The Lichenologist 36(1): 37–54 (2004) 2004 The British Lichen Society DOI: 10.1017/S0024282904013933 Printed in the United Kingdom Phylogenetic relationships and species concepts in Parmelia s. str. (Parmeliaceae) inferred from nuclear ITS rDNA and -tubulin sequences María del Carmen MOLINA, Ana CRESPO, Oscar BLANCO, H. Thorsten LUMBSCH and David L. HAWKSWORTH Abstract: The phylogenetic relationships of 16 species of Parmelia s. str. are presented based on sequences of nuITS rDNA from 56 specimens, and -tubulin gene sequences from 29 collections. Parmelia serrana sp. nov. a Mediterranean species morphologically very close to P. saxatilis is described. Parmelia ernstiae is the sister-group to P. saxatilis s. str., and a further undescribed North American species of the P. saxatilis complex may require recognition. The isidiate P. squarrosa is closely allied to the sorediate P. sulcata, which is paraphyletic. Japanese samples of P. cochleata form a monophyletic group but too few collections of these were studied to reach firm conclusions regarding their relationships. An epitype is selected for the lectotype of Lichen saxatilis to unequivocally fix the application of that epithet. Key words: Ascomycota, cryptic species, Lecanorales, lichens, Linnean typification Introduction 1999), but not always where no such correlations exist (Clauzade & Roux 1985; The family Parmeliaceae is one of the largest Eriksson & Hawksworth 1986; Purvis et al. lichen-forming ascomycete families; in the 1992; Nimis 1993; Santesson 1993), wide sense it encompasses around 2300 pending clarification by molecular or other species placed in about 85 genera, 49 of studies. Molecular analyses have been them being described in the last two decades carried out to determine phylogenies in (Hawksworth et al. 1995). The segregates parmelioid genera using several regions of have been generally accepted when they DNA: nuclear ITS and 5·8S (Mattsson & were based on relationships between Wedin 1998; Crespo & Cubero 1998); anatomical features of the sexual or conidial nuclear SSU and ITS rDNA (Wedin et al. structures as well as morphological features 1999); mitochondrial SSU rDNA (Crespo of the thallus (Mattsson & Wedin 1998, et al. 2001); and ITS and -tubulin (Crespo et al. 2002; Thell et al. 2002). Overwhelming molecular evidence is now starting to M. C. Molina (corresponding author): Departamento result in the synonymization of some genera de Matemáticas y Física Aplicadas y Ciencias introduced in recent decades, for example Naturales, Escuela Superior de Ciencias Experimen- tales y Tecnología, Universidad Rey Juan Carlos, Neofuscelia and Paraparmelia into Xantho- Madrid 28933, Spain. parmelia (Elix 2003; Hawksworth & Crespo A. Crespo, O. Blanco & D. L. Hawksworth: Departa- 2002). mento de Biología Vegetal II, Facultad de Farmacia, Molecular information is also assisting in Universidad Complutense, Madrid 28040, Spain. species delimitation in the family, and the H. T. Lumbsch: Department of Botany, The Field Museum, 1400 S. Lake Shore Drive, Chicago, Illinois developing species concept in lichenology 60605, USA. has been extensively discussed (e.g. Clerc 38 THE LICHENOLOGIST Vol. 36 1998; Grube & Kroken 2000). Tradition- regions and also included collections ally, differences in morphological, and to from more Atlantic sites in extrapolar some extent anatomical, characters have regions. The second monophyletic group been used to separate species. In addition, (the Mediterranean population) included geographic origin or apparent differences in samples from more continental environ- secondary chemistry were accorded great ments in the Mediterranean region. Subse- importance, and many allopatric popu- quently, the new species P. ernstiae has been lations were named as separate species segregated from P. saxatilis on minor mor- where there was little or no other discernible phological features and differences in the difference. A chemical species concept that internal transcribed spacer (ITS) sequences assumes a lack of variation at the species of the nuclear rDNA (Feuerer & Thell level was widely applied in macrolichen gen- 2002). era in the 1960s and 1970s (Culberson The present study has two objectives. 1986), but it is now recognized that chemi- First, to ascertain if the groups of P. saxatilis cal characters are best used a posteriori rather described as Atlantic and Mediterranean than a priori when characterizing lichen populations by Crespo et al. (2002) should species (Lumbsch 1998a, b). be considered as separate species, based on Increasingly, phylogenetic species con- molecular characters and also morphological cepts based on molecular (DNA) characters and ecological features. Second, to test the are being employed to delimit species, to validity of the recently described P. ernstiae determine a posteriori which types of pheno- by comparison with sequences from a larger typic characters are good predictors of number of collections of the complex. The phylogenetic species, and demonstrate how phylogeny of the group has been analyzed these characters evolve in lichenized fungi using phylogenetic methods, using data (Mallet 1995; Grube & Kroken 2000). from the ITS of the nuclear rDNA, and These molecular data have led to the recog- sequences of the protein-coding -tubulin nition of cryptic species (Kroken & Taylor genes. 2001), and conversely also to the union of species traditionally regarded as distinct (Myllys et al. 2001; Articus et al. 2002; Material and Methods Molina et al. 2002). Parmelia s. str. comprises around 57 Lichen material species, 40 from Asia, seven from Europe Fifty-six specimens representing 16 species, includ- and ten from North America (Hale 1987; ing all but one (P. discordans*) of the European species Kurokawa 1994a, b, c, d, e; Crespo et al. of Parmelia s. str., were used in this study. Details of the 2002). Crespo & Cubero (1998) included material, area of collection, and where samples are deposited, are presented in Table 1. Specimens were three species of Parmelia s. str., viz. P. air-dried and stored at room temperature. saxatilis, P. sulcata and P. omphalodes,in a molecular approach to Parmelia s. lat. DNA extraction and PCR amplification phylogeny. They showed that P. omphalodes and P. saxatilis were more closely related to Total DNA was extracted using the DNeasy Plant Mini Kit (Qiagen) with minor modifications as each other than to P. sulcata. In addition, described by Crespo et al. (2001). Sequences from the two species have been studied at population ITS region and the 5·8S gene of the rDNA were used level in the genus, P. saxatilis (Crespo et al. for almost all taxa. Partial sequences of the protein- 2002) and P. sulcata (Crespo et al. 1999). coding -tubulin gene were successfully amplified for These two Parmelia species are amongst the 31 of the samples. Amplification of the internally tran- scribed spacer region of the rDNA gene cluster was most widespread macrolichens on Earth. undertaken with the primers ITS1F (Gardes & Bruns Crespo et al. (2002) reported that P. saxatilis *Material was obtained and DNA extracted, but the populations from five continents belonged in sequences obtained were found to be due to a licheni- two monophyletic groups. One (the Atlantic colous fungus evidently growing in the lichen, probably population) occurred in Arctic and Antarctic Athelia arachnoidea. 2004 Phylogenetic relationships in Parmelia s. str.—Molina et al. 39 1993) and ITS4 (White et al. 1990). Amplification of appeared in the consensus trees. Bootstrap values were the partial -tubulin sequence was carried out using estimated from 1000 replicates, and phylogenetic trees Bt3-LM and Bt10-LM primers (Myllys et al. 2001). were drawn using TREEVIEW (Page 1996). Each PCR reaction contained the following ingredi- ents: 27·75 ldHO, 5 l10PCR buffer where the Bayesian analyses. The data of the combined 2 matrix were also analysed using a Bayesian approach MgCl2 2 mM was already included (10 mM Tris-HCl, pH 8·0; 50 mM KCl; 1 mM EDTA (ethylene diamine (Huelsenbeck et al. 2000; Larget & Simon 1999). tetra-acetate); 0·1% Triton X-100; 59% glycerol v/v), Posterior probabilities were approximated to by 1 l dNTP 10 mM (1·25 mM each of dATP, dCTP, sampling trees using a Markov Chain Monte Carlo dGTP and dTTP), 2·5 lofa10M dilution of each of (MCMC) method. The posterior probabilities of each the primers and 1·25 l DNA Polymerase (1 unit/l, branch were calculated by counting the frequency of Biotools). This cocktail was mixed with 10 lofthe trees that were visited during the course of the MCMC DNA template. The PCR amplification ran for 30 analysis. This analysis was performed assuming the cycles: denaturation at 94(C for 60 sec, annealing at general time reversible model (Rodriguez et al. 1990) 54(C (ITS) and 55(–58(C(-tubulin) for 60 sec, including the estimation of invariant sites and assuming and extension at 72(C for 90 sec. The reaction was a discrete gamma distribution with six rate categories carried out in an automatic thermocycler (Techne (GTR+I+G) for the combined analyses. A run with Progene). 1 000 000 generations starting with a random tree and PCR products were purified through a Biotools Bio- employing eight simultaneous chains was executed, and clean DNA purification column kit according to the every 100th tree saved into a file. We used the default manufacturer’s specifications. Sequencing was per- settings for the priors on the rate matrix, branch formed on both strands using the ABI PRISM Dye lengths, gamma shape parameter, and the proportion of terminator cycle Sequencing Ready Reaction Kit (PE invariable sites. A Dirichlet distribution was assumed Biosystems), with the amplification primer. The follow- for the base frequency parameters, and an uninforma- ing cycling profile was used: denaturation for 3 min at tive prior was used for the topology (default settings). 94(C, then 25 cycles with 10 sec at 96(C, 5 sec We plotted the log-likelihood scores of sample points at 50(C and 4 min at 60(C. Sequencing reactions against generation time and determined that station- were electrophoresed on an ABI PRISM 377 DNA arity was achieved when the log-likelihood values sequencer (Applied Biosystems).