The Lichenologist 41(6): 571–604 (2009) © British Lichen Society, 2009 doi:10.1017/S0024282909008317 The taxonomy of the Caloplaca citrina group (Teloschistaceae) in the Black Sea region; with contributions to the cryptic species concept in lichenology Jan VONDRÁK, Pavel Rˇ ÍHA, Ulf ARUP and Ulrik SØCHTING Abstract: A new taxonomy of the Caloplaca citrina group in the Black Sea region is presented. It is based on the nrDNA ITS molecular data, chemistry (anthraquinone contents) and 20 morphological characters. Six species previously known in the region are accepted: Caloplaca arcis, C. calcitrapa, C. dichroa, C. flavocitrina, C. geleverjae, C. limonia. Five new species are described: Caloplaca arcisproxima, C. austrocitrina, C. communis, C. confusa and C. nigromarina. Seven further species, Caloplaca britannica, C. citrina, C. marina, C. maritima, C. microthallina, C. ora and C. phlogina are also treated briefly. Some maritime species known from the Atlantic coast of Europe are absent from the region, and, surprisingly, Caloplaca citrina s. str. could not be confirmed from the study area. A key to the species present in the region is provided, although morphological characters are of very limited value in this group. The variability and taxonomic importance of particular features are discussed. No significant differences in secondary chemistry were observed among the species. Many examples of convergence and some semi-cryptic species were revealed by molecular data. The term ‘semi-cryptic species’ is introduced here into lichenology for those species which cannot be clearly diagnosed by their morphology, but which are determined by other characters, mainly by their ecology and distribution. We propose to describe formally such species, in spite of difficulties with subsequent morphological identification. Key words: alveolate cortex, Caloplaca citrina clade, lichens, nrDNA ITS, semi-cryptic species, Teloschistales Introduction (1997), Harada (2004) or McCarthy (1991), A number of lichenological papers, dealing and Xanthoria in Lindblom & Ekman with taxonomy or biodiversity, have included (2005). Marine and maritime lichens are lichen species inhabiting seashore habitats well-known in some regions, for instance in but only some of these have dealt with par- the British Isles, where some ecological and ticular taxonomic groups occurring specifi- generalized studies have been made (e.g. cally on coastal cliffs, e.g. Ramalina in Sheard Fletcher 1973a,b, 1975a,b). The genus Calo- (1978), Roccella in Tehler et al. (2004), Ver- placa has been intensively investigated on rucaria in for example Brodo & Santesson North American shores (Arup 1992a,b, 1993a,b, 1994, 1995a,b, 1997b) and in Europe (e.g. Tavares 1956; Laundon 1992; J. Vondrák: Department of Botany, Faculty of Science, Roux & Navarro-Rosinés 1992; Navarro- University of South Bohemia, Branišovská 31, Cˇeské Rosinés & Roux 1993, 1995; Arup 1997a). Budeˇjovice, CZ-370 05, Czech Republic. Email: In the East Mediterranean and the Black Sea [email protected] P. Rˇ ´ha:ı Department of Zoology, Faculty of Science, region, some papers, mainly contributions on University of South Bohemia, Branišovská 31, Cˇeské lichen biodiversity, partially cover maritime Budeˇjovice, CZ-370 05, Czech Republic. species, including Caloplaca (e.g. Szatala U. Arup: Botanical Museum, Lund Univeristy, Östra 1943a, b;Veˇzda 1975; Roux & Navarro- Vallgatan 18, SE-223 61 Lund, Sweden. U. Søchting: Section for Ecology and Evolution, Rosinés 1992; Güvenc & Öztürk 1999; Department of Biology, University of Copenhagen, Ø. Sipman & Raus 1999, 2002; Yazici 1999; Farimagsgade 2D, DK-1353 Copenhagen, Denmark. Khodosovtsev 2001, 2002, 2003; Redchenko 572 THE LICHENOLOGIST Vol. 41 2002; Khodosovtsev et al. 2003, 2007; John & Material and Methods Breuss 2004; Sipman et al. 2005; Vondrák & Material from the Atlantic coast of Europe used for Slavı´ková-Bayerová 2006; Vondrák et al. comparison (mainly of Caloplaca britannica, C. littorea, 2008a;), but otherwise knowledge of mari- C. marina, C. maritima,andC. microthallina) was ob- time species of this area remains sparse. tained on loan from BM, CBFS, LD, and hb. A. Aptroot While working in the Black Sea region, one (ABL). Material from Central Europe, the Black Sea region and the Mediterranean was obtained on loan of us (JV) found that current W European from B, BP, CBFS, GZU, KHER, PRM, SAV and W. literature for the identification of lichens The main part of the material from the Black Sea region (species in the genera Caloplaca, Candelari- was collected by the first author during field excursions ella, Catillaria, Lecania and Toninia) was in July 2004 (Bulgaria), July and November 2005 (Romania, Bulgaria, European part of Turkey), June inadequate. This was particularly true for 2006 (Ukraine: Crimea), and April, May 2007 the genus Caloplaca, which appeared to be (Romania, Bulgaria, Turkey, Georgia, Russia, Ukraine). extremely species-rich in the area. Currently, Vouchers are deposited in CBFS (the numbers follow- we estimate that more than 50 species occur ing this abbreviation are herbarium accession numbers). in coastal habitats around the Black Sea. Citations of specimens examined are abbreviated and for common species only selected specimens are pre- This huge, but largely unknown diversity, led sented. Data on additional samples and full sample us to initiate a complex project on the bio- information are available on the web page: http:// diversity of the genus Caloplaca in the Black botanika.bf.jcu.cz/lichenology/data.php. Sea region and the present study focuses on the taxonomy of the Caloplaca citrina group in Morphology and anatomy the area. Arup (2006a) has already clarified A total of 20 characters was measured and used in the the taxonomy of this group in Scandinavia detailed characterization of the Caloplaca citrina group, but only 11 traits were important for species separation: and his results are utilized and developed width of areoles, thickness of thallus and cortex/alveolate further here. This group seems to be rather cortex, size of vegetative diaspores, size of apothecia, species rich and according to phylogenetic exciple width, hymenium height, size of ascospores, studies it is closely related to the C. saxicola width of spore septa, width of paraphyses tips and size of group, various clades of Xanthoria species conidia. Sections for morphological examination were cut by and the C. holocarpa group (Arup & Grube hand and observed in water, but paraphysis tips and 1999; Gaya et al. 2008).This paper describes cortical tissues were observed after pretreatment with the species diversity in the C. citrina group. KOH. Measurements were made to an accuracy of The taxonomy proposed in this study is 0·5 µm for cells (e.g. ascospores, conidia and para- physes), 1 µm for thickness of the cortex and 10 µm for based on molecular data using the single larger structures (e.g. hymenium thickness and exciple locus, ITS nrDNA. This locus is usually well width). All measurements of cells included their walls. suited for taxonomic studies at the species Measurements are given as (min.–) x ± SD (–max.), level, and a number of published papers where x = mean value and SD = standard deviation. The using only this region has given meaningful total numbers of measurements (n) are given in paren- theses. At least five measurements were taken from all results in the genus Caloplaca (Tretiach et al. specimens available; in the morphologically indistin- 2003; Arup 2006a, 2009; Arup et al. 2007; guishable species (Caloplaca confusa, C. nigromarina), Søchting & Figueras 2007; Muggia et al. only specimens confirmed by molecular data were used 2008; Vondrák et al. 2008b,c). The only for measurements. Morphological terminology follows Ryan et al. (2002) study in Teloschistaceae so far using a multi- and Bungartz (2002). The term ‘alveolate cortex’ is locus approach is one dealing with taxonomy proposed here for a hyaline tissue formed by living at higher than species level (Søchting & fungal cells among dead algal cells or in gaps left by dead Lutzoni 2003). Our investigations have some algal cells. It is similar to a phenocortex (sensu Ryan et al. general implications exceeding the frame- 2002: 23), but differs in fungal cells, which are living, and not dead as in a phenocortex. Being situated be- work of a regional taxonomic study since tween algal and epinecral layers, it replaces a true cortex the molecular results have revealed several in some species. distinct groups, which are often morphologi- cally hardly distinguishable. This phenom- DNA extraction and amplification. enon, termed semi-cryptic speciation, is Direct PCR was used for DNA extraction and PCR- discussed at the end of the paper. amplification of the nuclear ITS regions including the 2009 Caloplaca citrina group in the Black Sea region—Vondrák et al. 573 T 1. Voucher specimens and GenBank accession numbers of the ITS sequences used in the phylogenetic analysis. Accession numbers in bold represent new sequences produced during this study Species & Herbarium Source GenBank Accession No. Accession No. Caloplaca arcis Austria. (Arup 2006a) DQ173213 C. arcis Sweden. (Arup 2006a) DQ173214 C. arcis Great Britain, England. (Arup 2006a) DQ173215 C. arcis W1990-00525 Italy, Sardinia (coll. W. Brunnbauer 1986) EU563454 C. arcis CBFS JV3036 Bulgaria, Black Sea coast (coll. Vondrák 2005) EU563395 C. arcis CBFS JV4985 The Netherlands, Noordoostpolder (coll. A. Aptroot EU563453 59871, 2004) C. arcis CBFS JV4986 The Netherlands, Gelderland