Journal of Biogeography (J. Biogeogr.) (2010) 37, 1262–1276
ORIGINAL
ARTICLE
The extreme Beringian/Atlantic
disjunction in Saxifraga rivularis
(Saxifragaceae) has formed at least twice
Kristine Bakke Westergaard1,2*, Marte Holten Jørgensen3, Tove M.
Gabrielsen2,4, Inger Greve Alsos4 and Christian Brochmann2
1
Tromsø University Museum, University of
Tromsø, NO-9037 Tromsø, Norway, 2National
Centre for Biosystematics, Natural History
Museum, University of Oslo, PO Box 1172
Blindern, NO-0318 Oslo, Norway,
3
Department of Biology, Centre for Ecological
and Evolutionary Synthesis (CEES), University
of Oslo, PO Box 1066 Blindern, NO-0316 Oslo,
Norway, 4University Centre in Svalbard,
PO Box 156, NO-9171 Longyearbyen, Norway
ABSTRACT
Aim The oceanic Saxifraga rivularis L. presents one of the most extreme
disjunctions known in the arctic flora: it has a small amphi-Beringian range and
a larger amphi-Atlantic one. It was recently suggested to have had a single
allopolyploid origin in Beringia at least one glacial cycle ago, followed by gradual
expansion in a more humid period and differentiation into two allopatric
subspecies (the Atlantic ssp. rivularis and the Beringian ssp. arctolitoralis). Here
we explore the history of its extreme disjunction.
Location The amphi-Beringian and northern amphi-Atlantic regions.
Methods We obtained amplified fragment length polymorphisms (AFLPs) and
chloroplast DNA sequences from 36 populations (287 individuals) and 13
populations (15 individuals), respectively. The data were analysed using principal
coordinates analyses, Bayesian clustering methods, and analyses of molecular
variance.
Results Two distinctly divergent AFLP groups were observed, corresponding
to the two described subspecies, but, surprisingly, four of the West Atlantic
populations belonged to the supposedly Beringian endemic ssp. arctolitoralis. This
was confirmed by re-examination of their morphological characteristics. The
overall AFLP diversity in the species was low (26.4% polymorphic markers),
and there was no variation in the five investigated chloroplast DNA (cpDNA)
regions. There was little geographic structuring of the AFLP diversity within each
subspecies, even across the extreme disjunction in ssp. arctolitoralis, across
the Bering Sea, and across the Atlantic Ocean, except that most plants from the
arctic Svalbard archipelago formed a separate genetic group with relatively
high diversity.
Main conclusions The extreme disjunction in S. rivularis has evidently formed
at least twice. The first expansion from Beringia was followed by allopatric
differentiation into one Beringian and one Atlantic subspecies, which are distinctly
divergent at AFLP loci but still harbour identical cpDNA haplotypes, suggesting
that the expansion was quite recent but before the last glaciation. The next
expansion from Beringia probably occurred by means of several long-distance
dispersals in the current interglacial, resulting in the colonization of the western
Atlantic region by ssp. arctolitoralis. The poor geographic structuring within each
subspecies suggests frequent long-distance dispersals from two main Weichselian
refugia, one Beringian and one western-central European, but it is possible that
the genetic group in Svalbard originates from an additional refugium.
*Correspondence: Kristine Bakke Westergaard,
Tromsø University Museum, University of
Tromsø, NO-9037 Tromsø, Norway.
E-mail: [email protected]
1262
Keywords
AFLPs, Beringia, biogeographical disjunction, long-distance dispersal, North
Atlantic region, Quaternary, refugia, Saxifraga rivularis.
www.blackwellpublishing.com/jbi
doi:10.1111/j.1365-2699.2010.02278.x
ª 2010 Blackwell Publishing Ltd
Extreme arctic disjunction in Saxifraga rivularis
INTRODUCTION
The history of arctic disjunctions can be expected to be
difficult to unravel, because glaciation-induced range shifts
must have been more frequent and dramatic in this area than
in more southern latitudes. The repeated advances of large ice
sheets in North America and Europe certainly led to the
fragmentation of previously continuous species distributions.
Hultén (1937, 1958) proposed that many arctic species were
unable to re-form their former ranges during interglacials
because of limited dispersal ability, often resulting in vicariant
disjunctions. However, several recent studies have demonstrated that long-distance dispersals occur frequently and
across vast distances in the Arctic (Alsos et al., 2007; Eidesen
et al., 2007a; Ehrich et al., 2008; Skrede et al., 2009).
The northern Atlantic region was extensively glaciated
during the Last Glacial Maximum (LGM; 25,000–10,000 years
ago; Andersen & Borns, 1997; Svendsen et al., 2004). Fossil
evidence shows that many arctic and alpine plant species
survived south and/or east of the ice sheet covering northern
Europe (Birks, 1994, 2008). In addition, some plants may have
survived in ice-free uplands or on mountain tops protruding
from the ice (nunataks), but there is so far no definite fossil or
molecular evidence supporting this hypothesis (Brochmann
et al., 2003). In contrast, the northern amphi-Beringian region
(called Beringia by Hultén, 1937) remained only partially
glaciated, and fossil, molecular and phytogeographical evidence show the importance of this area as a northern refugium
for arctic plants throughout the Quaternary (Hultén, 1937;
Abbott & Brochmann, 2003). Beringia experienced cycles of
sea-level changes, expanding the available terrestrial habitat
with the formation of the Bering Land Bridge during glacials,
and imposing a dispersal barrier when the land bridge was
flooded during interglacials (Hopkins, 1959; DeChaine, 2008).
Hultén (1937, 1958) suggested that most arctic plants
initially radiated east- and westwards from Beringia, and
reached a circumpolar distribution before the onset of the
Quaternary glaciations. He further proposed that arctic plants
persisted in Beringia during the glacial cycles, while their
distribution ranges were repeatedly fragmented and re-formed
elsewhere. He was, however, convinced that arctic plants
spread very slowly and thus were unable to re-form their full
distribution from Beringia during each of the short interglacials, and that the current, large distributions of many arctic
plants therefore have to be explained by expansion from
several additional refugia. Hultén’s hypothesis has recently
been tested for some circumpolar species using molecular and
fossil data. In Saxifraga oppositifolia, the existence of two
divergent chloroplast DNA (cpDNA) clades, one broadly
Beringian and one broadly Atlantic, fits nicely with Hultén’s
hypothesis of early radiation from Beringia and subsequent
persistence in separate refugia (Abbott et al., 2000; Abbott &
Brochmann, 2003; Abbott & Comes, 2003). In Vaccinium
uliginosum, the presence of three cpDNA lineages (one amphiBeringian, one amphi-Atlantic, and one circumpolar) and five
major genetic (amplified fragment length polymorphism –
Journal of Biogeography 37, 1262–1276
ª 2010 Blackwell Publishing Ltd
AFLP) groups also suggest early divergence in Beringia and
subsequent persistence in separate refugia (Alsos et al., 2005;
Eidesen et al., 2007b). However, analyses of Cassiope tetragona
(Eidesen et al., 2007a) and Rubus chamaemorus (Ehrich et al.,
2008) gave surprisingly different results. Although 2.5- to 2.0Myr-old fossils of C. tetragona are known from North
Greenland (Bennike & Böcher, 1990), this species showed no
cpDNA variation and a shallowly structured AFLP diversity
decreasing in a gradual, leading-edge fashion from Beringia to
the Atlantic region. This pattern suggests that the current
Atlantic populations originated from a recent, probably postglacial expansion from Beringia, and that earlier emigrants of
the species have gone extinct (Eidesen et al., 2007a). A similar
scenario was invoked for Rubus chamaemorus (Ehrich et al.,
2008).
Molecular studies of biogeographical disjunctions in the
arctic flora have so far focused on the disjunction across the
Atlantic Ocean, both in circumpolar and in amphi-Atlantic
species. Close trans-Atlantic relationships have been demonstrated in many species, suggesting a post-glacial origin of their
disjunctions through long-distance dispersal (summarized in
Abbott & Brochmann, 2003; Brochmann et al., 2003; Brochmann & Brysting, 2008). However, the most extreme
biogeographical disjunction observed in the arctic flora is
found between Beringia and the amphi-Atlantic region
(Fig. 1). Only a few of the species that are restricted mainly
to the Arctic show this type of disjunction (e.g. Luzula arcuata,
Beckwithia glacialis, and Saxifraga rivularis; Elven, 2007
onwards). These species are reported to be differentiated into
amphi-Beringian and amphi-Atlantic subspecies (Hultén,
1968; Lipkin & Murray, 1997; Jørgensen et al., 2006),
suggesting separation for a longer time span than the current
interglacial. Notably, the three species have in common that
they are confined to the most humid and least winter-cold
parts of the Arctic, bordering the northernmost Atlantic and
Pacific Oceans. They typically occur in sites with a thick,
insulating and late-melting snow cover, protecting against
winter and spring frost (Hultén, 1968; Lid & Lid, 2005).
In their recent study of the entire Saxifraga rivularis
complex, based on flow cytometry, morphology and AFLP
markers, Jørgensen et al. (2006) recognized four species. The
tetraploid S. rivularis L. was suggested to have originated from
a single alloploidization event in Beringia involving the amphiPacific, diploid Saxifraga bracteata lineage and the circumpolar, diploid Saxifraga hyperborea lineage (see also Brochmann
et al., 1998; Guldahl et al., 2005). They found distinct genetic
(based, however, on a single Beringian population of three
individuals only) and morphological differentiation within
S. rivularis, and recognized two subspecies, the amphi-Atlantic
ssp. rivularis and the amphi-Beringian ssp. arctolitoralis (Jurtz.
& V.V.Petrovsky) Jørgensen & Elven, suggested to have
diverged in allopatry after expansion from Beringia at least
one glacial cycle ago (Jørgensen et al., 2006). Their time
scenario is apparently consistent with findings of late
Weichselian macrofossils south of the ice sheet in Europe
(Birks & Willis, 2008), but it cannot be excluded that these
1263
K. B. Westergaard et al.
Figure 1 Geographic distribution (outlined), sampling for AFLP analysis (circles)
and cpDNA analysis (diamonds), and results
of structure analyses of the AFLP data for
Saxifraga rivularis (map in North Pole
Orthographic projection). The structure
analyses shown here were carried out separately for each subspecies (see text), with
K = 3 for ssp. rivularis (red) and K = 4 for
ssp. arctolitoralis (blue). Different shades of
red and blue represents different structure
groups. The pie diagrams show the frequency
of individuals belonging to the various
groups.
fossils rather represent the diploid S. hyperborea or originate
from late glacial dispersal of S. rivularis from Beringia.
Saxifraga rivularis is a strongly autogamous (Brochmann &
Håpnes, 2001), small (2–7 cm tall) perennial herb reproducing
through both seeds and short rhizomes. The species has shortrange ballistic seed dispersal, but no obvious morphological
adaptations to dispersal over longer distances except that the
seeds are small and light. It is obviously very cold-hardy; it has
been observed growing in bryophyte cushions deposited on a
glacier in Svalbard (I.G. Alsos, pers. obs.). Subspecies rivularis
has a long, glabrous or sparsely hairy flowering stem and short,
sparse glandular hypanthium hairs with non-coloured or
weakly coloured partition walls, whereas ssp. arctolitoralis has
a short, sparsely to densely hairy flowering stem and long, dense
glandular hypanthium hairs with purple partition walls
(Jørgensen et al., 2006). The subspecies also appear to differ
in habitat preferences. Subspecies rivularis is found in moist
and nutrient-rich habitats such as snow-beds, springs and birdmanured cliffs, whereas ssp. arctolitoralis seems to be limited to
clay and silt on seashores. Populations of both subspecies occur
fragmented throughout their distribution areas.
Jørgensen et al. (2006) hypothesized that the extreme
biogeographical disjunction in S. rivularis was caused by the
disruption of a previous continuous distribution during a
more humid climatic period. The Polar Sea was non-frozen
and the open sea caused a more oceanic climate along the
coasts of arctic Canada and northern Siberia during the
previous interglacial (120,000 yr bp; Frenzel et al., 1992; Miller
et al., 1999; Koerner & Fisher, 2002), which may have allowed
more continuous circumpolar distributions of species that
need snow protection in winter and moist conditions in
summer. Because the Polar Sea was frozen during the
glaciations and still more or less is in the current interglacial,
the more continental climate in arctic Canada and northern
1264
Siberia may have caused extinction in these areas and restricted
the populations to coastal regions of the North Pacific and
North Atlantic Oceans, which are characterized by more
oceanic climates.
As Jørgensen et al. (2006) had only limited material of
S. rivularis available for their AFLP analysis, especially from
the Beringian (one population only; their only representative
of ssp. arctolitoralis) and the West Atlantic areas, their
conclusions must be considered preliminary. We will therefore
re-address the history of the extreme biogeographical disjunction in this species based on more intensive sampling
throughout its distribution area, an extended AFLP dataset,
and DNA sequences from five chloroplast regions. We ask: (1)
whether the main disjunction originated from vicariance and/
or recent long-distance dispersal events; (2) whether there are
local, divergent genetic groups, possibly with high genetic
diversity, indicating a long in situ history; and (3) whether
there is genetic evidence for recent long-distance dispersal, in
particular trans oceanic dispersal, within the main disjunction
areas. We expect that the patterns of geographic structuring
and levels of genetic diversity within and between the current
ranges of the species will reflect the origin and history of the
disjunction. If the species colonized the amphi-Atlantic region
post-glacially from a Beringian source, we would expect to find
a shallow genetic structure at this geographic scale, and that
the disjunction originated from long-distance dispersal (given
the lack of suitable oceanic habitat in the intermediate regions
since the previous interglacial). If the disjunction is older, and
the species persisted in separate Beringian and Atlantic refugia
during the last glaciation, we would expect to find a stronger
genetic structure with distinctly divergent genetic groups. In
the latter case it will, however, be difficult to distinguish
between a vicariance hypothesis and a dispersal hypothesis
with certainty.
Journal of Biogeography 37, 1262–1276
ª 2010 Blackwell Publishing Ltd
Extreme arctic disjunction in Saxifraga rivularis
MATERIALS AND METHODS
Sampling and DNA isolation
Forty-five populations of S. rivularis covering most of the
distribution area of the species were included in this study.
Thirty-two of these populations were previously included by
Alsos et al. (2007) as part of a multi-species dataset used to
address colonization of the Arctic Svalbard archipelago.
Initially, 35 populations were referred to ssp. rivularis and 10
populations to ssp. arctolitoralis based on their geographic
origin (Table 1). From 36 of the populations, 11 plants (if
possible) were collected 25 m apart along a 250-m transect,
and fresh leaves were dried in silica gel for AFLP analysis.
Voucher specimens from these populations are deposited at
the Natural History Museum (NHM) in Oslo, and silica
samples and DNA extracts are stored in the DNA bank at the
National Centre for Biosystematics (NCB). Unfortunately, in
spite of considerable efforts we were not able to obtain new
field-collected plant material for AFLP analysis from Northwest Russia, where the species is very rare in most areas. For
the cpDNA sequencing, we initially included silica-dried leaf
material from eight individuals of ssp. rivularis and four
individuals of ssp. arctolitoralis (deposited at NHM in Oslo),
and leaf material from three herbarium specimens of ssp.
arctolitoralis obtained from the Russian Academy of Sciences,
St Petersburg (Table 1). Because this initial test revealed
identical cpDNA sequences, although specimens spanned the
entire geographic range of the species, we concluded that
sequencing of additional specimens was unnecessary.
Total genomic DNA was extracted using the DNeasyTM
Plant Mini Kit (Qiagen, Hilden, Germany) for the AFLP
analysis and the cetyl trimetyl ammonium bromide (CTAB)
method of Doyle & Doyle (1987) for the cpDNA sequencing.
One cm2 of dried leaf material was ground using tungsten
carbide beads for 2 min in a mixer mill (MM301; Retsch
GmbH & Co., Haan, Germany) at 20 Hz prior to the addition
of AP1 or CTAB, as appropriate. The quality of the extracted
DNA was checked on 1% agarose gels.
cpDNA sequencing
Five chloroplast regions were amplified using polymerase chain
reactions (PCRs). The trnL (UAA) intron and the trnL–trnF
intergenic spacer were amplified using the universal primers of
Taberlet et al. (1991), and the psbA–trnH region was amplified
using the primers psbAH and trnHR of Sang et al. (1997). The
psbC–trnS (UGA) region was amplified using the reverse
primer trnS of Demesure et al. (1995) combined with a new
forward primer (psbCMes, 5¢-GATCTTCGTGCTCCATGGTT). The trnS (GSU)–trnG (UCC) region was initially
amplified using the trnS and trnG primers of Hamilton
(1999), but because a long mononucleotide stretch disrupted
the sequence profiles in the 5¢ end, a new set of primers was
designed to amplify this region (trnSMes, 5¢-CAAGTTTTATCAGCAATTCYAA and trnGMes, 5¢-TGYRTTGTGCAAJournal of Biogeography 37, 1262–1276
ª 2010 Blackwell Publishing Ltd
GAATCCAG). All new primers were designed using Primer
3 (Rozen & Skaletsky, 2000).
PCRs were performed in volumes of 25 lL containing 1·
AmpliTaq buffer (Applied Biosystems, Foster City, CA, USA),
2.5 mm MgCl2, 1 mm of each dNTP, 0.04% bovine serum
albumen (BSA), 0.01% tetramethylammonium chloride
(TMACl), 0.8 lm of each primer, 1 U AmpliTaq polymerase
(Applied Biosystems) and 2 lL of genomic DNA. Amplifications were performed in a PTC-100 thermocycler (MJ
Research, Watertown, MA, USA) using the following conditions: for psbA–trnH, 4 min at 95 C was followed by six cycles
of 20 s at 95 C, 1 min at 58 C and 1 min at 72 C, and 35
cycles of 20 s at 95 C, 1 min at 52 C and 1 min at 72 C,
ending with a final 5-min extension at 72 C. For the other
four regions, 5 min at 94 C was followed by 25 cycles of 20 s
at 94 C, 20 s at 55 or 60 C and 40 s at 72 C, ending with a
final 7-min extension at 72 C. PCR products were purified
using a QIAquick PCR purification kit (Qiagen) or using
exonuclease I and shrimp alkaline phosphatase (ExoSAP-IT;
GE Healthcare, Chalfont St Giles, UK).
Cycle sequencing reactions were performed using the Big
Dye Terminator Cycle Sequencing kit v. 3.1 (Applied
Biosystems) in volumes of 10 lL containing 4 lL of Big
Dye, 0.1 lm primer and 3 lL of cleaned PCR product. The
sequencing reactions were run in a GeneAmp PCR system 9700
(Applied Biosystems) according to the protocol of the
manufacturer. Sequenced products were precipitated in
ethanol and sodium acetate to remove excess dye terminators
before running them on a capillary sequencer (ABI PRISM
3100; Applied Biosystems).
Sequences were assembled and edited manually using
Sequencher 4.1.4 (Gene Codes, Ann Arbor, MI, USA). The
sequences were submitted to GenBank with accession numbers
GU301250–GU301254.
AFLP fingerprinting
The AFLP analyses followed Gaudeul et al. (2000) with a few
modifications (Alsos et al., 2007). Sixty-four primer combinations were tested on four plants from different geographical
areas, and the following three selective primer combinations
were selected for the full analysis because they yielded most
variation (fluorescent dye in parenthesis): EcoRI AGA (6FAM)–MseI CAA; EcoRI AGG (NED)–MseI CAA; and EcoRI
AGG (VIC)–MseI CTG. For each individual, 1.3 lL of 6-FAM, 2 lL of NED-, and 1.3 lL of VIC-labelled selective PCR
products were combined with 0.2 lL of GeneScan ROX 500
(Applied Biosystems) and 11.8 lL of HiDi formamide, and
run on a capillary sequencer (ABI PRISM 3100; Applied
Biosystems). Raw data were collected and aligned with the
internal size standard using the ABI PRISM Genescan 3.7
analysis software (Applied Biosystems). Unambiguous fragments in the size range of 50–500 bp were scored using
Genographer 1.6 (available at: http://hordeum.oscs.montana.edu/genographer). The data were exported as a presence/
absence matrix.
1265
1266
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
riv
SUP03-634 Russia
AK-248
AK-281
CB99-25
CB99-45
CB99-47
AK-811
AK-846
AK-867
AK-416
AK-441
AK-456
AK-470
AK-499
AK-516
AK-745
AK-784
AK-109
AK-100
AK-101
AK-553
AK-554
AK-559
AK-740
AK-1239
TMG37-3
AK-494
AK-1336
TMG-43
Locality
Urals Federal District, Yamal, Harasavey
Newfoundland, Northern Pen,
Cape Onion
Greenland
Paamiut
Greenland
Nanortalik, Storefjell
Greenland
Ittoqqortoormiit, Uunarteq (Kap Tobin)
Greenland
Ammassallip, Tasiilaq, Blomsterdalen
Greenland
Ammassallip, Tasiilaq
Iceland
Vestfir d i, Hólmavik
Iceland
Vesturland, Akrafjall
Iceland
Nor d urland Eystra, Akureyri
Norway
Buskerud, Ål, Reineskarvet
Norway
Oppland, Lom, Vardhø
Norway
Hordaland, Odda
Norway
Sør-Trøndelag, Oppdal, Leirtjønnkollen
Norway
Sør-Trøndelag, Oppdal, Vinstradalen
Norway
Hordaland, Finse
Norway
Troms, Tromsø, Fløya
Norway
Troms, Storfjord, Lavkajavre
Norway (Jan Mayen) Blinddalen, Shertzegga
Norway (Svalbard)
Haakon VII Land, Mayerbukta
Norway (Svalbard)
Oscar II Land, Kjærstranda
Norway (Svalbard)
Nordenskiöld Land, Platåberget
Norway (Svalbard)
Nordenskiöld Land, Adventdalen, Gruve 7
Norway (Svalbard)
Nordenskiöld Land, Colesdalen
Norway (Svalbard)
Haakon VII Land, Blomstrandhalvøya
Norway (Svalbard)
Nordenskiöld Land, Vårsolbukta
Norway (Svalbard)
Oscar II Land, Alkhornet
Norway (Bjørnøya)
Kvalrossbuktdeltaet
Scotland
Highlands, Grampian Mts, Loch Nagar
Scotland
Highlands, Argyll, Bidean nam Bian
Canada
riv
riv
SUP-3034
Country
A priori A posteriori
det
det
Pop ID
Herb
PBE, GHJ
PBE, GHJ
CB, JN, PBE, SK
IGA, LL
IGA, LL
IS, LL, SK
IS, LL, SK
IS, LL, SK
MHJ, KBW, SK
PBE, IS, GHJ
PBE, IS, GHJ
PBE, IS, GHJ
AKB
MHJ, IS, GHJ
IGA, KBW
IGA, KBW
AW
IGA, BES
IGA, BES
IGA, KBW
IGA, KBW
IGA, KBW
IGA, BES
KBW
TMG
RS
PBE, IS
TMG, SWS,
KTH, MT, KW
?
SVER
IGA, AKB
Collectors*
ca. 70.25
61.99
60.15
70.41
65.87
65.85
65.74
64.33
65.81
60.75
61.67
59.84
62.44
62.00
60.61
69.64
69.21
71.00
79.26
78.90
78.23
78.16
78.10
78.99
77.76
78.22
74.38
56.96
56.67
51.58
Latitude
ca. 73.27
–
1
–
–
–
–
–
–
–
–
–
–
–
–
–
1
1
–
–
–
–
–
–
–
–
–
1
–
–
1
–
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
–
1
0
0
–
0.061
0.049
0.038
0.012
0.077
0.051
0.037
0.031
0.013
0.013
0.042
0.077
0.070
0.041
0.059
0.030
0.023
0.050
0.109
0.077
0.038
0.115
0.047
0.096
–
0.068
0.038
0.022
–
0.922
1.786
0.697
0.727
0.810
1.206
1.144
0.933
0.810
1.047
0.846
0.982
0.905
0.932
2.293
1.194
0.922
1.576
1.437
1.506
1.574
2.759
1.900
1.701
–
2.277
1.047
1.168
10
11
5
5
2
10
11
10
9
11
11
5
7
10
11
11
11
11
6
6
8
8
11
11
–
11
11
16
)49.65
)45.29
)21.94
)36.96
)36.98
)22.11
)21.93
)17.99
8.33
8.04
7.06
9.73
9.40
7.53
19.01
20.46
)8.50
12.14
11.50
15.47
16.03
15.22
11.97
14.39
13.83
19.18
)3.24
)5.00
N/A
N/A
0
1
)55.70
–
DW
n
n
No. private
Longitude (AFLP) (cpDNA) markers
D
Table 1 Collection information and AFLP diversity in the populations of Saxifraga rivularis analysed. A priori and a posteriori taxonomic determination of populations are abbreviated as
arc (S. rivularis ssp. arctolitoralis) and riv (S. rivularis ssp. rivularis); Pop ID refers to the collection code or the herbarium registration code for the populations; number of individuals per
population used for AFLP analysis [n (AFLP)] and cpDNA variation [n (cpDNA)]; number of private AFLP markers (no. private markers); average AFLP diversity (D), and frequency-downweighted-marker values (DW). If not stated otherwise, herbarium vouchers, silica-dried leaf samples and DNA extracts for each population are deposited at the Natural History Museum,
University of Oslo (ALA, University of Alaska, Museum of the North, Fairbanks, USA; LE, V. L. Komarov Botanical Institute, Russian Academy of Sciences, St Petersburg, Russia; SVER,
Herbarium, Institute of Plant and Animal Ecology, Ural Centre, Russian Academy of Sciences, Ekaterinburg, Russia).
K. B. Westergaard et al.
Journal of Biogeography 37, 1262–1276
ª 2010 Blackwell Publishing Ltd
Journal of Biogeography 37, 1262–1276
ª 2010 Blackwell Publishing Ltd
riv
arc
arc
arc
arc
arc
arc
arc
arc
arc
arc
arc
arc
arc
arc
riv
riv
riv
riv
riv
arc
arc
arc
arc
arc
arc
arc
arc
arc
arc
BE-1080
BE-1090
RE01-11
TC03-202
TC03-279
68-09K
71-17-1
70-160y
BE-691
SUP04-36
CB99-39
AK-4615
AK-1148
BE-56
FJL1996
Pop ID
USA
USA
USA
USA
USA
(Alaska)
(Alaska)
(Alaska)
(Alaska)
(Alaska)
Russia
Russia
Russia
Russia
Canada
Greenland
Greenland
Greenland
Russia
Russia
Country
Northwest Federal District, Franz
Josef Land
Baffin Island, Iqaluit
Ittoqqortoormiit
Upernavik, Tasiusaq
Ilulissat, Saqqaq
Far Eastern Federal District, Chukchi
Peninsula, Novo Chaplino
Far Eastern Federal District, Chukchi
Peninsula, Lavrentiya Bay
Far Eastern Federal District, E Chukchi,
Leymin Cape, Kitulinveem River
Far Eastern Federal District, Wrangel Island,
Somnitelnaya harbour
Far Eastern Federal District, W Chukchi,
Chaunskaya Bay
Barrow
Barrow
Seward Peninsula, Shishmaref Inlet S
Demascation Point Q, Jay Reef
Point Barrow Peninsula
Locality
Levitsev, Korobkov,
Yurtsev
HS, GK
RE
RE, HS
Meyers, Bliedman
Glacher, McPherson,
Galeski
Kozhevnikov,
Nechaev, Yurtsev
Petrovsky, Taraskina
HS, RE
SA
CB, JN, PBE, SK
KBW
KBW
HS, RE
H Strøm?
Collectors*
ALA
ALA
LE
LE
LE
Herb
71.30
71.30
66.53
69.76
71.27
69.00
72.50
66.00
65.63
63.73
70.49
73.37
70.01
64.50
ca. 80.33
Latitude
–
)170.00
–
5
5
–
–
–
170.00
)156.73
)156.73
)164.77
)141.62
)156.83
–
5
)171.22
180.00
2
4
6
6
4
–
n
(AFLP)
)68.50
)21.95
)56.05
)51.96
)172.83
ca. 55.59
Longitude
–
–
2
1
1
1
1
1
–
1
–
–
–
2
n
(cpDNA)
0
0
–
–
–
–
–
–
1
0
0
0
0
5
–
No. private
markers
0.046
0.027
–
–
–
–
–
–
0.050
0.038
0.010
0.065
0.065
0.147
–
D
1.399
1.304
–
–
–
–
–
–
2.295
1.034
1.120
1.239
1.626
6.884
–
DW
*Collectors: AKB, A. K. Brysting; AW, A. Wollan; BES, B. E. Sandbakk; CB, C. Brochmann; GHJ, G. H. Jacobsen; GK, G. Kihlberg; HS, H. Solstad; IGA, I. G. Alsos; IS, I. Skrede; JG, J. Glacher; JN, J. Nyléhn;
KBW, K. B. Westergaard; KTH, K. T. Hansen; KW, K. Watson; L, Levitsev; LL, L. Lund; MHJ, M. H. Jørgensen; MT, M. Tebbitt; PBE, P. B. Eidesen; RE, R. Elven; RS, R. Solheim; SA, S. Aiken; SK, S. Kjølner;
SWS, S. W. Steen; TMG, T. M. Gabrielsen.
A posteriori
det
A priori
det
Table 1 Continued
Extreme arctic disjunction in Saxifraga rivularis
1267
K. B. Westergaard et al.
The dataset was checked for linked markers by replacing
individuals with markers in the matrix and constructing a
neighbour-joining tree using simple matching as distance
coefficient in the program treecon 1.3b (Van de Peer & De
Wachter, 1994). Eleven linked markers found in ssp. rivularis
were removed (cf. Bonin et al., 2007). Negative controls and
replicates were included to test for contamination and
reproducibility, based on the same as well as on new DNA
extracts. Reproducibility was estimated as the average proportion of correctly replicated markers (Bonin et al., 2004), and
markers with low reproducibility were excluded from further
analyses. Markers with a proportion of presences and absences
lower than or similar to the error rate were checked once more,
and kept if they had clear peaks.
dependent among populations when analysing all populations
together, and correlated when analysing the two subspecies
separately. Similarity coefficients and DK values (Evanno et al.,
2005) comparing the resulting assignments were calculated
using structure-sum (Rosenberg et al., 2002; Ehrich, 2006)
in R 2.5.0 (R Development Core Team, 2007). baps 3.2 uses a
stochastic optimization algorithm when identifying the
optimal number of clusters (Corander & Marttinen, 2006).
We clustered the individuals using the population mixture
analysis, and the maximum number of clusters was set to 18–
22 in three independent runs with otherwise default settings.
To evaluate the structure identified by the various methods,
the different groupings were analysed using analyses of
molecular variance (AMOVAs) based on pairwise differences
in Arlequin 3.11 (Excoffier et al., 1992, 2005).
AFLP data analysis
Gene diversity (D) was estimated as the average number of
pairwise differences between individuals for each population
(Kosman, 2003). To quantify the amount of rare markers, we
calculated frequency-down-weighted-marker values (DW) as a
measure of divergence (Schönswetter & Tribsch, 2005) as
follows. For each population, each AFLP marker was divided
by the total number of occurrences of that particular marker in
the total dataset, and these relative values were added to obtain
the rarity index for this population. Calculations were carried
out using AFLPdat (Ehrich, 2006) in R 2.5.0 (R Development
Core Team, 2007).
The AFLP data were subjected to parsimony analyses in tnt
(Goloboff et al., 2008). Heuristic searches were performed with
10,000 random addition sequences and tree bisection–
reconnection (TBR) branch swapping, saving three trees per
replication. The resulting trees were swapped with TBR, saving
up to 30,000 trees altogether. The collapsing rule was set to
minimum length = 0, and random seed was set to ‘time’.
Goodness-of-fit statistics were calculated using the consistency
index (CI), retention index (RI) and rescaled consistency index
(RC) (Kluge & Farris, 1969; Farris, 1989). Jackknife (36%
deletion, Farris et al., 1996) and bootstrap (Felsenstein, 1985)
resampling analyses were performed with 1000 replicates.
The variation in the AFLP dataset was visualized using
principal coordinates analysis (PCoA) in NTSYSpc 2.02
(Rohlf, 1999) based on simple matching similarity.
To identify molecular structure in our dataset, the Bayesian
non-hierarchical clustering methods structure and baps
were used. Both assume Hardy–Weinberg equilibrium and that
the loci are unlinked and at linkage equilibrium. structure
2.2 (Falush et al., 2007) calculates a logarithmic probability for
the data being assigned to a given number of clusters, based on
minimizing linkage among clusters and maximizing linkage
within them. Using an admixture model, 10 replicates of each
value of K (the number of groups, set to 1–20) were run for
various selections of samples under the recessive allele model
with a burn-in period of 100,000 and 1,000,000 iterations
using the Bioportal at the University of Oslo (http://
www.bioportal.uio.no). Allele frequencies were assumed in1268
RESULTS
cpDNA sequences
The cpDNA alignment consisted of 2046 nucleotides in total.
The trnL intron region was 565 bp long, and the intergenetic
spacers were 412 bp (trnL–trnF), 211 bp (psbA–trnH), 549 bp
(psbC–trnS) and 309 bp (trnS–trnG) long. No variation was
observed in any of these five investigated chloroplast regions
among the 15 individuals sequenced, which represented both
subspecies and most of their distribution area (Fig. 1).
AFLP data
In total, 287 individuals from 36 populations were successfully
analysed for AFLPs. Based on 81 replicates, the reproducibility of
the AFLP markers was 97.25%. The three primer combinations
resulted in a final dataset of 197 markers, after removal of linked
markers, markers with low reproducibility and markers with
frequency below the error rate (see Materials and Methods), of
which only 52 were polymorphic (26.4%).
All analyses of the AFLP data (see Figs 1 & 2; and Fig. S1a,b
in the Supporting Information) revealed a distinct division
into two groups of populations, one amphi-Atlantic and one
mainly Beringian. Surprisingly, four of the populations from
the western Atlantic area grouped with the Beringian
populations. We re-examined the herbarium vouchers of these
four Atlantic populations using the key and morphological
descriptions given by Jørgensen et al. (2006) and found that
they clearly belonged to the supposedly Beringian endemic ssp.
arctolitoralis (small plants with short, sparsely to densely hairy
flowering stem and long, dense glandular hypanthium hairs
with purple partition walls). We also re-examined all other
vouchers and confirmed that the Beringian populations
belonged to ssp. arctolitoralis and all remaining amphi-Atlantic
ones to ssp. rivularis, consistent with the grouping based on the
AFLP data. The re-determined AFLP dataset thus consisted of
250 individuals from 28 populations of ssp. rivularis and
37 individuals from eight populations of ssp. arctolitoralis
(Table 1).
Journal of Biogeography 37, 1262–1276
ª 2010 Blackwell Publishing Ltd
Extreme arctic disjunction in Saxifraga rivularis
(a)
(b)
Figure 2 Principal coordinates analysis of 287 individuals of Saxifraga rivularis based on 52 polymorphic AFLP markers and simple
matching similarity. (a) Individuals referred to population, (b) individuals referred to groups according to separate structure analyses of
each subspecies (blue, ssp. arctolitoralis; red, ssp. rivularis).
The PCoA of the total AFLP dataset resulted in two wellseparated groups along the first axis, which extracted as much
as 25.0% of the variation (Fig. 2). One group (to the right in
Journal of Biogeography 37, 1262–1276
ª 2010 Blackwell Publishing Ltd
Fig. 2) corresponded to ssp. rivularis and the other to ssp.
arctolitoralis. The second axis (14.0%) spanned the variation
within ssp. rivularis, partly separating the Svalbard populations
1269
K. B. Westergaard et al.
from the rest. The third axis (8.2%) gave no additional
information. Separate PCoAs of each subspecies revealed no
further structure (not shown).
The parsimony analysis of the AFLP markers resulted in at
least 30,000 most parsimonious trees of length 283 (CI = 0.18,
RI = 0.81, RC = 0.15). The strict consensus tree resolved two
clades (78% bootstrap and 87% jackknife support) corresponding to the two subspecies (not shown). No further
subdivision was supported by more than 50%, except for parts
of individual populations.
The structure analysis of the total AFLP dataset with K = 2
resulted in groups corresponding exactly to the two subspecies
as identified based on morphology. However, the largest value
of K giving a combination of high likelihood and similarity
coefficients for the total dataset was six (Fig. S1a,b). One of the
six groups contained all populations of ssp. arctolitoralis; the
other five contained the populations of ssp. rivularis (not
shown). Calculating DK (Evanno et al., 2005) to determine the
optimal K for our dataset was not appropriate, as the standard
deviation of L¢(K) for most values of K was zero, and DK was
therefore infinitely large. In accordance with the maximum
parsimony and the PCoAs, we therefore chose K = 2 as the most
appropriate division of the total dataset and performed separate
structure analyses for each subspecies (Fig. 1). For ssp.
Table 2 Analyses of molecular variance of Saxifraga rivularis based on 52 AFLP markers. ‘Total’ treats the dataset as one group; ‘Total two
groups’ splits the dataset according to the two groups inferred by the principal coordinates analysis and maximum parsimony analyses;
‘Total structure groups’ splits the dataset into the six groups identified by structure; ‘Total baps groups’ splits the dataset into the
eight groups identified by baps. The two subspecies were also analysed separately based on the groups identified by structure and baps.
When assigning individuals to groups, populations were assigned as a unit based on the population mean.
Source of variation
d.f.
Sum of
squares
Variance
components
Percentage
of variation
Total
Among populations
Within populations
Among groups
Among populations within groups
Within populations
Total structure groups
Among groups
Among populations within groups
Within populations
Total baps groups
Among groups
Among populations within groups
Within populations
ssp. rivularis
Among populations
Within populations
ssp. rivularis structure groups
Among groups
Among populations within groups
Within populations
ssp. rivularis baps groups
Among groups
Among populations within groups
Within populations
ssp. arctolitoralis
Among populations
Within populations
ssp. arctolitoralis structure groups
Among groups
Among populations within groups
Within populations
ssp. arctolitoralis baps groups
Among groups
Among populations within groups
Within populations
724.631
318.401
1043.031
244.141
480.490
318.401
1043.031
474.174
250.457
318.401
1043.031
532.333
192.298
318.401
1043.031
396.945
275.351
672.296
175.912
221.034
275.351
672.296
279.463
117.483
275.351
672.296
83.545
43.050
126.595
58.325
25.219
43.050
126.595
47.925
35.619
43.050
126.595
2.45050
1.26853
3.71903
3.62793
1.60805
1.26853
6.05451
2.06572
0.89834
1.26853
4.20672
2.15359
0.70212
1.26853
4.12424
1.51486
1.24032
2.75518
0.95499
0.86345
1.24032
3.05876
1.20837
0.50492
1.24032
2.95361
2.28218
1.48448
3.76667
1.44509
1.08322
1.48448
4.01279
1.58973
1.23130
1.48448
4.30551
65.89
34.11
Total two groups
35
251
286
1
34
251
286
6
29
251
286
7
28
251
286
27
222
249
2
25
222
249
6
21
222
249
7
29
36
3
4
29
36
2
5
29
36
1270
55.78
24.72
19.50
48.81
21.22
29.97
52.22
17.02
30.76
54.98
45.02
31.22
28.23
40.55
40.91
17.10
41.99
60.59
39.41
36.01
26.99
36.99
36.92
28.60
34.48
Journal of Biogeography 37, 1262–1276
ª 2010 Blackwell Publishing Ltd
Extreme arctic disjunction in Saxifraga rivularis
rivularis, K = 3 was chosen as the most appropriate number of
groups based on a combination of high likelihood and similarity
coefficients (Fig. S1c,d; K = 6 and 7 also gave high likelihood
and similarity coefficients, but many individuals were divided
between groups). One of the three groups in ssp. rivularis
contained most of the plants from Svalbard and Bjørnøya, one
was mainly northern amphi-Atlantic, and one was mainly
southern amphi-Atlantic (Fig. 1). For ssp. arctolitoralis, K = 4
was considered most appropriate (Fig. S1e,f). One of these four
groups contained a single Chukotka population (BE-56), and
each of the other three contained individuals from Beringia as
well as from the western Atlantic area (Fig. 1).
The eight groups obtained from the baps analysis of the
total dataset (not shown) mostly corresponded to those from
structure. Subspecies rivularis was divided into five baps
groups: two corresponding to the two amphi-Atlantic
structure groups, and three corresponding to the structure group restricted to Svalbard and Bjørnøya. Subspecies
arctolitoralis was divided into three baps groups corresponding
to two of the structure groups (populations BE-056, and
AK-4615, BE-691 and SUP04-36) and a collapsing of the last
two (populations AK-1148, BE-1080, BE-1090 and CB-99-39).
A two-level AMOVA of the total dataset resulted in 65.89%
among-population variation and 34.11% within-population
variation (Table 2). Grouping of populations according to the
two subspecies gave 55.78% among-group variation; grouping
according to the seven structure groups gave 48.81%; and
grouping according to the eight baps groups gave 52.22%.
When analysing the two subspecies separately, 54.98% of the
variation in ssp. rivularis was among populations, and the
corresponding value for ssp. arctolitoralis was 60.59%. Subdivision of ssp. rivularis according to the three structure
groups explained 31.22% of the variation, whereas the five
baps groups explained 40.91%. Subdivision of ssp. arctolitoralis according to the four structure groups explained 36.01%
of the variation, and that according to the three baps groups
explained 36.92% of the variation.
Eight of the AFLP markers were private to ssp. rivularis.
Four of them were distributed all over Svalbard, but limited to
this archipelago, and the other four were widespread. Nine
markers were private to ssp. arctolitoralis, of which five were
limited to a single population from Chukotka (BE-56). Each of
the four West Atlantic populations of ssp. arctolitoralis
harboured different subsets of AFLP markers found in the
Beringian populations.
Overall AFLP gene diversity in S. rivularis was low
(0.052 ± 0.03) and quite similar in the two subspecies
(Table 1, Fig. 3). Gene diversity in ssp. rivularis varied from
0.012 in one population from East Greenland (CB99-45) to
0.115 in one population from Svalbard (AK-559), and was on
average 0.051 ± 0.028. Gene diversity in ssp. arctolitoralis
varied from 0.010 in one population from East Greenland
(CB99-39) to 0.147 in one population from Chukotka (BE-56),
and was on average 0.056 ± 0.041. A similar pattern appeared
from the distribution of DW as a measure of population
divergence (Table 1). In ssp. rivularis, the DW value ranged
from 0.727 in one East Greenlandic population (CB99-45) to
2.759 in one Svalbard population (AK-559), and was on
average 1.300 ± 0.534. In ssp. arctolitoralis, the DW value
ranged from 1.120 in one East Greenlandic population (CB9939) to 6.884 in one population from Chukotka (BE-56), and
was on average 2.113 ± 1.968.
DISCUSSION
The extreme disjunction in S. rivularis has formed
at least twice
Our AFLP analysis confirms that the extremely disjunct
S. rivularis contains two distinctly divergent genetic groups
Figure 3 Intra-population AFLP diversity
(D) in Saxifraga rivularis (map in North Pole
Orthographic projection). The populations
of ssp. arctolitoralis are shown by a thick
outline and ssp. rivularis by a thin outline.
One population (Cape Onion, Canada; SUP3034) was not included in the AFLP diversity
analysis as it was represented by a single
individual.
Journal of Biogeography 37, 1262–1276
ª 2010 Blackwell Publishing Ltd
1271
K. B. Westergaard et al.
corresponding to ssp. rivularis and ssp. arctolitoralis as
described based on morphology, in agreement with Jørgensen
et al. (2006). Surprisingly, however, in contrast to their report
of ssp. arctolitoralis as an exclusively Beringian endemic, we
found that both subspecies occur in the Atlantic region. Four of
the West Atlantic populations analysed here clearly belonged to
the genetic group corresponding to ssp. arctolitoralis. This was
confirmed by re-examination of their morphological characteristics. Notably, each of these four Atlantic populations
contained different subsets of AFLP markers found in Beringia,
and three of the four structure groups identified in ssp.
arctolitoralis occurred both in Beringia and in the West Atlantic
region, suggesting several very recent connections across the
vast Canadian Arctic (Fig. 1). Even though Atlantic populations of the two subspecies occur in close proximity today (e.g.
in Ittoqqortoormiit, East Greenland), the AFLP data do not
indicate any hybridization between them.
Thus, our new molecular data demonstrate that the extreme
disjunction in S. rivularis has formed at least twice. The first
expansion from Beringia was apparently followed by allopatric
differentiation into one Beringian and one Atlantic subspecies.
The fact that the two subspecies are distinctly divergent at
AFLP loci but still share a single, identical cpDNA haplotype
suggests that the first expansion from Beringia occurred quite
recently, but before the last glaciation. In spite of the genetic
distinctness of the two subspecies, the overall AFLP diversity at
the species level was low (only 26.4% polymorphic markers
were obtained, in spite of extensive primer testing to maximize
variation). This finding corroborates the hypothesis that
S. rivularis originated a single time through allopolyploidization (Jørgensen et al., 2006). A single polyploidization event
imposes a very strong genetic bottleneck because alleles are
sampled from only one individual of each diploid progenitor
lineage (or possibly two individuals in the case of a two-step
process involving backcrossing of a triploid hybrid to another
individual of one of the progenitor species; cf. Soltis & Soltis,
2009). Finding low variation in a polyploid derivative species
also suggests that insufficient time has elapsed since the
polyploidization event for new mutations to accumulate at
nuclear loci. This bottleneck and the absence of new mutations
are also evident in the cpDNA genome of S. rivularis. The
single cpDNA haplotype we observed across its entire range in
this study is identical to one of several haplotypes observed in
the Beringian range of one of its diploid progenitor lineages,
the circumpolar S. hyperborea lineage (T.M. Gabrielsen &
C. Brochmann, unpublished data). In conclusion, we find it
very likely that the first expansion from Beringia occurred
before the last glaciation, although the available data are
insufficient to determine whether this first expansion occurred
by means of direct long-distance dispersal from Beringia to the
Atlantic region or through gradual expansion and subsequent
extinction in the Canadian Arctic and/or northern Siberia (i.e.
vicariance; further discussed below).
The next expansion from Beringia probably occurred
through several long-distance dispersals in the current postglacial period, resulting in the colonization of the western
1272
Atlantic region by ssp. arctolitoralis. We argue that this
expansion has been very recent because the West Atlantic
populations of this subspecies are both morphologically and
genetically indistinguishable from the Beringian ones, and still
harbour different subsets of the AFLP markers occurring today
in Beringia (Figs 1 & 2). There are two factors favouring longdistance dispersal over a vicariance hypothesis in the case of
this most recent expansion. First, the ecological characteristics
of the subspecies (as well as of the species) suggest that it will
have been unable to grow in most of Arctic Canada and Siberia
in the current inter-glacial period, because of the continentality
of the climate imposed by the frozen Polar Sea in these areas
(Frenzel et al., 1992; Miller et al., 1999; Koerner & Fisher,
2002). Second, S. rivularis evidently has high dispersal ability
in spite of its lack of obvious morphological adaptations, as
previously demonstrated for several arctic plant species
(Abbott & Brochmann, 2003; Alsos et al., 2007; Brochmann
& Brysting, 2008). This can be deduced from the high genetic
similarity among populations of S. rivularis from different
sides of the Atlantic and the generally poor geographic
structuring of the genetic variation within each subspecies
(Figs 1 & 2). The small seeds of S. rivularis may favour wind
dispersal, which can be particularly efficient over snow and ice
in the treeless arctic environment (cf. Savile, 1972; Ryvarden,
1975).
Thus, it is neither necessary nor plausible to invoke
vicariance as an explanation for the extreme Beringian–
Atlantic disjunction within ssp. arctolitoralis. We therefore
conclude that long-distance dispersal across the vast Canadian
Arctic, probably as several independent events, in the current
post-glacial period is the most likely explanation for the
disjunction in this subspecies. Our finding of four populations
of ssp. arctolitoralis in the West Atlantic area has motivated a
new revision of all available herbarium material from northeastern North America and Greenland. According to M.
Blondeau (Herbier Louis-Marie, Université Laval, Québec,
pers. comm.), ssp. rivularis also dominates in these geographic
areas, but there are also a considerable number of collections
definitely belonging to ssp. arctolitoralis from scattered sites
along the West Greenlandic coast and the north-eastern
Canadian coast. This corroborates our hypothesis that this
subspecies has colonized the West Atlantic area by means of
several long-distance dispersal events.
It is more difficult to determine whether the first, probably
pre-Weichselian, expansion from Beringia was more gradual or
also in this case involved direct long-distance dispersals to the
Atlantic region. Jørgensen et al.’s (2006) hypothesis that the
oceanic S. rivularis had a more continuous circumpolar
distribution during the last interglacial (c. 130,000 years
ago), when the climate was more oceanic along the Polar
Sea, can tentatively gain some support from the fact that the
species today is very rare in most of its north-western Russian
range (Fig. 1), which may suggest a scenario of gradual
extinction in northern Russia because of continentality.
However, we tend to favour a long-distance dispersal
hypothesis also in this case because the demonstrated high
Journal of Biogeography 37, 1262–1276
ª 2010 Blackwell Publishing Ltd
Extreme arctic disjunction in Saxifraga rivularis
long-distance dispersal ability of the species makes a vicariance
hypothesis superfluous.
descend from a separate glacial refugium, and if so, where this
was located.
Glacial refugia
CONCLUSIONS
The overall poor geographic structuring of the AFLP diversity
within each subspecies suggests that they expanded postglacially from two main Weichselian refugia, one Beringian
and one Atlantic. In addition, it is possible that the genetic
group identified in Svalbard originates from a separate
refugium. The main Weichselian refugium for ssp. rivularis
may have been located on the tundra south of the North
American and/or the northern European ice sheets (Frenzel
et al., 1992; Tarasov et al., 2000). The fossil findings reported
as S. rivularis from LGM and Weichselian late-glacial deposits
in northern and central Europe (Birks & Willis, 2008) may
indicate that its main Atlantic refugium was located in this
region, but it cannot be excluded that the fossils represent one
of its diploid progenitors, S. hyperborea, which is morphologically very similar to S. rivularis. The importance of the
Central European lowlands as an LGM refugium has recently
been demonstrated for another arctic–alpine species, Salix
herbacea (Alsos et al., 2008). The extensive fossil record of this
species shows that it was widely distributed in the Central
European lowlands during the last glaciation.
Surprisingly, we found that the genetic diversity in
S. rivularis was as high in the formerly extensively glaciated
Svalbard archipelago (including Bjørnøya) as in the formerly
partially ice-free Beringia. The Svalbard populations also
formed a separate AFLP group (Fig. 1) with high DW values
(Table 1), which may suggest that they originated from a
separate Weichselian refugium. Recent evidence suggests that
small ice-free areas existed in Svalbard during the late
Weichselian glaciation (e.g. Landvik et al., 2003; Ottesen et al.,
2007), which could have served as refugia for extremely hardy
species such as S. rivularis. It is, however, questionable whether
such small nunatak areas could harbour sufficiently large
populations to sustain high levels of genetic diversity (cf.
Widmer & Lexer, 2001). Another possible source area for the
Svalbard populations could be East Greenland, where ice-free
uplands existed during the LGM (Landvik, 1994; Funder et al.,
1998). The origins of Svalbard populations of two other arctic–
alpine plants, Empetrum nigrum and Cassiope tetragona, have
been traced to East Greenland (Alsos et al., 2007; Eidesen
et al., 2007a). The main source region for several other
Svalbard species has, however, been identified as north-western
Russia (Alsos et al., 2007), but, in spite of considerable efforts,
we were not able to obtain plant material of S. rivularis from
this area for AFLP analysis. The species is quite common on
Novaya Zemlya and the arctic archipelago of Franz Josef Land,
which are difficult to access; otherwise, it only occurs as very
scattered along the arctic coast from the Kola Peninsula to
Taimyr (author observations based on vouchers in several
herbaria). In conclusion, we cannot determine with certainty
based on the available data whether the Svalbard populations
Our AFLP and cpDNA data show how the extreme disjunction
in S. rivularis has formed at least twice. The first, preWeichselian expansion from Beringia was followed by
allopatric differentiation into the Beringian ssp. arctolitoralis
and the Atlantic ssp. rivularis. The subspecies are distinctly
divergent at AFLP loci but share the same cpDNA haplotypes,
which is consistent with Jørgensen et al.’s (2006) hypothesis
that the species had a recent, but pre-Weichselian origin in
Beringia. The next expansion from Beringia probably occurred
through several long-distance dispersals across the vast
Canadian Arctic in the current interglacial, resulting in
colonization of the western Atlantic region by ssp. arctolitoralis. The origin of this striking ‘double’ disjunction (within the
species and within one of its subspecies) through vicariance
cannot be ruled out, but it is not necessary to invoke, given the
high long-distance dispersal ability demonstrated by the
molecular data. The poor geographic structuring within each
subspecies suggests frequent long-distance dispersals from two
main Weichselian refugia, one Beringian and one westerncentral European, but it is possible that the genetically distinct
group in Svalbard originates from a separate refugium.
Journal of Biogeography 37, 1262–1276
ª 2010 Blackwell Publishing Ltd
ACKNOWLEDGEMENTS
We thank our colleagues and field assistants for help with
providing plant samples; the authorities in various regions for
permits to collect samples and help with field logistics; Liv
Guro Kvernstuen for laboratory assistance; Andreas Tribsch
and Dorothee Ehrich for statistical advice and discussions;
Marcel Blondeau for informing us on his revision of Canadian
material of S. rivularis and S. hyperborea; and Torstein
Engelskjøn and two anonymous referees for comments on an
earlier version of the manuscript. The study was supported by
grants 150322/720 and 170952/V40 to C.B. from the Research
Council of Norway. Additional grants to K.B.W. were obtained
from Roald Amundsen’s Centre for Arctic Research, K. and H.
Jakobsens Fund, King Haakon VII Educational Fund, Kometen, and Tromsø University Museum.
REFERENCES
Abbott, R.J. & Brochmann, C. (2003) History and evolution of
the arctic flora: in the footsteps of Eric Hultén. Molecular
Ecology, 12, 299–313.
Abbott, R.J. & Comes, H.P. (2003) Evolution in the Arctic: a
phylogeographic analysis of the circumarctic plant, Saxifraga
oppositifolia (Purple saxifrage). New Phytologist, 161, 211–
224.
Abbott, R.J., Smith, L.C., Milne, R.I., Crawford, R.M.M.,
Wolff, K. & Balfour, J. (2000) Molecular analysis of plant
1273
K. B. Westergaard et al.
migration and refugia in the Arctic. Science, 289, 1343–
1346.
Alsos, I.G., Engelskjøn, T., Gielly, L., Taberlet, P. & Brochmann, C. (2005) Impact of ice ages on circumpolar molecular diversity: insights from an ecological key species.
Molecular Ecology, 14, 2739–2753.
Alsos, I.G., Eidesen, P.B., Ehrich, D., Skrede, I., Westergaard,
K., Jacobsen, G.H., Landvik, J.Y., Taberlet, P. & Brochmann,
C. (2007) Frequent long-distance plant colonization in the
changing Arctic. Science, 316, 1606–1609.
Alsos, I.G., Alm, T., Normand, S. & Brochmann, C. (2008) Past
and future range shifts and loss of diversity in dwarf willow
(Salix herbacea L.) inferred from genetics, fossils, and
modelling. Global Ecology and Biogeography, 35, 801–814.
Andersen, B.G. & Borns, H.W., Jr (1997) The ice age world. An
introduction to Quaternary history and research with emphasis
on North America and Northern Europe during the last 2.5
million years. Scandinavian University Press, Oslo.
Bennike, O. & Böcher, J. (1990) Forest-tundra neighboring the
North pole: plant and insect remains from the Plio-Pleistocene Kap København formation, North Greenland. Arctic,
43, 331–338.
Birks, H.H. (1994) Plant macrofossils and the nunatak theory of
per-glacial survival. Dissertationes Botanicae, 234, 129–143.
Birks, H.H. (2008) The Late-Quaternary history of arctic and
alpine plants. Plant Ecology & Diversity, 2, 135–146.
Birks, H.J.B. & Willis, K.J. (2008) Alpines, trees, and refugia in
Europe. Plant Ecology & Diversity, 1, 147–160.
Bonin, A., Bellemain, E., Eidesen, P.B., Pompanon, F.,
Brochmann, C. & Taberlet, P. (2004) How to track and
assess genotyping errors in population genetics studies.
Molecular Ecology, 13, 3261–3273.
Bonin, A., Ehrich, D. & Manel, S. (2007) Statistical analysis of
amplified fragment length polymorphism data: a toolbox for
molecular ecologists and evolutionists. Molecular Ecology,
16, 3737–3758.
Brochmann, C. & Brysting, A.K. (2008) The Arctic – an evolutionary freezer? Plant Ecology and Diversity, 1, 181–195.
Brochmann, C. & Håpnes, A. (2001) Reproductive strategies
in some arctic Saxifraga (Saxifragaceae), with emphasis on
the narrow endemic S. svalbardensis and its parental
species. Botanical Journal of the Linnean Society, 137, 31–49.
Brochmann, C., Xiang, Q-Y., Brunsfeld, S.J., Soltis, D.E. &
Soltis, P.S. (1998) Molecular evidence for polyploidy origins
in Saxifraga (Saxifragaceae): the narrow endemic S. svalbardensis and its widespread allies. American Journal of
Botany, 85, 135–143.
Brochmann, C., Gabrielsen, T.M., Nordal, I., Landvik, J.Y. &
Elven, R. (2003) Glacial survival or tabula rasa? The history
of North Atlantic biota revisited. Taxon, 52, 417–450.
Corander, J. & Marttinen, P. (2006) Bayesian identification of
admixture events using multilocus molecular markers. Molecular Ecology, 15, 2833–2843.
DeChaine, E. (2008) A bridge or a barrier? Beringia’s influence
on the distribution and diversity of tundra plants. Plant
Ecology & Diversity, 1, 197–207.
1274
Demesure, B., Sodzi, N. & Petit, J. (1995) A set of universal
primers for amplification of polymorphic non-coding
regions of mitochondrial and chloroplast DNA in plants.
Molecular Ecology, 4, 129–134.
Doyle, J.J. & Doyle, J.L. (1987) A rapid DNA isolation procedure for small amounts of fresh leaf tissue. Phytochemical
Bulletin, 19, 11–15.
Ehrich, D. (2006) AFLPDAT: a collection of R functions for
convenient handling of AFLP data. Molecular Ecology Notes,
6, 603–604.
Ehrich, D., Alsos, I.G. & Brochmann, C. (2008) Where did the
northern peatland species survive the dry glacials: cloudberry (Rubus chamaemorus) as an example. Journal of Biogeography, 35, 801–814.
Eidesen, P.B., Carlsen, T., Molau, U. & Brochmann, C. (2007a)
Repeatedly out of Beringia: Cassiope tetragona embraces the
Arctic. Journal of Biogeography, 34, 1559–1574.
Eidesen, P.B., Alsos, I.G., Popp, M., Stensrud, O., Suda, J. &
Brochmann, C. (2007b) Nuclear vs. plastid data: complex
Pleistocene history of a circumpolar key species. Molecular
Ecology, 16, 3902–3925.
Elven, R. (ed.) (2007 onwards) Checklist of the Panarctic Flora
(PAF) Vascular Plants, Version: May 2007. Available at:
http://www.binran.ru/infsys/paflist/index.htm.
Evanno, G., Regnaut, S. & Goudet, J. (2005) Detecting the
number of clusters of individuals using the software
STRUCTURE: a simulation study. Molecular Ecology, 14,
2611–2620.
Excoffier, L., Smouse, P.E. & Quattro, J.M. (1992) Analysis of
molecular variance inferred from metric distances among
DNA haplotypes: application to human mitochondrial DNA
restriction data. Genetics, 131, 479–491.
Excoffier, L., Laval, G. & Schneider, S. (2005) Arlequin ver.
3.0: an integrated software package for population genetics
data analysis. Evolutionary Bioinformatics Online, 1, 47–
50.
Falush, D., Stephens, M. & Pritchard, J.K. (2007) Inference of
population structure using multilocus genotype data:
dominant markers and null alleles. Molecular Ecology Notes,
7, 574–578.
Farris, J.S. (1989) The retention index and the rescaled
consistency index. Cladistics, 5, 417–419.
Farris, J.S., Albert, V.A., Källersjö, M., Lipscomb, D. & Kluge,
A.G. (1996) Parsimony jackknifing outperforms neighborjoining. Cladistics, 12, 99–124.
Felsenstein, J. (1985) Confidence limits on phylogenies: an
approach using the bootstrap. Evolution, 4, 783–791.
Frenzel, B., Pécsi, M. & Velichko, A.A. (1992) Atlas of paleoclimates and paleoenvironments of the Northern Hemisphere.
Late Pleistocene–Holocene. Geographical Research Institute,
Hungarian Academy of Sciences. Gustav Fischer Verlag,
Budapest.
Funder, S., Hjort, C., Landvik, J.Y., Nam, S.I., Reeh, N. &
Stein, R. (1998) History of a stable ice margin – Greenland
during the Middle and Upper Pleistocene. Quaternary
Science Reviews, 17, 77–123.
Journal of Biogeography 37, 1262–1276
ª 2010 Blackwell Publishing Ltd
Extreme arctic disjunction in Saxifraga rivularis
Gaudeul, M., Taberlet, P. & Till-Bottraud, I. (2000) Genetic
diversity in an endangered alpine plant, Eryngium alpinum
L. (Apiaceae), inferred from amplified fragment
length polymorphism markers. Molecular Ecology, 9,
1625–1637.
Goloboff, P., Farris, J. & Nixon, K. (2008) TNT, a free program
for phylogenetic analysis. Cladistics, 24, 774–786.
Guldahl, A.S., Gabrielsen, T.M., Scheen, A.-C., Borgen, L.,
Steen, S.W., Spjelkavik, S. & Brochmann, C. (2005) The
Saxifraga rivularis species complex in Svalbard: molecules,
ploidy and morphology. Flora, 200, 207–221.
Hamilton, M.M. (1999) Four primer pairs for the amplification of chloroplast intergenic regions with intraspecific
variation. Molecular Ecology, 8, 521–523.
Hopkins, D.M. (1959) Cenozoic history of the Bering Land
Bridge. Science, 129, 1519–1528.
Hultén, E. (1937) Outline of the history of arctic and boreal biota
during the Quaternary period. Lehre J. Cramer, New York.
Hultén, E. (1958) The amphi-Atlantic plants and their phytogeographical connections. Kungliga Svenska Vetenskapsakademiens Handlingar, Fjärde Serien, 8, 1–275.
Hultén, E. (1968) Flora of Alaska and neighboring territories. A
manual of the vascular plants. Stanford University Press,
Stanford, CA.
Jørgensen, M.H., Elven, R., Tribsch, A., Gabrielsen, T.M.,
Stedje, B. & Brochmann, C. (2006) Taxonomy and evolutionary relationships in the Saxifraga rivularis complex.
Systematic Botany, 31, 702–729.
Kluge, A.G. & Farris, J.S. (1969) Quantitative phyletics and
evolution of anurans. Systematic Zoology, 18, 1–32.
Koerner, R.M. & Fisher, D.A. (2002) Ice-core evidence for
widespread Arctic glacier retreat in the Last Interglacial and
the early Holocene. Annals of Glaciology, 35, 19–24.
Kosman, E. (2003) Nei’s gene diversity and the index of
average differences are identical measures of diversity within
populations. Plant Pathology, 52, 533–535.
Landvik, J.Y. (1994) The last glaciation of Germania Land and
adjacent areas, northeast Greenland. Journal of Quaternary
Science, 9, 81–92.
Landvik, J.Y., Brook, E.J., Gualtieri, L., Raisbeck, G., Salvigsen,
O. & Yiou, F. (2003) Northwest Svalbard during the last
glaciation: ice-free areas existed. Geology, 31, 905–908.
Lid, J. & Lid, D.T. (2005). Norsk Flora, 7th edn (ed. by R.
Elven) Det Norske Samlaget, Oslo.
Lipkin, R. & Murray, D.F. (1997) Alaska rare plant field guide.
US Department of the Interior, Office of Equal Opportunity,
Washington, DC.
Miller, G.H., Mode, W.N., Wolfe, A.P., Sauer, P.E., Bennike,
O., Forman, S.L., Short, S.K. & Stafford, T.W. (1999) Stratified interglacial lacustrine sediments from Baffin Island,
Arctic Canada: chronology and palaeoenvironmental
implications. Quaternary Science Reviews, 18, 789–810.
Ottesen, D., Dowdeswell, J.A., Landvik, J.Y. & Mienert, J.
(2007) Dynamics of the Late Weichselian ice sheet on
Svalbard inferred from high-resolution sea-floor morphology. Boreas, 36, 286–306.
Journal of Biogeography 37, 1262–1276
ª 2010 Blackwell Publishing Ltd
R Development Core Team (2007) A language and environment for statistical computing. R Foundation for Statistical
Computing, Vienna.
Rohlf, F. (1999) NTSYS-pc. Numerical taxonomy and multivariate analysis system. Exeter Software, New York.
Rosenberg, N.A., Pritchard, J.K., Weber, J.L., Cann, H.M.,
Kidd, K.K., Zhivotovsky, L.A. & Feldman, M.W. (2002)
Genetic structure of human populations. Science, 298, 2381–
2385.
Rozen, S. & Skaletsky, J. (2000) Primer 3 on the WWW for
general users and for biologist programmers. Bioinformatics
methods and protocols: methods in molecular biology (ed. by
S. Krawetz and S. Misener), pp. 365–386. Humana Press,
Totowa, NJ.
Ryvarden, L. (1975) Studies in seed dispersal II. Winterdispersed species at Finse, Norway. Norwegian Journal of
Botany, 22, 21–24.
Sang, T., Crawford, D.J. & Stuessy, T.F. (1997) Chloroplast
DNA phylogeny, reticulate evolution and biogeography of
Paeonia (Paeoniaceae). American Journal of Botany, 84,
1120–1136.
Savile, D.B.O. (1972) Arctic adaptations in plants. Monograph
No. 6. Canada Department of Agriculture, Ottawa.
Schönswetter, P. & Tribsch, A. (2005) Vicariance and dispersal
in the alpine perennial Bupleurum stellatum L. (Apiaceae).
Taxon, 54, 725–732.
Skrede, I., Borgen, L. & Brochmann, C. (2009) Genetic structuring in three closely related circumpolar plant species:
AFLP versus microsatellite markers and high-arctic versus
arctic–alpine distributions. Heredity, 102, 293–302.
Soltis, P.S. & Soltis, D.E. (2009) The role of hybridization
in plant species. Annual Review of Plant Biology, 60, 561–
588.
Svendsen, J.I., Alexanderson, H., Astakhov, V.I. et al. (2004)
Late Quaternary ice sheet history of northern Eurasia.
Quaternary Science Reviews, 23, 1229–1271.
Taberlet, P., Gielly, L., Patou, G. & Bouvet, J. (1991) Universal
primers for amplification of three non-coding regions
of chloroplast DNA. Plant Molecular Biology, 17, 1105–
1109.
Tarasov, P.E., Volkova, V.S., Webb, T., Guiot, J., Andreev,
A.A., Bezusko, L.G., Bezusko, T.V., Bykova, G.V., Dorofeyuk, N.I., Kvavadze, E.V., Osipova, I.M., Panova, N.K. &
Sevastyanov, D.V. (2000) Last glacial maximum biomes
reconstructed from pollen and plant macrofossil data
from northern Eurasia. Journal of Biogeography, 27, 609–
620.
Van de Peer, Y. & De Wachter, R. (1994) TREECON for
Windows: a software package for the construction and
drawing of evolutionary trees for the Microsoft Windows
environment. Computer Applications in the Biosciences, 10,
569–570.
Widmer, A. & Lexer, C. (2001) Glacial refugia: sanctuaries for
allelic richness, but not for gene diversity. Trends in Ecology
and Evolution, 16, 267–269.
1275
K. B. Westergaard et al.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article:
Figure S1 Summary of structure analyses of Saxifraga rivularis.
As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are
peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from
supporting information (other than missing files) should be addressed to the authors.
BIOSKETCH
The National Centre for Biosystematics (NCB) at the Natural History Museum, University of Oslo, integrates basic research and
education in the taxonomy and systematics of plants, fungi and animals. Of particular importance is the merging of expertise in
molecular systematics with expertise in field- and collection-based taxonomy. K.B.W., M.H.J., I.G.A. and T.M.G. are current
or former members of C.B.’s research group at the NCB; members work mainly on the biogeography and evolution of the arctic–
alpine flora.
Author contributions: All authors were involved in conceiving the ideas and collecting the data; K.B.W. and M.H.J. carried out the
AFLP analyses; T.M.G. carried out the chloroplast analyses; all authors contributed to the interpretation of the data; and the
manuscript was drafted by K.B.W. and C.B. with input from the other authors.
Editor: Malte Ebach
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Journal of Biogeography 37, 1262–1276
ª 2010 Blackwell Publishing Ltd