Phylogenetic Relations of 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 -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 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 ; 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 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 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. PCR amplifications for se- traction protocol was as follows: Lichen herbarium quence analysis were performed in a volume of material (0.1 g) was ground to a fine powder in 50 μl containing 200 ng of genomic DNA, 5 μlof liquid nitrogen. Prewarmed extraction buffer μ 10 ¥ reaction buffer, 5 μlof25mm MgCl2,5 lof [50 mm Tris [(tris(hydroxymethyl) amino metha- 2.5 mm dNTPs, 200 ng of each 4 mm of ITS1F ne]-HCl (pH 8), 50 mm EDTA, 10 ml LiCl (4 m), primer and ITS4 primer, and 1 U Taq DNA poly- 1% CTAB, 2% PVPP (addition of PVPP is op- merase (Promega, Madison, USA). Amplification tional)] in the amount of 1 ml was added to the was performed in a Techne Progene thermal cycler samples and ground once more in the buffer. After (Techne, Barloworld Scientific, Stanfordshire, the samples were taken to the 1.5-ml Eppendorf U.K.). The reaction mixtures were heated in an μ tubes, 10 l -mercaptoethanol were added. The initial step of 94 ∞C for 2 min and then subjected solution was incubated in a water bath at 65 ∞C for to 35 cycles of the following program: 94 ∞C for 15 min. Following these incubation period, sam- 30 s, 55 ∞C for 1 min, 72 ∞C for 1 min 45 s. After ples were cooled to room temperature, 0.5 ml the last cycle, the temperature was maintained at chloroform/isoamyl alcohol [24:1 (v/v)] was added 72 ∞C for 8 min. The amplification products were and mixed well (no vortex). Then, samples were analyzed by electrophoresis in 1.2% agarose gel centrifuged at 17,000 ¥ g (14,000 rpm) for 2 min. containing ethidium bromide and the product sizes The supernatant was transferred to a fresh tube were determined on gels by nucleotide size marker (~ 0.8 ml) and an equal volume of isopropanol was (100 bp ladder, Fermentas, Vilnius, Litvanya). The added; the mixture was gently mixed by inversion PCR products were sequenced by the cycle se- several times. Samples incubated for at least for quencing method using a dye terminator cycle 15 min on ice increased the efficiency of DNA sequencing kit (Amersham Biosciences Corp., Pis- yield. The samples were then centrifuged for 2 min cataway, USA) according to the manufacturer’s at 17,000 ¥ g (14,000 rpm). The supernatant was protocol and an OpenGeneO` automated DNA se- discarded. 1 ml 70% ethanol was added. The sam- quencing system (Visible Genetics, Piscataway, ples were then centrifuged for 1 min at 17,000 ¥ g USA). (14,000 rpm). The pellet was once more washed with 70% ethanol optionally and air-dried until all Sequence analysis ethanol was removed. To obtain nucleic acids, the pellet was dissolved in an appropriate amount of The amplified fragments with the primers ITS1F Ј TE buffer [10 mm Tris-HCl (pH 8), 1 mm EDTA] and ITS4 comprising the 3 end of small subunit Ј (30Ð60 μl). The nucleic acids dissolved in TE gene, ITS1, the 5.8 S gene, ITS2 and the 5 termi- buffer were treated with ribonuclease (RNase, nus of the large subunit gene were sequenced 10 mg/ml) and stored at Ð 20 ∞C until use. (Fig. 1). Ambiguities arising from reading errors DNA was quantified via spectrophotometric were resolved by comparing the complementary measurement of UV absorption at 260 nm strands with the program Clustal W 1.83. The se- (Specord UV-200). DNA was also quantified by quences were also aligned using the same program means of agarose gel electrophoresis with ethi- and adjusted visually. Ambiguous aligned sites dium bromide fluorescence; a 100-bp DNA ladder were excluded from analysis and gaps were treated was used (Fermentas) as the DNA size marker. as missing data. ITS sequences for eight other dif- ferent samples from the Rhizoplaca genus and for six different species of Lecanora, available from PCR amplification and sequencing GenBank (Table I) were included into multiple Internally transcribed spacer region of the alignments. (Data can be obtained from the corre- rDNA gene cluster was amplified with the primers sponding author.) A sequence matrix of 727 nucle- ITS1F (Gardes and Bruns, 1993) and ITS4 (White otide positions was analyzed and 298 conservative, 408 D. Cansaran et al. · Phylogenetic Relations in Rhizoplaca

Rhizoplaca melanopthalma

GCGAACCTGATCCGAGGGGTTTTTGGACAATGGGGGCTTCGGCCCCCGCATCTCTTCACCCTAAGTTTACAGG

TACCTTCGTTGCTTTGGCGGCTTTGTCTTGGGGCTCGCCCCCTAGCCGGCTGCTGGCCTCGTGCCCCGAGCCG

GTCCGTGCCCGCCTGGGGCCCATCAAATTCCGTTCATCAGTGCCGTCCGAGCAAAACCACAATAGTAAAAACT

TTCAACAACGATGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATTCGTGAATT

GCAGAATTCAGTGAATCCGTTATCGAATCTTTGAACGCACATTGCGCCCCTCGGTATTCCGGGGGGCATGCCT

GTTCGAGCGTCATTGACACCCCTCAAGCTTAGCTTGGTATTGGGTCTTCGCCCCAACGTGGCGGGCCCGAAAG

TCAGAATGGCGAAGTTCGGCGCGACTTCGAGCGTAGTAACTCTTCTCCCGATCTGGAAGGTCGCGCTGGGCCA

TAAACGCAATCTTTCAA

Rhizoplaca chrysoleuca

TTGTTGCTTTGGGCGGGCCTTGGGGTCCGCCCCATCCCGGCCACGGGCCTCGCGCCCGGGCCAGGCCGCGCCG

TCCGGGGCCCATTCAAACTATGTTTACCAGTGCTGTCCGAGTGAATACCACAATAGTAAAAACTTTCAACAAG

GGATCTCTGGGTTCTCGCAATCGAATGAAGAAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCA

GTGAAATCATCGAATCTTTGAACGCACATTAGAGCCCCTTTGGGTATTCACGAGGGGCATGCCTGTTCGAGCG

TCATTGAACACTCACCCCTCAACGCTCCGCTTGCCGTGTTAGGCCGAGACCCCCGCGTAGGGCCGTACCGAAA

TCCAGTGGCGGGCCGTACCGAAATCCAGTGGCGGTCCGGCGTGACTTCGAGCGTAGTAGGGATCCTATCGCTC

GTTGGAAGCTCGGCGTCGGGGCCGGCCACGAACGCCCTCAATTTCATTTCCGA

Rhizoplaca peltata

AGTTTCCGAGTGACCTGCGAAAGATCATATGGAGAGGTCTTCGGACCTCGGGCTCGGCCCCCAGTTCTCACCC

CCTGTTCACGTATCATGTTGCTTGACGGGCCCCGGAGGCTGCCCCCTCGATGGCTGGCGACTTCGCGTCCCGG

CCTGTTCGCGCCGTCCGAGGCCTATCAACCCGACTTACCGTGCCGTCCGAGTACAACCATAATAGTATAAAAC

TTTCAACAACGGATCTCTGGGTCTGGGCATCGATGAAGAACGCAGCGAATGCGATAAGTAATGTGAATTGCAG

AATTCAGTGAGTCATCGAATCTTGAACGCACATTGCGCCCCTCGGTATTCCGGGGGGCATACCTGTTCGAGCG

TCATTACACCCCTCAAGCTCGGCTTGGTACTGGGTCTTCGCCCCGGGGGCGGGCCCGAAAGTCAGTGGTGGTT

CGGCGCGACTTCGAGCGTAGTAAAACTTCTCCCGCTCTGGAAGTCGCCGCGTGACCGGCCAGACGCCCAAATC

TTGGAATGATGAGCTCGATCAGGTACGCCA Fig. 1. The sequence information of the three different species from Turkey. The sequences comprise the 3Ј end of small subunit gene, ITS1, the 5.8 S gene, ITS2, and the 5Ј terminus of the large subunit gene.

325 variable, 164 parsimony informative sites were The target sequences generated by PCR amplifi- detected. All data were analyzed by MEGA 3 cation were sequenced in both directions. The se- (Kumar et al., 2004) and a bootstraped dendogram quencing reactions for species of Rhizoplaca was generated. yielded alignable sequences of 727 nucleotides long. Within these sites 298 were conservative and 325 were variable whereas 164 sites were parsi- Results mony informative. ITS sequences for eight other DNA was extracted with a new, improved different samples from the Rhizoplaca genus and CTAB based protocol for this study. The sequen- for six different species of Leconora, available cing reactions were conducted with this DNA of from GenBank (Table I) were included into multi- enough purity, suitable for PCR amplification. ple alignments. D. Cansaran et al. · Phylogenetic Relations in Rhizoplaca 409

Table I. The aligned Species or specimen GenBank accession No. Origin species, localities and their GenBank acces- Lecanora achariana A. L. Sm. AF070019 Sweden sion numbers. Lecanora garovaglii (Kölber) AF189718 Austria Zahlbr. Lecanora novomexicana H. AF159945 Arizona Magn., U 363 Lecanora pruinosa Chaub. AF070018 Italy Lecanora reuteri Schaer AF070026 Australia Lecanora swartzii subsp. AY541273 Australia Nylanderi Protoparmalia badia (Hoffm.) AF070023 Australia Hafellner Protoparmaliopsis muralis AF159922 Australia (Schreb.) Rabenh. Rhizoplaca chrysoleuca (Sm.) DQ321748 Turkey Zopf Rhizoplaca chrysoleuca (Sm.) AF159924 Arizona Zopf, U192 Rhizoplaca chrysoleuca (Sm.) AF159940 Kazakhstan Zopf, U302 Rhizoplaca chrysoleuca (Sm.) AY530884 China Zopf, GenBank Rhizoplaca melanopthalma DQ321747 Turkey (DC.) Leuck. & Poelt Rhizoplaca melanopthalma AF159929 Arizona (DC.) Leuck. & Poelt, U219 Rhizoplaca melanopthalma AF159934 Arizona (DC.) Leuck. & Poelt, U278 Rhizoplaca melanopthalma AF159935 Austria (DC.) Leuck. & Poelt, U281 Rhizoplaca peltata (Ram.) DQ321749 Turkey Leuck. & Poelt, MGH Rhizoplaca peltata (Ram.) AF159925 Arizona Leuck. & Poelt, U198 Rhizoplaca peltata (Ram.) AF159936 British Columbia Leuck. & Poelt, U282 Rhizoplaca peltata (Ram.) AY509803 China Leuck. & Poelt, GB

Nine most parsimonous trees were generated by branch to this assamblage which is supported by a using farinacea as outgroup. One of 100% bootstrap value. these trees generated by maximum parsimony is Analysis with neigbour joining (Fig. 3) and mini- shown in Fig. 2. The trees yield similar topology mum evolution (Fig. 4) displayed trees with similar indicating only slight rearrangements within the topology with slight differences among groups groups (aÐe). Multiple alignments of samples of (aÐe) and within groups. Rhizoplaca species with different Lecanora species (obtained from GenBank) yielded the information in the dendogram that Rhizoplaca species do not Discussion constitute a monophyletic branch. R. melano- pthalma species group with L. novomexicana DNA sequence analysis of lichens has yielded which is supported by a 84% bootstrap value. The valuable information for more than a decade. The other umblicate species from Anatolia, R. chryso- ITS region is particularly suitable for this purpose leuca and R. peltata, appeared elsewhere in the as it comprises two non-coding spacers, ITS1 and tree (Fig. 2; groups e and b, respectively), apart ITS2, which are less constrained than the coding from the assamblage of R. melanopthalma and Le- regions of rDNA and therefore more likely to ac- canora species. R. peltata remained as a sister cumulate mutations (White et al., 1990). 410 D. Cansaran et al. · Phylogenetic Relations in Rhizoplaca

Fig. 2. Phylogenetic relations of three Rhizoplaca species from Anatolia and other Rhizoplaca and Leconora species. One of the nine equally parsimonious trees by maximum parsimony analysis is shown. Numbers at the nodes are bootstrap frequencies above 50%. Group placement is indicated as aÐe.

Using the thallus morphology in lichen system- lichen materials collected from the volcanic rocks atics to distinguish taxa at different levels works of Mount Erciyes located in the middle Anatolian well for many species. For example, separation of province of Kayseri revealed similar sequence the crustose Buelliaceeae from the foliose to fruti- data information. The three species which have cose Physciaceae almost completely depends on umblicate thalli are the only species found in Ana- thallus morphology. Such examples are also found tolia. When the phylogenetic tree was examined, within the large family in which the R. melanopthalma from Turkey, two different genus Rhizoplaca is classified. In this family, two samples of R. melanopthalma from Arizona lobate genera Arctopeltis Poelt (Poelt, 1983) and (AF159934, AF159929) and a sample from Austria Rhizoplaca (Leuckert et al., 1977) have been sepa- (AF159935) appeared on the same branch. The lo- rated from the genus Lecanora mainly by their bate species L. novomexicana (AF159945) appears umbilicate thalli with well developed upper and in the same branch with R. melanopthalma as it lower cortices (Arup and Grube, 2000). was pointed earlier by Arup and Grube (2000) as Does the higher organization with an umblicate an interesting outcome. Also R. peltata and R. to foliose thallus represent a natural group? Arup chrysoleuca from Anatolia are located in two and Grube (2000) tested repetitive taxa of Rhizo- other branches of the tree that forms sister groups placa that vary in thallus morphology, chemistry, with the samples of the same species from differ- disc color and ecology and concluded that the gen- ent countries. On the other hand although R. pel- erally accepted concept of Rhizoplaca as a genus tata remained on the same branch with other R. separate from Lecanora can be rejected. The data peltata samples from other countries it took place obtained from this study support this idea as the in a different branch within the group and longer D. Cansaran et al. · Phylogenetic Relations in Rhizoplaca 411

Fig. 3. Neighbor joining analysis inferred from ITS region sequences. branch length might point the importance of geo- pruinose in R. melanopthalma. While the medulla graphical distribution or some other characteristics gives P+ orange reaction (pannarin) in R. peltata, unique to that ecology. the medulla gives P+ bright yellow or less fre- When the three species from Anatolia were quently PÐ (with or without psoromic acid) in R. considered alone, it is noticed that Rhizoplaca me- melanopthalma. lanopthalma and Rhizoplaca peltata are phyloge- netically closer to each other than Rhizoplaca Acknowledgements chrysoleuca; the morphological characteristics also This study was supported by Ankara University, support this result. From the examined species, Management of Scientific Research Projects Rhizoplaca chrysoleuca differs from Rhizoplaca (no. 2004-07-05-093) and also by Ankara Univer- peltata and Rhizoplaca melanopthalma by having sity, Biotechnology Institute (project no. 171 and pruinose, orange apothecial disks. The apothecial partially projects nos. 61 and 8). Finally authors disks are yellowish brown and not pruinose in R. the are thankful to Prof. Dr. Ender Yurdakulol for peltata, but yellow-brown to greenish or black and his support in every aspect of the study. 412 D. Cansaran et al. · Phylogenetic Relations in Rhizoplaca

64 Rhizoplaca melanopthalma Turkey

78 Rhizoplaca melanopthalma Arizona Rhizoplaca melanopthalma Austria 96 93 Rhizoplaca melanopthalma Arizona AF159929

Lecanora novomexicana Arizona 32

81 Rhizoplaca chrysoleuca China Rhizoplaca chrysoleuca Kazakhstan

100 Rhizoplaca chrysoleuca Arizona 99 80 Rhizoplaca chrysoleuca Turkey

98 Lecanora pruinosa Italy Lecanora reuteri Australia

99 Lecanora achariana Sweden 3742 Protoparmaliopsis muralis Australia

Lecanora garovaglii Austria

46 Rhizoplaca peltata Turkey

Rhizoplaca peltata China 100

82 Rhizoplaca peltata Arizona 90 Rhizoplaca peltata British Columbia

Lecanora swartzii Australia

Ramalina farinacea

0.02 Fig. 4. Analysis by minimum evolution according to the data obtained by ITS region sequences from Anatolia and by the data obtained from GenBank.

Arup U. and Grube M. (2000), Is Rhizoplaca (Lecano- Leuckert C., Poelt J., and Hahnel G. (1977), Zur Chemo- rales, lichenized ) a monophyletic genus? taxonomie der eurasischen Arten der Flechtengattung Can. J. Bot. 78, 318Ð327. Rhizoplaca. Nova Hedwigia 28,71Ð129. Brodo I. M., Sharnoff S. D., and Sharnoff S. (2001), Li- Lutzoni F. and Vilgalys R. (1995), Integration of mor- chens of North America. Yale University Press, Lon- phological and molecular data sets in estimating fun- don, pp. 1Ð795. gal phylogenies. Can. J. Bot. 73, S649ÐS659. DePriest P. T. (1993), Small subunit rDNA variation in Poelt J. (1958), Die lobaten Arten der Flechtengattung a population of lichen fungi due to optional group I Lecanora Ach. in der Holarktis. Mitt. Bot. Staats- introns. Gene 134,67Ð74. samml. München, 411Ð573. Gardes M. and Bruns T. D. (1993), ITS primers with en- Poelt J. (1983), Über den Formenkreis der Flechte Le- hanced specifity for basidiomycetes-application to the canora contractual. Int. J. Mycol. Lichenol. 1, 143Ð identification of mycorrhizae and rusts. Mol. Ecol. 2, 160. 113Ð118. Purvis W. O., Coppins B. J., Hawksworth D. L., James Gargas A., DePriest P. T., Grube M., and Tehler A. P. W., and Moore D. M. (1992), The Lichen Flora of (1995), Multiple origins of lichen symbioses in fungi Great Britain and Ireland. Natural History Museum suggested by SSU rDNA phylogeny. Science 268, Publications, London, England. 1492Ð1495. Ryan B. D. and Nash T. H. III. (1997), Placodioid taxa Hafellner J. (1984), Studien in Richtung einer natürli- of Lecanoraceae sensu Zahlbr. (lichenized Ascomy- cheren Gliederung der Sammelfamilien Lecanoraceae cotina) in North America: taxa excluded from Le- and Lecideaceae. Nova Hedwigia 79, 241Ð371. canora subgen. Placodium. Nova Hedwigia 64, 393Ð Hawksworth D. L. (1988), The variety of the fungal-algal 420. symbiosis, their evolutionary significance, and the na- White T. J., Bruns T. D., Lee S., and Taylor J. (1990), ture of lichens. Bot. J. Linn. Soc. 96,3Ð20. Amplification and direct sequencing of fungal ribo- Kumar S., Tamura K., and Nei M. (2004), MEGA 3. In- somal RNA genes for phylogenetics. In: PCR Proto- tegrated software for molecular evolutionary genetics cols (Innis M. A., Gelfand D. H., Sninsky J. J., and analysis and sequence alignment. Briefings in Bioin- White T. J., eds.). Academic Press, San Diego, pp. 315Ð formatics 5, 150Ð163. 322.