulose 0.2–1 mm 70–130 µm, to olive persistent with blackened × 1.3–1.7 µm abundant, one areas type white, beige white 29–45 hyaline, B. phacodes warted to scurfy 0.2–0.5 mm paler than disc or pale to grey-green × 1.5–2 µm one type orange-pink ~50 µm, filmy thin, thinly marginate, pale yellowish dull greenish to 25–35 hyaline to pale B. pseudoisidiata fine granular to 0.2–0.4 mm becoming to yellowish grayish green × 1.1–1.8 µm brownish, one coralloid immarginate orange type pale grey, pale pale yellow, hyaline, wrinkled or 31–49 B. ramea yellow-grey or 0.4–0.6 mm thin, thalline pale pink to ~100 µm, warted to areolate × 1.6–2.2 µm pale green-gray orange brown one type flat, deeply incised concolorous hyaline, 45–48 B. squamellosa squamules, lobes pink 0.3–0.5 mm with disc, finally pale pink 75–100 µm, × 1.3–1.4 µm 20–30 µm wide excluded three types dull yellow- paler than disc, white gray to 24–46 white, B. sulphurella finely granular 0.3–0.7 mm green to fawn white-pruinose grey-brown × 1–2 µm one type 72°C. For ITS the annealing phase was 52°C for 45 sec; using MAFFT v. 7.305b with default parameters (Katoh for RPB1, 1 min at 56°C; and for mtSSU, 1 min at 58°C. & Standley 2013) and further inspected by eye. Ambigu- PCR fragments were purified using EXO-SAP (VWR ous regions were delimited manually and excluded from International, West Chester, PA, USA) and sequenced the analyses. by Macrogen Europe (Amsterdam, The Netherlands). Sequence fragments were assembled using Geneious Phylogenetic analyses v. 10.2.3 (Biomatters LTD). Matrices were assembled in We downloaded ITS, mtSSU and RPB1 alignments Mesquite v. 3.11 (Maddison & Maddison 2016), aligned from Kistenich et al. (2018). We pruned them to keep P. P. G. van den Boom & N. Magain: Three new lichen species and a new genus from Macaronesia 169 a maximum of two representatives per genus, following sequences from the other specimen. We obtained mtSSU the new classification from Kistenich et al. (2018), and and RPB1 sequences from one B. pallidocarpa specimen, favouring type species, then taxa with all or most of the and only mtSSU for the other specimen. We obtained ITS three loci, except in clades where sequences exhibited and RPB1 sequences from the two DNA extractions of high similarity with our sequences, where all represent- Tylocliostomum viridifarinosum (Table S1). atives were kept. Catillaria contristans, Catillaria ery- siboides, Glyphopeltis ligustica, Protoblastenia rupestris Phylogenetic analyses and Psora decipiens were selected as outgroups and other The final 3-locus alignment consisted of 131 taxa and outgroup taxa were further removed (Table S1).