Phylogenetic Relations of Rhizoplaca Zopf. from Anatolia Inferred from ITS Sequence Data Demet Cansarana, Sümer Arasb,*,I˙rfan Kandemirc, and M. Gökhan Halıcıd a Botany Section, Department of Biology, Faculty of Science, University of Ankara, Tandogan, Turkey b Biotechnology Section, Department of Biology, Faculty of Science, University of Ankara, Tandogan 06100 Ankara, Turkey. Fax: +90-3122232395. E-mail: [email protected] c Department of Biology, University of Karaelmas, Zonguldak, Turkey d Department of Biology, University of Erciyes, Kayseri, Turkey * Author for correspondence and reprint requests Z. Naturforsch. 61c, 405Ð412 (2006); received September 19/November 2, 2005 Like many lichen-forming fungi, species of the genus Rhizoplaca have wide geographical distributions, but studies of their genetic variability are limited. The information about the ITS rDNA sequences of three species of Rhizoplaca from Anatolia was generated and aligned with other species from other countries and also with the data belonging to Lecanora species. The examined species were collected from the volcanic rocks of Mount Erciyes which is located in the middle of Anatolia (Turkey). The sequence data aligned with eight other samples of Rhizoplaca and six different species of Lecanora were obtained from GenBank. The results support the concept maintained by Arup and Grube (2000) that Rhizoplaca may not be a genus separate from Lecanora. According to the phylogenetic tree, Rhizoplaca melanopthalma from Turkey with two different samples of R. melanopthalma from Arizona (AF159929, AF159934) and a sample from Austria formed a group under the same branch. R. peltata and R. chrysoleuca samples from Anatolia located in two other branches of the tree formed sister groups with the samples of the same species from different countries. Although R. peltata remained on the same branch with other samples of the same species from other countries it was placed in a different branch within the group. When the three species from Anatolia were considered alone, it was noticed that Rhizoplaca melanopthalma and Rhizoplaca peltata are phylogenetically closer to each other than Rhizoplaca chrysoleuca; the morphological characteristics also support this result. Key words: Rhizoplaca, Phylogeny, ITS Introduction The thallus morphology has been used as a con- The genus Rhizoplaca belongs to the Lecanora- ventional character in lichen systematics to differ- ceae within the order Lecanorales and suborder entiate many different taxa. For example, separa- Lecanorineae with Trebouxia as photobiont tion of the crustose Buelliaceeae from the foliose (Hawksworth, 1988). The members of this genus to fruticose Physciaceae almost completely de- grow on siliceous or calcareous rocks or unat- pends on thallus morphology. This is also the case tached (vagrant) on soil, in open, especially dry for the Leconoraceae family. In this family, two sites. The growth forms of the Rhizoplaca mem- lobate genera Arctopeltis Poelt (Poelt, 1983) and bers are mostly umbilicate foliose but also rarely Rhizoplaca (Leuckert et al., 1977) have been sepa- crustose or fruticose (Ryan and Nash, 1997). The rated from the genus Lecanora mainly by their colors of the thalli in the genus Rhizoplaca are umbilicate thalli with well developed upper and frequently yellowish green to yellow-gray, but lower cortices (Arup and Grube, 2000). sometimes only gray. That thalli are usually However, in the species, which have intermedi- rounded and often lumpy, 10Ð30 mm in diameter; ate thallus characters, classification by using tra- the lower surface is pale to black, without rhizines. ditional generic concepts is usually difficult. Medulla often contains placodiolic or pseudopla- Recently, ascomatal characters became very im- codiolic acid and other depsides, depsidones and portant in classification of families and genera triterpenes in various combinations (Brodo et al., (Hafellner, 1984). The genus Rhizoplaca has le- 2001). canorine apothecia, colorless, 1-celled spores and 0939Ð5075/2006/0500Ð0405 $ 06.00 ” 2006 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D 406 D. Cansaran et al. · Phylogenetic Relations in Rhizoplaca Lecanora-type asci as in the genus Lecanora. As In molecular studies of lichen, analysis of nu- mentioned above, the genus Rhizoplaca is sepa- clear ribosomal DNA (rDNA) has become an rated from the genus Lecanora mainly by its thal- important tool. The availability of non-algal lus morphology. Another related genus, Squama- primers allows the amplification of fungal target rina has the same ascomatal characters with Le- DNA without any contamination with the algal canora and Rhizoplaca, but has Bacidia-type asci partner. The sequence data accumulated with (Purvis et al., 1992). these studies shed light to phylogenetic relation- Rhizoplaca species were previously classified in ships (Lutzoni and Vilgalys, 1995), population the Leconora genus as section Omphalodina in the structures (DePriest, 1993), and origin of lichens subgenus Placoduim (Poelt, 1958). But according (Gargas et al., 1995). to evaluation of many characteristics of the group In this study we obtained ITS sequence data for it was raised to a generic level by Leuckert et al. three species of Rhizoplaca found in Turkey. The (1977) using the name Rhizoplaca once again. In examined species were collected from the volcanic addition to its umblicate thallus structure, Rhizo- rocks of Mount Erciyes which is located in the placa could also be distinguished by distinct upper middle of Anatolia (Turkey). The sequence data cortex, thick lower cortex and relatively loose me- aligned with eight other samples of Rhizoplaca dulla. Although this situation seems to be the final and six different species of Lecanora were ob- decision about the discrimination between Leco- tained from GenBank. The results support the nora and Rhizoplaca, Ryan and Nash (1997) have concept maintained by Arup and Grube (2000) pointed the necessity of further studies in order to that Rhizoplaca may not be a genus separate from set the definite boundaries. Lecanora. Besides this, the study constitutes a con- The ITS sequence data for many different Rhi- tribution to the phylogenetic studies based on ITS zoplaca species have been documented and their sequence data of lichens from Turkey. In this re- differentiation from Leconora have been argued spect this study constitutes the first data from Tur- by Arup and Grube (2000). According to phyloge- key, and from this point of view it may show off netic analysis they indicated that the generally ac- the presence of variation in populations of Rhizo- cepted concept of Rhizoplaca as a genus separate placa species growing in different geographical from Leconora might be rejected. areas and ecologies. Rhizoplaca genus comprise more than nine spe- cies, distributed throughout the world, only three Materials and Methods of them are found in Turkey. Among the species distributed around the world, such as R. chryso- Plant material leuca (Sm.) Zopf, R. glaucophana (Hasse) W. We- Three Rhizoplaca species were collected from ber, R. haydenii (Tuck.) W. Weber, R. marginalis around Kayseri province. The three species might (Hasse) W. Weber, R. melanopthalma (DC.) be regarded as a represention of the whole popu- Leuckert & Poelt, R. peltata (Ramond) Leuck. & lation, as these were the only species grown in Tur- Poelt, R. subdiscrepans (Nyl.) R. Sant., R. asido- key. phora, R. priestleyi (Dodge), the species found in The samples were dried at room temperature Turkey are R. chrysoleuca (Sm.) Zopf, R. melano- and foreign matters were removed prior to grind- pthalma (DC.) Leuckert & Poelt, R. peltata (Ra- ing. The lichen samples are stored in the Herba- mond) Leuckert & Poelt. rium of Erciyes University (Erciyes University, A common characteristic of the three species Department of Botany, Kayseri, Turkey). The col- found in Turkey is their direct anchorage to the lection localities were as follows: Rhizoplaca me- rocks with an umbilicus structure of their thallus. lanopthalma, Turkey, (Prov.) Kayseri (38) Erciyes While in R. chrysoleuca the lower surface is rough, Mountain, western slope of Erciyes Mountain broken into areoles with cracks, the apothecial (along the telepherics), 38∞32ЈN, 35∞30ЈE, 2500Ð disks are yellowish brown and medulla P+ (para- 2600 m (leg. & det. M. G. Halıcı); Rhizoplaca chry- phenylendiamine) orange (pannarin), in R. peltata soleuca, Turkey, (Prov.) Kayseri (38) Erciyes and in R. melanopthalma the lower surface is Mountain, western slope of Koc¸ Mountain, smooth, apothecial disks are yellowish-brown to 38∞32ЈN, 35∞32ЈE, 2200Ð2300 m (leg. & det. M. G. greenish or black, pruinose and also their medulla Halıcı); Rhizoplaca peltata, Turkey, (Prov.) Kayseri are bright yellow with psoromic acid P+. (38) Erciyes Mountain, north of Perikartın (north- D. Cansaran et al. · Phylogenetic Relations in Rhizoplaca 407 ern slope of Erciyes Mountain), 38∞35ЈN, 35∞27ЈE, et al., 1990). ITS1F primer was designed specificly 2300 m (leg. & det. M. G. Halıcı). for fungal sequences at the 3Ј end of the small subunit gene of the rDNA, overlapping with ITS5, DNA extraction whereas ITS4 was described as an universal primer corresponding to the 5Ј end of the large subunit Total DNA was extracted from thalli according gene (White et al., 1990). to an improved CTAB (cetyl-trimethyl ammonium Different parameters were tested for optimiza- bromide) based extraction method. Briefly the ex- tion of PCR reactions.