PHYLOGENETIC RELATIONSHIPS AMONG THE TAXA OF THE GENUS JOHRENIA DC . ( APIACEAE ) FROM TURKEY BASED ON MOLECULAR METHOD

In the present study, ISSR markers were employed to determine the phylogenetic relationships among the taxa of Johrenia. The genera Angelica and Xanthogalum were selected as outgroups. Unweighted pair group method with arithmetic mean (UPGMA) and Principal Coordinate Analyses were conducted to view the molecular relationships. Johrenia alpina, J. depauperatum and J. aurea are transferred to the genus Dichoropetalum. The infrageneric and intergeneric phylogenetic relationship among the Johrenia and Dichoropetalum genera are determined.


Introduction
The Apiaceae family is represented by approximately 400 genera and 3500 species worldwide (Constance, 1971;Pimenov and Leonov, 1993).The family consists of 102 genera and 434 species in Turkey (Erik and Tarıkahya, 2004).The Apiaceae includes many commonly grown vegetables (carrot, parsnip) and condiments (chervil, cumin, parsley, dill).They owe their distinctive flavour largely to diverse essential oil compounds in the fruits, seeds and leaves.The family also encompasses widespread weeds and toxic plants (Downie et al., 2000).
A molecular approach has contributed much to understanding the evolutionary relationships of Apiaceae.Phylogenetic analyses of the family using chloroplast DNA (cpDNA) sequences (Downie et al., 1996), cpDNA restriction sites (Plunkett and Downie, 1999), and nuclear ribosomal DNA internal transcribed spacer (ITS) sequences (Downie and Katz-Downie, 1996) provided an alternative classification of Drude (1898).Revision based on molecular data provide better resolution of the systematic positions.The taxonomic problems at the species level have begun started to be solved with DNAbased molecular analyses which are not affected by environmental conditions, in contrast to the phenotypical analyses.
The aim of our study was to determine the infrageneric and intergeneric phylogenetic relationship among the Johrenia and Dichoropetalarum genera emplyomg ISSR method.Also, we selected Angelica sylvestris (M.Bieb.)Sprengel and Xanthogalum purpurascens Lallem.to resolve their controversial status by using a DNA based molecular marker system.

Materials and Methods
Plant materials: Johrenia and Dichoropetalum specimens were collected by the authors from Amasya, Mersin, Adana, Niğde, Kahramanmaraş, Bursa, Konya, Osmaniye and Kayseri provinces between of 2003-2008 (Fig. 1).The Flora of Turkey (Chamberlian, 1972), Flora Iranica (Rechinger, 1987), and Flora Europaea (Tutin, 1968), were used to identify the collected plant samples.Specimens are kept in Selçuk University Education Faculty Herbarium.The specimens' localities (Fig. 1) and examined representative specimens are in the appendix.The genera Angelica and Xanthogalum were selected as outgroups.These genera are closest to Johrenia in respect to phylogeny in Turkish flora.
DNA isolation: Nuclear DNA was isolated from leaves both from herbarium and fresh materials using CTAB method (Sambrok et al., 1989).Total DNA was obtained from 50-75 mg dried leaf tissue from 10 different individuals.DNAs were isolated with the Easy Nucleic Acid Isolation Kit (OMEGA) and concentrations were determined by Nanodrop.Sample DNAs were diluted to 25 ng/µl.Stock DNAs were kept at -86ºC.
ISSR amplifications: ISSR primers (Galvan et al., 2003) were amplified in a PCR thermal cycler.The characteristics of the primers used are given in Table 1.Each PCR reaction contained 25 µl containing 2.5 µl PCR buffer (10 mM TRIS/50 mM KCl buffer, pH 8.0), 3 µl 25 mM MgCl, 0.5 µl of each primer, 0.5 µl of dNTP mix, 0.4 µl Taq DNA polymerase, 4 µl of each DNA and 14.1 µl distilled water.After a pre-denaturation step of 3 min at 94 0 C, amplification reactions were cycled 40 times at 94ºC for 1 min, at annealing temperature (Table 1) for 1 min and 72ºC for 1 min and a final extension was allowed for 10 min at 72ºC in an Eppendorf Mastercycler Gradient Thermocycler.Upon completion of the reaction, 15 µl aliquots of the PCR products were mixed with 3 µl of loading dye (50% glycerol, 0.25% bromophenol blue and 0.15% xylene cyanol) and loaded onto a 2% agarose, 1X TRIS-Borate-EDTA gel and electrophoriesed at 4V cm -1 .Amplified fragments were visualized under a UV transiluminator and photographed using a gel documentation system (Vilbert Lourmat, Infinity model).
Data analysis: All the fragments amplified were treated as dominant genetic markers.Each DNA band generated was visually scored as an independent character or locus ('1' for presence and '0' for absence).Qualitative differences in band intensities were not considered.Every gel was scored in triplicate (independent scorings) and only the fragments consistently scored were considered for analysis.A rectangular binary data matrix was prepared and all the data analysis was performed using the Numerical Taxonomy System, NTSYS-pc version 2.02 (Applied Biostatistic, Exeter Software, Setauket, New York, USA).Similarity coefficient method was used.In cluster analysis of the samples the unweighted pair-group method with arithmetic mean (UPGMA) procedure was followed (Rohlf, 1992).Genetic distances calculated with the Simple Mathing coefficient.In order to determine the ability of ISSR data to display the interrelationships among the samples analysis was conducted using NTSYS-pc package.

Results and Discussion
From an initial screening of 25 ISSR primers, nine primers revealed high levels of polymorphisms.These primers generated 90 highly polymorphic fragments that were consistently amplified in repeated experiments.The GC percentages of the selected primers were within the range of 38.9-66.7%(five of them being 52.6%).In total, the average number of polymorphic fragments per primer used was roughly 11.Genetic distances calculated with the SM coefficient ranged from 0.46 to 0.99.
As a result of the evaluation of the ISSR data, Dichoropetalum depauperatum showed 95% similarity with and D. aytachii.
The clade composed of J. dichotoma and J. porteri are very similar according to the morphological characters.These similarities are supported with the ISSR data and these species show much more similarity in the dendogram than any other species (Fig. 2).Pimenov et al. (2007) selected 33 morphological characters for phenetic analyses of Johrenia, Dichoropetalum, Zeravschania and related genera.Selected characters were used to perform UPGMA analysis, and the resulting phenogram showed the relationships among Johrenia, Dichoropetalum, Zeravschania, and related genera.In the phenogram, three major groups matched with one of the three genera.The molecular dendogram of the genera Dichoropetalum and Johrenia showed parallelism among the major groups like the phenogram of Johrenia, Dichoropetalum, Zeravschania genera of Pimenov et al. (2007).The present study based on ISSR data revealed four clades, each clade matching with one of the four genera, viz.Johrenia, Dichoropetalum, Angelica and Xanthogalum.The genus Angelica with Xanthogalum, and Dichoropetalum with Johrenia shows much more molecular phylogenetic similarity.There is a correlation between the morphologic diagnostic characters and molecular taxonomic classification.
The concept of the "Diagonal" was first proposed by Davis (1971), who defined it as an oblique belt running from the north east, south to the Anti-Taurus; it then divides into two, with one branch to the Amanus (Amanos Mountains), and the other to the Cilician Taurus (Fig. 1).Thirty three percent of the total species growing in Turkey are found along the diagonal, while 5% are more or less restricted to it (Ekim and Güner, 1986).
The spread of genus Dichoropetalum is on the Anatolian diagonal (Fig. 1).The genus occurs in the Anti-Taurus region and branch (Aladağ, Amanos and Bolkar mountains) of the Anatolian diagonal.In Anatolia, almost all mountain peaks so far examined abound in endemics (Zohary, 1973).All species of Dichoropetalum are grown on mountain peaks and endemics in Anatolia.The Anatolian Diagonal and its adjacent areas are one of the most important centers of genetic diversity in Turkey.The Amanos mountain range is an interesting area, occupying an intersection of the Mediterranean phytogeographical region and the Anatolian Diagonal, with many Euro-Siberian phytogeographical region enclaves (Ekim and Güner, 1986).The area is very rich in paleo and neo endemic plants.2).The related species were clearly separated by the principal coordinate analysis (Fig. 3).
Although the genus Johrenia is distributed primarily in the East Mediterranean region, some species maintain an interrupted spread in the ecotone zone in Central Anatolia.They grow on the Anatolian diagonal or western side of the diagonal except for J. dichotoma ssp.sintenisii.This taxon is only known from the type locality and is a very local endemic.This genus represents six species in the world and five species in Turkey (J.distans spread in Greece and F.Y.R. Macedonia).Four species of Johrenia endemic to Turkey are found on the Anatolian Diagonal.Taxa of Johrenia are poorly occurring and have local population in Anatolia.Opposite to the genus Dichoropetalum, the genus Johrenia is distributed in the lowest altitude of the mountains in Turkey.As a result the Johrenia and Dichoropetalum taxa in Turkey are classified according to the molecular data.J. alpina, J. depauperatum and J. aurea species have been transferred to the genus Dichoropetalum.Therefore, Johrenia and Dichoropetalum contain five and three species, respectively as revealed from molecular phylogenetic studies.