Molecular Ecology (2005) 14, 2739–2753
doi: 10.1111/j.1365-294X.2005.02621.x
Impact of ice ages on circumpolar molecular diversity:
insights from an ecological key species
Blackwell Publishing, Ltd.
I . G . A L S O S ,*‡ T . E N G E L S K J Ø N ,* L . G I E L L Y ,† P . T A B E R L E T † and C . B R O C H M A N N ‡
*Tromsø Museum, University of Tromsø, NO-9037 Tromsø, Norway, †Laboratoire d’Ecologie Alpine, CNRS UMR 5553, Université
Joseph Fourier, BP 53, F-38041 Grenoble cedex 09, France, ‡National Centre for Biosystematics, Natural History Museum, University
of Oslo, PO Box 1172 Blindern, NO-0318 Oslo, Norway
Abstract
We address the impact of the ice age cycles on intraspecific cpDNA diversity, for the first
time on the full circumboreal-circumarctic scale. The bird-dispersed bog bilberry (or arctic
blueberry, Vaccinium uliginosum) is a key component of northern ecosystems and is here
used to assess diversity in previously glaciated vs. unglaciated areas and the importance of
Beringia as a refugium and source for interglacial expansion. Eighteen chloroplast DNA
haplotypes were observed in and among 122 populations, grouping into three main lineages which probably diverged before, and thus were affected more or less independently
by, all major glaciations. The boreal ‘Amphi-Atlantic lineage’ included one haplotype
occurring throughout northern Europe and one occurring in eastern North America, suggesting expansion from at least two bottlenecked, glacial refugium populations. The boreal
‘Beringian lineage’ included seven haplotypes restricted to Beringia and the Pacific
coast of USA. The ‘Arctic-Alpine lineage’ included nine haplotypes, one of them fully
circumpolar. This lineage was unexpectedly diverse, also in previously glaciated areas, suggesting that it thrived on the vast tundras during the ice ages and recolonized deglaciated
terrain over long distances. Its largest area of persistence during glaciations was probably
situated in the north, stretching from Beringia and far into Eurasia, and it probably also
survived the last glaciation in southern mountain ranges. Although Beringia apparently
was important for the initial divergence and expansion of V. uliginosum as well as for continuous survival of both the Beringian and Arctic-Alpine lineages during all ice ages, this
region played a minor role as a source for later interglacial expansions.
Keywords: Arctic-Alpine, boreal, chloroplast DNA, glacial refugia, molecular diversity, Vaccinium
uliginosum
Received 29 November 2004; revision received 22 March 2005; accepted 12 April 2005
Introduction
The contemporary genetic make-up and diversity within
individual species can be used as signatures to infer the
effects of the recurrent large-scale climate changes that
occurred during the Quaternary (Hewitt 1996, 2004; Avise
2000; Schaal & Olsen 2000). Such changes have repeatedly
induced shifts in the distribution of species and extinction
of populations and genotypes. Isolation in refugia during
the long-lasting ice ages and expansions during the shortterm interglacials have resulted in conspicuous differences
Correspondence: Inger Greve Alsos, Fax: +47 22 85 18 35; E-mail:
[email protected]
© 2005 Blackwell Publishing Ltd
among geographical areas in levels and spatial distribution
of intraspecific diversity.
Preserving biodiversity requires knowledge of its
geographical distribution as well as the mechanisms that
sustain and develop it over long periods of time (Cowling
& Pressye 2001). A particular challenge is to identify Quaternary glacial refugia, which may represent long-term reservoirs of a species’ genetic variation where evolution has
produced unique genotypes and high levels of diversity
(Willis & Whittaker 2000; Liepelt et al. 2002; Taberlet &
Cheddadi 2002). Temperate plants and animals often have
low diversity in formerly glaciated northern areas and
high diversity in their southern refugia, which served as
sources for inter- and postglacial expansions (Soltis et al.
2740 I . G . A L S O S E T A L .
1997; Taberlet 1998; Hewitt 1999; Petit et al. 2002). Typically, repeated bottlenecks during ‘leading edge’ colonization after deglaciation led to reduced genetic diversity
northwards in temperate organisms, except in areas where
lineages expanding from different refugia met (Hewitt
1996, 1999).
Much less is known about organisms with more northern present-day distributions. Many northern plant species
in particular have enormous ranges, spanning the entire
circumboreal and/or circumarctic regions and also occurring
in more southern mountains, thus presenting considerable logistic obstacles for representative sampling of intraspecific diversity. From the fossil record it is known that
several such species were distributed at lower latitudes
during the glaciations. However, large high-latitude areas
in northern Asia and northwestern North America were
never or only partly glaciated (Fig. 1), and there is now
good evidence that the Beringian region, especially, served
as a major northern refugium for several boreal and arctic
plants and animals (Goetcheus & Birks 2001; Abbott &
Brochmann 2003). However, so far not a single circumboreal
plant species and only two nondominant circumarctic ones,
Saxifraga oppositifolia (Abbott et al. 2000; Abbott & Comes
2004) and Saxifraga cernua (cf. Bronken in Brochmann et al.
2003), have been studied for molecular variation in the
entire distributional range. In S. oppositifolia, there is divergence between Beringian and European/amphi-Atlantic
lineages dating back to Pliocene (Abbott & Comes 2004).
A similar divergence has been found in several arctic
small mammals dating back 0.1–1.0 million years ago (Ma)
(Hewitt 2004). However, even if Beringia served as a
major northern refugium, its role as a source for interand postglacial expansions is poorly known. Whereas the
two saxifrage species as well as several animals, for example,
probably expanded out of Beringia, two lemming species
apparently did not (Fedorov & Stenseth 2002; Fedorov et al.
2003).
We selected Vaccinium uliginosum L. sensu lato (bog bilberry, also named arctic blueberry) as an ecologically significant representative of the plant species that currently
occur throughout both the circumboreal and the circumarctic regions (Fig. 1). It also occurs in many southern
mountain areas and often plays a dominant role in several
boreal, arctic and alpine ecosystems, ranging from boreal
mires, shrublands, forests, and dry uplands to arctic and
alpine tundras, heaths, and ridges. Vaccinium uliginosum is
a long-lived, insect-pollinated, mainly outcrossing, and
animal/bird-dispersed shrub (Vander Kloet 1988; Jacquemart 1996). Some 30 taxa at various taxonomic levels have
been described in this morphologically variable, diploid–
polyploid complex, but it is now usually recognized as a
single species (but with a disputed number of subspecies,
see Hultén 1970; Young 1970; Vander Kloet 1988). Differences in ploidal level have been used for taxonomic
distinction even at the species level, but we have recently
shown that tetra- and hexaploids probably originated
repeatedly at different scales in time and space (Alsos 2003;
Brochmann et al. 2004). Fossil records have shown that
V. uliginosum occurred south of its present range during
the late Wisconsian/Weichselian (e.g. Lower Peninsula of
Michigan 12 500 –13 300 bp, Miller & Benninghoff 1969), and
some of its northernmost present outposts were probably
reached during the early Holocene (Greenland, 10 500 bp,
Bennike 1999) or during the Holocene hypsithermal (the
Arctic archipelago of Svalbard, Alsos et al. 2002).
We have addressed the impact of repeated isolations
in glacial refugia and interglacial expansions in this species by examining chloroplast DNA (cpDNA) variation in
field-sampled populations from throughout the geographical distribution, supplemented by herbarium material
from various museums. We have focused on the potential
differences in molecular diversity among arctic, alpine,
and boreal populations in previously glaciated vs. nonglaciated areas, on the location of refugia, and on the role of
Beringia as a northern refugium and source for interglacial
expansions.
Materials and methods
Materials
Fresh leaves were collected from each of five samples from
71 populations and dried in silica gel. Leaves were also
sampled from herbarium (69 populations) and cultivated
material (eight populations). These 148 populations were
analysed for two cpDNA regions and sequences were
obtained from 122 populations, representing the entire
distribution range (Fig. 1, Appendix). Sequencing of one
or both cpDNA regions was successful for 86%, 48%,
and 0% of herbarium material collected 1–30, 31–50, and
71–90 years ago, respectively. All populations analysed
for cpDNA variation in this study have previously been
analysed for variation in morphology and ploidy (Alsos
2003). We used Vaccinium myrtillus (section Myrtillus) and
Vaccinium vitis-idaea (section Vitis-idaea) as outgroups based
on information in Vander Kloet (1988), who placed
Vaccinium uliginosum in the monotypic section Vaccinium
(traditionally placed in section Myrtillus). Leaves from
outgroup species were collected in Belledonne, France,
and dried in silica gel.
cpDNA analysis
DNA was extracted with the DNeasy Plant Mini Kit
(QIAGEN) following the manufacturer’s instructions.
Noncoding cpDNA regions were initially amplified and
sequenced for five samples using six primer pairs (c – d and
e –f from Taberlet et al. 1991), and trnH-psbA, trnS-trnG,
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 2739–2753
C I R C U M P O L A R M O L E C U L A R D I V E R S I T Y 2741
Fig. 1 Vaccinium uliginosum and the
maximum extent of the late Weichselian/
Wisconsian ice sheets (white) and tundra
(dark grey; compiled from Frenzel et al.
1992; CAFF 2001; Abbott & Brochmann
2003; Svendsen et al. 2004). Above: Current
geographical distribution of V. uliginosum
according to Hultén & Fries (Hultén &
Fries 1986). Below: Geographic distribution
of cpDNA haplotypes and the three major
cpDNA lineages identified in V. uliginosum
(cf. Fig. 2, Appendix). Upper-case letters
denote combined (two-region) haplotypes,
lower case letters denote single-region
haplotypes (based on the trnL/F or on the
trnS/G region; cf. Table 1). Single-region
haplotypes that unambiguously could
be referred to a combined haplotype (see
Table 1) are indicated as, e.g. K′. Each letter
represents 1–5 populations.
rpl 20–5 ′ rps 12, and psb B- psb F from Hamilton 1999). The
intergenic spacer regions separating trnL and trnF
(primers e–f) and trn S and trn G were most variable
and selected for full analysis. Most poly merase chain
reactions (PCR) contained 3 µL DNA extract, 2.5 µL buffer
(10 × QIAGEN buffer or Buffer II from PerkinElmer),
2.5 mm MgCl2, 0.2 mm of each dNTP, 1 µm of each primer,
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 2739–2753
and 1 U Taq polymerase (QIAGEN Taq or AmpliTaq Gold,
PerkinElmer) in a total volume of 25 µL. Some herbarium
samples were amplified in reactions containing 3 µL DNA
extract and 22 µL PCR Promega Master mix. The cycling
profile was 5–10 min at 95 °C followed by 35 cycles of 30 s
at 95 °C, 30 s at 50 °C, and 60–120 s at 72 °C. The PCR
products were purified with QIAquick PCR Purification
2742 I . G . A L S O S E T A L .
Kit (QIAGEN) and sequenced in both directions with
Dye Terminator Cycle Sequencing Ready Reaction with
AmpliTaq DNA Polymerase (PerkinElmer), abi prism BigDye
Terminator Cycle Sequencing Ready Reaction Kit 2.0
(Applied Biosystems), or DYEnamic ET Dye Terminator
Cycle Sequencing Kit for MegaBACE. The sequence PCR
profile was 25 cycles of 30 s at 96 °C, 30 s at 50 °C, and
4 min at 60 °C. The samples were run on an ABI Prism
377 DNA sequencer or on a MegaBACE 500. Sequence
ambiguities were resolved by comparing complementary
strands. Alignment was straightforward. Because we usually detected little variation between geographically close
populations, only one sample was analysed from all except
three populations.
Data analyses
The incongruent length difference test (Mickewich &
Farris 1981) as implemented in paup 4.0b10 (Swofford
2003) was performed using 1000 random replicates to
assess the possibility of partition heterogeneity before
combining the cpDNA data. The data were analysed using
maximum parsimony and maximum likelihood in paup.
Characters were considered unordered and weighted
equally. The best-fit model of nucleotide substitution for
the maximum-likelihood analysis was selected using the
hierarchy of likelihood-ratio tests implemented in modeltest 3.06 (Posada & Crandall 1998). A heuristic search
employing 10 random stepwise additions and tree-bisection–
reconnection (TBR) branch swapping was performed. Nodal
support was estimated using the bootstrap approach
(Felsenstein 1985) with 1000 replicates. When DNA sequences
differ by few substitutions only, conventional phylogenetic
methods may perform poorly (Crandall 1996). Therefore,
we also carried out statistic parsimony analysis using the
network algorithm of Templeton et al. (1992) as implemented
in the tcs program (Clement et al. 2000). This method estimates the unrooted haplotype network and a 95% plausible
set of all haplotype lineages in that network. Tip haplotypes
connected to the network by only one connection were
assumed to be younger than interior haplotypes connected
by two or more connections (Castelloe & Templeton
1994).
The populations were grouped into geographical
regions according to geographical isolation and pattern
of glaciation during the Quaternary (see Fig. 1b for the delimitation of the regions). Gene and nucleotide diversities
per geographical region were calculated using arlequin
version 2.000 (Schneider et al. 2000). A Mantel test comparing
geographical distances Dgeo and genetical distances Dgen
was performed in genetix 4.03 (Belkhir et al. 1996–2004;
http://www.University-montp2.fr/∼genetix/genetix/
genetix.htm) with 10 000 permuations. Geographical distances between all pairs of samples were calculated using
the general formula for a globe. Genetic distances were
calculated as pairwise sequence divergences between all
samples using the best-fit maximum-likelihood model as
implemented in paup 4.0b10 (Swofford 2003).
A molecular clock hypothesis was tested with a maximum-likelihood ratio test (MLR) upon the estimated tree
and best-fit model (Felsenstein 1981). The oldest macrofossils of V. uliginosum have been reported from Pliocene
(1.7–6 Ma) strata in France (Laurent 1904–05) and Bulgaria
(Stoyanoff & Stefanoff 1929). Pairwise sequence divergences
between V. uliginosum and the two outgroup species were
calculated using the best-fit maximum-likelihood model
as implemented in paup 4.0b10 (Swofford 2003). The upper
and lower range of the minimum divergence rates were
calculated as average sequence divergence divided by 1.7
and 6 Myr, respectively.
Results
The sequences of the trnL/F spacer were 423 base pairs
(bp) long with 10 (2.4%; excl. outgroups) and 21 (5.0%; incl.
outgroups) substitutions. In the ingroup, the sequences of
the trnS/G spacer were 677 bp long with 12 substitutions
(1.8%) and one indel. The indel (8 or 9 A at bp 519–528) was
neglected because it varied inconsistently with the substitutions and because indels typically mutate more frequently
than substitutions. When aligned with the outgroups, the trnS/
G spacer sequences were 706 bp long with 17 substitutions
(2.4%). In Vaccinium uliginosum, sequences were obtained
of both cpDNA regions from 92 samples, sequences of the
trnL/F region from 31 additional samples, and sequences of
the trnS/G region from three additional samples, totalling
126 samples representing 122 populations (Appendix).
Eighteen combined (two-region) haplotypes were identified (Fig. 1, Table 1). The sequences are deposited in the
GenBank database (Accession nos DQ073105–DQ073326).
For 17 of the samples for which we only were able to obtain
a sequence of one of the two regions, this sequence could
unambiguously be referred to one particular combined
haplotype (Fig. 1, Table 1). There was no evidence for heterogeneity (P = 0.71) and the analyses were performed on
the combined data set. The best-fit model of nucleotide
substitution was the HKY + G model (Hasegawa et al. 1985),
including rate variation among sites as modelled by the
gamma distribution (Yang 1996). The estimated transition/
transversion ratio was 0.9868 and the gamma shape parameter
(a) was 0.0173. The average relative contents of nucleotides
were A = 0.3283, C = 0.1750, G = 0.1375, and T = 0.3592.
The maximum-parsimony analysis identified 18 most
parsimonious trees of length 44. The strict consensus tree
(Fig. 2) was similar to the maximum-likelihood tree and
three medium- to well-supported major clades were
identified: the two boreal ‘Amphi-Atlantic’ and ‘Beringian’
lineages and the ‘Arctic-Alpine’ lineage. Their relationships
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 2739–2753
C I R C U M P O L A R M O L E C U L A R D I V E R S I T Y 2743
Table 1 Variable sites recorded in the trnL/F and trnS/G cpDNA spacer region sequences in Vaccinium uliginosum. Designation of singleregion haplotypes and combined (two-region) haplotypes are shown. Nucleotide position refers to the number of bases from the first
position of the region. Bases at the corresponding sites are also shown for the two outgroup species
Combined
haplotype
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
V. myrtillus
V. vitis-idaea
trnL/F (423 bp)
trnS/G (677 bp)
trnL/F
trnS/G
haplotype haplotype 49 51 52 104 152 178 183 185 205 262 86 159 241 245 342 402 404 425 605 612 639 644
a
a
a
b
b
b
b
b
c
d
e
f
i
a
j
d
h
g
n
o
q
n
p
q
r
s
l
m
l
k
v
w
x
t
y
u
A
A
A
A
A
A
A
A
C
C
C
C
C
A
C
C
C
C
C
C
T
T
T
T
T
T
T
T
G
G
G
T
T
T
G
G
G
G
G
G
A
A
A
C
C
C
C
C
A
A
A
A
A
A
A
A
A
A
C
A
T
T
T
T
T
T
T
T
T
C
T
T
T
T
C
C
T
C
T
T
C
C
C
C
C
C
C
C
C
C
C
C
C
C
T
C
C
C
C
C
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
A
A
C
C
C
C
C
C
C
C
C
A
C
A
A
C
A
A
A
A
C
C
were unresolved. The well-supported (bootstrap 85–
88%) boreal Amphi-Atlantic lineage only contained two
very similar haplotypes (differing by one transversion),
one European-Greenlandic (K) and one eastern North
American (I). Haplotype K was found in boreal, formerly
glaciated areas (throughout northern continental Europe
and in Iceland) as well as in formerly unglaciated areas
eastwards to the southern Urals (K′ in Fig. 1). It also occurred
sparsely in southeastern Greenland and the northern Alps,
regions dominated by the Arctic-Alpine lineage. Unexpectedly,
this boreal haplotype not only occurred in the lowlands but
also, with a single exception (North Cape in northernmost
Norway), exclusively in the mountains in Scandinavia.
Haplotype I was found in boreal, formerly glaciated areas
in eastern-central Canada and eastern USA.
The moderately supported (66 – 75%) boreal Beringian
lineage consisted of seven haplotypes, of which six were
restricted to boreal areas on both sides of the Bering Strait
and one divergent one to western North America (California
and Nevada).
The well-supported (89 –90%) Arctic-Alpine lineage
was extremely widespread and included nine haplotypes,
several of them with very wide distributions. The most
common haplotype (C) was fully circumarctic and also
occurred in the Carpathian Mountains and in several
mountains in Central and East Asia. Only a single ArcticAlpine haplotype (N) was restricted to Beringia, and three
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 2739–2753
A
A
A
A
A
A
A
A
A
A
A
G
A
A
A
A
A
A
A
A
G
G
G
G
G
G
G
G
A
G
G
G
G
G
G
G
G
G
G
A
G
G
G
G
G
G
G
G
C
G
C
G
G
G
G
G
G
G
G
G
T
T
T
T
T
T
T
T
G
G
G
G
?
T
G
T
G
G
G
G
A
A
A
A
A
A
A
A
A
T
A
A
A
A
T
T
A
A
A
A
C
C
C
C
C
C
C
C
C
C
C
T
C
C
C
C
C
C
C
C
G
G
G
G
G
G
G
G
G
G
G
T
G
G
G
G
G
G
G
G
T
T
T
T
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
A
G
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
G
T
G
T
T
T
T
G
G
G
G
G
T
G
G
G
G
G
G
A
A
A
A
A
A
A
A
A
A
A
A
T
A
A
A
A
A
A
A
C
C
C
C
C
C
C
C
T
C
T
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
T
T
C
C
C
C
C
C
C
C
C
C
C
C
A
A
A
A
A
A
A
A
A
C
A
C
C
A
C
C
C
C
A
A
C
C
C
C
C
C
C
T
C
C
C
C
C
T
C
C
C
C
C
C
haplotypes (A, B, and D) were restricted to the southern
European mountains.
The same three major lineages were recognized in
the network analysis, connected via haplotypes M and Q
of the Beringian lineage and several inferred missing
intermediates (Fig. 2). Haplotypes separated by up to
14 mutational steps had probabilities greater than 0.95
of being connected in a parsimonious fashion. A single
loop of ambiguities was resolved by assuming that the
southern European haplotype D was most closely related
to haplotype A, which was found in the same geographical
region.
The Arctic-Alpine lineage had high gene diversity and
intermediate nucleotide diversity (Table 2). Unexpectedly,
the levels of diversity in this lineage were similar in previously glaciated and unglaciated regions (Fig. 3). Whereas
the gene and nucleotide diversities were strikingly low in
the boreal Amphi-Atlantic lineage, very high diversities
were observed in the boreal Beringian lineage (Table 2).
When calculated for each geographical region, the highest
gene diversity was observed in Beringia, where the Beringian
and the Arctic-Alpine lineages co-occurred (Table 2), but
the Arctic-Alpine lineage per se did not have higher diversities in Beringia than in other geographical areas. High
genetic diversity was also found in the southern European
mountains, where the Arctic-Alpine and the Amphi-Atlantic
lineages co-occurred.
2744 I . G . A L S O S E T A L .
Fig. 2 Phylogenetic relationships inferred among the 18 combined (two-region) cpDNA haplotypes (A–R) in Vaccinium uliginosum.
Left: Strict consensus of 18 equally most parsimonious trees. The numbers on the branches are bootstrap support values obtained in
maximum-parsimony and maximum-likelihood analyses, respectively, separated by a slash. Thirteen characters were parsimony informative
in the ingroup. Consistency and retention indices were 0.760 and 0.815, respectively. Right: The 95% plausible set of haplotype networks.
Lines represent the mutational pathway interconnecting the haplotypes; dots represent inferred intermediate haplotypes not observed
in the samples. The size of each circle is determined by the square root of the sample size.
The Mantel test showed no significant correlation
between geographical and genetic distances within the
Beringian lineage (P = 0.089). There was a positive correlation between geographical and genetic distances within
the Arctic-Alpine lineage (P = 0.0001, Pearson r = 0.313), but
this was no longer significant when haplotypes A, B, and
D from the southern European mountains were excluded
(P = 0.0927).
The MLR tests did not reject the null hypothesis that
the trnL/F and trnS/G sequences evolved in a clockwise
fashion (χ2 = 21.22, d.f. = 16, P = 0.17). The mean pairwise
differences between the V. uliginosum cpDNA haplotypes
and those of the outgroup species were 1.55% and 1.62%
for V. vitis-idaea and V. myrtillus, respectively (mean 1.58%).
Thus, the estimated upper and lower ranges of the minimum divergence rates were 0.26% and 0.92% per Myr,
respectively, depending on dating of the oldest fossil to
6 or 1.7 Myr. The mean pairwise differences between the
three major lineages in V. uliginosum were similar to each
other (0.64%, 0.67%, and 0.77%), and thus they probably
diverged 0.7–3.0 Ma.
Discussion
Deep phylogenetic split in Vaccinium uliginosum
In this first full-scale cpDNA analysis of a widespread
species occurring over the entire circumarctic and
circumboreal regions, we identified 18 haplotypes that
grouped into three divergent main lineages: two disjunct
boreal ones (the Beringian and the Amphi-Atlantic) and
one extremely widespread which covered much of the
species’ range (the Arctic-Alpine). Chloroplast lineages
may not always be identical to organism lineages because
of chloroplast transfer (Wendel & Doyle 1998), but this
can be addressed based on independent data from
morphology, ploidy, and nuclear molecular markers. In
V. uliginosum, eight morphological characters showed
significant differences between the three cpDNA lineages,
suggesting that the cpDNA lineages represent organism
lineages (Alsos 2003). The Amphi-Atlantic cpDNA lineage
exclusively contained tetraploids (38 samples), whereas
the Arctic-Alpine cpDNA lineage mainly contained
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 2739–2753
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 2739–2753
Table 2 Frequency of the combined (two-region) cpDNA haplotypes, gene diversity (H), and nucleotide diversity in the 11 geographical regions (cf. Fig. 1) and in the three major cpDNA
lineages (cf. Fig. 2) in Vaccinium uliginosum. Gene and nucleotide diversities were only calculated for geographical regions represented by at least five populations. Standard errors are
given in parentheses
Geographical regions
1
2
3
4
C & E Asia N Russia N &
S European
E Europe mts.
5
6
7
8
9
10
11
ArcticIceland E Greenland Svalbard E America W Greenland W America Beringia Alpine
and Canada
Amphi- Beringian
Atlantic
3
3
9
9
25
25
10
10
7
7
6
6
2
4
8
8
14
14
2
2
19
21
45
47
44
44
16
18
—
—
0.667
—
—
0.333
—
—
—
—
—
—
—
—
—
—
—
—
2
0
1
—
—
—
0.444
—
—
0.111
—
0.222
—
—
0.222
—
—
—
—
—
—
—
4
0
1
0.778
(0.110)
0.164
(0.164)
—
—
0.040
—
—
—
—
—
—
—
0.960
—
—
—
—
—
—
—
2
0
2
0.080
(0.072)
0.022
(0.022)
0.500
0.100
0.100
0.100
—
—
—
—
—
—
0.200
—
—
—
—
—
—
—
5
3
2
0.756
(0.130)
0.108
(0.073)
—
—
—
—
—
—
—
—
—
—
1.000
—
—
—
—
—
—
—
1
0
1
0.000
—
—
—
—
—
0.167
0.667
—
—
—
0.167
—
—
—
—
—
—
—
3
0
2
0.600
(0.215)
0.130
(0.092)
—
—
1.000
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1
0
1
—
—
—
—
—
0.125
—
—
0.125
0.750
—
—
—
—
—
—
—
—
—
3
0
2
0.464
(0.200)
0.190
(0.120)
—
—
0.429
—
0.143
0.071
0.214
—
0.143
—
—
—
—
—
—
—
—
—
5
0
1
0.780
(0.085)
0.136
(0.085)
—
—
—
—
—
—
—
—
—
—
—
1.000
—
—
—
—
—
—
1
1
2
—
—
0.190
—
—
—
—
—
—
0.333
—
—
0.048
0.048
0.048
0.048
0.048
0.238
8
7
2
0.824
(0.054)
0.182
(0.106)
0.106
0.021
0.489
0.021
0.064
0.064
0.149
0.064
—
—
—
—
—
0.021
—
—
—
—
9
—
—
0.729
(0.060)
0.063
(0.044)
—
—
—
—
—
—
—
—
0.182
—
0.818
—
—
—
—
—
—
—
2
—
—
0.304
(0.075)
0.014
(0.016)
—
—
—
—
—
—
—
—
—
0.389
—
0.111
0.056
—
0.056
0.056
0.056
0.278
7
—
—
0.813
(0.067)
0.144
(0.088)
—
0.000
(0.000)
—
—
—
C I R C U M P O L A R M O L E C U L A R D I V E R S I T Y 2745
# Pop.
# Ind.
Haplotype
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
# Haplotypes
# Local haplotypes
# Major clades
Gene
diversity H
Nucleotide
diversity
cpDNA linages
2746 I . G . A L S O S E T A L .
history of the species. According to this scenario, the three
lineages were affected more or less independently by all
major Pleistocene glaciations, although they currently coexist
in some geographical regions (Fig. 1).
Unexpectedly, the widespread Arctic-Alpine lineage
was highly diverse, not only in previously nonglaciated
areas but remarkably so also in the heavily glaciated ones,
suggesting that it thrived in large populations on the vast
tundras during the ice ages and that many genotypes efficiently recolonized deglaciated terrain over long distances.
In contrast, the two boreal lineages were depauperate in all
regions except Beringia, probably because of bottlenecking
in small refugial populations south of the ice in Europe as
well as in eastern and western North America.
Fig. 3 Gene diversity (H) in the Arctic-Alpine lineage of Vaccinium
uliginosum in previously glaciated and unglaciated regions, calculated from the cpDNA data. Sample sizes (number of populations)
are given above; bars represent standard errors.
diploids (39 samples) and only a few tri- to hexaploids (Alsos
2003). Thus, these two cpDNA lineages can generally also
be distinguished by ploidy level (the Beringian cpDNA
lineage is less studied but appears to be variable in this respect). Our preliminary analyses of amplified fragment
length polymorphism (AFLP) (Eidesen et al., unpublished)
and Internal Transcribed Spacer (ITS) sequence diversity
(Alsos et al., unpublished) also confirm the split between the
Arctic-Alpine and Amphi-Atlantic cpDNA lineages, but it
is less clear how the Beringian cpDNA lineage relates to the
others.
Our cpDNA data suggest that the initial divergence
among the V. uliginosum lineages took place before the
onset of the major Quaternary glaciations (> 700 000 bp).
The estimated minimum rate of 0.26 – 0.92% cpDNA divergence per Myr in V. uliginosum is higher than the average
estimated for plants in general (0.024 – 0.116%, Hewitt 2000).
It is also higher than for Arctic Cerastium species, where no
variation was found in the trnL/F spacer (Sheen et al. 2004).
The lack of haplotypes inferred as missing intermediates
between the three lineages in the network analysis (Fig. 2)
also suggests that the split in V. uliginosum is old.
The geographical origin of the three lineages is uncertain
due to the lack of resolution in the parsimony and
maximum-likelihood analyses (Fig. 2). However, the central position of Beringian haplotypes in the network analysis, as well as the diploid level of haplotype Q from the
Seward Peninsula (Alsos 2003; Brochmann et al. 2004), may
indicate that the species originated in Alaska/Beringia. The
highest diversity of temperate Vaccinium species is found
in North America (Vander Kloet 1988), which may have
served as a secondary centre of speciation in the genus. It
is therefore possible that V. uliginosum originated in Beringia,
from where it expanded towards the east, south and/or
west to attain a circumpolar distribution early in the
Depauperate in huge areas: the boreal Amphi-Atlantic
lineage
It is notable that the boreal Amphi-Atlantic lineage, which
corresponds to the widespread boreal Eurasian-eastern
North American subspecies uliginosum in a wide sense
(Alsos 2003), only contained two closely related haplotypes
in spite of its huge distribution area. One haplotype is
European (possibly Eurasian) with extension to Greenland
and the other is eastern North American. Their different
geographical distributions suggest that they diverged before
the last glaciation. This depauperate lineage probably
experienced independent, heavy bottlenecks leading to
extinction of genotypes in small glacial refugia on both
sides of the Atlantic, located south of the last ice sheets
in Europe and eastern North America. Its generally boreal
ecology may have caused a much heavier bottleneck in
this lineage compared to the Arctic-Alpine lineage. The
rare occurrence of the European haplotype in southeastern
Greenland suggests trans-Atlantic dispersal after the last
glaciation, as also inferred for Saxifraga oppositifolia and many
other plant species (Brochmann et al. 2003). Depauperate
and phylogenetically shallow lineages have been reported
in several other plants and animals in the Atlantic region
(Abbott et al. 2000; Brochmann et al. 2003).
Diverse but isolated: the boreal Beringian lineage
This lineage contained six, some of them rather divergent,
haplotypes in boreal areas of Beringia and one single divergent haplotype in California/Nevada (L, Fig. 2), suggesting
continuous northern survival during all ice ages as well as
survival in a bottlenecked refugial population south of
at least the last west American (Cordilleran) ice sheet.
The Beringian lineage is also morphologically diverse and
probably represents several subspecies (Alsos 2003). The
northern part of Beringia was mainly unglaciated throughout
the Quaternary (Andersen & Borns 1997). The high gene
and nucleotide diversities in the Beringian cpDNA lineage of
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 2739–2753
C I R C U M P O L A R M O L E C U L A R D I V E R S I T Y 2747
V. uliginosum (as well as for the species in general in this
geographical region; Table 2), suggest long-term persistence
of large populations in Beringia, as also suggested for
S. oppositifolia and several animals (Hewitt 2004), highlighting the value of this region for preserving northern
biodiversity.
However, the only expansion of the Beringian lineage
appears to have occurred southwards along the Pacific
coast, probably several glacial cycles ago, as inferred from
the isolated occurrences of the divergent L haplotype in
California and Nevada. Notably, the populations in this area
have been referred to a distinct subspecies (ssp. occidentale;
Hultén & Fries 1986). Our sample size in this area is low,
but the occurrence of the same haplotype in California and
Nevada may suggest bottlenecking in a small refugial
population south of the Cordilleran ice sheet, similar to
what we inferred based on much larger sample sizes for
the boreal Amphi-Atlantic lineage. Glacial refugia south
of the Cordilleran ice sheet have been suggested for a
number of species (Soltis et al. 1997; Abbott & Comes 2004;
DobeS et al. 2004).
The boreal Beringian and the Arctic-Alpine lineages
both occur in Beringia, but the Beringian lineage appears to
be more associated with an oceanic climate with high levels
of precipitation; all populations analysed of this lineage are
situated in areas characterized by coexisting oceanic and
continental vegetation complexes (Yurtsev 1997). This may
explain why the only expansion of the Beringian lineage
has occurred southwards along the Pacific.
Diverse all over: the Arctic-Alpine lineage
This lineage, which corresponds to subspecies microphyllum in a wide sense (Alsos 2003), occurs in the
entire circumarctic including Beringia as well as in alpine
regions in Central Asia, southern Europe and eastern
North America. Even several individual haplotypes are
extremely widespread; one of them has a full circumarctic
distribution with extensions to Central Asia and the
Carpathians. About half of the present area of this lineage
was repeatedly glaciated during the Quaternary, but
nevertheless contained high levels of molecular diversity,
similar to the levels in nonglaciated areas (Fig. 3). This
result contrasts sharply with the patterns observed in most
temperate plants and animals, which typically have low
levels of diversity in previously glaciated areas because of
repeated bottlenecks during colonization (Soltis et al. 1997;
Taberlet 1998; Hewitt 1999, 2004). The most reasonable
explanation for the high diversity in the Arctic-Alpine
lineage in previously glaciated areas is efficient and broadfronted recolonization from large and diverse populations
on the tundras surrounding the ice, as well as from more
distant populations. The wide distribution of individual
haplotypes, the few local Arctic haplotypes, and the lack of
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 2739–2753
correlation between geographical and genetic distances in
the Arctic populations, indicate that long-distant source
populations frequently contributed to recolonization.
Thus, in contrast to several other circumarctic animals
and plants analysed so far (Hewitt 2004), there was no
subdivision of the fully circumarctic Arctic-Alpine V. uliginosum
lineage into divergent Beringian and European/AmphiAtlantic groups. Frequent long-distant dispersal, probably
via birds, has probably prevented such divergence in the
V. uliginosum. The two circumarctic saxifrages analysed
(S. oppositifolia, Abbott et al. 2000 and S. cernua, cf. Brochmann
et al. 2003), on the other hand, show more distinct geographical structuring at this large scale, probably because
they have no typical mechanisms for long-distance dispersal.
The Arctic-Alpine lineage was probably even more
dominant than today during the periods of glaciation,
thriving in a vast area including Beringia and large parts of
Northern Asia. Other areas of persistence, at least during
the last glaciation, were situated in the southern European
mountains and in the Central Asian mountains, and possibly
south of the eastern American ice sheet. Glacial persistence
in the Canadian High Arctic or Greenland has been suggested for very hardy species such as S. oppositifolia (Abbott
et al. 2000) and Dryas integrifolia (Tremblay & Schoen 1999),
but appears less probable for the more thermophilous
V. uliginosum, which has a more fragmented distribution
and impeded sexual reproduction in the High Arctic
(Hultén & Fries 1986; Alsos et al. 2002; Alsos et al. 2003). It
appears therefore most likely that Greenland and northern
Canada were recolonized by V. uliginosum from the south,
possibly also from the east and west.
The three Arctic-Alpine haplotypes in the European
Alps and the Pyrenées were not observed elsewhere,
suggesting isolation in this region at least since the last
glaciation. It is likely that the species survived on the periglacial plains north and/or south of the mountains. Glacial
persistence in the southern European mountains as well as
in their periglacial surroundings has also been suggested
for a number of Alpine species (Stehlik 2003; Tribsch &
Schönswetter 2003). It is unclear why the Alpine populations
of V. uliginosum did not contribute to northwards recolonization of Europe. It is possible that northwards postglacial
expansion of the Arctic-Alpine lineage from this area for
some reason was blocked by the expanding boreal AmphiAtlantic lineage, which today, although depauperate,
occupies virtually all of Europe including the Scandinavian
mountains. Notably, populations belonging to the boreal
lineage also occur today in the northern Alps (Fig. 1).
Conclusions
The initial divergence among the Vaccinium uliginosum
lineages probably took place before the onset of the
major Quaternary glaciations, possibly in Beringia, from
2748 I . G . A L S O S E T A L .
where they initially expanded. If this dating is correct,
the depauperate Amphi-Atlantic lineage, the diverse but
geographically restricted Beringian lineage, and the
highly diverse and widespread Arctic-Alpine lineage were
affected independently by all of the major glaciations.
The inferred impact of the ice ages on the intraspecific
diversity in V. uliginosum differed conspicuously between
geographical regions: northern survival and extensive
postglacial migration in the Arctic, isolation of southern
European Alpine populations, bottlenecking in previously
glaciated boreal amphi-Atlantic areas, and diversification
in Beringia. The effects of the Pleistocene glaciations
can thus vary to a large extent even within the same
species. We conclude that although Beringia probably was
important for the initial divergence and expansion of
V. uliginosum as well as for continuous in situ survival of
both the Beringian and the Arctic-Alpine lineages during
all glaciations, this region unexpectedly appears to have
played a minor role as a source area for later inter- and
postglacial expansions.
Acknowledgements
We thank M. Aasen, C. Aedo, S. Aiken, T. Alm, I. Alvarez,
G. Arnesen, M. R. Bauert, J. L. Benito, P. B. Eidesen, A. K. Brysting,
K. A. Bråthen/Tundra Northwest Expedition 1999, S. M. Coleman,
S. Dalmarsdottir, R. Elven, J. Feilberg, K. Helskog, U. R. B. Gamst,
H. H. Grundt, J. Jorgensen, L. Lund, J. Mangerud, H. Meltofte,
J. A. Olsen, C. L. Parker, M. Piirainen, V. Razzhivin, B. E. Sandbakk,
D. Soden, H. Solstad, G. Søvik/Otto Sverdrup Centennial Expedition, S. Vander Kloet, and V. Vange for collecting material and/
or for field assistance. We also thank V. Razzhivin for inviting
I. G. Alsos to the Komarov Herbarium and for translating Russian
herbarium labels, A. Batten for providing a large selection of
herbarium specimens from ALA, V. T. Ravolainen, P. B. Eidesen,
and S. Kjølner for laboratory assistance, and T. M. Gabrielsen,
C. Printzen, and D. Ehrich for discussions on data analysis. S. Vander
Kloet, R. J. Abbott, M. Koch and two anonymous referees gave
valuable comments on the manuscript. The study was supported
by Tromsø Museum and by grants 135652/730 and 146515/420 to
C. Brochmann from the Research Council of Norway. Additional
support was obtained by grants to I. G. Alsos from the Norwegian
Polar Institute (1998), the Center for Women’s Studies and Women
in Research (1998), the Roald Amundsen Center for Arctic
Research (A21/98, A36/99), A/S Norsk Varekrigsforsikringsfond
1999, Kjellfrid og Helge Jakobsens Fund (1999), Inger Haldorsens
Legacy (1999), and the Nansen Foundation (61/99, 51/2000).
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Oechel WC, Callaghan T, Gilmanov T et al.), pp. 229 – 244.
Springer, New York.
This study formed a part of the PhD thesis of Inger Greve Alsos,
which was supervised by Christian Brochmann and Torstein
Engelskjøn. Alsos is now working as a postdoctoral fellow on a
project entitled Effects of climate change on ecosystems in Svalbard:
past and future immigration of thermophilous key species. Professor
Christian Brochmann is working at the National Centre for Biosystematics where he leads a 5-year research programme entitled
Migration and evolution of arctic plants in response to Quaternary climate
changes. Torstein Engelskjøn is associate professor at Tromsø
Museum and investigates arctic and antarctic phytogeography.
Pierre Taberlet is the Director of the Laboratoire d’Ecologie
Alpine, and focuses on the conservation genetics and molecular
ecology of many different plant and animal species. Ludovic
Gielly is a research engineer at the Laboratoire d’Ecologie Alpine.
He taught Alsos molecular methods and was responsible for the
automated DNA sequencing.
Details of the 121 Vaccinium uliginosum populations successfully analysed for variation in the cpDNA regions trnL/F (n = 122) and trnS/G (n = 95). Material: f, fresh leaves from cultivated
material; h, leaves from herbarium material; s, silica-dried leaves from field-collected material
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 2739–2753
Population
Material
Location
V86
V87
V80
V85
V82
V84
V79
V46a
V150
V77
V81
V151
V152a
V152c
V83
V73
V78
V47a
h
h
h
h
h
h
h
s
s
h
h
s
s
s
h
h
h
f
V88
V137
V139
V142
V128
V147
V48a
h
f
f
f
h
s
f
V130
V140
V138
V141
V131
V52a
V50a
V148
V133
V129
h
f
f
f
h
s
s
s
h
h
USA, Alaska, Aleutian Is., Unalaska Quad, Umnak I.
USA, Alaska, Aleutian Is., Adak Quad, Adak I.
USA, Alaska, Arctic Coastal Plain, Oumalik Test Well
USA, Alaska, Aleutian Is., Atka Is.
USA, Alaska, Ketchikan Quad, Quartz Hill area
USA, Alaska, Ketchikan Quad, Ella Lake, NE shore
USA, Alaska, Killik R. Quad, vic. Lake Kaniksrak
USA, Alaska, N of Brooks Range, along Sag river
USA, Alaska, Noatak Quad, Sheshalik Split
USA, Alaska, Alaska Range, Rainbow Mt.
USA, Alaska, Selawik Quad, Waring Mts.
USA, Alaska, Seward Pen., Cowpack Inlet
USA, Alaska, Seward Pen., Teller Road
USA, Alaska, Seward Pen., Teller Road
USA, Alaska, Skagway Quad, St. Elias Mts.
USA, Alaska, Umiat
USA, Alaska, Unalakleet Quad, Nulato Hills
USA, NE California, Sierra Nevada N, Adams Peak
(Cult. Tromsø Bot. Garden)
USA, Nevada, Ormsby Co., Carson Range
USA, New Hampshire, White Mts., Mt. Pierce
USA, New Hampshire, White Mts., Mt. Pierce
USA, New Hampshire, Mt. Clinton, Rt302
Canada, Manitoba, Churchill
Canada, Manitoba, Churchill, Northern Study Centre
Canada, Newfoundland, Bay du Nord Wilderness
(Cult. Tromsø Bot. Garden)
Canada, Newfoundland, Bonavista
Canada, Newfoundland, Cape Spean, Avalon Pen.
Canada, Newfoundland, Holyrood, Hawke Hills
Canada, Newfoundland, St. John’s, Signal Hill
Canada, Nova Scotia, Cape Breton I., White Point
Canada, Nunavut, Ellesmere I., Grise Fiord
Canada, Nunavut, Melville I.
Canada, Nunavut, Rankin Inlet
Canada, Nunavut, Baffin I., Resolution I.
Canada, Nunavut, Keewatin, Eskimo Point/Arviat
m a.s.l.
131
55
1160
76
575
200–300
2900
360–400
980
370–520
2370
Combined
haplotype
Longitude/latitude
Herbarium
53°23′N, 167°50 W
51°50′N, 176°35′W
69°50′N, 156°0′W
52°04′N, 174°35′W
55°30′N, 130°25′W
55°29′N, 131°04′W
68°11′N, 154°09′W
69°19′N, 148°43′W
67°01′N, 162°57′W
63°18′N, 145°28′W
66°58′N, 159°41′W
66°21′N, 164°57′W
64°41–43′N, 165°45′W
64°41–43′N, 165°45′W
59°37′N, 135°28′W
69°25′N, 152°20′W
63°48′N, 160°23′W
40°N, 120°W
ALA
ALA
ALA
ALA
ALA
ALA
ALA
TROM
O
ALA
ALA
O
O
O
ALA
ALA
ALA
TROM
R
R
C
M
R′
R
39°N, 119°W
44°N, 71°30′W
44°N, 71°30′W
44°N, 71°30′W
58°46′N, 94°01′W
58°44′N, 93°49′W
48°N, 55°W
ALA
AKAD/TROM
AKAD/TROM
AKAD/TROM
AKAD
TROM
L
H
48°38′N, 53°08′W
47°N, 53°W
47°23′N 53°08′W
47°N, 53°W
45°52′N, 59°59′W
76°27′N, 82°40′W
75°N, 110°W
62°48′N, 92°06′W
61°30′N, 64°40′W
61°08′N, 94°08′W
AKAD
AKAD/TROM
AKAD/TROM
AKAD/TROM
AKAD
TROM
TROM
TROM
AKAD
AKAD
C
O
J
J
J
Q
R′
C
P
L
I′
I′
I′
I
I′
I′
E′
I′
I′
G
E
G′
trnL/F
trnS/G
g
g
a
i
g
g
a
a
j
a
d
d
d
h
g
a
d
f
u
u
q
v
f
b
b
c
c
c
c
k
s
u
q
x
m
m
m
y
q
t
k
l
c
c
p
c
c
b
b
b
a
r
p
r
2750 I . G . A L S O S E T A L .
Appendix
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 2739–2753
Appendix Continued
Material
Location
V49a
V149
V51a
V60a
V61a
V62a
V56a
V57a
V58a
V59a
V63a
V64a
V53a
V54a
V55a
V65a
V4a
V11a
V8a
V9a
V7a
V1a
V6a
V5a
V2a
V2-V10a
V2-V3a
V25a
V27a
V26a
V70
V18a
V17a
V68
V14a
V69
V21a
V22a
V23a
V24a
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
h
s
s
h
s
h
s
s
s
s
Canada, Nunavut, Victoria I., Wollaston Pen.
Canada, NWT, Mackenzie R. Delta, Tuktoyaktut
Canada, Québec, Ungava Pen.
Greenland, Anmagssalik, Kulusuk
Greenland, Anmagssalik, Tasilak
Greenland, Anmagssalik, Kummiut
Greenland, Inglefield Land, Firfinger Sø
Greenland, Inglefield Land, Hiawatha
Greenland, Inglefield Land, Marshall Bugt
Greenland, Inglefield Land, Spejlsø
Greenland, Jameson Land, Constable Point
Greenland, Liverpool Land, Scoresbysund
Greenland, Qeqertarsuaq (Disko), Fortune Bay
Greenland, Upernavik, Langø
Greenland, Upernavik, Upernavik Ø
Greenland, Zackenberg
Iceland, Austurland, Egilsta3ir
Iceland, Austurland, Rey3arfjör3ur
Iceland, Nor3urland Eystra, Myvatn
Iceland, Nor3urland Eystra, Vesturhei3i
Iceland, Nor3urland Vestra, Kjölur
Iceland, Vesturland, Akranes, Akrafjall
Iceland, Vesturland, Holtavör3uhei3i
Svalbard, Dickson Ld., Idodalen
Svalbard, Nordenskiöld Ld., Colesdalen
Svalbard, Nordenskiöld Ld., 40 m above V2
Svalbard, Nordenskiöld Ld., 50 m below V2
Norway, Oppland, Dovre, Geitryggen
Norway, Oppland, Dovre, Gråsida, Verkenssætri
Norway, Hedmark, Folldal, Råtåsjøhøi
Norway, Finnmark, Berlevåg, Kongsfjord
Norway, Finnmark, Nordkapp, Dår’kavck’ka
Norway, Finnmark, Nordkapp, Duken
Norway, Nordland, Hemnes, Okstindan, Okskalvan
Norway, Nordland, Lofoten, Hadsel, Grunnførfjorden
Norway, Nordland, Narvik, Frostisen
Norway, Nordland, Vesterålen, Bø, Bufjellet
Norway, Nordland, Vesterålen, Sortland, Holand
Norway, Nordland, Vesterålen, Andøy, Måtind
Norway, Nordland, Vesterålen, Hadsel, Storheia
m a.s.l.
50
20
10
ca 200
50
50
170
10–15
280
520
578
430
50–80
100
30
1225
1330
1225
214
1280
1290
Longitude/latitude
Herbarium
Combined
haplotype
trnL/F
trnS/G
69 °20′N, 114°50′W
69°26′N, 133°01′W
62°22′N, 73°45′W
65°34′N, 37°10′W
65°37′N, 37°40′W
65°51′N, 36°59′W
78°59′N, 67°10′W
78°50′N, 67°18′W
78°50′N, 68°50′W
79°04′N, 66°24′W
70°44′N, 22°41′W
70°29′N, 21°56′W
69°15′N, 53°45′W
72°46′N, 56°05′W
72°47′N, 56°07′W
74°28′N, 20°33′W
65°21′N, 14°30′W
65°01′N, 14°04′W
65°34′N, 16°57′W
65°59′N, 17°52′W
64°56′N, 19°30′W
64°21′N, 21°54′W
64°52′N, 21°12′W
78°35′N, 15°23′E
78°06′N, 15°08′E
78°06′N, 15°08′E
78°06′N, 15°07′E
62°12′N, 09°29′E
62°03′N, 09°26′E
62°16′N, 09°48′E
69°43′N, 29°17′E
70°50′N, 25°47′E
71°02′N, 25°48′E
66°00′N, 14°13′E
68°25′N, 14°32′E
68°12′N, 17°18′E
68°46′N, 14°28′E
68°38′N, 15°15′E
69°15′N, 15°53′E
68°32′N, 14°52′E
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
C
C
E
G
F
K
G
C
C
C
G
G
F
C
C
G
K
K
K
K
K
K
K
C
C
C
C
K
K
K
K′
K
C
K
K
K
K
K
K
K
a
a
b
b
b
e
b
a
a
a
b
b
b
a
a
b
e
e
e
e
e
e
e
a
a
a
a
e
e
e
e
e
a
e
e
e
e
e
e
e
q
q
p
r
q
l
r
q
q
q
r
r
q
q
q
r
l
l
l
l
l
l
l
q
q
q
q
l
l
l
l
q
l
l
l
l
l
l
l
C I R C U M P O L A R M O L E C U L A R D I V E R S I T Y 2751
Population
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 2739–2753
Population
Material
Location
V12a
V13a
V16a
V15a
V20a
V19a
V29a
V28a
V136
V134
V135
V33a
V34a
V67a
V32a
V66a
V30a
V31b
V93
V143
V37a
V36a
V35a
V108
V43a
V43e
V116
V115
V44a
V120
V98
V38a
V99
V95
V96
V124
V122
V118
s
s
s
s
s
s
s
s
h
h
h
s
s
s
s
s
s
s
s
h
s
s
s
h
s
s
h
h
s
h
h
s
h
h
h
h
h
h
Norway, Troms, Tromsø, Fløya
Norway, Troms, Kvænangen, Kvænangsfjellet
Norway, Troms, Målselv, Skakteråsen
Norway, Troms, Målselv, Skakteråsen
Norway, Troms, Storfjord, Adjit
Norway, Troms, Storfjord, Adjit
Scotland, Coire An T-Sneachda, Cairn Gorm
Scotland, Liathach, Wester Ross
Spain, Granada, Capileira, Trancada de Aguas Verde
Spain, Granada, Sierra Nevada, Laguna Hondera
Spain, Granada, Trevelez
Spain, Huesca, Bielsa, valle del Trigoniero
Spain, Huesca, Canfranc, Somport Frontier
Spain, Pyrénées, Lérida, Barruera
France, Pyrénées, Gabás, Vallée d’Ossau
France, Rhône-Alpes, Isère, Belledonne, Chamrousse
France, Provence-Alpes, Hautes-Alpes, Col du Lautaret
France, Rhône-Alpes, Isère, Belledonne, Grandes Rousses
Switzerland, Appenzell Assuer-Rhoden, Schwägalp, Hungbuel
Switzerland, St. Gallen, Altenberg
Finland, Kainuu, Kajaani, Koivukylä
Finland, Kainuu, Suomussalmi
Finland, Uusimaa, Sipoo, Pohjois-Paippinene
Russia, Amur Distr, Zeya R. drainage, Tukuringra Range
Russia, Chukotka, Anadyr Bay
Russia, Chukotka, Anadyr Bay
Russia, Chukotka, Anadyr distr., NW Rarytkin Range
Russia, Chukotka, Bilibino, Besimyanny settlement
Russia, Chukotka, Lavrentia village
Russia, Chukotka, N Koryak, Udachnaya R. mouth
Russia, E Altai, Kurkure Range, Kuzulun R.
Russia, Kola Pen., Drozdovka
Russia, Krasnoyarsk district, Khatanga settlement
Russia, Polar Ural, Khadata settlement
Russia, Polar Ural, Kharp railway station
Russia, Ryazan region, Tumsky distr, near Akulovo
Russia, S Ural, Iremel Mts., Belaya R. drainage
Russia, Taymyr Pen., Ragozinka R. mouth
m a.s.l.
400
624
450
926
3050
2950
2180
1670
2300
1800
1800
2300
2080
1380
840
148
205
50
1200
2000
Longitude/latitude
Herbarium
69°36′N, 19°01′E
69°54′N, 21°34′E
68°46′N, 19°40′E
68°46′N, 19°40′E
69°21′N, 20°22′E
69°22′N, 20°23′E
57°07′N, 3°40′W
57°33′N, 5°30′W
36°58′N, 3°21′W
37°N, 03°W
36°53′N, 3°20′W
42°42′N, 0°14′E
42°46′N, 0°32′W
42°31′N, 0°51′E
42°50′N, 0°31′W
45°7′N, 05°54′E
45°04′N, 06°23′E
45°05′N, 06°04′E
47°15′N, 09°20′E
47°N, 9°E
64°01′N, 27°27′E
64°57′N, 29°20′E
60°27′N, 25°12′E
50°N, 138°E
64°37′N, 177°27′E
64°37′N, 177°27′E
65°N, 171°E
68°N, 166°E
65°30′N, 171°02′E
62°N, 175°E
50°N 85°E
67°47′N, 40°32′E
71°57′N, 102°24′E
65°N, 66°E
65°N, 66°E
54°N, 39°E
54°N, 57°E
75°N, 102°E
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
GDA
GDAC
GDA
TROM
TROM
TROM
TROM
TROM
TROM
TROM
TROM
ETH
TROM
TROM
TROM
LE
TROM
TROM
LE
LE
TROM
LE
LE
TROM
LE
LE
LE
LE
LE
LE
Combined
haplotype
K
K
K
K
K
K
K
K
A
A
B
A
A
A
D
K
K′
K
K
K
C
J
J
J′
N
J
F
K
F′
C
K′
K′
H
trnL/F
trnS/G
e
e
e
e
e
e
e
e
a
a
a
a
a
a
a
a
a
b
e
e
e
e
e
a
d
d
d
a
d
a
b
e
l
l
l
l
l
l
l
l
a
a
e
e
b
n
n
o
n
n
n
n
l
l
l
l
q
m
m
w
m
q
l
q
q
s
2752 I . G . A L S O S E T A L .
Appendix Continued
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 2739–2753
Appendix Continued
Population
Material
Location
m a.s.l.
Longitude/latitude
Herbarium
V101
V121
V102
V42a
V41a
h
h
h
s
s
Russia, Tuva, W Sayan, Khor-Taiga Range
Ukraine, Carpathian Mts., Zakarpatskaya Region, Perechinsky
Russia, upland between Angara R and Lena R.
Russia, Ural, Hadata
Russia, Vorkuta, Ileymusyur
2000
52°N, 95°E
48°N, 24°E
56°N, 102°E
67°35′N, 66°19′E
67°08′N, 62°35′E
LE
LE
LE
TROM
TROM
V40a
V39a
V100
V119
V105
V106
V114
V94
Vm
Vv
s
s
h
h
h
h
h
h
s
s
Russia, Vorkuta, Lyasmylk
Russia, Vorkuta, Seyda
Russia, W Sayan Mts., Us R. valley, Aradana
Russia, W Taymyr, Efremov Kamen′ Bay
Russia, Yakutia, Allaikha distr., lower Indigirka R.
Russia, Yakutia, Indigirka R. drainage, Verkhny Tuguchan
Russia, Yakutia, lower Indigirka drainage, Shandrin R.
Korea, Ryang-gang do, Mt. Paekdu-san
V. myrtillus, France, Rhône-Alpes, Isère, Belledonne, Chamrousse
V. vitis-idaea, France, Rhône-Alpes, Isère, Belledonne, Chamrousse
67°28′N, 62°50′E
67°23′N, 62°55′E
53°N, 90°E
75°N, 79°E
62°N, 129°E
62°N, 129°E
62°N, 129°E
42°N 128°06′E
45°7′N, 05°54′E
45°7′N, 05°54′E
TROM
TROM
LE
LE
LE
LE
LE
TROM
TROM
TROM
150
1800
1800
Combined
haplotype
C
C
C
C
H
C
trnL/F
a
a
a
a
a
a
b
a
a
a
a
a
a
trnS/G
q
q
q
q
s
q
C I R C U M P O L A R M O L E C U L A R D I V E R S I T Y 2753