Molecular Ecology (2004) 13, 167 – 178
doi: 10.1046/j.1365-294X.2003.02034.x
Extensive sharing of chloroplast haplotypes among
European birches indicates hybridization among Betula
pendula, B. pubescens and B. nana
Blackwell Publishing Ltd.
A . E . P A L M E ,* Q . S U ,*‡ S . P A L S S O N † and M . L A S C O U X *
*Department of Conservation Biology and Genetics, Evolutionary Biology Centre, Uppsala University Norbyvägen 18 D,
752 36 Uppsala, Sweden, †Institute of Biology, University of Iceland, Grensásvegur 12, IS 108 Reykjavík, Iceland
Abstract
Extensive sharing of chloroplast haplotypes among the silver birch, Betula pendula Roth.,
the downy birch, B. pubescens Ehrh., and the dwarf birch, B. nana L., was discovered using
polymerase chain reaction–restriction fragment length polymporphism markers. The geographical component of the genetic variation was stronger than the species component: the
species were not significantly different while 11% of the variation could be attributed to
differentiation between the two main regions studied, Scandinavia and western Russia. All
haplotypes occurring in more than 2% of the individuals were shared among the species
and the introgression ratios were quite large: 0.79 between B. pubescens and B. pendula and
0.67 between B. pubescens and B. nana. The data also indicate that B. pendula individuals
are more similar to sympatric B. pubescens than to B. pendula individuals from nearby forests.
However, this trend is not as pronounced when B. pubescens is considered, suggesting that
introgression is not symmetrical. The haplotype sharing among the three Betula species
is most likely caused by hybridization and subsequent cytoplasmic introgression.
Keywords: Betula pendula, Betula pubescens, Betula nana, chloroplast, hybridization, introgression
Received 31 August 2003; revision received 3 October 2003; accepted 3 October 2003
Introduction
It has become increasingly clear that many pairs of plant
species or species complexes share chloroplast haplotypes
across the species boundaries (e.g. Quercus spp., Petit et al.
2002; Juniper spp., Terry et al. 2000; Rorippa spp., Bleeker &
Hurka 2001) and that this is generally caused by introgression, the incorporation of genes of one species into the
gene pool of another.
It is also becoming clear that introgression plays a key
part in the evolution of plant species and that it can have
both positive and negative evolutionary effects. Introgression can result in new gene combinations and larger variation in fitness-related traits. This is a valuable quality as
adaptation to new environments may be crucial for the
survival of the species. In some cases hybrids can be better
Correspondence: M. Lascoux. Fax: (46) 18 471 64 24; E-mail:
[email protected]
‡Present address: Bioengineering Department, Dalian University
of Technology, Dalian, Liaoning Province, China.
© 2004 Blackwell Publishing Ltd
adapted to new environments that are created by humans.
For example, a Viola hybrid in Germany has a higher fitness
than either of the parental species in a local area seriously
affected by pollutants (Neuffer et al. 1999). Introgression
can also facilitate species dispersal. In a study of two eucalyptus species, Potts & Reid (1988) suggest that hybridization could play an important part in the dispersal of a
species if seed transport is limited. Similarly, introgression
was important during the postglacial recolonization of
Europe by Quercus robur and Q. petraea (Petit et al. 1993;
Dumolin-Lapègue et al. 1997; Petit et al. 2001), Q. petraea
taking advantage of the good colonizing ability of Q. robur
seeds. Introgression can also have detrimental effects
when cultivars that are highly specialized to artificial agricultural conditions hybridize with their wild relatives and
the wild species are ‘drowned’ by the genetic material of
the cultivars (Allendorf et al. 2001; Wolf et al. 2001).
Betula has long been known for its high levels of hybridization. Hybrids between the three species studied here,
two diploid species, the silver birch (Betula pendula Roth.,
2n = 28) and the dwarf birch (B. nana L., 2n = 28), and one
168 A . E . P A L M E E T A L .
tetraploid species, the downy birch (B. pubescens Ehrh.,
2n = 56), are mentioned in most floras ( Jonsell 2000; Tutin
et al. 1964) and it is also suggested that the subspecies B.
pubescens ssp. tortuosa (Ledeb) Nyman (mountain birch)
inherited its characteristic traits from B. nana via introgression (Elkington 1968; Jonsell 2000). The Betula species are
monoecious, wind-pollinated, and have small winddispersed seeds (Jonsell 2000). Both B. pendula and B. pubescens
are trees that have wide distributions in Europe and are
also found in parts of northern Asia (Hegi 1957; Atkinson
1992). Betula nana is a dwarf shrub with a nearly circumpolar distribution (Polunin 1959). It is primarily found at
northern latitudes, but small isolated populations also
occur further south, in Scotland and Central Europe (Tutin
et al. 1964).
There is evidence of barriers to interspecific gene flow in
Betula, for example between B. pendula and B. pubescens
(Hagman 1971) and between B. occidentalis and B. papyrifera
(Williams et al. 1999), but these barriers are only partial.
Experiments show that the success of interspecific crosses
varies extensively among individual pairs, but that crosses
where B. pubescens is the maternal parent are generally
less successful than the reciprocal crosses or interspecific
crosses among diploids (Hagman 1971; Kallio et al. 1983).
Genetic evidence does not only confirm that hybridization occurs, but it also suggests that introgression is
relatively frequent, at least at high latitudes. The fact that
triploid progeny of crosses between B. nana and B. pubescens produce offspring when backcrossed with B. pubescens
(Anamthawat-Jónsson & Tomasson 1990), demonstrates
the potential for introgression and relatively high frequencies of triploid plants have been found (9 and 14%, respectively) in two Icelandic mixed birch forests (B. pubescens
and B. nana). Furthermore, both morphological and genetic
data indicate that bi-directional introgression occurs in
Icelandic populations (Thórsson et al. 2001). In contrast no
triploid descendants of B. pendula and B. pubescens were
found in British forests (Gill & Davy 1983). The morphology of the tetraploid individuals (B. pubescens) in the
British forest was, however, highly variable, ranging from
morphology typical for B. pendula to that regarded as
typical for B. pubescens, and the two species could not
be distinguished with molecular markers (Howland et al.
1995).
The level of hybridization often varies across the distribution range of a given species. Picea rubens and Picea
marina frequently hybridize on a coastal island in Maine,
but hybridization is limited in the mountain populations
on Mount Lafayette and Mount Washington in New
Hampshire (Bobola et al. 1996). In Betula, the extent of
hybridization is suggested to vary with latitude, with
higher hybridization frequencies in the subarctic zone —
Scandinavia, Iceland, the Scottish Highlands, and Alaska —
than in other regions (Kallio et al. 1983). This could be the
result of shorter growing seasons in the north that reduce differences in flowering times among species (Kallio et al. 1983).
In the present study, we investigated cytoplasmic introgression in the three main European Betula species: Betula
pendula, B. pubescens and B. nana. More specifically we
wanted to address the following questions. Do the three
species share chloroplast haplotypes? If this is the case, to
what extent do they share haplotypes and do the three species pairs differ? Is the pattern of haplotype sharing different between the two main regions studied, namely
Scandinavia and Russia? According to Kallio et al. (1983)
increased hybridization in the north should be expected.
Finally, the geographical pattern of the haplotypes is studied and we evaluate which conclusions can be drawn
about past hybridization and introgression between these
species.
Materials and methods
Sampling
Material for this study was collected in forests where
at least two Betula species were present. The two main
sampling areas are northern Scandinavia and European
Russia, but three populations were also sampled outside
these two areas. In total Betula pendula, B. pubescens and/or
B. nana were sampled in 21 locations across Europe
(Table 1 and Fig. 1). In each population an average of eight
individuals per species were sampled, amounting to a total
sample size of 363. To avoid sampling clones or close
relatives the individuals sampled within the same species
were separated by at least 200 m.
DNA extraction
DNA was extracted with a protocol adapted from Doyle
& Doyle (1990). Leaf or bud tissue was ground in liquid
nitrogen and about 10 mg of this powder was added to
800 mL of extraction buffer. The extraction buffer includes
alkyltrimethammonium bromide (20 g/L), ethylenediaminetetraacetic acid (EDTA; 0.02 m, pH 8), Tris–HCl (0.1 m,
pH 8), NaCl (1.4 m) and Polyvinylpyrrolidone (PVP) (10 g/
L), to which 1,4-dithiothreitol was added just before use.
Polymerase chain reaction–restriction fragment length
polymorphism (PCR-RFLP) analysis
The chloroplast primers chosen for this analysis, CD, AS
and TF, were selected because they were variable in B.
pendula (Palmé et al. 2003a). These primers were developed
by Demesure et al. (1995) and Taberlet et al. (1991) from the
complete chloroplast sequences of several other species
and were chosen on the basis of maximum consensus across
species. PCR was performed in 1 × PCR buffer (Fermentas),
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 167–178
H Y B R I D I Z A T I O N A M O N G B I R C H E S 169
Table 1 Sampling locations of Betula pendula, B. pubescens and B. nana. The longitude and latitude do not in all cases correspond exactly to
the sampling location, but to a nearby town
Species
Location
Country
Latitude, Longitude
B. pubescens, B. pendula
Tofta
Brunflo
Lake District
Freiburg
Steinkjer
Borgan
Storjord
Voronez
Overjata
Divja
Langasovo
Korolevo
Konkovo-Bitsevsky
Sweden
Sweden
Great Britain
Germany
Norway
Norway
Norway
Russia
Russia
Russia
Russia
Russia
Russia
57°87′ N, 11°70′ E
63°05′ N, 14°49′ E
54°27′ N, 3°00′ W
47°96′ N, 7°83′ E
64°01′ N, 11°30′ E
64°58′ N, 10°54′ E
66°49′ N, 15°23′ E
51°83′ N, 39°50′ E
58°12′ N, 56°27′ E
58°12′ N, 56°27′ E
58°41′ N, 49°47′ E
56°25′ N, 43°52′ E
55°55′ N, 37°38′ E
B. pubescens, B. pendula, B. nana
Svererka
Russia
56°27′ N, 60°35′ E
B. nana, B. pubescens
Gällivare
Tänndalen
Svansjön
Vauldalen
Saltfjellet
Store Haugfjell
Sweden
Sweden
Sweden
Norway
Norway
Norway
67°08′ N, 20°42′ E
62°33′ N, 12°19′ E
62°33′ N, 12°13′ E
62°40′ N, 12°00′ E
66°45′ N, 15°25′ E
68°27′ N, 17°54′ E
B. pendula, B. nana
Ullatti
Sweden
67°01′ N, 21°49′ E
1.8 mm MgCl2 (Fermentas), 0.2 mg/µL bovine serum albumin
(Fermentas), 0.1 mm dNTP (Roche), 0.2 µm of each primer
and 0.016 units/µL Taq DNA polymerase (Fermentas). A
touch-down PCR program was used for the amplification:
an initial cycle of 4 min at 94 °C, then 14 –20 cycles of 45 s
at 94 °C, 45 s at 57/51 °C decreasing 0.5 °C with each cycle
to 50/41 °C, 3/4 min at 68/70 °C, then 15–20 cycles of 45 s
at 94 °C, 45 s at 50/41 °C, 3/4 min at 68/70 °C and finally
10 min at 68/70 °C. Specific annealing temperatures (touchdown range), elongation times and temperatures were as
follows for the three fragments: CD 57–50 °C, 4 min, 70 °C;
AS 57–50 °C, 4 min, 68 °C; TF 51–41 °C, 3 min, 68 °C.
The following combinations of PCR fragments and
restriction enzymes were analysed: CD HinfI, CD TaqI, AS
TaqI, TF HinfI and TF TaqI (known to be variable in a rangewide study of B. pendula (Palmé et al. 2003a). The restriction
reactions contained 15 µL PCR product, 2 µL H2O,
1 × buffer (Fermentas) and 3 U enzyme. After mixing these
components the tubes were placed over night at a temperature optimal for each restriction enzyme: 37 °C for HinfI,
and 65 °C for TaqI. After the restriction, 15 µL of stop-solution
(formamide with 3 mg/mL xylencyanol, 3 mg/mL bromophenol blue and 10 mm EDTA pH 8) was added to each
tube. Just before loading the restriction mix was heated
to 85 °C for 4 min and then placed on ice. Of this mix 2.4 µL
was loaded onto the gel. The restriction fragments were
analysed on a denaturing 6% acrylamide gel (43 cm × 35 cm
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 167–178
× 0.4 mm), which were run at 2000 V for 5000–6000 Volthours in a 1 × TBE buffer. The gel was then silver-stained and
scanned.
Data analysis
Calculations of gene diversity within each population,
hierarchical analysis of molecular variance, exact test of
population differentiation and construction of a minimum
spanning tree were performed using the arlequin 2.000
package (Schneider et al. 2000). Comparisons of species
were made for the species pairs B. pendula/B. pubescens
and B. nana/B. pubescens, while the B. pendula/B. nana pair
was excluded because these two species only co-occurred
in two populations and the sampling size would therefore be too small to draw any meaningful conclusions.
To test for the presence of phylogeographic structure
within species, GST and NST, were calculated according to
Pons & Petit (1996) and a test to determine if NST was
significantly larger than GST was performed according
to Burban et al. (1999) with the program permut 2 (http://
www.pierroton.inra.fr/genetics/labo/software). If NST,
which takes the genetic differences between the haplotypes
into account, is higher than GST, this indicates the presence
of a phylogeographic structure (Pons & Petit 1996), i.e. closely
related haplotypes are more often found in the same
geographical area than would be expected by chance.
170 A . E . P A L M E E T A L .
Borgan
YQ
Fig. 1 Geographical distribution and frequencies of chloroplast haplotypes (A, B, C, D, F, H, S, T, Q, Y). For each population the haplotype
frequencies are given for the pair of species occurring at this location. () indicates that the left-hand column gives the haplotype
frequencies in Betula pendula and the right-hand one those in B. nana. Similarly, () is associated with the haplotype frequencies of B.
pubescens (on the left) and B. nana (on the right) and finally, () with those of B. pubescens and B. pendula. In the population Severka the three
columns correspond, from left to right, to B. pubescens, B. pendula and B. nana. Local haplotypes such as F, R, S, Y, Q are indicated.
The introgression ratio, IG, and the expected introgression ratio, IGe, were calculated according to Belahbib et al.
(2001) [see also Dumolin-Lapègue et al. (1999) for additional information]. The introgression ratio, IG, reflects the
amount of locally shared haplotypes between two species
and is expected to be one when there is no difference
between the species and zero when they are totally different. The expected introgression ratio, IGe, is the expected
value if the sharing of haplotypes is not geographically
structured. Despite the name ‘introgression ratio’ these
ratios simply mirror the similarity between two species
and do not on their own say anything about the reason for
this similarity (convergence, ancestral polymorphism, or
introgression). The standard errors and confidence intervals of the IG values were estimated by nonparametric
bootstrapping, with the programme boot in the r package
(Ihaka & Gentleman 1996). Bootstrap samples were
obtained by resampling 1000 individuals within the subpopulations of each species. 95% confidence intervals were
obtained using the bias corrected and accelerated (BCa)
method (Efron 1987; Carpenter & Bithell 2000).
To assess better the geographical scale on which sharing
of haplotypes occurs a new parameter was defined:
IGR = IG(xi,xj)/IG(xi,yi)
where IG(xi, xj) is the ‘introgression ratio’ among populations of species x (called the focal species) in forest i
and in forest j, and IG(xi,yi) is the introgression ratio between species x and y in forest i. If the ratio is larger than
one then individuals of species x in forest i are more
similar to individuals of the same species in forest j than to
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 167–178
H Y B R I D I Z A T I O N A M O N G B I R C H E S 171
Table 2 The PCR-RFLP haplotypes, designated by capital letters, and the variable restriction fragments associated with each haplotype
(columns)
CD HinfI
CD TaqI
AS TaqI
TF HinfI
TF TaqI
Haplotype
Found in*
1
2
3
5
6
8
1
2
3
1
1
2
1
2
A
B
C
D
F
H
Q
S
T
Y
BP, BPU, BNA
BP, BPU, BNA
BP, BPU, BNA
BP, BPU
BPU
BP
BP
BP
BP, BPU, BNA
BP
1
1
1
1
1
1
1
1
2
1
1
2
1
1
1
2
1
1
1
1
2
2
1
1
1
2
1
1
2
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
2
1
1
1
1
1
3
3
1
1
1
2
1
1
3
1
1
1
1
1
3
1
1
1
1
1
1
2
1
1
1
2
1
1
1
1
1
2
3
4
4
2
4
4
1
3
1
1
1
3
1
1
4
4
1
5
1
2
1
1
1
2
1
1
1
1
1
2
1
1
1
2
1
1
1
1
1
1
1
1
1
1
3
3
1
4
The numbers (1–5) in each column signify the different states found of each restriction fragment, that is the different positions of each band
on the gel
*BP, Betula pendula; BPU, B. pubescens; BNA, B. nana.
individuals of the other species (y ) in the same forest (i).
We then plotted IGR over the geographical distance that separated the forests. The significance of the relationship was
tested by Mantel test. This parameter was calculated for
populations containing B. pendula and B. pubescens, but not
for the other species pairs because of low sample sizes.
Results
Molecular variation
Variation was identified with all five primer–enzyme
combinations. The CD fragment was the most variable,
displaying six and three polymorphic fragments when cut
with HinfI and TaqI, respectively (Table 2). TF was less
variable, but two polymorphic fragments showed variation
when cut with both enzymes. The AS primer pair only displayed one polymorphic fragment. The variation in these
markers allowed for the definition of 10 haplotypes (Table 2).
All haplotypes are rather similar and they are all fairly
closely related to the two most common haplotypes, A and
C (Fig. 2).
Hierarchical partitioning of variation: within and among
species
Most of the variation found in Betula pubescens and B.
pendula can be attributed to variation within populations
(68–74%), but there is also considerable variation among
populations with FST equal to 0.26 and 0.32, respectively. In
B. nana, on the other hand, most of the variation could be
attributed to differentiation between populations (FST = 0.58).
When two or three species were considered simultaneously, most of the variation was found within populations
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 167–178
Fig. 2 Minimum spanning tree based on chloroplast PCR-RFLP.
The sizes of the circles are roughly proportional to the haplotype
frequencies. Haplotypes found in Betula pendula (Palmé et al.
2003a) in populations that are not included in this study are
symbolized by dotted circles. Haplotypes that are only found in
one species are marked with grey (B. pubescens) or black (B. pendula).
or between populations within species, but species were
not significantly differentiated as shown by values of FCT
(proportion of variation that can be attributed to differentiation between species) that are never significantly different
from zero (Table 3). The differentiation between the two
main regions analysed, Scandinavia and Russia, was also
investigated. When all three species were included 11% of
the variation was the result of differentiation between
these two regions (27% among populations within regions,
62% within populations).
172 A . E . P A L M E E T A L .
Table 3 Hierarchical partitioning of variation among the species
Species
No. of
locations
Percentage of
variation within
populations
Percentage of
variation among
populations
Percentage of
variation among
species (FCT)
Betula pubescens/B. nana
B. pendula/B. pubescens
7
14
65%
66%
39%
35%
−3.5%NS
−1.1%NS
NSNot
significant (P = 0.75 and 0.52, respectively). Tested using a nonparametric permutation method according to Excoffier et al. (1992).
Geographic structure
Haplotypes A and C are present in all three species over
most of the investigated range (Fig. 1, Table 4). The other
haplotypes and haplotype groups are more restricted
geographically. The closely related haplotypes H and B
are only found in northwestern Europe and haplotype
T is restricted to northern Fennoscandia. Haplotype D is
present both in Scandinavia and Russia while the haplotypes
S and Q are only found in Russian populations close to the
Ural Mountains.
Comparisons of NST and GST did not reveal any significant phylogeographic structure. NST was not significantly
larger than GST in any of the three species (B. nana, GST =
0.538, NST = 0.539; B. pubescens, GST = 0.288, NST = 0.252;
B. pendula, GST = 0.383, NST = 0.425). NST and GST were not
significantly different when the three data sets were combined either. This is both true if different species from the
same location were treated as different populations and
when they were treated as the same population. The
population introgression ratios, IGs, were not significantly associated with either latitude or longitude (data
not shown). Neither was the correlation between withinpopulation level of variation (gene diversity and number
of alleles) and latitude significant (data not shown).
Sharing of haplotypes among species
The common haplotypes are shared among the three species
(Fig. 3). The most common haplotype, C, dominates all three
species, 50%, 59% and 65% of the investigated individuals
carry this haplotype in B. pendula, B pubescens and B. nana,
respectively. A couple of less common, but still frequent,
haplotypes, A, B and T, are also found in all three species.
Haplotype D, which is as common as B (3%), is shared only
between B. pendula and B. pubescens, but was absent in B.
nana. The other haplotypes are all rare (less than 2%), geographically restricted, and only observed in one species.
This general sharing of haplotypes among the species is
accompanied by a tendency for populations to share the
commonly occurring haplotypes in a given region. This is
particularly clear with haplotype T, which is only present
in northern Fennoscandia and is found in all three Betula
species in this area. In the Russian populations west of the
Urals haplotype C is the most common haplotype in populations of both B. pendula and B. pubescens while A seems
to be most common in the British Isles. Haplotype B occurs
only in a few western European populations, but it is
present in all three species.
This pattern of sharing the regionally common haplotypes is also, although perhaps only partly, mirrored at the
more local scale. The samples of different species from the
same forest always share some haplotypes, but usually not
all and the frequencies may differ. An exact test showed
that B. pendula and B. pubescens differed significantly with
respect to haplotype composition in two (14%) of the investigated forests while B. nana and B. pubescens were different
in three of the seven comparisons performed (43%). This
can be compared with the differentiation within species
where 34%, 49% and 54% of the population pairs were significantly different in B. pubescens, B. pendula and B. nana,
respectively. Nonetheless, the introgression ratios, IGs
(Table 5) are larger than zero in both species comparisons,
indicating that there is local sharing of haplotypes.
The tendency for B. pubescens to differ more from B. nana
than from B. pendula is also mirrored in the introgression
ratios (Table 5). Since there are differences between the
regions studied, the same comparisons were done within
Scandinavia, the only region where B. nana was sampled to
a sufficient extent. Within this region the introgression
ratio was larger between B. pubescens and B. nana than
between B. pubescens and B. pendula, but the 95% confidence intervals are overlapping (Table 5).
Constraints to free gene flow may contribute to the patterns in Fig. 4. Both when B. pendula is the focal species
(Fig. 4a) and when B. pubescens is the focal species (Fig. 4b)
the correlation between IGR and distance was significantly
negative (P < 0.001, 0.005, respectively). The decrease in
IGR with distance was most pronounced when B. pendula
was the focal species, and a larger number of high IGR
values were observed at small distances in B. pendula than in
B. pubescens. At short distances, individuals from the focal
species were genetically closer to individuals of the same
species from neighbouring forests than from individuals of
the other species. As distance increases between the populations of the same species, they become genetically more
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 167–178
H Y B R I D I Z A T I O N A M O N G B I R C H E S 173
Table 4 Haplotype distribution
Haplotypes
Location
Species*
N
n
H
A
B
C
D
F
H
S
T
Q
Y
Tofta
BPU
BP
BPU
PB
BPU
BP
BPU
BP
BPU
BP
BPU
BP
BPU
BP
BPU
BP
BPU
BP
BPU
BP
BPU
BP
BPU
BP
BPU
BP
BPU
BP
BNA
BPU
BNA
BPU
BNA
BPU
BNA
BPU
BNA
BPU
BNA
BPU
BNA
BNA
BP
9
12
11
10
7
10
4
8
7
2
3
3
10
7
10
12
11
12
10
11
4
12
10
11
6
11
10
10
11
10
9
4
4
9
9
10
7
7
8
10
9
3
10
2
4
4
3
1
2
2
3
3
1
3
1
2
2
1
1
1
2
1
3
2
2
2
2
1
2
2
3
1
3
1
2
1
3
1
3
2
2
4
2
2
2
3
0.5
0.71
0.75
0.73
0
0.53
0.5
0.61
0.67
0
1.00
0
0.56
0.29
0
0
0
0.55
0
0.47
0.5
0.53
0.47
0.33
0
0.18
0.47
0.62
0
0.64
0
0.67
0
0.42
0
0.62
0.57
0.29
0.75
0.53
0.22
0.67
0.51
—
6
—
4
7
6
—
2
2
2
1
3
5
6
—
—
—
—
—
—
1
7
3
2
—
1
—
6
—
5
—
2
—
1
—
2
4
1
2
—
—
—
2
—
1
1
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—
4
—
—
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—
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—
—
4
—
—
—
—
6
3
3
3
—
—
3
5
1
—
—
—
5
—
10
12
11
6
10
8
3
5
7
9
6
10
7
2
11
4
9
2
4
7
9
6
3
6
1
6
1
1
7
3
2
2
—
—
—
—
—
—
—
1
—
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1
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1
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1
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6
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—
—
—
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—
—
—
5
3
—
—
—
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4
—
1
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—
1
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—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1
—
2
—
—
1
4
8
2
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
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—
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3
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Brunflo
Lake District
Freiburg
Steinkjer
Borgan
Storjord
Voronez
Overjata
Divja
Langasovo
Korolevo
Konkovo-Bitsevsky
Svererka
Gällivare
Tänndalen
Svansjön
Vauldalen
Saltfjellet
Store Haugfjell
Ullatti
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
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—
—
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—
—
—
—
—
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—
—
—
—
—
—
No.
shared
alleles
Exact
test
Population
IG ± SE
2
+
0.56 ± 0.217
2
−
0.84 ± 0.239
1
−
0.82 ± 0.172
1
−
1.05 ± 0.221
1
−
0.43 ± 0.232
1
−
0.67 ± 0.380
1
−
0.74 ± 0.200
1
−
1
1
+
0.69 ± 0.185
1
−
0.95 ± 0.092
2
−
0.95 ± 0.278
2
−
1.04 ± 0.126
1
1
0
−
−†
+‡
+§
1.00 ± 0.090
0.31 ± 0.192†
0.29 ± 0.190‡
0.91 ± 0.105§
1
+
0.59 ± 0.207
1
−
0.75 ± 0.301
1
−
0.98 ± 0.091
2
−
0.92 ± 0.235
2
+
0.30 ± 0.218
2
−
0.68 ± 0.239
2
−
0.73 ± 0.413
N, the number of individuals; n, number of haplotypes found in a population (for haplotype definitions see Table 2); H, gene diversity; IG,
introgression ratio. Exact test (according to Raymond & Rousset 1995): + indicates a significant difference between the two species in allele
distribution (significance level 0.05). The standard error of the IG coefficient was estimated by bootstrapping.
*BP, Betula pendula; BPU, B. pubescens; BNA, B. nana.
†Comparison between B. pendula and B. pubescens.
‡Comparison between B. pendula and B. nana.
§Comparison between B. nana and B. pubescens.
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 167–178
174 A . E . P A L M E E T A L .
Table 5 Introgression ratios (IG and IGe) between Betula pendula and B. pubescens and between B. nana and B. pubescens in different
geographical regions (mean value and 95% confidence interval within parenthesis)
Species pair
B. pubescens/B. pendula
B. pubescens/B. nana
All populations
Scandinavia
Russia
IG
0.791 (0.7436–0.8954)
0.619 (0.4155–0.9228)
IGe
0.616 (0.6059–0.6841)
0.596 (0.5657–0.7218)
0.847* (0.7638– 9657)
0.937† (0.8529– 1.0160)
0.770* (0.7100–0.9000)
IG
IGe
0.671 (0.5466–0.8868)
0.750 (0.7053–0.8949)
0.717 (0.5724–0.9321)
0.595 (0.5501–0.7377)
—‡
—‡
The confidence intervals were estimated by bootstrapping.
*With population Severka.
†Without population Severka.
‡Only one location with B. nana and B. pubescens was studied in this area.
Fig. 3 Distribution of chloroplast haplotypes in Betula pubescens,
B. nana and B. pendula. The frequency refers to the haplotype
frequency in each species separately.
different from each other, resulting in IGRs below one. In
B. pubescens the trend at all distances is for the IGR to be
below one, indicating that B. pubescens is generally more
similar to sympatric B. pendula than to B. pubescens from
other forests.
separated by one or two mutations (Fig. 2, Table 2). Assuming neutrality and similar mutation rates, this suggests that
the gene genealogies for the various species were of similar
lengths.
Because of extensive haplotype sharing, geographical
location rather than species was the main factor determining the haplotype composition of a population.
Although 11% of the total variation could be attributed to
differences between the two main regions studied (Russia
and Scandinavia), none of it could be attributed to differences between the species. The geographical distribution
of chloroplast haplotypes in European B. pendula was recently
studied and a strong geographical component, caused by
differences between regions, was identified (Palmé et al.
2003a). There was however, no significant isolation by distance pattern (Palmé et al. 2003a). The postglacial history of
the species was suggested to have played an important role
in shaping the genetic structure and this is probably also
true for the other two Betula species. The geographical pattern found in B. pubescens and B. nana in this study largely
resembles the geographical pattern found in B. pendula.
Why do the Betula species share haplotypes?
Discussion
Overall genetic variation and geographical structure
The level of variation in chloroplast DNA found within the
three Betula species is similar to that found in many single
plant species (e.g. Calluna, Rendell & Ennos 2002; Hedera,
Grivet & Petit 2002). In a large-scale study of B. pendula, 13
haplotypes were identified (Palmé et al. 2003a). Only two
new haplotypes (F and Y) were found when B. pubescens
and B. nana were added to the analysis, showing that the
variation found in one of the Betula species includes most
of the variation found in the others. As is also the case in
other species from the same geographical area (Grivet &
Petit 2002; Rendell & Ennos 2002), the Betula haplotypes were very similar to one another and generally only
There are three main causes of species sharing haplotypes: (i) convergence, (ii) ancestral polymorphism, or (iii)
hybridization/introgression. Convergence appears to be
the least likely explanation in this case as identical mutations are rare events, and all the haplotypes occurring in
more than 2% of the individuals are shared among the
species. Ancestral polymorphism is possible, but unlikely
to suffice to explain the observed pattern. Haplotypes A
and C are probably the most ancient haplotypes because
they are the most common (Watterson & Guess 1977) and
are therefore the most likely to have been present in the
common ancestor of the three Betula species. However,
common ancestry is much less likely for shared haplotypes
that are both less frequent and more peripheral in the
haplotype network, such as haplotypes B, D and T.
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 167–178
H Y B R I D I Z A T I O N A M O N G B I R C H E S 175
Fig. 4 Relationship between IGR and geographical distance (measured in km) when (a) Betula pendula is the focal species and when
(b) B. pubescens is the focal species. The local regression line is shown.
The fact that the haplotypes, both common and rare
ones, show similar geographical patterns in all three species is a strong argument for the presence of recent, or at
least postglacial, hybridization. If the species only shared
haplotypes as a result of ancestral polymorphism their
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 167–178
geographical patterns should be independent of each
other. In regions where one of the common haplotypes is
dominating, such as the British Isles (A) and western Russia (B), it is common in both species investigated (Fig. 1).
The rare haplotype T can be found in northern Scandinavia
in all three species, but not in any of the other areas investigated in this study or in a geographically more extensive
study on B. pendula (Palmé et al. 2003a). Similarly, haplotype B is present in all three species, but it is restricted to
northwestern Europe and haplotype D is only found in
Russia and Sweden but in two different species. In fact
none of the haplotypes that are shared among species are
found in different geographical regions in different species. Such a pattern is most likely to be caused by local
hybridization resulting in gene flow among the species.
This argument is strengthened by the fact that IGR tend to
be below one (Fig. 4), which means that B. pendula and
B. pubescens from the same location tend to be more similar
to each other than to individuals of the same species at a
different location.
Another factor that argues for hybridization being a
more important cause of the haplotype sharing than ancestral polymorphism, is that geography is more important in
influencing the haplotype composition of a population
than species identity. Eleven per cent of the variation could
be attributed to differences between Russia and Scandinavia but none to differentiation among the species. A reproductive isolation among the species would most likely
result in allele frequency differences between the species, if
not in a more pronounced phylogenetic differentiation.
Taken together, the arguments above indicate that a
local transfer of haplotypes between the species (hybridization) is the main cause of the haplotype sharing. There is
also plenty of independent evidence that hybridization
does occur between the three Betula species of this study
(see Introduction and Anamthawat-Jónsson & Tomasson
1990; Tutin et al. 1964; Jonsell 2000; Thórsson et al. 2001;
Anamthawat-Jónsson 2003). The analysis of the IGR suggests that introgression is somewhat asymmetrical (Fig. 4),
indicating that introgression from B. pendula to B. pubescens
is more common than introgression in the opposite direction. Only in a very few cases was B. pubescens more similar
to neighbouring B. pubescens than to sympatric B. pendula
while the equivalent situation was much more frequent in
B. pendula. This result seems consistent with the asymmetric direction of cross-compatibility observed between
B. pendula and B. pubescens, B. pendula × B. pubescens crosses
being generally less successful when B. pubescens is the
maternal parent in such crosses. It should therefore be relatively easier for B. pubescens to capture B. pendula cytoplasm
through ‘the pollen swamping’ mechanism proposed to
explain local chloroplast DNA haplotype sharing between
Quercus robur and Q. petraea (Petit et al. 2001) than for B.
pendula to do so.
176 A . E . P A L M E E T A L .
In tetraploid species there is an additional process that
can result in sharing of haplotypes between the tetraploid
and its parent species. When a tetraploid species is created
it will of course share alleles with its parent species but in
time they will look increasingly different, making this
process similar to ancestral polymorphism discussed
above and therefore not likely to be the main cause of the
haplotype sharing among the birch species. However, this
description only holds if the tetraploid species originated
in one event or in a limited number of events that were
restricted in time but if there is a recurrent formation of the
tetraploid species, alleles will be transferred from the parent species in each of these events. There is increasing evidence in the literature that many species have multiple
origins and that this is actually more common than single
origins (Soltis & Soltis 1993; van Dijk & Bakx-Schotman
1997; Segraves et al. 1999).
There are several hypotheses about the origin of B. pubescens, but they generally involve B. pendula. It has been suggested that B. pubescens is an allotetraploid with B. pendula
as one parent and possibly B. humilis or a now extinct Betula
species as the other parent (see Howland et al. 1995). Alternatively B. pubescens could be an autotetraploid of B. pendula, but the question of the origin of B. pubescens is not yet
resolved. If B. pubescens originated from B. pendula, and if
it has several local origins across Europe, this will of course
increase the similarity of the two species. Multiple origins
have been suggested as the cause of extensive haplotype
sharing among diploids and polyploids in other species,
for example in Heuchera grossulariifolia (Segraves et al.
1999), but a single origin and subsequent hybridization
could potentially cause similar patterns of haplotype sharing as multiple origins and limited or no hybridization.
The two scenarios are therefore difficult to separate, and in
the case of B. pubescens and B. pendula a combination of
multiple origins of B. pubescens and hybridization between
the two species cannot be excluded.
Introgression levels
The introgression ratio, IG, which mirrors the amount
of locally shared haplotypes, is of the same order of
magnitude between B. nana and B. pubescens, and Quercus
suber and Q. ilex (IG = 0.63), which were also studied with
chloroplast DNA PCR-RFLP markers (Belahbib et al. 2001).
Similar IG values were also found between B. pendula and
B. pubescens, and Q. robur and Q. pubescens (IG = 0.82,
Belahbib et al. 2001). However both are lower than those
found in two other pairs of oak species: Q. robur/Q. petraea
(0.97) and Q. petraea/Q. pubescens (0.96) (Belahbib et al.
2001) and between Salix caprea and S. cinerea (1.05) (Palmé
et al. 2003b). In all these cases the pattern of haplotype
sharing was explained by extensive introgression between
the species. Since the introgression ratio has only recently
been defined it has not been widely estimated and it is
difficult to compare the extent of haplotype sharing and
potential hybridization among different studies. There are
a number of other studies though, that show that chloroplast DNA haplotypes are shared among species (e.g.
Wagner et al. 1987; Terry et al. 2000; Bleeker & Hurka 2001)
and that the amount of sharing varies extensively from
case to case. Introgression can, in contrast to the examples
discussed above, be very restricted geographically, as
between lodgepole pine (Pinus contorta) and jack pine (P.
banksiana). In these species a few individuals combining
the morphology of one of the two species and the chloroplast
haplotype of the other are confined to a restricted area of
sympatry (Wagner et al. 1987).
While IG values were of the same order of magnitude in
Betula and in Quercus the expected introgression ratios
(IGe) were much higher in the former than in the latter. IGe
ranged from 0.26 to 0.36 in the different Quercus pairs studied by Belahbib et al. (2001) while in this study the overall
IGe were 0.62 and 0.75 for B. pubescens/B. pendula and B.
pubescens/B. nana, respectively. IGe is based on the overall
frequencies in each species, and not as IG on the average of
the interspecific identities of the mixed-species forests. IGe
therefore does not take into account the geographical
structure of haplotype sharing. Consequently a high value
of IG relative to IGe would suggest that haplotype sharing
is the result of the species’ sympatry, whereas similar values
would suggest that the current sympatry of the species is not
crucial to the genetic similarity of the species.
Introgression ratios were high and did not differ much
between geographical areas or species (Table 5). There
were some trends, however, because the confidence intervals were in most cases large and overlapping the interpretation of these trends should be made cautiously. Kallio
et al. (1983) suggested that hybridization in Betula would
be more common in the north than in the south because the
shorter growing seasons bring the flowering times closer
together. However, no significant relationships were
found between IG ratios and latitude and the introgression
ratio among B. pendula and B. pubescens was, if anything,
higher in Russia than in Scandinavia (Table 5). It should
however, be noted that the two main groups of populations representing high and low latitudes probably had
different histories, for example the higher introgression
ratio in Russia may simply reflect a longer period of sympatry between the two species.
Conclusions
In this study we have investigated the maternal lineages
of B. pendula, B. pubescens and B. nana and shown that
they are extensively shared among the species. Hybridization and cytoplasmic introgression are suggested as the
main causes of the pattern. However, it is not advisable to
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 167–178
H Y B R I D I Z A T I O N A M O N G B I R C H E S 177
extrapolate this information to include the nuclear genome.
There are naturally some morphological differences between
the three species, especially between the shrub species
B. nana and the two tree species, and even though these
differences are not apparent in the chloroplast genome
they should be mirrored by at least some nuclear genes. To
estimate the total amount of genetic similarity among the
species a number of unlinked genetic markers will be
essential.
Acknowledgements
We thank Vladimir Semerikov for help with sampling in Russia.
We also thank all the members of the CYTOFOR project for invaluable assistance with sampling. The study has been carried out
with financial support from the Commission of the European
Communities, Agriculture and Fisheries (FAIR) specific RTD programme, CT97–3795, ‘CYTOFOR’ and the Carl Tryggers Foundation. Su Qiao was supported by a grant from the Swedish Institute.
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Anna Palmé is a researcher at Uppsala University working with genetic variation in natural plant populations and Su Qiao is a researcher at the Dalian University of Technology. Snæbjörn Pálsson is
a researcher at the University of Iceland, working mainly on genetic
variation in marine fish populations. Martin Lascoux is a population geneticist at Uppsala University working primarily with plants.
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 167–178