The phylogenetic relationships of the Abies species in European and Turkish regions have frequently been an object of controversial conclusions. Therefore, we compared the genetic structures of 21 populations belonging to six Abies species which are native to Ã‚Â entral, eastern and south-eastern European regions and Ã‚Â different areas in the Turkey (Asia minor). We used the allele frequency distribution of five isozyme gene locialready showing a high discriminatory power to assess the genetic differentiation among the Abies populations. The UPGMA-dendrogram based on genetic distances showed a clear discrimination between the Turkish Abies species and A. alba from Europe. The particular allele frequency distributions at the isozyme loci PGI-A and 6PGDH-Aconfirmed a great genetic dissimilarity between A. alba and the Turkish Abies species.These results contradict the phylogenetic relationships of the Abies species postulated inother studies

Bergmann, F.

Hosius, B.

Leinemann, L.

English

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 Research in Plant Biology, 3(4): 27-32, 2013                                                            ISSN : 2231-5101  www.resplantbiol.com  Received: 10.4.2013; Revised: 17.7.2013; Accepted: 22.7.2013  Short Communication Genetic differentiation and phylogenetic relationships among six Abies species from European and Turkish areas  F. Bergmann1*, B. Hosius2 and L. Leinemann1  1Department of Forest Genetics and Tree Breeding of University of Göttingen, Germany and 2ISOGEN, Göttingen  The phylogenetic relationships of the Abies species in European and Turkish regions have frequently been an object of controversial conclusions. Therefore, we compared the genetic structures of 21 populations belonging to six Abies species which are native to central, eastern and south-eastern European regions and different areas in the Turkey (Asia minor). We used the allele frequency distribution of five isozyme gene loci already showing a high discriminatory power to assess the genetic differentiation among the Abies populations. The UPGMA-dendrogram based on genetic distances showed a clear discrimination between the Turkish Abies species and A.alba from Europe. The particular allele frequency distributions at the isozyme loci PGI-A and 6PGDH-A confirmed a great genetic dissimilarity between A. alba and the Turkish Abies species. These results contradict the phylogenetic relationships of the Abies species postulated in other studies.  Key Words: Genetic divergence, isozyme gene markers, biogeography, Abiesspecies  In contrast to other forest tree species (e.g. Norway spruce), Abies alba has not been artificially regenerated in most cases, thus preserving a great deal of their native genetic structures. As a consequence, the genetic composition and diversity of most present-day fir populations are the result of their distribution during the last glaciation and their postglacial expansion to the present natural areas (Bergmann 1991). Hence, comparisons between the genetic structure of Abies populations within or between different species may uncover reliable data on genetic similarities, differentiation and probably phylogenetic relationships within the genus Abies occurring in Europe and Asia Minor. Since the phylogenetic relationships of the Abies species in these regions have frequently been an object of controversial conclusions (Fady and Conkle 1993, Scaltsoyiannes et al. 1999, Ziegenhagen et al. 2005, Liepelt et al. 2012), it appears worthwhile to compare the genetic structures of Abies populations belonging to six species which are native to central, eastern and south-eastern European mountains and different mountainous regions in the Turkey. In contrast to other studies, we used specific isozyme gene markers of such enzyme systems, which have already indicated a high discriminating power among populations of silver fir (Konnert and Bergmann 1995). The objectives of the present paper are to show the genetic similarities among populations of the same Abies species and the genetic differentiation among populations of different Abies species. The latter data were used to evaluate some phylogenetic relationships, especially Bergmann et al. / Research in Plant Biology, 3(4): 27-32, 2013 28  between European and Turkish Abies species.  Materials and Methods The plant material used in this study consisted of 10 Abies alba populations from central, eastern and south-eastern Europe, each of two populations of Abies bornmülleriana, Abiesequi-trojani and Abies cilicica from different regions of the Turkey. Four populations of Abies nordmanniana are located in Russia (1), Georgia (2) and Turkey (1). Furthermore, one population of the so-called interspecific hybrid Abies borisii-regis belongs to the Slovjanska mountains in Bulgaria. The origin of the tree samples and the type and sample size of the test material are listed in Table 1. Extracts from single seed endosperms (=macrogametophytes) or dormant buds were subjected to horizontal starch-gel electrophoresis. After electro-phoretic separation the gel slabs were stained for the enzyme systems (gene loci in brackets) phosphoglucose isomerase(PGI-A, PGI-B), menandione reductase (MNR-A) and 6phosphogluconate dehydrogenase (6PGDH-A, 6PGDH-B). The experimental methods and the genetic analyses were already published in an earlier study on Abies alba (Hussendörfer et al. 1995).  Statistical methods Data analysis was carried out using the program GSED Version 3.0 beta, (Gillet 1994). The dendrogram, illustrating the genetic distances between populations is based on the software NTSYS, Version 2.01, Applied Biostatistics. The genetic distance between all pairs of populations was based on the allele frequencies of the five isozyme gene loci. We used the genetic distance d0 (Gregorius, 1974) which is based on a simple and better interpretableformula:  0121d p pP P k kkn( , ' ) '= −=∑   The arithmetic mean of d0-values over single loci was calculated to get the gene pool distance which was used for the calculation of the UPGMA-dendrogram (Sneath and Sokal, 1973). For simplification of the figures the genetic data of the four populations of Abies nordmanniana and each of two populations of A. bornmülleriana and A. equi-trojani have been pooled. Similarly the three populations of A. alba from Bulgaria were also combined since the provenance localities possess similar environmental conditions and a short geographical distance.  Results and Discussion A reliable assessment of the genetic differentiation of a species, genus or family requires the utilization of those gene markers which exhibit geographically variable allele frequencies and/or possess area-specific allelic variants. Both properties can be attributed to the five isozyme loci examined in 21populations belonging to six Abies species (see Table 1). In this study, two data sets based on allele frequencies were used to arrive at the above given objections. One was the measure of genetic distance between all population pairs and the construction of a UPGMA-dendrogram based on gene pool distance values. The other refers to the distribution of particular area-specific allelic variants at two isozyme loci which were traced across the European and Turkish range of the Abies genus.  The clustering analysis (depicted as dendrogram in Fig. 1) revealed patterns which were not in agreement with results in earlier studies (Fady and Conkle 1993, Scaltsoyiannes et al. 1999) of this genus, which will be explained later. With the exception of the species A. cilicica all other populations clustered into two major groups, of which one includes only populations of A. alba, whereas the second major group further divided into two subgroups. One of these subgroups Bergmann et al. / Research in Plant Biology, 3(4): 27-32, 2013 29  comprises all other Turkish Abies species, whereas the other subgroup contains A. borisii-regis and A. alba from Bulgaria. This subgrouping corresponds exactly to a transition zone (Bergmann and Gagov, 2001) between European and Turkish Abies species where area-specific alleles still occur in low frequencies. The pooled populations of A. cilicica clustered greatly from the two major groups indicating a nearly complete genetic differentiation from the other Turkish Abies species. For instance, the A. cilicica populations possess the allele PGI-B1 with frequencies of 80% or 90%, whereas in all other fir species from Turkey PGI-B1 reached only 8-10% (data not shown). By far more genetic difference occurs at the isozyme locus MNR-A, since only A. cilicica possesses the allelic variant A4 in moderate frequencies (20 – 60%), which indicated a total isolation of this fir species (see also Scaltsoyiannes et al. 1999).  Table 1: Origin, test material and sample size of the analyzed populations Location Origin Test Material Size of Sample A. nordmanniana Apcheronsk Russia bulk seed lot 100 seeds Ambrolauri Georgia bulk seed lot 100 seeds Bordjomi Georgia bulk seed lot 100 seeds Savsat Turkey bulk seed lot 100 seeds  A. bornmülleriana Ulu-dag Turkey dormant buds  42 trees Bolukökez Turkey bulk seed lot 100 seeds  A. equi-trojani Kazdagi Turkey dormant buds  69 trees Endremit Turkey bulk seed lot 100 seeds  A. cilicica Antalya Turkey bulk seed lot 20 trees Maras Turkey bulk seed lot 20 trees  A. borisii-regis Slovjanka Mts. Bulgaria single tree seed lots 52 trees  A. alba Ribarica Bulgaria single tree seed lots 55 trees Jundula Bulgaria single tree seed lots 29 trees Jenda Bulgaria single tree seed lots 79 trees Toplita Romania bulk seed lot 100 seeds Sinai Romania bulk seed lot 100 seeds Postojna Slovenia dormant buds 86 trees Siegsdorf Germany single tree seed lots 30 trees Lichte Germany  dormant buds 50 saplings Nagold Germany  single tree seed lots 20 trees Ottenhöfen Germany  single tree seed lots 30 trees  Supporting evidence for the genetic dissimilarity between European populations of A. alba and Turkish populations of A. nordmanniana, A. bornmülleriana and A. equi-trojani resulted from the distribution of area-specific allelic variants. For instance, the allele A1 of the locus PGI-A occurs in all populations of the Turkish fir species with considerable frequencies (60 – 90%), but could not be Bergmann et al. / Research in Plant Biology, 3(4): 27-32, 2013 30  detected in nearly all A. alba populations (Fig. 2). Only the Bulgarian populations of A. borisii-regis and A. alba contain this allele in lower frequencies (15 – 25 %), which indicated some gene flow between Abies populations of Asia minor and Europe and a relatively narrow transition zone which is represented by the Bulgarian populations (Fig. 2). A similar genetic differentiation has been observed at this isozyme locus by Fady and Conkle (1993), even though the allele numbering was different.    Figure 1: UPGMA-dendrogram of d0 genetic distances between 11 populations of 6 Abies species (A. nordmanniana= NOR, A. bornmülleriana = BOM, A. equitrojani = EQI, A. cilicica = CIL, A.borisiregis = BOR, A. alba = AL-**)     Figure 2: Distribution of the allele frequencies at PGI-A across European and Turkish areas. The black region of the pie diagrams represents the proportion of allele A1, the white region represents the allele A2 Bergmann et al. / Research in Plant Biology, 3(4): 27-32, 2013 31   On the other hand, the area-specific allelic variant A2 of the locus 6PGDH-A occurs in all populations of A. alba with intermediate frequencies (20 – 50%), but could not be observed in the populations of Turkish Abies species (based on the size of the tree samples, frequencies lower than 1% were not detectable) (Fig. 3). Again, the Bulgarian populations of A. alba and A. borisii-regis were found to exhibit the lowest frequencies of this allele A2, which is typical for a so-called transition zone. Although we presented the genetic data for only 10 European Abies alba populations in this study, the allozyme (allelic isozyme) patterns at PGI-A and 6PGDH-A conform closely to patterns in much more populations of this Abies species from central, eastern and southeastern European regions (Konnert and Bergmann 1995, Longauer et al. 2003).   Figure 3: Distribution of the allele frequencies at 6PGDH-A across European and Turkish areas. The black region of the pie diagrams represents the proportion of allele A2, the white region represents allele A3  With respect to the frequency differences of PGI-A1 and 6PGDH-A2 between Abies alba in Europe and the four Abies species in the Turkey, it is unlikely that either stochastic events or natural selection could result in such large genetic differentiation, if all Abies species originated from one ancient mega population (Fady et al. 1992). Rather, this allozyme differentiation may have occurred during and after the last glaciation indicating a complete separation of these two species groups in glacial refugia and postglacial migration routes. This conclusion is not in agreement with the phylogenetic hypothesis of Scaltsoyiannes et al. (1999), who postulated a close relationship between A. alba, A. bornmülleriana and A. nordmanniana. Interestingly, the results of Ziegenhagen et al. (2005) using a mitochondrial DNA marker are more concordant with our data, since they found two haplotypes which occurred only in Abies alba and one other haplotype which shared the three Abies species from northern Turkey and A. cephalonica. The most recent study of Liepelt et al. (2012) employed cpDNA markers with high and low mutation rates in order to distinguish between interspecific genetic contact before speciation and after Bergmann et al. / Research in Plant Biology, 3(4): 27-32, 2013 32  speciation. Applying several statistical methods they concluded that in most cases no or only minimal genetic contact had occurred between the different Mediterranean Abies species after speciation of this genus, which supports the phylogenetic interpretation of our allozyme patterns based on specific isozyme loci.  References Bergmann, F. 1991. Causes and consequences of species-specific genetic variation patterns in European forest tree species: Examples with Norway spruce and silver fir. In: Müller-Stark, G. and M. Ziehe (eds.) Genetic Variation in European Populations of Forest Trees. J.D. Sauerländer’s Verlag, Frankfurt/M, pp.192-205. Bergmann, F., Gagov V. 2001. Genetische Diversität und Differenzierung von Tannenpopulationen der Balkanh-albinsel. In: IUFRO-Proceedings of the 9th International European Silver Fir Symposium, Skopje, Macedonia, 1-4. Fady, B., Conkle, M. T. 1993.Allozyme variation and possible phylogenetic implications in Abies cephalonica Loudon and some related eastern Mediterranean firs. SilvaeGenetica42, 351-359. Fady, B., Arbetz, M. and Marpeau, A. 1992. Geographic variability of terpene composition in Abies cephalonica Loudon and Abies species around the Aegean: hypotheses for their possible phylogeny from the Miocene. Trees 6, 162 - 171. Gillet, E. 1994. GSED-Genetic Structures from Electrophoresis Data.Version 1.1a, 1994. Abt. für Forstgenetik und Forstpflanzenzüchtung, Universität Göttingen, Germany. Gregorius, H.-R. 1974. Genetischer Abstand zwischen Populationen. Zur Konzeption der genetischen Abstandsmessung. SilvaeGenetica 23: 22-27.     Hussendörfer, E., Konnert, M. and Bergmann, F. 1995. Inheritance and linkage of isozyme variants of silver fir (Abies alba Mill.). Forest Genetics 2, 29-40.  Konnert, M. and F. Bergmann. 1995. The geographical distribution of genetic variation of silver fir (Abies alba, Pinaceae) in relation to its migration history. Plant Syst. & Evol 196, 19-30.     Liepelt, S., Mayland-Quellhorst, E., Lahme, M. and Ziegenhagen, B. 2012. Contrasting geographical patterns of ancient and modern genetic lineages in Mediterranean Abies species. Plant Syst. & Evol. 04 141-151. Longauer, R., Paule, L. and Andonoski, A. 2003. Genetic diversity of southern populations of Abies alba (Mill). Forest Genetics 10, 1 - 9 Scaltsoyiannes, A. Tsaktsira, M. and Drouzas, A. D. 1999. Allozyme differentiation in the Mediterranean firs (Abies, Pinaceae). A first comparative study with phylogenetic implications. Plant Syst. & Evol. 216, 289-307. Sneath, P.H.A. and Sokal, R.R., 1973. Numerical Taxonomy. H.W. Freeman, San Francisko, 573 pp. Ziegenhagen, B. Fady, B., Kuhlenkamp, V. and Liepelt, S. 2005.Differentiating Groups of Abies Species with a Simple Molecular Marker. Silvae Genetica 54, 123 - 126.  

Genetic differentiation and phylogenetic relationships among six Abies species from European and Turkish areas

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Genetic differentiation and phylogenetic relationships among six Abies species from European and Turkish areas

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