Plant Syst Evol (2009) 277:197–205 DOI 10.1007/s00606-008-0123-y
ORIGINAL ARTICLE
Genetic variation of Pinus uncinata (Pinaceae) in the Pyrenees determined with cpSSR markers A. Dzialuk Æ E. Muchewicz Æ A. Boratyn´ski Æ J. M. Montserrat Æ K. Boratyn´ska Æ J. Burczyk
Received: 30 June 2008 / Accepted: 31 October 2008 / Published online: 21 January 2009 Ó Springer-Verlag 2009
Abstract The genetic variation within and between 13 populations (385 individuals) of Pinus uncinata was analyzed with ten chloroplast microsatellite markers. Both the infinite allele mutation and stepwise mutation model (SMM) have been applied to the analysis of the genetic structure and the geographical distribution of haplotypic variation. High level of genetic diversity and low but significant differentiation among compared population were found. Three marginal populations, Sierra de Cebollera, Margaride Mountains and Sierra de Gu´dar are strongly differentiated from the rest. Mutations following SMM-like process contributed significantly to the regional differentiation. The pattern of genetic structure observed in mountain pine is common in conifers with a wide distribution range. Lack of significant genetic structuring may be a result of a recent fragmentation of a historically larger population and/or interspecific hybridization and introgression. The southernmost populations from the Sierra Cebollera and the Sierra de Gu´dar are the most genetically distinct. This suggests a long period of spatial isolation and/or origin from different ancestral populations.
A. Dzialuk J. Burczyk Department of Genetics, Kazimierz Wielki University, Chodkiewicza 30, 85-064 Bydgoszcz, Poland E. Muchewicz A. Boratyn´ski (&) K. Boratyn´ska Polish Academy of Sciences, Institute of Dendrology, Parkowa 5, 62-035 Ko´rnik, Poland e-mail:
[email protected] J. M. Montserrat Institut de Cultura de Barcelona, Jardı´ Bota`nic de Barcelona, C/Font i Quer 2, 08038 Barcelona, Spain
Keywords Chloroplast microsatellites Genetic diversity Mountain pine Pleistocene migrations Phylogeography Pinus uncinata Pyrenees
Introduction The levels and distribution of genetic diversity within species may have significant effects on survival and evolution of the species in changing environment. The genetic differences between isolated plant populations depend on the geographic distance and the period of isolation, which restricted gene flow, and on drift, mutations, selection processes and historical events (Hewitt 1996; Hamrick and Nason 2000; Savolainen and Kuittinen 2000; Yeh 2000; Hewitt 2004). The effect of population history is especially significant for the species that have survived the Quaternary ice ages, because their current distribution and biodiversity is the result of successive range shifts during glacial and interglacial cycles. The northward expansion from southern refugia following Holocene climatic warming for European trees has been postulated (Hewitt 2004). Pinus uncinata Ramond has been treated as an independent species or as subspecies and even as a variety of P. mugo Turra (for a review see Sandoz 1980, 1982; Christensen 1987; Businsky´ 1999; Minghetti and Nardi 1999; Boratyn´ska 2004; Businsky´ and Kirschner 2006). At present it is treated as a subspecies of P. mugo Turra (Christensen 1987; Bolo`s and Vigo 1984) or as a species (Amaral Franco 1986; Businsky´ 1998; Businsky´ and Kirschner 2006). We have adopted the latter concept in the present paper. P. uncinata occurs in the Pyrenees and Western Alps, with several more or less isolated localities around these centers, as in the Sierra de Gu´dar and Sierra Cebollera to
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the south of the Pyrenees, and the Massif Central, Jura and Vosges between the Pyrenees and Alps (Jalas and Suominen 1973). In the Alps, it occurs together with P. mugo and intermediate forms are formed there (Christensen 1987), known as P. rotundata Link and in the Sudetes as P. uliginosa Nemann (for review see Christensen 1987; Boratyn´ska and Bobowicz 2001; Businsky´ and Kirschner 2006; Marcysiak and Boratyn´ski 2007). Hybridization can also occur with Pinus sylvestris L. (Neet-Sarqueda et al. 1988; Neet-Sarqueda 1994; Lewandowski et al. 2000). P. uncinata is a mountain species, which forms the subalpine forest at altitudes of between 1,400 and 2,700 m (Amaral Franco 1986; Ozenda 1988). This type of contemporary distribution (Jalas and Suominen 1973) results from the restriction during the Holocene of its previous, probably much greater range (Burga 1988; Ramil-Rego et al. 1998; Braggio et al. 2000; Field et al. 2000; Gandouin and Franquet 2002; Ali et al. 2006). The isolation of particular populations and restricted gene flow among them have been repeated several times during warm periods of the Pleistocene, when P. uncinata was shifted up to the high mountain locations. The range of the species was larger during cold periods, overlapping lower portions of the mountains (Burga 1988; Ramil-Rego et al. 1998; Field et al. 2000). Presently isolated localities, especially those distant from the main centers of occurrence in the Pyrenees and Alps are to be considered as relicts (Tardif et al. 2003). Genetic diversity of P. uncinata has been studied only fragmentary. The difference between two Pyrenean samples of the species, compared by Lewandowski et al. (2000) using isozymes, was found to be low. The variation of P. uncinata within its range in the Pyrenees has been tested on the morphology of cones. Differences among eight compared samples from six populations were found to be slight (Marcysiak 2004). Similarly low differences were found in the five Pyrenean samples based on anatomy of needles analyzed by Boratyn´ska and Bobowicz (2000, 2001) and in the 12 samples from the western and central parts of the species range in the form of needle sclerenchyma types verified by Boratyn´ska and Boratyn´ski (2007). Moreover, Monteleone et al. (2006) found absence of differentiation among 15 populations of P. mugo and P. uncinata in the Alps. This suggests an extensive hybridization between these taxa and is consistent with the hypothesis of a recent fragmentation of a historically larger population, which occurred in the late Tertiary and during the Quaternary interglacial periods (Gonza´lez-Sampe´riz et al. 2005; Robledo-Arnuncio et al. 2005; Cheddadi et al. 2006). In the present paper, we report the first evidence of molecular variation in P. uncinata beyond the natural range of P. mugo. Chloroplast microsatellites (cpSSRs) we used, are inherited paternally in many species of
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Pinaceae (reviewed in Mogensen 1996) and show lower mutation rate than nuclear genome (Provan et al. 1999). Because they do not recombine, they are very sensitive to genetic drift and hence to changes in population size, and therefore are quality recent-history markers (Petit et al. 2005). The main objective of this study was to provide a detailed picture of neutral genetic variation in 13 P. uncinata populations in the southernmost range of the species. We check for the conservation of the cpSSRs designed for pine species (Vendramin et al. 1996; Provan et al. 1998) to mountain pine, evaluate their usefulness to study genetic variation within and among natural populations, as well as to investigate whether there is a phylogeographical structure in this variation. We test the hypothesis that the southernmost populations of P. uncinata in Spain, especially in the Sierra de Gu´dar, were isolated for longer period of time than populations between the Pyrenees and Alps. The impact of Pleistocene climatic changes is discussed to explain the observed levels of variation and differentiation. Moreover, we address questions about genetically homogenous zones within the southernmost P. uncinata range that may be used to define the conservation and/or breeding units. The data obtained are basic to establishing a strategy of management and conservation of this species.
Materials and methods Plant material The present study is based on the 385 individuals of P. uncinata sampled in 13 southernmost, morphologically the most typical populations (Table 1). We collected needles in the main part of the P. uncinata range in the Pyrenees (eight core populations) and in four marginal populations dispersed around this mountain range. Additionally, one population was collected in the Western Alps. Generally, material has been sampled outside the range of P. mugo to exclude the possible influence of this species. After collection, fresh needles were preserved in 70% ethanol, then stored at 20°C. DNA extraction Total genomic DNA was extracted from 50 mg of needle tissue using standard CTAB procedure described by Doyle and Doyle (1990) after grinding with Mixer Mill (MM301, Retsch). The concentration of DNA was measured using DNA calculator (BioPhotometer, eppendorf) and 10 ng/ll dilutions were prepared. Ten pairs of chloroplast microsatellite primers: Pt26081, Pt36480, Pt45002, Pt71936,
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Table 1 Geographic location and genetic diversity estimates for the Pinus uncinata populations No. Population Location
Latitude Longitude Altitude N (m)
Nh
Nhp fa
1
42°580 N 00°460 W
Pyrenees 1 Belagua, Spain
0
0
Np
Ne
He
D2sh
PD (%)
1,800
30
22
4
0.10 8
17.3 0.98
6.74 73.3
2
Pyrenees 2 Benasque, Spain
42°37 N 00°39 E
2,000
30
23
4
0.07 9
16.1 0.97
5.76 76.7
3
Pyrenees 3 Vall de Ransol, Andorra
42°340 N 01°290 E
2,000
30
26
11
0.03 9
23.7 0.99
5.37 86.7
4
Pyrenees 4 Col de Jau, France Pyrenees 5 Vall de Nu´ria, Spain
42°390 N 02°150 E
1,500
30
24
6
0.17 8
16.7 0.97
4.57 80.0
42°240 N 02°090 E
2,200
30
23
8
0.07 9
18.0 0.98
3.72 76.7
2,000
30
22
6
0.07 8
18.0 0.98
4.70 73.3
5 6
Pyrenees 6 San Miguel de Engolasters, Andorra
42°310 N 01°340 E
7
Pyrenees 7 Port de la Bonaigua, Spain
42°390 N 00°570 E
2,100
30
21
8
0.07 8
15
0.97
4.48 70.0
8
Pyrenees 8 La Trapa, Spain Gudar Sierra de Gu´dar, Spain
42°410 N 00°320 W
1,720
30
19
2
0.13 9
14.1 0.96
5.57 63.3
40°230 N 00°360 W
2,000
30
13
5
0.03 8
5.1 0.83
8.42 43.3
9
0
0
10
Cebollera
Sierra de Cebollera, Spain
2,100
30
25
21
0.00 8
21.4 0.99 16.62 83.3
11
Massif 1
Col de la Croix de Morand, France 45°410 N 03°030 E
41°59 N 02°38 W
1,400
30
26
13
0.00 9
20.5 0.98
12
Massif 2
Margaride Mountains, France
45°090 N 03°210 E
1,400
25
21
15
0.00 8
18.9 0.99 12.37 84.0
13
Alps
Claviere, Italy
44°560 N 06°440 E
1,800
30
21
9
0.00 9
14.5 0.96
5.03 70.0
8.6 0.06 8.5 16.9 0.97
8.43 74.4
Average
29.62 22
Total
385
174
7.27 86.7
0.99
N sample size, Nh number of haplotypes, Nhp number of private haplotypes, fa frequency of the most common haplotype H1, Np number of polymorphic loci, Ne effective number of haplotypes, He unbiased haplotype diversity, D2sh within population genetic distance between haplotypes, PD proportion distinguishable
Pt15169, Pt30204 (Vendramin et al. 1996), PCP1289, PCP41131, PCP87314, PCP102652 (Provan et al. 1998) were analyzed using multiplex PCR protocol (Dzialuk and Burczyk 2004, modified). The DNA amplification was carried out in 10 ll volumes using a PTC-200 thermocycler (MJ Research). The PCR started with the denaturation phase at 94°C for 5 min. We then performed 30 cycles with denaturation at 94°for 30 s, annealing at 50°C for 1 min and extension at 72°C for 1 min (10 min for the last one). The reactions consisted of 30 ng of template DNA, 1x Qiagen PCR buffer, 4.0 mM MgCl2, 0.2 mM each of dNTP, 40–300 nM each of forward and revers primers, 5 lg/ul of BSA and 0.25 U of Taq Polymerase (Qiagen). The fluorescence-labelled amplification products were resolved using capillary electrophoresis on ABI 310 Genetic Analyser (Applied Biosystems) and fragment sizes were calculated with GENESCAN software ver. 3.7 (Applied Biosystems).
Data analyses Genetic diversity estimates For each chloroplast microsatellite, the total number of the size variants (no) and the effective number of the size variants (ne) were calculated. Each individual chloroplast haplotype was defined as the unique combination of size variants across the microsatellite regions within an
individual. The genetic diversity of each population was assessed by computing the number of haplotypes (No), the number of private haplotypes (Nhp), the frequency of the most common haplotype (fa), the number of polymorphic loci (Np), the effective number of haplotypes (Ne), the unbiased haplotype diversity (He), the proportion distinguishable (PD), and the within population genetic distance between tree haplotypes D2sh ; as defined by Vendramin et al. (1998), which assumes a stepwise mutation model (SMM) for microsatellite loci. Population genetic structure The population genetic structure was investigated using analysis of variance framework (AMOVA) based on the pairwise genetic differentiation coefficients, both FST (under the infinite allele mutation, IAM model) and RST (under SMM), computed for all pairs of populations using the program SPAGeDi (Hardy and Vekemans 2002). The potential benefit of using allele-size-based statistics as Rst, compared with traditional F statistics, which are alleleidentity-based statistics, is the possibility to use the extra information in knowing not just that alleles are different, but also how different the alleles are. Hierarchical AMOVA to partition the total genetic variation among groups and among populations within groups was estimated based on 1,000 permutations using Arlequin ver. 3.1 (Excoffier et al. 2005). The possible presence of geographic structure of genetic variation in cpSSR P. uncinata was evaluated by (a)
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comparing RST to pRST (permuted) after 10,000 random permutations using the SPAGeDi program, (b) by Mantel tests with 1,000 permutations as implemented in Arlequin ver 3.01 (Excoffier et al. 2005), and (c) by spatial analysis of molecular variance (SAMOVA) based on FST and RST for 2–6 groups using software Samova 1.0 (Dupanloup et al. 2002). Grouping of the populations was carried out by a principal coordinates analysis (PCA) performed on Nei’s genetic distance matrix (Ds, Nei, 1978), using the program GenAlEx (Peakall and Smouse, 2006). Based on pairwise coancestry genetic distance (DR, Reynolds et al. 1983), an UPGMA dendrogram of the 13 P. uncinata populations was constructed. Node consistency was evaluated by running 5,000 bootstrap replicates using TFPGA software (Miller 1997). Additionally, the boundaries of sharp change in allelic frequencies using Monmonier’s algorithm applied on a Delaunay triangulation were identified with the software BARRIER 2.2 (Manni et al. 2004) based on 100 bootstrap matrices of Goldstein’s pairwise genetic distances (dl)2.
analyzed, 174 different haplotypes were identified. All haplotypes had a frequency below 0.06, averaged over the total set of 385 trees. The most abundant haplotype (H1) was found in 22 trees from nine different populations (overall frequency 0.06, Table 1). The majority of haplotypes were detected only once (64.4% of population-private haplotypes), 18.4% were detected twice. On average, 38.5% of the haplotypes found in each population were unique to it. We found seven most abundant haplotypes common to 105 individuals (H1–H7 on Fig. 1). Table 1 shows the genetic characteristics of chloroplast haplotypes based on nine cpSSR loci in the 13 P. uncinata populations analyzed. Estimates of the effective number of haplotypes (Ne) and haplotype diversity (He) averaged across all the populations were 16.9 and 0.97, respectively. The lowest values were noted in Gudar (5.1 and 0.83, respectively) and the highest in Pyrenees 3 (23.7 and 0.99, respectively). Values of mean genetic distances between tree haplotypes within populations D2sh varied greatly among populations, from a minimum of 3.72 in Pyrenees 5 to 16.62 in Cebollera, with a mean of 6.97 (Table 1).
Results Phylogeographical structure of variation Genetic diversity The plastid genome of P. uncinata contains ten sites that can be amplified using multiplex PCR protocol. Nine of the cpSSRs used in this study were polymorphic. The monomorphic locus (Pt71936) was excluded from the further analyses. From 3 to 13 size variants were identified at each locus, yielding a mean of 7 and the effective number of ‘‘alleles’’ (ne) ranged from 1.1 to 3.4, with an average of 2.0. Of the 62 alleles detected, 15 were unique to particular populations: three private alleles in population Gudar, two in populations Massif 1, Alps, Pyrenees 7, Cebollera and one in populations Pyrenees 1, Pyrenees 2, Pyrenees 8, Massif 2. When alleles at each of the nine loci were jointly Table 2 Analysis of molecular variance (AMOVA) based on FST and RST among Pinus uncinata populations: (a) assuming no population structuring, (b) assuming population structuring based on isolation in Pyrenees (Pyrenees 1–8) and other regions with one population each (Gudar, Cebollera, Massif 1, Massif 2, Alps)
The analysis of molecular variance (AMOVA) based both on FST and RST, showed that the proportion of genetic variation attributable to differences among populations was low but significant (7.21 and 21.85%, respectively), with most of the total genetic variation residing within population (Table 2). When the populations were divided into six groups (Pyrenees, Gudar, Cebollera, Massif 1, Massif 2 and Alps), the hierarchical AMOVA showed that a small but significant amount of genetic variation (1.3 and 9% of the total, respectively) is due to differences among groups and that another, larger significant amount (6.5 and 16.3% of the total, respectively) is due to differences among populations within groups.
Source of variance
Variance component
Variation (%)
P
FST Among populations
12
0.1664
7.21
\0.001
Within populations
372
2.1406
92.79
\0.001
1
0.0306
1.32
\0.001
11
0.1508
6.49
\0.001
Within populations
372
2.1406
92.19
\0.001
(a)
Among populations Within populations
12 372
2.5493 9.1181
21.85 78.15
\0.001 \0.001
(b)
Among groups
(a) (b)
Among groups Among populations within groups
RST
Among populations within groups Within populations
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df
1
1.0952
8.97
\0.001
11
1.9921
16.32
\0.001
372
9.1181
74.71
\0.001
Genetic variation of Pinus uncinata
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Fig. 1 Map of the location, haplotypic distribution and genetic boundaries computed on 100 bootstrap (dl)2 genetic distance matrices of the sampled populations of Pinus uncinata (numbers in Pyrenees as in Table 1). Private haplotypes (only found in one population) and shared haplotypes (found in less than five populations) were pooled in a single category. Symbols (dots and triangles) show genetically different groups according to spatial analysis of molecular variance (SAMOVA) based on RST index and a K = 2. The robustness of computed barriers is shown as a percentage of supporting resampled bootstrap matrices and the thickness of each edge. The shading area represents the native range of the P. uncinata
With permutation procedures, phylogenetically similar alleles were found in the same populations more often than randomly chosen alleles, indicating a signal of phylogeographic structure in a total sample (RST = 0.22 [ pRST = 0.07, P = 0.002). The test of isolation by distance (Mantel test) revealed the positive relationship between Nei’s (1978) genetic distance matrix (Ds) and geographic distance matrix (R = 0.38), but not significant (P = 0.07). The lack of a clear geographic structure among 13 populations of P. uncinata was confirmed by the results of SAMOVA analysis based on RST (Fig. 1). Two groups of phylogeographically homogenous populations that maximize the among-groups variation were detected when populations Cebollera and Massif 2 were mixed (FCT = 0.48, triangles on Fig. 1). Grouping of populations revealed that three marginal populations, Cebollera, Margaride Mountains (Massif 2) and Sierra de Gu´dar (Gudar) are strongly isolated from the rest. A clear separation of these populations was revealed by high bootstrap values in the UPGMA dendrogram calculated from DR genetic distance matrix (Fig. 2), FST values significantly different from zero (Table 3) and by PCA, where the first two factors explained more than 93% of the variation found in the Ds genetic distance matrix (data not shown). Based on bootstrap matrices, the Monmonier’s algorithm identified four boundaries defining zones of maximum genetic differences within the network of 13 P. uncinata populations. The most significant genetic boundaries (a and b in Fig. 1) separated the two easternmost populations, the Massif 2 in Massif Central and Claviere in the Alps (Alps) from south-western range of the species. The westernmost population from Cebollera and the southernmost population
Fig. 2 UPGMA dendrogram of Pinus uncinata populations calculated from Raynolds genetic distance (DR). Numbers indicate bootstrap support of the respective nodes
from Sierra de Gu´dar (Gudar) were separated by barriers c and d, respectively. Among marginal populations of P. uncinata, only Massif 1 is not separated, showing a close similarity to those from the Pyrenees (Fig. 1). The presence of these genetic barriers was confirmed by analysis with single overall matrix (data not shown).
Discussion Genetic diversity The cpSSRs indicate that P. uncinata appears to maintain a very high level of genetic diversity (He = 0.986), similar to those observed in Spain for P. sylvestris (He = 0.978;
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Table 3 Matrix of pairwise FST between Pinus ucinata populations No.
Population
1
2
3
4
1
Pyrenees 2
ns
2
Pyrenees 3
ns
ns
3
Pyrenees 4
ns
ns
4
Pyrenees 5
ns
5
Pyrenees 6
ns
6
Pyrenees 7
7
5
6
7
8
9
10
ns
ns
ns
ns
ns
ns
ns
0.027
ns
ns
ns
ns
0.029
Pyrenees 8
ns
ns
0.038
0.037
ns
ns
0.056
8
Gudar
0.182
0.129
0.156
0.224
0.208
0.151
0.214
0.181
9
Cebollera
0.125
0.126
0.141
0.156
0.187
0.105
0.173
0.153
0.209
10
Massif 1
ns
ns
ns
ns
ns
ns
ns
ns
0.174
0.139
11 12
Massif 2 Alps
0.096 ns
0.084 ns
0.106 ns
0.119 ns
0.108 ns
0.056 ns
0.142 ns
0.08 0.027
0.239 0.211
0.056 0.174
11
12
0.071 ns
0.111
ns values not significantly different from zero (P C 0.05) as shown by a permutation test (1,000 permutations of haplotypes between populations
Robledo-Arnuncio et al. 2005), but higher than values reported for other Iberian and Macaronesian populations of pine species, as for example P. halepensis Mill. (He = 0.580; Gomez et al. 2005), P. pinaster Aiton (He = 0.930; Gomez et al. 2005) and P. canariensis C. Sm. (He = 0.730, Gomez et al. 2003). However, higher genetic diversity in P. uncinata may result from a different set and number of cpSSR loci used in particular studies. Much lower genetic diversity in P. uncinata was reported by Monteleone et al. (2006), who used RAPD markers (He = 0.333), but this difference could be due to the methodology applied and to the different regions of sampling. We found a high level of variation among D2sh values observed in the 13 P. uncinata populations (Table 1). Terrab et al. (2006) found the correlation of D2sh with population size, observing the lowest D2sh values in small and isolated populations of Cedrus atlantica Manetti. We found a completely different relation, the isolated populations having the highest values of D2sh among all of tested (Table 1). The AMOVA analysis shows a low but significant differentiation in P. uncinata. Most of the variation in this species lies within populations. Similar, low and not significant differentiation between P. mugo and P. uncinata populations in the Alps, was reported by Monteleone et al. (2006). This lack of clear geographic pattern in chloroplast markers is common in conifers and can be explained in several ways. First, by the absence of strong barriers to gene exchange among populations. However, at least for populations from Cebollera and Massif 2, the genetic similarity revealed by SAMOVA analyses (Fig. 1) is difficult to explain by extensive gene flow through pollen, because these populations are more than 580 km distant and separated by Pyrenees. Similar, while we found that genetic differentiation between populations was
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independent of geographical distances, the hypothesis of P. uncinata acting as single large population seems hardly likely. An alternative explanation may be the effect of homoplasy within cpSSRs, where the same number of repeats may evolve in two different microsatellite lineages through independent mutational events (Liepelt et al. 2001; Navascues and Emerson 2005). The mutational mechanism of chloroplast microsatellites seems to be different from genera to genera or even from species to species, because different number of size variants in some genera/species is found (Hansen et al. 2005). High mutation rates may be regarded as homogenizing factor explaining inability of the cpSSRs to detect among population differentiation. Another hypothesis to explain the low differentiation is the history of the species. The isolation of P. sylvestris populations at the southern limit of the species’ geographic range during last ice age resulted in morphological and genetic differentiation (Staszkiewicz 1993; Prus-Głowacki and Stephan 1994; Boratyn´ska and Hinca 2003; PrusGłowacki et al. 2003; Marcysiak 2005; Robledo-Arnuncio et al. 2005; Labra et al. 2006; Marcysiak 2006; Marcysiak and Boratyn´ski 2007). In the case of P. uncinata the isolation took place from the Holocene, but this also occurred during each earlier warm interglacial period, similarly to P. sylvestris (Gonza´lez-Sampe´riz et al. 2005, RobledoArnuncio et al. 2005; Cheddadi et al. 2006). A recent fragmentation of a historically larger population, which occurred in the late Tertiary (Neogene) and during the Pleistocene interglacial periods, has been postulated for P. uncinata (Monteleone et al. 2006). Having the same ancestor population, there has been neither time nor possibility to generate substantial differentiation among the populations. However, we found that marginal populations (Gudar, Cebollera and Massif 2) are the most genetically
Genetic variation of Pinus uncinata
distant from the rest, which suggests independent evolutionary processes at least since the Last Glacial Maximum (LGM). The genetic similarities between 14 samples of P. sylvestris from the mountain regions of Spain and two from the Massif Central in France, calculated on the basis of 11 isoenzymatic polymorphic loci (Prus-Głowacki et al. 2003), revealed a somewhat similar differentiation, as found among samples of P. uncinata from the Pyrenees and Massif Central (Fig. 1). It is noteworthy that P. sylvestris from the Margaride Mountains (Massif 2) revealed a greater genetic distance than those collected from the northern Massif Central (Massif 1), as in our finding on P. uncinata. This indicates that the southern part of the Massif Central can have been populated with pines from another Pleistocene refugium (Prus-Głowacki et al. 2003). When the result from Monmonier’s algorithm is included in our study, the most probable source would be from the Maritime Alps. This, however, shall be verified in a separate study. In P. uncinata, similar to Cedrus atlantica (Terrab et al. 2006) and Pinus nigra J. F. Arnold (Afzal-Rafii and Dodd 2007), observed RST was significantly greater than permuted RST, indicating that mutations following an SMM-like process contributed significantly to regional differentiation. While in Cebollera the percentage of private haplotypes is high, supporting independent evolution from the Pyrenees, the frequency of private haplotypes in Gudar is quite low. Thus, the differences between the latter and Pyrenean samples come from the frequency of some haplotypes also present in the Pyrenees (Fig. 1). Hence, it seems that Gudar population was related in the past with the Pyrenean pool, so sub-recent colonization is an alternative hypothesis in this case. The gene flow between the Alpine and Pyrenean populations was not so restricted because of the possibility of a wider potential range of P. uncinata during glacial periods (Field et al. 2000; Gandouin and Franquet 2002; Hardy et al. 2003; Ali et al. 2006), also during the last glaciation. The Sierra de Gu´dar was isolated from the Pyrenees by the deep depression of the Ebro river, which can be compared with the isolation of the Alps from the Apennines by the river Po basin in Italy and its influence on differentiation of P. sylvestris (Labra et al. 2006). The examination of the mitochondrial DNA from 106 populations of P. sylvestris described similarities between the East-Pyrenean and West-Alpine samples (Soranzo et al. 2000; Cheddadi et al. 2006). P. sylvestris migration routes during Late Glacial and early Holocene (Cheddadi et al. 2006) also confirm the possibility of similar migrations of P. uncinata. The map constructed mostly on the presence of Pinus pollen and P. sylvestris macrofossils (Fig. 7 in Cheddadi et al. 2006) could in fact include the migration of P. uncinata. The
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latter species is more tolerant of low temperatures than P. sylvestris and so the first Pinus migration wave could have been formed by P. uncinata, when taking into consideration its ecological characteristics (Villar et al. 1997; Tardif et al. 2003). The possibility of the natural occurrence of P. uncinata in the Cebollera is justified by the presence of the species in the Sierra de Neila during Late Glaciation times (Pen˜alba et al. 1997) and also by study on population structure (Ceballos 1968; Camarero et al. 2005). The population of the species in the Sierra de Gu´dar is geographically more isolated and separated from the Pyrenees than those of the Cebollera, mostly because the Ebro basin with its continental Mediterranean climate is deeper and broader in the eastern part. The Ebro basin and coastal region of the Maestrazgo, where the Sierra de Gu´dar is located, has been recognized as a potential Pleistocene refugium of P. sylvestris (Gonza´lez-Sampe´riz et al. 2005, Cheddadi et al. 2006). It is very probable that P. uncinata has been able to remain and survive Holocene (van Andel 2002) in the Sierra de Gu´dar and Cebollera, which are the most elevated mountains of the Cordillera Iberica. The differences between populations Cebollera and Gudar (Fig. 2, Table 3) also suggested a large period of spatial isolation and/or origin from different ancestral populations. The possibility of survival of several tree species, including P. uncinata, was recently justified by predictive modelling on the Iberian Peninsula during the LGM and Holocene (Benito Garzo´n et al. 2007). The larger than present area of distribution of this species during the LGM and mid-Holcene (Benito Garzo´n et al. 2007) confirms the expansion of P. uncinata’s range in the lower mountain parts and hence a gradual retreat with climate warming to the uppermost parts. The sufficiently high level of genetic distances between samples of P. uncinata from the Pyrenees and from the Sierra de Gu´dar and Cebollera found in our study, confirm this suggestion. Acknowledgments Material for the study was gathered as a result of bilateral cooperation between Polish Academy of Sciences and Consejo Superior de Investigaciones Cientificas. The study was partly financed by the Polish Ministry of Sciences and Higher Education grant (2P04C 018 29). During this study, E. Muchewicz benefited from a doctoral fellowship for the Institute of Dendrology. We thank all institutions for their support. We also thank Samuel Pyke for his great effort to linguistic improvement.
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