GENETIC DIVERSITY AND DIFFERENTIATION OF PINUS SYLVESTRIS L . FROM THE IUFRO 1982 PROVENANCE TRIAL REVEALED BY AFLP ANALYSIS

DNA markers have become effective tools in genetic diversity studies of forest trees. However, molecular marker analyses are associated with laborious and costly effort. One of the possibilities to overcome these constraints is to analyze bulked samples per population, rather than individual plants. We have used bulked DNA-based AFLP analysis to investigate genetic variation in Pinus sylvestris L. (Scots pine) from the IUFRO 1982 provenance trial in Kórnik (western Poland). Four AFLP primer combinations yielded a total of 309 bands, of which 208 (67.31%) were polymorphic. Thirty-six (11.65%) unique alleles were deployed randomly among the populations. Estimated genetic diversity and differentiation was high, as expressed by He = 0.238 and I = 0.356, and by genetic distance values which ranged from 0.154 to 0.363. A geographic pattern of interpopulation differentiation was observed, pointing at the individual character of populations from northeastern Europe. In the light of available data, we discuss the influence of historical migration routes, gene flow and human activity on observed genetic diversity and differentiation of Scots pine in Europe. Our results indicate that the AFLP method applied to DNA templates extracted from bulked leaf samples provides an efficient approach to elucidate genetic diversity and relationships among Scots pine populations.


INTRODUCTION
Scots pine (Pinus sylvestris L.) is one of the most important components of the boreal forests across Europe and Asia.The species is characterized by the most extensive continuous range in the Pinaceae family and spreads over a distance of 14000 km, from beyond the Arctic Circle in Scandinavia to southern Spain, and from western Scotland to the Okhotsk Sea in eastern Siberia (Boratyński, 1991).Scots pine populations are found at elevations from sea level up to 2500 m, with the elevation generally increasing from north to south (Mirov, 1967), but also in areas characterized by high levels of groundwater, like peat bogs or fresh water swamp forests, as well as in dry, lichen Scots pine forests (Cladonio-Pinetum).It is tolerant to poor soils and extreme temperatures such as the hot summers in Spain and freezing winters in Siberia (Pravdin, 1964).Therefore, the Scots pine is characterized by a high level of phenotypic plasticity and genetic variation, which together with wide ecological tolerance have contributed to the success of the species.
Present genetic structure of Scots pine is influenced by an ancient origin and immigration history of certain populations, natural selection, an extensive gene flow caused by pollen dispersal, genetic drift due to small population size and human activities such as deforestation or industrial pollution (Oleksyn et al., 1994).In particular, the Holocene postglacial history of Pinus sylvestris appears to be very interesting, with putative refugial areas located in the Balkans, the Alps and the Iberian Peninsula and on the Russian Plains (Pravdin, 1964;Bennett et al., 1991;Naydenov et al., 2007).Postglacial expansion of Scots pine is likely to have taken place both from the remaining local populations as well as from the northward expansion of southern refugial populations following the retreat of the ice sheets.
The large natural variation that exists within the extensive range of the natural distribution of Scots pine has been the subject to many studies based on variation of morphological traits and later also on analyses conducted with the use of isoenzymatic and molecular markers.The use of DNA technology appeared to be effective in the characterization of forest trees, including Scots pine, based on genetic information contained in nuclear, chloroplast and mitochondrial DNA (e.g.Szmidt et al., 1996;Sinclar et al., 1999;Pyhäjärvi et al., 2008;Wachowiak et al., 2011).Among all the available genetic markers, the AFLP technique (Vos et al., 1995) has become an extensively used tool in Scots pine genome mapping (e.g.Lercetau at al., 2000;Yin et al., 2003), with less popularity, however, in genetic diversity studies (e.g.Lerceteau and Szmidt, 1999;Kuchma and Finkeldey, 2011).Despite the many advantages of the AFLP technique (for review see Bonin et al., 2005), its usefulness in studies of the genetic diversity of Pinus sylvestris may be limited by the large number of populations and individuals that need to be processed when the whole or a significant part of the species distribution range is concerned.
One of the possibilities to overcome this constraint is to analyze one or several bulked samples per population, rather than individual plants.This approach can reduce considerably the financial cost; however, bulking of DNA samples also results in the potential non-detection of rare alleles and the loss of information concerning the amount of heterozygosity within samples (Reif et al., 2005).Nevertheless, there are many examples of application of molecular markers, including AFLP, in the detection of DNA polymorphism in bulked samples of many plant species (e.g.Goto et al., 2001;Herrmann et al., 2005;Reif et al., 2005;Chuang et al., 2010).This study, to the best of our knowledge, is the first attempt to use AFLP bulked analyses in genetic diversity studies of Scots pine.
One of the most convenient sources of material for examining Scots pine variability is a provenance trial.In the present study, we investigated genetic diversity of the Scots pine from the IUFRO 1982 provenance trial in Kórnik (Poland).Previous analyses of this material provide valuable information concerning growth, plasticity and productivity, survival, susceptibility to biotic and abiotic factors (e.g.Stephan and Liesbach, 1996;Barzdajn, 2008;Oleksyn et al., 2001), as well as morphological variation and the physiological properties of investigated populations (e.g.Oleksyn, 1988;Oleksyn et al., 1992Oleksyn et al., , 1999Oleksyn et al., , 2003;;Reich et al., 1994;Androsiuk et al., 2011a).
The molecular data concerning genetic differentiation of Scots pine from the IUFRO 1982 provenance trial related to geographic or racial aspects remain largely incomplete (Androsiuk et al., 2011b;Androsiuk and Urbaniak, 2014).Therefore, the objectives of the study were (i) to investigate the usefulness of the AFLP technique with bulked DNA samples in analyses of genetic variation of Pinus sylvestris, (ii) to study how the AFLP-based grouping correlates with previously reported morphological and molecular diversity of the species from IUFRO 1982 provenance trial, and (iii) to discuss the implications of the results for Scots pine genetic diversity studies.

Plant material
The 1982 IUFRO provenance trial in Kórnik is a permanent plot of the Institute of Dendrology, Polish Academy of Sciences, in an experimental forest located in central Poland (52°15`N and 17°04`E).The analyzed provenance trial initially consisted of twenty populations of Scots pine (Pinus sylvestris L.) that represent almost the entire European distribution range of that species (Table 1, Fig. 1).However, in the following analysis, the IUFRO 1982 in Kórnik is represented by only 19 Scots pine populations because Bolewice provenance did not survive on the plot and was not represented by any tree when populations were sampled during the winter 2004.In our GENETIC DIVERSITY OF P. sylvestris FROM IUFRO 1982 analyses, each population was represented by 10 trees.Only the Turkish provenance Catacik (20) consisted of six trees, the last which survived on this plot.The material for our investigation consisted of Scots pine needles, which were stored at -20°C after being collected and cleaned.

DNA extraction
Total DNA was extracted from the needles using the CTAB procedure (Murray and Thompson, 1980) with some modifications as described by Polok (2007) and further adjustment for Pinus sylvestris.DNA was extracted from frozen Scots pine needles previously cleaned and sterilized with 70% ethanol and white spirit (Shell).Briefly, the liquid nitrogenground needles were thoroughly mixed with 2 mL of a preheated CTAB isolation buffer (2% CTAB, 100

AFLP analysis
The AFLP procedure was based on Vos et al. (1995), with some modifications (Polok, 2007).Sixty-four AFLP primer pairs were initially screened, of which four primer pairs that were polymorphic and reproducible were used for AFLP analysis in the 19 Scots pine populations.The general AFLP primers without any selective nucleotides are: ecoRI: 5'-GACTGCG-TACCAATTC-3' and MseI: 5'-GATGAGTCCTGAG-TAA-3' The details of the used AFLP primers are shown in Table 2. Genomic DNA (180 ng) was digested with 3 units each of MseI and ecoRI restriction endonucleases at 37°C for 3 h.The DNA fragments were ligated with MseI and ecoRI adapters with T4 DNA ligase for 12 h at 20°C to generate template DNA for amplification.The ligated DNA was diluted to 1:9 in deionized and sterilized water and stored at -20°C.PCR was performed in two consecutive reactions: a preselective and selective amplification.In the preselective PCR, genomic DNA was amplified using an AFLP primer pair, each having one selective nucleotide (MseI + n and ecoRI + n, where n is a selective nucleotide A, G, C or T, respectively).Accordingly, a 1.5-µl diluted ligation product, 0.25 µM each of MseI + n and ecoRI + n pre-selective primers, 250 µM of dNTPs, 0.750 µl 20x PCR buffer, 1.5 mM of MgCl 2 , 1.5 µl 10x Enhancer containing betaine were mixed with 0.5 U of Tfl DNA polymerase (Epicentre Technology) and used for preselective amplification in a total volume of 15 µL.PCR amplification was performed using the following protocol: denaturation at 94°C for 1 min, annealing at 56°C for 1 min, elongation at 72°C for 1 min, repeated for 30 cycles, followed by final elongation at 72°C for 7 min.The efficiency of preselective PCR amplification was verified on 1% agarose gel containing 0.5 µg/mL ethidium bromide separated in 1x TBE buffer (Tris-Borate-EDTA;Sambrook et al., 1989).The amplified products were diluted to 1:49 in deionized and sterilized water and used as a template for the selective amplification using AFLP primers, each containing three selective nucleotides.Selective PCR amplification was performed in a total volume of 10 µl containing 2.5-µl preselective template DNA, 0.5 µM each of MseI and ecoRI selective primers, 250 µM of dNTPs, 0.75 µl 20x PCR buffer, 1.5 mM of MgCl 2 , 1.0 µl 10x Enhancer containing betaine and 0.5 U of Tfl DNA polymerase (Epicentre Technology).The following cycling parameters were used for selective amplification: 94°C for 30 s, 65°C for 30 s reducing by 0.7°C/cycle to 56°C, 72°C for 1 min for 13 cycles, followed by 94°C for 30 s, 56°C for 30 s and 72°C for 1 min for 28 cycles.PCRs were performed separately for each primer pair and the products were denatured with formamide at 94°C for 5 min.Electrophoresis of the PCR-amplified DNA fragments was carried out on 6% denaturing polyacrylamide gels at 45 W constant power until the xylene cyanol reached two-thirds of the total length of the gel.The vertical electrophoresis was conducted with the use of a Dual Dedicated Height Nucleic Acid Sequencer 20(w) x 52 cm(l) (C.B.S. Scientific Company).The gels were developed using the silver nitrate staining technique.

Data analysis
Each band that could be reliably read was treated as a separate putative locus and was scored either present (1) or absent (0).The AFLP data were analyzed using different parameters such as the PIC (polymorphic information content) and Rp (resolving power).The PIC i was calculated as PIC = 2∫ = i (1 -∫ = i ); where PIC i is the PIC of marker 'i' , ∫ = i is the frequency of the amplified allele (band present), and 1 -∫ = i is the frequency of the null allele (Roldan-Ruiz et al., 2000).The Rp of a primer was estimated using the formula Rp = Σ lb, where lb describes relative band informativeness and takes the value 1 -(2 × |0.5 -p|), and p is the proportion of the samples containing the band (Prevost and Wilkinson, 1999).Fischer's LSD multiple range test was applied to test the significance level of differences between obtained PIC values.
Following the results of previous studies (e.g.Oleksyn et al., 1992, Androsiuk et al., 2011a), Scots pine populations were found in three groups representing the Northern, Central and Southern range of the species in Europe, for which parameters describing genetic diversity and differentiation were estimated.
The genetic variation parameters were calculated using POPGENE 1.32 software (Yeh et al., 2000).They include the mean number of alleles per locus (N a ), effective number of alleles (N e ) and percentage of polymorphic loci (P).The number of unique alleles was also estimated.A unique allele was a band that was either present (1) or absent (0) in only one specific population.Genetic diversity was estimated using Shannon's diversity (I) and Nei's gene diversity statistic, i.e. gene diversity (H e ).In order to test whether N e , H e and I values vary among the three groups of populations, Fischer's LSD multiple range test was performed.
The data were also tested for the presence of population structure by analysis of molecular variance (AMOVA) using Arlequin 3.5 software (Excoffier et al., 2005).For this purpose, the analysed Scots pine provenances were also separated according to their origin into three groups of populations (North, Cen-tral and South).For this analysis, the AFLP data were treated as haplotypic, comprising of a combination of alleles at one or several loci (Excoffier et al., 2005).The significance of the fixation indices was tested using a non-parametric permutation approach according to Excoffier et al. (1992).
The genetic similarities among all populations were estimated using Nei's coefficient of similarity (I N ) (Nei, 1972) and Jaccard's similarity coefficient (I J ) (Jaccard, 1908), calculated with the use of POP-GENE 1.32 and MVSP 3.2 software (Kovach, 2005), respectively.Moreover, pairwise geographical distances were computed as great-circle distances from the geographic coordinates and then transformed to natural logarithms.Afterward, a Mantel test implemented in GenAlEx 6.2 (Peakall and Smouse 2006) was used to estimate the strength of association between matrices of Nei's pairwise genetic distances and geographical distances for the sampled populations.Nei's genetic distances were calculated in POPGENE 1.32.The matrix of Jaccard's similarity coefficient was used to generate a dendrogram showing the clustering of analyzed Scots pine provenances using a UPGMA (unweighted pair-group method with arithmetical averages) algorithm, implemented in Statistica 7.1 software.

Efficiency of AFLP primers
The four AFLP primer pairs that were used to compare 19 Scots pine populations from the IUFRO 1982 provenance trial in Kórnik yielded a total of 309 The majority of Pinus sylvestris populations shared the same bands; however, 36 unique alleles were found (Table 2 and 3).The highest number of unique alleles (12) was revealed by ecoI + ACC and MseI + CTG primer combinations.The high number of unique alleles was accompanied by the highest Rp values.The lowest number of unique alleles (5) was characteristic for ecoI + AGG and MseI + CTT (Table 2).At the population level, the population from Supraśl (6) was found to be the most abundant in unique alleles (6), whereas populations Roshtshinsaya Datsha (1), Silene (4), Spała (7), Rychtal (8) and Ardennes (13) had only one unique allele, and the four populations Lampertheim (12), Zahorie (16), Pornóapáti (17) and Prusačka Rijeka (19) did not present any unique alleles.When the populations were grouped according to the latitude of their origin (North, Central, South) the total number of unique alleles for each group and the average number of unique alleles per population in each group were as follows: 11 and 2.2 for North, 18 and 1.8 for Central, 7 and 1.8 for South (Table 3).Unique alleles appeared to be randomly distributed among populations showing no geographic pattern.
The average PIC value for Pinus sylvestris populations from the Central group (0.203) was significantly higher than for Scots pines from the two other groups (0.168 for North and 0.160 for South), suggesting a higher polymorphic level of AFLP markers in that group.

Genetic diversity and differentiation
AMOVA (Table 4) revealed the presence of population structure when populations were clustered into three groups (North, Central and South) according to the latitude of their origin (F ST = 0.053, p = 0.001).Based on AMOVA analysis, the differences among populations were significant, but greater variance was recorded among populations within groups, which accounted for 94.7% of the total variance, while among-group variance accounted for 5.3%.
The effective number of alleles, gene diversity and Shannon's diversity index (Table 5) showed that the Central group of Scots pine provenances differs significantly from the other two groups.However, this can be explained as an effect of the relatively high level of polymorphism (58.58%) found for the Central  group, which is a reflection of the number of Scots pine populations forming the group (10), which is the highest among all three sets of provenances.
Comparison of pairwise Jaccard and Nei coefficient of similarity values revealed exactly the same relationships between analyzed populations, even though similarity values were different.Genetic similarity values calculated with Jaccard's coefficient of similarity ranged from 0.649 to 0.818 with the average value of 0.729, whereas the Nei's genetic similarity ranged from 0.696 to 0.858 and on average equaled 0.773 (Table 6).Simultaneously calculated values of pairwise genetic distances (Nei, 1972) ranged from 0.154 to 0.363 with the average value of 0.259 (data not shown).

Group
The remaining four clusters, cluster I (Supraśl (6)), cluster II (Betzhorn ( 11)), cluster III (Neuhaus (10)) and cluster V (Sumpberget ( 15)) were composed of only one population each, occupying solitary clusters.In a second step, to estimate a degree of differentiation among the three distinctive groups of Scots pine provenances (North, Central and South), average Jaccard's genetic similarities for populations belonging to the same and/or different groups, were calculated.The highest average similarity values are located on the diagonal of Table 7, indicating that the populations belonging to the same group tend to be more similar than populations belonging to different groups.These findings suggest the presence of some degree of differentiation among groups, which is in agreement with previously described AMOVA results.
Significant correlation between genetic and geographic distances (r = 0.354, p = 0.0003) provided evidence that AFLP markers differentiate between populations according to their geographic origin.This means that, in general, the more that populations are geographically distant, the larger are the observed genetic differences.

DISCUSSION
AFLP analysis based on bulked needle samples allowed us to detect high genetic variation of the investigated Pinus sylvestris populations as expressed by the high values of Shannon's diversity (I = 0.356) and Nei's gene diversity (H e = 0.238).The level of genetic diversity of Scots pine revealed in our paper is concordant with data obtained previously for the species by the means of isoenzymes (H e = 0.211 to 0.365) (Ledig, 1998) and with the use of RAPD (H e = 0.192 to 0.356) (Szmidt et al., 1996) or ISSR markers (H T = 0.262) (Li. et al., 2005).However, molecular diversity of Scots pine revealed by methods based on polymorphism of DNA sequences could be even two-fold higher, as reported for example by Karhu et al. (1996) for RFLPs (H T = 0.54) and microsatellites (H T = 0.74) or by Pyhäjärvi et al. (2008) for mitochondrial genes (H T = 0.58).
The high number of polymorphic bands obtained for all primer pairs in the present study shows the suitability of AFLP markers in discriminating Scots pine populations.The application of four primer pairs turned out to be enough to differentiate the studied populations, since each of them generated specific molecular profiles.The total proportion of 67.3% of polymorphic loci (on average 52 per primer combination) reported herein for Scots pine points to comparatively high polymorphic rates when compared to other studies where AFLP markers were used.For example, in Norway spruce Paglia and Morgante (1998) found on average 12.6 polymorphic fragments per primer combination; Segovia-Lerma et al. ( 2003)  1.
Table 7.Average Jaccard similarity coefficient (standard deviation) between pairs of populations belonging to the same and to different groups.All AFLP markers were included in calculations.found on average 40% of polymorphic AFLP fragments in alfalfa; Sensi et al. (2003) found 59.8% of polymorphic loci in cultivated accessions of Olea europaea.In the case of Scots pine, a similar level of polymorphism (61.9%) revealed by AFLP markers was found by Lerceteau and Szmidt (1999) for 13 P. sylvestris individuals from Sweden.However, it needs to be emphasized that the number of alleles and level of polymorphism revealed for each Scots pine provenance from the IUFRO 1982 provenance trial in Kórnik may be even higher since the bulking strategy applied in our analyses decreased the chance for detecting low-frequency alleles (Reif et al., 2005).

North
The high discriminating power of AFLP markers is also confirmed by the values of genetic distance (D N ) estimated among the analyzed populations of Scots pine, which are higher than those reported previously for the IUFRO 1982 provenance trial in Kórnik by the means of isoenzymes (0.0027 to 0.031 based on genotype frequencies; Wójnicka-Półtorak, 1997) and also higher than those observed for the same experimental area using B-SAP markers (0.0 to 0.252, average of 0.069) (Androsiuk et al., 2011b) or RAPD and ISJ markers (0.0 to 0.240) (Androsiuk and Urbaniak 2014).
The application of four AFLP primer combinations revealed the presence of 36 population-specific alleles.More detailed analysis of their distribution showed that they do not show any geographic pattern but are distributed randomly among populations.However, previous studies on the genetic diversity of Scots pine from IUFRO 1982 performed with the use of B-SAP markers revealed six loci that divided populations into two groups according to their origin (Androsiuk et al., 2011b).These two groups, described as North and South, differed significantly also with respect to most genetic variation parameters.The plausible explanation of this geographic pattern is the putative origin of some B-SAP markers from sequences related to functional genes, not repetitive sequences.In such loci, mutations might not be neutral and may reflect the influence of some selective forces.For example, in grasses from the genus lolium several B-SAP markers are tightly linked with Per1 and Per3 loci encoding peroxidases (Polok, 2007).Furthermore, the link between B-SAP markers and another five enzymatic loci on the l.multiflorum x l. perenne genetic map implies the possibility of a similar link between important genes and B-SAP markers in other species including P. sylvestris (Androsiuk et al., 2011b).In the case of unique alleles revealed in this study by AFLP technique, the lack of clear geographic patterns in their distribution is perhaps not surprising, since most of these molecular markers are assumed to be neutral (Vos et al., 1995) and therefore not likely to be linked to the plant's phenotype, which undergoes adaptation or selection.Similarly, even though there are many examples of clinal variation in adaptive phenotypic traits (e.g., Hurme et al., 1997;García-Gil et al., 2003;Notivol et al., 2007), data are still lacking to confirm allele frequency clines in neutral genetic markers in Scots pine (Kujala and Savolainen, 2012).
The clustering of Scots pine populations into seven clusters revealed diverse relationships between the populations used in the study, which is also shown by significant genetic structure (F ST = 0.053) when populations were grouped based on their origin.
The group of five genetically close populations, Roshtshinsaya Datsha (1), Kondezhskoe (2) and Serebyanskoe (3) from Russia, Silene (4) from Latvia and Miłomłyn (5) from Poland, can be observed.The presence of closely related populations from northeastern Europe concurs with observations made for IUFRO 1982 in Kórnik based on B-SAP markers, which showed that north-European populations of the species form a separate, genetically very homogenous cluster (Androsiuk et al., 2011b).The similarity of Scots pine provenances from northern and northeastern Europe was also previously reported after isoenzymatic analyses (Prus-Głowacki et al., 1993;Stephan and Liesebach, 1996) and studies on interpopulation differentiation of morphological traits of seeds (Urbaniak, 1997) or physiological features like high N and P resorption efficiency from senescing foliage (Oleksyn et al., 2003), earlier bud set (Garcia-Gil et al., 2003) or lower average maximum photosynthetic rate (Luoma, 1997), which seems to explain the slower aboveground growth of northern popula-tions (Oleksyn et al., 1999).This geographical pattern of interpopulation differentiation of Scots pine is in agreement with the phenotype-based taxonomic division of the species (Pravdin, 1964;Giertych and Mátyás, 1991).Furthermore, UPGMA analyses revealed the population Supraśl (6) from Poland and two German populations Neuhaus (10) and Betzhorn (11) as the most distinct.However, since the majority of Polish and German Scots pine populations are not primeval, it cannot be excluded that the observed pattern of population differentiation could be the result of the diverse history/origin of their ancestral populations.
The next point of concern involves the Swedish population Sumpberget (15), whose position on the dendrogram suggests that it has more in common with populations from Central Europe than with other north-European provenances.Similar observations were made by Kullman (2008) andTollefsrud et al. (2008) for Norway spruce.According to the authors, similarities found among populations of that species from southern Sweden and provenances of Picea abies from Central Europe are a result of past gene flow/migration routes across the Baltic Sea, which led Norway spruce to southern Scandinavia after the last glaciation.However, according to Vendramin et al. (2000) it could be also a consequence of human activity as the non-autochthonous stands of the species have been reported to be planted in southern Sweden and the possible pollen movement between these and native stands may have influenced the observed pattern of differentiation.Since both coniferous species (Scots pine and Norway spruce) have the same life strategy (mainly allogamous, wind-pollinated and long-lived species), they generally share a similar postglacial history and they became the most economically important species in European silviculture, the abovementioned theories postulated by Vendramin et al. (2000), Kullman (2008) andTollefsrud et al. (2008) may also explain the position of the Swedish population.
What remains contentious is the character of the genetic relationships among southern European provenances of Scots pine represented in IUFRO 1982.
On the one hand, the similarity between the Turkish population Catacik (20) and Maočnica (18) from Montenegro can be observed, but on the other hand, two neighboring populations from Balkans, Maočnica (18) from Montenegro and Prusačka Rijeka (19) from Bosnia, which are separated by a distance of about 200 km, are found in two different clusters (Fig. 2).The similarity between the populations Catacik (20) and Maočnica (18) may reflect their common history after the last glaciations; however, according to previous data, Turkish populations of Scots pine originating from refugia in Asia Minor did not participate in the recolonization of Europe since they possess a number of private alleles not found in any other provenance (Naydenov et al., 2007;Pyhäjärvi et al., 2008).In addition, previous observations based on both morphological (Androsiuk et al., 2011a) and molecular markers (Androsiuk et al., 2011b; Androsiuk and Urbaniak 2014) did not point to high levels of similarity between the abovementioned pair of populations.Therefore, this issue remains ambiguous and needs further clarification.In the case of two Balkan provenances, Maočnica (18) and Prusačka Rijeka (19), their low genetic similarity is quite unexpected.The reason for such an occurrence may lie in the complex physiographic conditions of that mountainous area, which promote differentiation by limitation in the gene flow or/and by diversifying selection pressure associated with severe environmental conditions.
The current paper, like previous studies on Scots pine genetic structure and its geographical differentiation based on biochemical and molecular methods (Oleksyn 1988;Prus-Głowacki and Bernard 1994;Stephan and Liesbah 1996), proved that provenance trials might serve far beyond the initial purposes.This experimental area, established primarily to investigate how trees of various provenances adapt to certain climate conditions found in a particular provenance trial location, appeared also to be a valuable source of material for genomic studies.Our results, as well as previous observations based on morphological and molecular data obtained from the IU-FRO 1982 provenance trial (Androsiuk et al. 2011a(Androsiuk et al. , 2011b;;Androsiuk and Urbaniak 2014), showed that despite many years of continuous pressure of both natural and artificial selection, Scots pine populations from that experiment preserve their characteristic geographic pattern of differentiation.Even intense thinning, which reduced the number of individuals by even 46% (Oleksyn et al. 2000), or mortality rate, which may eliminate as much as 35% of individuals (Oleksyn et al. 1999), were not strong enough to erase the genetic imprint characteristic for the analyzed populations.This observation is of significance for further exploitation of this long-lasting experimental site, since populations gathered there represent carefully selected provenances, considered as a representative sample of species variation characteristics for a certain location, which may also constitute potentially interesting material for developing future breeding programs or conservation strategies.
The presented results indicate that bulked DNAbased AFLP analysis is capable of detecting genetic diversity among Scots pine populations.Nevertheless, it needs to be emphasized that the use of DNA-bulked samples in our study may reduce marker-based genetic distances compared with evaluation based on individual genotypes within populations, since it favored the detection of common alleles, whereas the lower-frequency or unique alleles present in only a few genotypes may have been diluted to such an extent that they were not amplified efficiently.Although the loss of some allelic information certainly has to be taken into account, the AFLP method applied to DNA templates extracted from bulked leaf samples provides an efficient approach to elucidate the genetic diversity of Scots pine populations.Moreover, this approach permits the sampling of a greater number of populations and higher number of individuals per population with comparable resources.Thus, as regards costs and time, it seems that the presented method is a good alternative for analyses based on individuals for large-scale diversity studies where information concerning genetic structure of populations is not essential.

Fig. 2 .
Fig. 2. Dendrogram showing clustering of 19 Scots pine populations from IUFRO 1982 based on AFLP markers.Population numbers correspond to those of Table1.

Table 1 .
Origin of Scots pine (Pinus sylvestris L.) provenances from the IUFRO 1982 provenance trial.Provenances are ordered and grouped by latitude of origin.

Table 2 .
AFLP primers, number of amplified fragments and level of polymorphism.Number of unique alleles presented as: total No of unique alleles (No of null alleles/No of bands present) *bands, of which 208 (67.31%) were polymorphic.The primer combinations produced from 64 (ecoI + ACT and MseI + CTG) to 95 (ecoI + ACT and MseI + CAT) bands, with an average number of bands per primer combination of 77.25.The AFLP primer pairs differed in their ability to detect polymorphism between populations.The highest number of polymorphic bands (64) was found for ecoI + ACC and MseI + CTG, and the lowest (30) for ecoI + AGG and MseI + CTT (Table2).

Table 3 .
Distribution of unique alleles among analyzed Scots pine populations divided into 3 groups: North, Central and South.Unique alleles presented as band present (1) or absent (0).Population numbers correspond to those of Table1.
The same letters means lack of statistically significant differences (Fischer's LSD multiple range test, p<0.05).

Table 6 .
Genetic similarity according to coefficients between Scots pine populations.Population numbers correspond to those of Table1.