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. Boratyński Æ
J. M. Montserrat Æ K. Boratyńska 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 Gúdar 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 Gúdar 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. Boratyński (&) K. Boratyńska
Polish Academy of Sciences, Institute of Dendrology,
Parkowa 5, 62-035 Kórnik, Poland
e-mail: borata@man.poznan.pl
J. M. Montserrat
Institut de Cultura de Barcelona, Jardı́ Botànic 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; Businský 1999; Minghetti and Nardi
1999; Boratyńska 2004; Businský and Kirschner 2006). At
present it is treated as a subspecies of P. mugo Turra
(Christensen 1987; Bolòs and Vigo 1984) or as a species
(Amaral Franco 1986; Businský 1998; Businský 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 Gúdar and Sierra Cebollera to
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198
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;
Boratyńska and Bobowicz 2001; Businský and Kirschner
2006; Marcysiak and Boratyński 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 Boratyńska 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 Boratyńska and Boratyński (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 (González-Sampériz
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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A. Dzialuk et al.
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 Gúdar, 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,
Genetic variation of Pinus uncinata
199
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
22
4
Pyrenees 1 Belagua, Spain
0
0
1,800
30
Np
0.10 8
Ne
He
17.3 0.98
D2sh
PD
(%)
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
42°390 N 02°150 E
1,500
30
24
6
0.17 8
16.7 0.97
4.57 80.0
5
Pyrenees 5 Vall de Núria, Spain
42°240 N 02°090 E
2,200
30
23
8
0.07 9
18.0 0.98
3.72 76.7
6
Pyrenees 6 San Miguel de Engolasters,
Andorra
42°310 N 01°340 E
2,000
30
22
6
0.07 8
18.0 0.98
4.70 73.3
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
42°410 N 00°320 W
1,720
30
19
2
0.13 9
14.1 0.96
5.57 63.3
9
Gudar
40°230 N 00°360 W
2,000
30
13
5
0.03 8
5.1 0.83
8.42 43.3
Sierra de Gúdar, Spain
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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A. Dzialuk et al.
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
Variation (%)
P
FST
(a)
(b)
Among populations
12
0.1664
7.21
\0.001
Within populations
372
2.1406
92.79
\0.001
Among groups
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
Among populations within groups
RST
Among populations within groups
Within populations
123
Variance component
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
201
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 Gúdar (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 Gúdar (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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202
A. Dzialuk et al.
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
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
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
0.029
0.209
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
123
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; Boratyńska and Hinca 2003; PrusGłowacki et al. 2003; Marcysiak 2005; Robledo-Arnuncio
et al. 2005; Labra et al. 2006; Marcysiak 2006; Marcysiak
and Boratyński 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 (González-Sampériz 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 Gúdar 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
203
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 (Peñalba
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 Gúdar 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 Gúdar is located, has been
recognized as a potential Pleistocene refugium of P. sylvestris (González-Sampériz 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 Gúdar 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 Garzón et al. 2007). The larger than
present area of distribution of this species during the LGM
and mid-Holcene (Benito Garzón 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 Gúdar 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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