Cryptic hybrids between Pinus uncinata and P. sylvestris

Abstract

We tested the performance of molecular markers and biometric traits in the identification of hybrids between closely related mountain pine (Pinus uncinata) and Scots pine (Pinus sylvestris). A plastid DNA marker and a set of morphological and anatomical needle traits were applied in analyses of individuals from several sympatric stands of the species and a single-species' population from southern Europe, used as a reference. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker from the plastid trnL–trnF region and morphological and anatomical traits clearly discriminated between the pure species. Significant differences were found between P. uncinata and P. sylvestris, mostly in the shape of epidermal cells and the number of stomata. Four putative hybrids with P. sylvestris morphology, but with P. uncinata plastid DNA haplotypes, were found in a population from Sierra de Gúdar near Valdelinares, the southernmost locality of the latter species in eastern Spain. Discrimination analyses between and within populations placed these individuals on the edge of an agglomeration of P. sylvestris individuals. The results suggest that hybridization between the species is rare, but can result in cryptic hybrids morphologically similar to the maternal species. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 473–485.

Full text

Cryptic hybrids between Pinus uncinata
and P. sylvestris
ANNA K. JASIN
´SKA1, WITOLD WACHOWIAK1, EWELINA MUCHEWICZ1,
KRYSTYNA BORATYN
´SKA1, JOSIP M. MONTSERRAT2and ADAM BORATYN
´SKI1*
1Polish Academy of Sciences, Institute of Dendrology, Parkowa 5, 62-035 Kórnik, Poland
2Institut de Cultura de Barcelona, Jardín Botànic de Barcelona, C/ Font i Quer 2, 08038 Barcelona,
Spain
Received 12 October 2009; revised 14 April 2010; accepted for publication 28 May 2010
We tested the performance of molecular markers and biometric traits in the identification of hybrids between
closely related mountain pine (Pinus uncinata) and Scots pine (Pinus sylvestris). A plastid DNA marker and a set
of morphological and anatomical needle traits were applied in analyses of individuals from several sympatric
stands of the species and a single-species’ population from southern Europe, used as a reference. A polymerase
chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker from the plastid trnL–trnF region
and morphological and anatomical traits clearly discriminated between the pure species. Significant differences
were found between P. uncinata and P. sylvestris, mostly in the shape of epidermal cells and the number of stomata.
Four putative hybrids with P. sylvestris morphology, but with P. uncinata plastid DNA haplotypes, were found in
a population from Sierra de Gúdar near Valdelinares, the southernmost locality of the latter species in eastern
Spain. Discrimination analyses between and within populations placed these individuals on the edge of an
agglomeration of P. sylvestris individuals. The results suggest that hybridization between the species is rare, but
can result in cryptic hybrids morphologically similar to the maternal species. © 2010 The Linnean Society of
London, Botanical Journal of the Linnean Society, 2010, 163, 473–485.
ADDITIONAL KEYWORDS: anatomy – biometry – introgressive hybridization – morphology – plant
variation – plastid DNA marker.
INTRODUCTION
Natural hybridization is an important evolutionary
process contributing to the adaptive variation and
speciation of living organisms (Arnold, 1997, 2006;
Mallet, 2008). Traditionally, putative hybrids between
closely related species have been recognized on the
basis of intermediate morphological characters rela-
tive to the hypothesized parental species. This type of
biometric information has been used with many
plants, including conifers (e.g. Szweykowski, 1969;
Christensen, 1987; Boratyn´ska & Bobowicz, 2000,
2001; Delgado et al., 2007). However, as a result of
phenotypic plasticity and, in many cases, a lack of
well-characterized diagnostic traits, the identification
of hybrid individuals is difficult. By combining bio-
metric, biochemical and molecular methods, a more
complete picture of evolutionary processes in natural
populations, including interspecific gene flow, can be
obtained (Senjo et al., 1999; Ma, Szmidt & Wang,
2006; Liston et al., 2007; Wachowiak & Prus-
Głowacki, 2008).
Mountain pine (Pinus uncinata Ramond) is a tree
about 15–20 m in height (Amaral Franco, 1986;
Ozenda, 1988; Villar, Sesé & Ferrández, 1997) which
has asymmetric cones about 5 cm in length with long
apophyses (Marcysiak & Boratyn´ski, 2007). Taxo-
nomically, P. uncinata is usually recognized as a
species (Amaral Franco, 1986; Marcysiak & Boratyn´-
ski, 2007) or a subspecies (Maier, 1993; Christensen &
Dar, 1997), and is included in the group of taxa
*Corresponding author. E-mail: borata@rose.man.poznan.pl
Botanical Journal of the Linnean Society, 2010, 163, 473–485. With 2 figures
© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 473–485 473

comprising the P. mugo Turra complex (Christensen,
1987; Businský & Kirschner, 2006).
Pinus uncinata occurs in the subalpine vegetation
belt, with a range that covers the Pyrenees, western
Alps and dispersed localities in the Massif Central,
Jura, Sierra de Gúdar and Sierra Cebollera (Jalas &
Suominen, 1973; Amaral Franco, 1986; Ozenda,
1988). The species is closely related to Scots pine
(Pinus sylvestris L.) (Lewandowski, Boratyn´ski &
Mejnartowicz, 2000; Gernandt et al., 2005), which has
the largest distribution of all pines (de Ferre, 1982;
Boratyn´ski, 1993). Both species frequently co-occur in
south-western Europe, but at slightly different eleva-
tions. In the Pyrenees, the upper elevational limits of
P. sylvestris overlap with the lowest elevational limits
of P. uncinata (Amaral Franco, 1986; Ozenda, 1988;
Villar et al., 1997).
Cross-pollination of P. uncinata with P. sylvestris
seems to be possible considering their geographical
proximity and phenology, as the growth periods of
reproductive organs usually overlap, a large amount
of pollen is produced and it is efficiently dispersed
over long distances (Chałupka, 1993; Sugita, Gail-
lard & Broström, 1999; Boratyn´ski et al., 2003).
Individuals with some phenotypic characteristics
intermediate between pure species have been
observed in populations on the south-facing slopes of
the Pyrenees within the altitudinal range of P.
sylvestris, but have not been recorded at the upper
forest line formed by P. uncinata, outside the
reported altitudinal range of P. sylvestris (e.g. Flous,
1933, 1934; Vigo i Bonada, 1983; Carreras Raurell
et al., 1993; Villar et al., 1997). Individuals showing
intermediate morphology between P. sylvestris and
the P. mugo complex are treated as P. ¥rhaetica
Brügger (e.g. Amaral Franco, 1986; Christensen &
Dar, 1997). The hybrid between P. uncinata and P.
sylvestris var. pyrenaica Svob. has been described
recently as P. ¥rhaetica notovar. bolosii Rivas Mart.,
M.J.Costa & P.Soriano, and that between P. unci-
nata and P. sylvestris var. iberica Svob. as
P. ¥rhaetica notovar. borgiae Rivas Mart., M.J.Costa
& P.Soriano (Rivas Martínez et al., 2002).
Field examinations in the Pyrenees have revealed a
few putative zones of hybridization, with intermedi-
ate individuals found exclusively where both species
grow together (e.g. Villar et al., 1997; Rivas Martínez
et al., 2002, also authors’ personal observations).
Many morphologically intermediate individuals were
observed in the two southernmost, isolated popula-
tions of P. uncinata in Spain, in the Sierra Cebollera
(Ceballos, 1968) and Sierra de Gúdar (Rivas Martínez
et al., 2002), both surrounded by extensive P. sylves-
tris forests. It is possible, however, that some
individuals in the mixed populations, which are rec-
ognized morphologically as representatives of pure
species, are really cryptic hybrids and could be iden-
tified as such using genetic markers.
Recent studies have provided some indication of the
direction and frequency of hybridization between P.
sylvestris and the taxa of the P. mugo complex, mainly
dwarf mountain pine (P. mugo Turra) and peat bog
pine (P. uliginosa G.E.Neumann ex Wimm.). Con-
trolled crossing experiments have indicated no repro-
ductive barriers between P. mugo as a pollen donor
(Kormuták et al. 2005; Wachowiak, Lewandowski &
Prus-Głowacki, 2005b) and free backcrosses of F1
hybrids (Wachowiak et al., 2006b). In natural popula-
tions, plastid DNA markers and isozymes have indi-
cated a low frequency of gene flow from taxa of the P.
mugo complex to P. sylvestris (Lewandowski et al.,
2002; Wachowiak et al., 2006a), despite a lack of phe-
nological barriers (Boratyn´ski et al., 2003). Potential
gene flow from an extensive population of P. sylvestris
surrounding a small population of P. uliginosa could
not be detected in morphological characters of needles
in that species (Boratyn´ska, Boratyn´ski & Lewan-
dowski, 2003; Boratyn´ska, Jasin´ska & Ciepłuch,
2008). Conversely, the seeds of the F1 generation and
hybrid trees resulting from gene flow from P. mugo
and P. uliginosa to P. sylvestris were found in several
mixed stands of the species (Wachowiak & Prus-
Głowacki, 2008). Thus, the results to date indicate
rather rare and unidirectional hybridization in this
group of closely related pine taxa (Wachowiak & Prus-
Głowacki, 2008).
Patterns of hybridization between P. uncinata and
P. sylvestris and their potential consequences are still
unknown. Morphologically intermediate individuals
between these two taxa can be observed in mixed
populations, suggesting quite frequent hybridization.
However, it is uncertain whether cryptic hybrids exist
that are morphologically similar to P. sylvestris or P.
uncinata. The aim of the study was to use molecular
markers and biometric traits to verify the taxonomic
status of individual trees in mixed stands of P. sylves-
tris and P. uncinata, determined in the field on the
basis of morphological characters as one of the two
species.
MATERIAL AND METHODS
STUDY AREA AND SAMPLING
Plant material was collected from natural populations
of P. sylvestris and P. uncinata in Spain, Andorra and
France. Allopatric reference populations of P. sylves-
tris originated from the Pyrenees and Sierra de Neila,
and of P. uncinata from the Pyrenees (Table 1). Mixed
stands of P. sylvestris and P. uncinata originated from
the Sierra de Gúdar, Pyrenees and Massif Central
(Fig. 1). Pinus sylvestris is a variable species with
474 A. K. JASIN
´SKA ET AL.
© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 473–485

more than 150 varieties and forms described within
its range. In the Iberian Peninsula, var. nevadensis
Christ (Sierra Nevada), var. olivicola Vayr.
(Cataluña), var. iberica (Sierra de Guadarrama), var.
pyrenaica (central and western Pyrenees) and var.
catalaunica Gaussen (north-eastern Cataluña)
(Amaral Franco, 1986) have been reported. We did
not, however, distinguish between varieties of P.
sylvestris during collection of materials in the field.
Plant material was collected from individuals deter-
mined in the field as P. sylvestris or P. uncinata on the
basis of morphological traits. Trunk form, trunk and
branch bark colour, cone form and placement, form of
cone scale apophysis, needle colour and length were
analysed during the determination of every individual
before sampling (Amaral Franco, 1986; Boratyn´ski,
1993; Villar et al., 1997; Businský & Kirschner, 2006).
The sampling was directed towards potentially cryptic
hybrid individuals, which did not show intermediate
morphological characteristics. For this reason, indi-
viduals of intermediate morphology between P. sylves-
tris and P. uncinata have been omitted.
Two-year-old needles from about 30 individuals
from each population were collected for biometric
and DNA analyses. Healthy needles without visible
discoloration or damage by insects and fungi were
collected from the southern part of tree crowns at a
height of 3–5 m. After collection, fresh needles were
preserved in 70% ethanol and stored at 20 °C. The
same material was used in DNA and biometric
analyses.
DNA HAPLOTYPE ANALYSIS
For the detection of species-diagnostic plastid DNA
haplotypes, we used a DNA marker in the trnF–trnL
region. Previous analysis of this region in P. sylvestris
and the P. mugo complex indicated the existence of
one point mutation, leading to the loss (for P. sylves-
tris) or gain (for P. mugo complex) of a DraI restric-
tion site (Wachowiak & Prus-Głowacki, 2008).
DNA was extracted from needles following the pro-
tocol of Doyle & Doyle (1990), modified as described in
Wachowiak et al. (2006b). PCR amplification was
carried out in a total volume of 15 mL containing
about 10 ng of template DNA, 2.5 mMMgCl2, 100 mM
of each deoxynucleoside triphosphate (dNTP), 0.2 mM
of each primer and 0.25 U Taq polymerase (Fermen-
tas, Lithuania) with the respective 1 ¥PCR buffer,
following the cycle profile and primers as reported
previously (Wachowiak & Prus-Głowacki, 2008). The
PCR products (10 mL) were subjected to overnight
restriction at 37 °C. After digestion, the samples were
separated on 2% agarose gel, stained with ethidium
bromide and analysed under UV light. Each sample
was scored for the presence of the diagnostic marker.
Table 1. Locations of the studied populations of Pinus uncinata and Pinus sylvestris
Species Location Acronym
Type of
population Longitude Latitude
Elevation
(m)
Number of
individuals
Pinus
uncinata
Andorra, Pyrenees, Ransol U_1 Isolated E 1°38′19″N 42°35′00″1850 32
Spain, Pyrenees, Pico de Aneto U_2 Isolated E 0°39′50″N 42°41′20″2000 32
Spain, Sierra de Gúdar, Valdelinares U_3 Mixed W 0°37′10″N 40°23′03″1950 31
France, Massif Central, de la Croix de Morand U_4 Mixed E 3°21′02″N 45°09′29″1350 31
Pinus
sylvestris
Spain, Pyrenees, below Tunel de Viella (var.
pyrenaica Svob.)
S_1 Isolated E 0°46′30″N 42°40′17″1550 32
Spain, Sierra de Neila (var. iberica Svob.) S_2 Isolated W 3°00′41″N 42°03′10″1400 32
Spain, Sierra de Gúdar, Valdelinares (var. iberica Svob.) S_3 Mixed W 0°37′10″N 40°23′03″1950 30
Andorra, Pyrenees, Sant Miguel d’Engolasters
(var. pyrenaica Svob.)
S_4 Mixed E 1°34′12″N 42°31′28″1500 32
CRYPTIC HYBRIDS BETWEEN PINUS UNCINATA AND P. SYLVESTRIS 475
© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 473–485

Pinus uncinata haplotypes were assigned as ‘M’ and
P. sylvestris haplotypes as ‘S’.
BIOMETRIC TRAIT ANALYSES
To describe the differences between individuals, popu-
lations and species and to evaluate the possible occur-
rence of putative hybrids in mixed stands, 17
morphological and anatomical characters were mea-
sured (Table 2) on 10 needles from each individual.
The length of needles was measured in the field
before the samples were preserved in 70% ethyl
alcohol. The number of stomatal rows on the convex
(abaxial) and flat (adaxial) sides of the needle, and
the number of stomata along a 2-mm-long section in
the central part, on the convex and flat sides of the
needle, were counted using a Nikon SMZ800 binocu-
lar microscope.
Anatomical traits were measured and/or evaluated
on the subpersistent preparations of cross-sections
from the central part of the needle, under a Jenamed
2 light microscope (Carl Zeiss, Jena). Traits 7 and 8
(Table 2) were examined under 50¥magnification and
traits 9, 10 and 11 under 320¥magnification
(Boratyn´ska & Bobowicz, 2000, 2001). Three types of
sclerenchyma cells were distinguished around the
resin canals: (A) fibre-like cells with thick cell walls
and a small lumen; (C) nonfibrous cells with walls of
medium thickness and a distinct lumen; (B) cells with
intermediate or mixed characteristics (Szweykowski,
1969). The cell type was determined for each resin
canal present in the needle cross-section, separately
for every sclerenchyma cell.
Four types of sclerenchyma cell were distinguished
between the vascular bundles, as described in
Boratyn´ska & Boratyn´ski (2007): (AA) fibrous cells
Figure 1. Location of the allopatric and mixed populations of Pinus uncinata and Pinus sylvestris.
476 A. K. JASIN
´SKA ET AL.
© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 473–485

Table 2. Average values for needle characters in populations of Pinus sylvestris (S_1–S_4) and Pinus uncinata (U_1–U_4)
No Character
Arithmetic means for populations
S_1 S_2 S_3 S_4 U_1 U_2 U_3 U_4
1 Needle length (mm) 43.92 50.78 44.02 41.57 56.05 60.78 45.59 71.32
2 Number of stomatal rows
on convex (abaxial) side
of needle
10.49 11.86 9.69 9.76 9.49 10.48 9.09 11.62
3 Number of stomatal rows
on flat (adaxial) side of
needle
9.49 10.61 8.20 8.38 7.08 7.92 7.16 8.58
4 Number of stomata per
2-mm section of needle,
on convex side
21.14 21.25 21.68 21.43 17.77 18.29 18.57 18.70
5 Number of stomata per
2-mm section of needle,
on flat side
20.59 21.43 21.47 21.18 17.77 18.24 18.85 18.76
6 Number of resin canals 7.36 9.15 8.52 6.75 3.83 3.73 4.58 5.25
7 Width of needle (mm) 1606.93 1643.72 1685.85 1483.98 1584.32 2106.51 1512.74 1804.13
8 Thickness of needle (mm) 818.71 832.14 877.29 790.74 912.82 1167.98 858.30 1031.45
9 Distance between vascular
bundles (mm)
214.75 217.37 221.47 191.60 153.14 185.15 127.98 173.79
10 Width of epidermal cells
(mm)
16.16 15.92 16.32 16.00 16.29 20.46 16.03 15.92
11 Thickness of epidermal
and hypodermal cells
(mm)
31.65 31.64 33.77 32.51 43.64 57.15 44.63 43.03
12 Marcet’s coefficient
(character 9*7/8)
425.07 432.93 430.24 361.29 269.09 337.10 228.89 306.77
13 Stomatal row ratio
(character 2/3)
1.12 1.13 1.20 1.21 1.37 1.34 1.33 1.38
14 Needle thickness/width
ratio (character 8/7)
0.51 0.51 0.52 0.53 0.57 0.56 0.57 0.57
15 Width/thickness of
epidermal cell ratio
(character 10/11)
0.52 0.51 0.49 0.50 0.38 0.36 0.36 0.37
16 Character of cells around
the resin canals
A – fibre-like cells with
thick walls and
restricted lumen (%)
89.63 56.45 78.07 86.13 15.91 37.03 46.25 39.96
B – intermediate cells (%) 8.19 27.68 15.20 11.08 59.94 53.24 44.25 49.19
C – cells with thin walls
and distinct lumen (%)
2.19 15.93 6.73 2.80 23.56 9.76 9.50 10.87
17 Character of cells between
vascular bundles
A – fibre-like cells 39.75 6.13 50.80 49.80 6.73 6.17 16.86 11.35
B – intermediate,
semifibrous cells
20.88 17.16 21.27 24.73 57.36 44.34 48.60 27.29
C – intermediate cells
between vascular
bundles
31.75 49.93 21.87 20.67 21.06 31.07 14.59 38.35
D – cells with thin walls
and large lumens
7.66 26.77 6.07 4.80 14.84 18.83 19.95 23.29
CRYPTIC HYBRIDS BETWEEN PINUS UNCINATA AND P. SYLVESTRIS 477
© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 473–485

with thick cell walls and a strongly restricted lumen
in numerous clusters; (BB) fibrous elements present
separately or in sparse clusters; (CC) elements
lacking typical fibrous cells, only cells with walls of
intermediate thickness and a distinct lumen; and
(DD) only cells with slightly and irregularly thickened
walls and a distinct lumen (Szweykowski, 1969).
The set of characters and the method of measure-
ment were based on earlier biometric studies of pine
needles generally accepted as diagnostic in distin-
guishing P. mugo s.l. from P. sylvestris (Staszkiewicz
& Tyszkiewicz, 1976; Boratyn´ska, 2002, 2004; Sobi-
erajska & Boratyn´ska, 2008). The width and thick-
ness of the needle, distances between the vascular
bundles, shape of epidermal cells, numbers of sto-
matal rows, numbers of resin canals, and types of
sclerenchyma cells around resin canals and between
vascular bundles have been indicated previously as
being the most discriminatory between P. sylvestris
and P. uncinata (Boratyn´ska & Bobowicz, 2000, 2001;
Boratyn´ska & Boratyn´ski, 2007).
The data were analysed statistically on the basis of
individual means. The differences between popula-
tions were analysed with Student’s t-test for unpaired
data and Tukey’s T-test with a separate assessment of
variance (post hoc comparison of means, analysis of
variance) to determine which needle characters dif-
ferentiated between species and among populations
at a statistically significant level. A discriminant
analysis was also performed on the synthetic (calcu-
lated) (12–15) and simple (did not include synthetic)
(6, 16 and 17) characters. The position of individuals
was examined along the first two or three discri-
minant variables to explore both intra- and inter-
populational and intra- and interspecific variation
(Morrison, 1990; Zar, 1999; Sokal & Rohlf, 2003).
STATISTICA 7.0 for Windows software (StatSoft)
and JMP software (SAS Institute Inc.) were used in
the analyses.
RESULTS
DNA HAPLOTYPE ANALYSIS
All analysed P. uncinata individuals from all popula-
tions had the Mplastid haplotype typical of the P.
mugo complex. Similarly, most of the analysed P.
sylvestris individuals had the Shaplotype. However,
four of 123 individuals classified morphologically as P.
sylvestris carried haplotype M, normally diagnostic of
P. uncinata. These individuals originated from a
mixed population of both species from the Sierra de
Gúdar.
BIOMETRIC ANALYSIS
Pinus uncinata populations were significantly differ-
ent from all populations of P. sylvestris for most traits
(Table 3). The number of stomata on the convex and
flat sides of the needle, number of resin canals, thick-
ness of epidermal cells, shape of needle and shape of
epidermal cell (characters 4, 5, 6, 11, 14 and 15,
respectively) differentiated between every population
of P. sylvestris and P. uncinata at a statistically sig-
nificant level (Pⱕ0.01). The distance between vascu-
lar bundles, thickness of needles and frequencies of
fibre-like and intermediate types of cell around resin
canals (characters 9, 8 16A and 16B, respectively)
differentiated between every population of P. uncinata
and P. sylvestris, but also between single populations
at the intraspecific level (Table 3). The width of epi-
dermal cells (character 10) did not differentiate
between taxa and populations. All other characters of
the needles differentiated between populations, but
not between species, except for the types of cell
between vascular bundles (character 17B), which dif-
ferentiated one sample of P. uncinata (U_4) from all
others of that species, but not from P. sylvestris
(except S_4) (Table 3).
The shapes of the epidermal cells and frequency of
intermediate cells around resin canals in the first
canonical variable and needle length in the second
canonical variable had the most discriminatory power
among the tested individuals and populations
(Table 4). The discriminatory analysis on the syn-
thetic (calculated) traits and those not included in the
synthetic characters showed two well-separated
groups of individuals (Fig. 2). The four individuals
with the P. sylvestris phenotype but P. uncinata
plastid DNA haplotype were located at the edge of an
agglomeration of individuals otherwise belonging to
the P. sylvestris group (Fig. 2).
DISCUSSION
DISCRIMINATORY POWER OF MOLECULAR MARKERS
AND BIOMETRIC TRAITS
The study shows that P. uncinata and P. sylvestris
from allopatric locations can be clearly differentiated
on the basis of molecular markers and several bio-
metric traits. All reference P. uncinata and P. sylves-
tris individuals had plastid DNA haplotypes typical of
their species (Mand S, respectively). Thus, as in
previous reports, we found no evidence of sharing (as
a result of common or independent origin) of the two
haplotypes in the species from numerous locations in
Europe (Wachowiak et al., 2000, 2006b; Wachowiak &
Prus-Głowacki, 2008). This result supports the value
of the trnF-trnL marker for discriminating between
species in this group.
On the basis of biometry, reference individuals of P.
uncinata differed at a statistically significant level
from P. sylvestris for most morphological and ana-
478 A. K. JASIN
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© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 473–485

Table 3. Tukey’s T- test of differences between eight populations of Pinus species; ¥significance at level P=0.05; ¥¥, significance at level P=0.01
Character 1 Character 2 Character 3
S1 S1 S1
S2 ¥¥ S2 ¥¥ S2 ¥¥
S3 ¥¥ S3 ¥¥¥ S3 ¥¥ ¥¥
S4 ¥¥ S4 ¥¥¥ S4 ¥¥ ¥¥
U1 ¥¥ ¥¥ ¥¥ ¥¥ U1 ¥¥ ¥¥ U1 ¥¥ ¥¥ ¥¥ ¥¥
U2 ¥¥ ¥¥ ¥¥ ¥¥ U2 ¥¥ ¥¥ U2 ¥¥ ¥¥ ¥¥
U3 ¥¥ ¥¥ ¥¥ ¥¥ U3 ¥¥ ¥¥ ¥¥ U3 ¥¥ ¥¥ ¥¥ ¥¥ ¥
U4 ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ U4 ¥¥ ¥¥ ¥¥ ¥¥ ¥ ¥¥ U4 ¥¥ ¥¥ ¥¥ ¥ ¥¥
S1 S2 S3 S4 U1 U2 U3 U4 S1 S2 S3 S4 U1 U2 U3 U4 S1 S2 S3 S4 U1 U2 U3 U4
Character 4 Character 5 Character 6
S1 S1 S1
S2 S2 ¥¥ S2 ¥¥
S3 S3 ¥¥ S3 ¥¥
S4 S4 ¥¥ S4 ¥¥ ¥¥
U1 ¥¥ ¥¥ ¥¥ ¥¥ U1 ¥¥ ¥¥ ¥¥ ¥¥ U1 ¥¥ ¥¥ ¥¥ ¥¥
U2 ¥¥ ¥¥ ¥¥ ¥¥ U2 ¥¥ ¥¥ ¥¥ ¥¥ U2 ¥¥ ¥¥ ¥¥ ¥¥
U3 ¥¥ ¥¥ ¥¥ ¥¥ U3 ¥¥ ¥¥ ¥¥ ¥¥ U3 ¥¥ ¥¥ ¥¥ ¥¥ ¥ ¥¥
U4 ¥¥ ¥¥ ¥¥ ¥¥ ¥ U4 ¥¥ ¥¥ ¥¥ ¥¥ ¥ U4 ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ ¥
S1 S2 S3 S4 U1 U2 U3 U4 S1 S2 S3 S4 U1 U2 U3 U4 S1 S2 S3 S4 U1 U2 U3 U4
Character 7 Character 8 Character 9
S1 S1 S1
S2 S2 S2
S3 ¥S3 ¥¥ ¥ S3
S4 ¥¥ ¥¥ ¥¥ S4 ¥¥¥ S4 ¥¥¥
U1 ¥¥ ¥¥ U1 ¥¥ ¥¥ ¥¥ U1 ¥¥ ¥¥ ¥¥ ¥¥
U2 ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ U2 ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ U2 ¥¥¥ ¥
U3 ¥¥¥¥¥ ¥¥ U3 ¥¥¥¥¥¥¥U3 ¥¥ ¥¥ ¥¥ ¥¥ ¥ ¥¥
U4 ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ U4 ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ U4 ¥¥ ¥¥ ¥¥ ¥¥
S1 S2 S3 S4 U1 U2 U3 U4 S1 S2 S3 S4 U1 U2 U3 U4 S1 S2 S3 S4 U1 U2 U3 U4
Character 10 Character 11 Character 12
S1 S1 S1
S2 S2 S2
S3 S3 ¥¥ ¥¥ S3
S4 S4 S4 ¥¥ ¥¥ ¥¥
U1 U1 ¥¥ ¥¥ ¥¥ ¥¥ U1 ¥¥ ¥¥ ¥¥ ¥¥
U2 ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ U2 ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ U2 ¥¥ ¥¥ ¥¥ ¥
U3 ¥¥ U3 ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ U3 ¥¥ ¥¥ ¥¥ ¥¥ ¥¥
U4 ¥¥ U4 ¥¥ ¥¥ ¥¥ ¥¥ U4 ¥¥ ¥¥ ¥¥ ¥¥ ¥¥
S1 S2 S3 S4 U1 U2 U3 U4 S1 S2 S3 S4 U1 U2 U3 U4 S1 S2 S3 S4 U1 U2 U3 U4
CRYPTIC HYBRIDS BETWEEN PINUS UNCINATA AND P. SYLVESTRIS 479
© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 473–485

Table 3. Continued
Character 13 Character 14 Character 15
S1 S1 S1
S2 S2 S2
S3 ¥¥ ¥ S3 ¥S3 ¥
S4 S4 ¥¥ ¥¥ S4
U1 ¥¥ ¥¥ ¥¥ ¥¥ U1 ¥¥ ¥¥ ¥¥ ¥¥ U1 ¥¥ ¥¥ ¥¥ ¥¥
U2 ¥¥ ¥¥ ¥¥ ¥ U2 ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ U2 ¥¥ ¥¥ ¥¥ ¥¥ ¥
U3 ¥¥ ¥¥ ¥ U3 ¥¥ ¥¥ ¥¥ ¥¥ U3 ¥¥ ¥¥ ¥¥ ¥¥
U4 ¥¥ ¥¥ ¥¥ ¥¥ U4 ¥¥ ¥¥ ¥¥ ¥¥ ¥ U4 ¥¥ ¥¥ ¥¥ ¥¥
S1 S2 S3 S4 U1 U2 U3 U4 S1 S2 S3 S4 U1 U2 U3 U4 S1 S2 S3 S4 U1 U2 U3 U4
Character 16A Character 16B Character 16C
S1 S1 S1
S2 ¥¥ S2 ¥¥ S2 ¥¥
S3 ¥¥ ¥¥ S3 ¥¥ ¥¥ S3 ¥¥ ¥¥
S4 ¥¥ ¥ S4 ¥¥ S4 ¥¥ ¥
U1 ¥¥ ¥¥ ¥¥ ¥¥ U1 ¥¥ ¥¥ ¥¥ ¥¥ U1 ¥¥ ¥¥ ¥¥
U2 ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ U2 ¥¥ ¥¥ ¥¥ ¥¥ U2 ¥¥ ¥¥ ¥¥
U3 ¥¥ ¥¥ ¥¥ ¥¥ U3 ¥¥ ¥¥ ¥¥ ¥¥ ¥¥ U3 ¥¥ ¥ ¥¥
U4 ¥¥¥ ¥¥¥¥¥¥ U4 ¥¥ ¥¥ ¥¥ ¥¥ ¥ U4 ¥¥ ¥¥ ¥¥
S1 S2 S3 S4 U1 U2 U3 U4 S1 S2 S3 S4 U1 U2 U3 U4 S1 S2 S3 S4 U1 U2 U3 U4
Character 17A Character 17B Character 17C
S1 S1 S1
S2 ¥¥ S2 S2 ¥¥
S3 ¥¥ S3 S3 ¥¥¥
S4 ¥¥ S4 S4 ¥¥¥
U1 ¥¥ ¥¥ ¥¥ U1 ¥¥ ¥¥ ¥¥ ¥¥ U1 ¥¥¥
U2 ¥¥ ¥¥ ¥¥ U2 ¥¥ ¥¥ ¥¥ ¥¥ ¥ U2 ¥¥ ¥
U3 ¥¥¥ ¥¥¥¥¥ ¥ U3 ¥¥ ¥¥ ¥¥ ¥¥ U3 ¥¥ ¥¥ ¥¥
U4 ¥¥ ¥¥ ¥¥ U4 ¥¥ ¥¥ ¥ ¥¥ U4 ¥¥ ¥¥ ¥¥ ¥¥
S1 S2 S3 S4 U1 U2 U3 U4 S1 S2 S3 S4 U1 U2 U3 U4 S1 S2 S3 S4 U1 U2 U3 U4
Character 17D
S1
S2 ¥¥
S3 ¥¥
S4 ¥¥
U1 ¥¥¥¥¥
U2 ¥¥ ¥¥ ¥¥
U3 ¥¥ ¥¥ ¥¥
U4 ¥¥ ¥¥ ¥¥
S1 S2 S3 S4 U1 U2 U3 U4
480 A. K. JASIN
´SKA ET AL.
© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 473–485

tomical traits. One of the most distinctive traits is the
shape of the epidermal cells (Boratyn´ska & Bobowicz,
2001; Boratyn´ska & Boratyn´ski, 2007; Muchewicz,
2007), which were found in our study to be more or
less rectangular for P. uncinata and almost square for
P. sylvestris (width of epidermal cells/thickness of
epidermal cells close to 0.5 and about 0.8, respec-
tively). The species differ significantly in distance
between vascular bundles (about 140–150 mm for P.
uncinata and >200 mm for P. sylvestris), and the dif-
ference is within the range reported for other allopat-
ric populations of the species (Boratyn´ska &
Bobowicz, 2001; Boratyn´ska & Hinca, 2003). Other
important traits discriminating between the species
Table 4. Discriminatory power tests for needle characters of Pinus uncinata and Pinus sylvestris
Character
Partial Wilks’
lambda FP
1. Needle length 0.5776 23.504 0.000
5. Number of stomata per 2-mm section of needle, on flat side 0.9089 3.222 0.002
6. Number of resin canals 0.7319 11.775 0.000
14. Needle thickness/width ratio (character 8/7) 0.8415 6.065 0.000
15. Width/thickness of epidermal cell ratio (character 10/11) 0.5518 26.110 0.000
16A. Fibre-like cells with thick walls and restricted lumen around
resin canals (%)
0.8413 6.063 0.000
16B. Intermediate cells around resin canals (%) 0.8841 4.215 0.000
17A. Fibre-like cells between vascular bundles 0.8464 5.832 0.000
17B. Intermediate, semifibrous cells between vascular bundles 0.8557 5.420 0.000
Figure 2. Ordination of the individuals of Pinus sylvestris and Pinus uncinata along discriminant functions U1and U2,
responsible for more than 91% of the total variation (population acronyms as in Table 1); numbers 3, 6, 12 and 17 refer
to hybrids with the plastid DNA haplotype diagnostic for P. uncinata.
CRYPTIC HYBRIDS BETWEEN PINUS UNCINATA AND P. SYLVESTRIS 481
© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 473–485

are the number of resin canals, the number of
stomata on the convex and flat sides of the needles,
stomatal row ratio and needle shape.
Our data did not confirm the high discriminatory
value of the frequencies of various types of scleren-
chyma cells between vascular bundles and around
resin canals, which appeared to be similar in P.
sylvestris from Sierra de Neila and the two reference
P. uncinata populations. However, it cannot be
excluded that variation of some biometric traits in P.
sylvestris from this area was influenced by ancient
hybridization with P. uncinata. The presence of P.
uncinata in the Sierra de Neila at the beginning of
the Holocene has been confirmed by macrofossil
analysis (Peñalba et al., 1997). Modelling studies of
the distribution of the species at the last glacial
maximum and during the Holocene suggest that P.
uncinata disappeared from that region during the
first millennium of the Holocene (Benito-Garzón,
Sánchez de Dios & Sáinz Ollero, 2007).
CRYPTIC HYBRIDS BETWEEN P. SYLVESTRIS
AND P. UNCINATA
Despite similarities in some biometric characters
between the species, discriminant analysis clearly
placed all P. sylvestris individuals from reference
and mixed stands into one cluster and all individu-
als of P. uncinata into another. A contrasting
pattern of molecular markers and biometric traits
was found in only four individuals originating from
sympatric populations of P. sylvestris from the
Sierra de Gúdar. These individuals were determined
in the field as P. sylvestris and clustered on the edge
of the P. sylvestris group on the basis of needle char-
acteristics. Because plastid DNA inheritance is
paternal in these and other pines (Wachowiak et al.,
2005b), the presence of a plastid DNA haplotype
diagnostic to P. uncinata in these individuals sug-
gests that they represent hybrids between P. sylves-
tris as the mother tree and P. uncinata as the pollen
donor. It is likely that these individuals are back-
crosses resulting from hybrid pollination of P. sylves-
tris individuals. Each of the four hybrids showed P.
sylvestris morphology without any intermediate or P.
uncinata-like traits.
These results also indicate the successful use of
macromorphological characters during material col-
lection to avoid individuals with intermediate traits
between P. sylvestris and P. uncinata. Trunk form,
bark colour, cone form and placement, cone scale form
and apophysis length, and needle colour and length
(Amaral Franco, 1986; Boratyn´ski, 1993; Villar et al.,
1997; Businský & Kirschner, 2006) appeared to be
correlated with morphological and anatomical char-
acters of the needles.
Previous studies have noted morphological interme-
diacy between P. uncinata and P. sylvestris (Staszk-
iewicz & Tyszkiewicz, 1972; Christensen, 1987; Neet-
Sarqueda, Plumettaz-Clott & Bécholey, 1988; Neet-
Sarqueda, 1994; Christensen & Dar, 1997). Hybrids
such as P. uncinata ¥P. sylvestris and/or P. mugo ¥P.
sylvestris with such characteristics have also been
reported in floristic works (e.g. Vigo i Bonada, 1983;
Amaral Franco, 1986; Carreras Raurell et al. 1993).
The intermediate phenotype of natural hybrids
between the species was suggested by Neet-Sarqueda
et al. (1988) and Neet-Sarqueda (1994), indicating the
possibility of introgression between the species in
mixed stands. Our own field observations also confirm
the existence of individuals of intermediate phenotype,
for example in populations from Sierra de Gúdar,
Sierra Cebollera and Sant Miguel d’Engolasters in the
Pyrenees. Further molecular studies are needed to
estimate the proportion of hybrids in the populations
and to investigate whether individuals with interme-
diate phenotypes are F1 or successive generation
hybrids.
EVOLUTIONARY CONSEQUENCES OF HYBRIDIZATION
Our results reveal the possible presence of individuals
containing genetic material from two parental taxa,
but preserving morphological and anatomical charac-
ters of one of them. In previous molecular and bio-
chemical studies, hybrids of P. sylvestris morphology
were found in sympatric Central European stands
containing P. mugo and P. uliginosa (Wachowiak &
Prus-Głowacki, 2008) trees, which were also involved
in controlled crosses (Wachowiak et al., 2006b). It
seems that cryptic hybrids may exist in contact
hybrid zones of closely related pine species (e.g. Senjo
et al., 1999; Ma et al., 2006; Delgado et al., 2007), as
observed in Quercus (Dodd & Afzal-Rafii, 2004) or
Gossypium (Álvarez & Wendel, 2006). Studies in
European species of Orchis L. and Schoenoplectus
(Rchb.) Palla have also demonstrated that plants
which morphologically resemble one species can be
demonstrated to be hybrids with a second species or
introgressed forms in many cases (e.g. Fay, Cowan &
Simpson, 2003; Bateman, Smith & Fay, 2008).
The present study provides no evidence of intro-
gression into P. uncinata populations via pollen flow
from P. sylvestris, and the results suggest unidirec-
tional hybridization towards P. sylvestris. Such asym-
metric gene flow is known for several plant species,
and has also been reported in conifers. Artificial cross-
ing experiments revealed barriers between P. sylves-
tris and P. mugo (Wachowiak et al., 2005b) and P.
sylvestris and P. montana (P. mugo complex)
(Wachowiak et al., 2006b). Hybrid seeds resulting
only from cross-pollination of P. sylvestris with P.
482 A. K. JASIN
´SKA ET AL.
© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 473–485

mugo (Wachowiak et al., 2006a) or P. uliginosa pollen
(Lewandowski et al., 2005) were found in contact
zones of the species. These results suggest the exist-
ence of incompatibility between the species with
potential evolutionary consequences.
Populations of P. uncinata seem to be resistant to the
invasion of P. sylvestris by pollen transport, similar to
its close relative, P. uliginosa (Lewandowski et al.,
2002; Boratyn´ska et al., 2003; Wachowiak, Celin´ski &
Prus-Głowacki, 2005a). Introgression and dispropor-
tional gene flow among other pine species have also
been found (e.g. Senjo et al., 1999; Ma et al., 2006;
Liston et al., 2007). The introgression of Pinus albicau-
lis Engelm. plastid DNA into P. lambertiana Douglas
(Liston et al., 2007), the mitochondrial introgression
from P. pumila Regel to P. parviflora var. pentaphylla
(Mayr) Businský (Senjo et al., 1999), the higher intro-
gression rate from P. montezumae Lamb. to P. pseu-
dostrobus Lindl., and hybrids P. montezumae ¥P.
pseudostrobus (Delgado et al., 2007) are good examples
of such a process. In addition, introgression of Q.
petraea (Matt.) Liebl. into populations of Q. robur L.
(Petit, 2004; Petit et al., 2004), or among other Quercus
taxa (Dodd & Afzal-Rafii, 2004), can be used as proof of
the high frequency of disproportional gene flow
between taxa.
Reproductive barriers seem to be advantageous for
successful protection and conservation of isolated
populations of P. uncinata, especially those of a relict
character in the Sierra de Gúdar, Sierra Cebollera
and the Massif Central. On the other hand, the influ-
ence of pollination of P. sylvestris individuals by
pollen of P. uncinata seems to be marginal for Scots
pine, as it is a common species with the largest
distribution of all pines.
ACKNOWLEDGEMENTS
We would like to thank Steven Cavers for correcting
the English and two anonymous referees for helpful
comments that significantly improved the manu-
script. The majority of the plant material was col-
lected thanks to cooperation between CSIC (Spanish
Research Council) and PAS (Polish Academy of Sci-
ences). The research was conducted within the statu-
tory research program of the Institute of Dendrology,
partly supported by The Polish Ministry of Science
(Contr. Nr: N N303 360535).
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