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Molecular Ecology (2012) doi: 10.1111/j.1365-294X.2012.05705.x
Tales of the unexpected: Phylogeographyof the arctic-alpine model plant Saxifragaoppositifolia (Saxifragaceae) revisited
MANUELA WINKLER,* ANDREAS TRIBSCH,† GERALD M. SCHNEEWEISS ,* SABINE
BRODBECK,‡ FELIX GUGERLI ,‡ ROLF HOLDEREGGER,‡ RICHARD J . ABBOTT§ and PETER
SCHONSWETTER–
*Department of Systematic and Evolutionary Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria, †Department
of Organismic Biology ⁄ Ecology and Diversity of Plants, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg,
Austria, ‡WSL Swiss Federal Research Institute, Zurcherstrasse 111, CH-8903 Birmensdorf, Switzerland, §School of Biology,
Harold Mitchell Building, University of St Andrews, Fife KY16 9TH, UK, –Institute of Botany, University of Innsbruck,
Sternwartestrasse 15, A-6020 Innsbruck, Austria
Corresponde
E-mail: manu
� 2012 Black
Abstract
Arctic-alpine biota occupy enormous areas in the Arctic and the northern hemisphere
mountain ranges and have undergone major range shifts during their comparatively
short history. The origins of individual arctic-alpine species remain largely unknown. In
the case of the Purple saxifrage, Saxifraga oppositifolia, an important model for arctic-
alpine plants, phylogeographic studies have remained inconclusive about early stages of
the species’ spatiotemporal diversification but have provided evidence for long-range
colonization out of a presumed Beringian origin to cover today’s circumpolar range. We
re-evaluated the species’ large-scale range dynamics based on a geographically extended
sampling including crucial areas such as Central Asia and the (south-)eastern European
mountain ranges and employing up-to-date phylogeographic analyses of a plastid
sequence data set and a more restricted AFLP data set. In accordance with previous
studies, we detected two major plastid DNA lineages also reflected in AFLP divergence,
suggesting a long and independent vicariant history. Although we were unable to
determine the species’ area of origin, our results point to Europe (probably the Alps) and
Central Asia, respectively, as the likely ancestral areas of the two main lineages. AFLP
data suggested that contact areas between the two clades in the Carpathians, Northern
Siberia and western Greenland were secondary. In marked contrast to high levels of
diversity revealed in previous studies, populations from the major arctic refugium
Beringia did not exhibit any plastid sequence polymorphism. Our study shows that
adequate sampling of the southern, refugial populations is crucial for understanding the
range dynamics of arctic-alpine species.
Keywords: AFLPs, arctic-alpine plants, geographic diffusion model, phylogeography, plastid
sequences, range dynamics, Saxifraga oppositifolia
Received 21 February 2012; revision received 10 May 2012; accepted 30 May 2012
Introduction
The present-day arctic biome is approximately only
3 million years old (Matthews 1979) and comprises
many species that occupy enormous areas in the Arctic
nce: Manuela Winkler, Fax: +43142779541;
well Publishing Ltd
and the northern hemisphere temperate mountain
ranges (e.g. Hulten & Fries 1986). These species have
likely undergone major range shifts during their com-
paratively short history, which was strongly affected by
Pleistocene climatic fluctuations (Hewitt 2004). More-
over, they are predicted to be more strongly impacted
by current and future global warming (Sala et al.
2000; Alsos et al. 2012) than other biota. From the
2 M. WINKLER ET AL.
biogeographic viewpoint, major questions concern the
origin of arctic-alpine taxa as well as range formation,
particularly with respect to disjunct occurrences in tem-
perate mountain systems. Whereas at least in western
Eurasia immigration from the north into southern areas
was initially considered most likely (e.g. Brockmann-
Jerosch & Brockmann-Jerosch 1926; for opposing views
on North America see Weber 1965, 2003), recent molec-
ular phylogeographic studies rather suggest that north-
wards range expansion prevails (Schonswetter et al.
2003; Ehrich et al. 2007), with sometimes rapid coloniza-
tion (Schonswetter et al. 2003) and a high frequency of
dispersal events (Alsos et al. 2007) occurring. Despite
these advances, there remains a need to investigate the
early stages of spatiotemporal diversification of key
arctic-alpine species to provide a deeper understanding
of the origins and evolution of present-day arctic and
alpine floras.
The Purple saxifrage, Saxifraga oppositifolia L., has
become an important model system for studying the
evolution, biogeography, ecophysiology and reproduc-
tion of arctic-alpine plants (e.g. Stenstrom & Molau
1992; Crawford et al. 1993, 1995; Crawford & Abbott
1994; Gugerli 1997; Abbott & Comes 2004; Crawford
2004). This common insect-pollinated species with high
colonizing ability is widely distributed in the Arctic and
additionally occurs disjunctly in many temperate high
mountain ranges of the northern hemisphere (Hulten &
Fries 1986). Pleistocene presence in intervening low-
lands as well as in the Arctic is documented by a com-
paratively rich macrofossil record (e.g. Bennike &
Bocher 1990; Matthews & Ovenden 1990; Birks 1994;
Goetcheus & Birks 2001). Despite an essentially continu-
ous current distribution in the Arctic, a clear separation
of two clades with an amphi-Atlantic (Eurasian Clade)
and an amphi-Pacific distribution (North American
Clade) has been found based on restriction fragment
length polymorphisms (RFLPs) of plastid DNA (Abbott
et al. 2000; Abbott & Comes 2004) or plastid DNA
sequences (Holderegger & Abbott 2003). Based on the
exclusive distribution of ancestral haplotypes of both
lineages in the Taymyr Peninsula (northern Siberia), it
was suggested that the species had its first arctic occur-
rence in western Beringia and migrated east- and west-
wards to finally embrace the entire Arctic (Abbott et al.
2000). The observed high haplotype diversity in Berin-
gia was attributed to an important glacial refugium,
whereas that in northern Greenland, where haplotypes
of both clades co-occur, was regarded as a secondary
contact zone. The Alps and the Pyrenees hosted haplo-
types otherwise widespread in the amphi-Atlantic Arc-
tic suggesting either origin from a common ancestor
surviving glaciations(s) south of the ice sheet (Abbott
et al. 2000; Abbott & Comes 2004) or recent immigra-
tion as supported by ITS sequence data (Vargas 2003).
In agreement with the generally low haplotype diver-
sity in the Eurasian Clade and high dispersal abilities,
no structuring of random amplified polymorphic DNA
(RAPD) diversity was evident within Scandinavia (Ga-
brielsen et al. 1997).
Our understanding of the large-scale range dynamics
of S. oppositifolia may, however, be compromised by the
lack of data from southern areas, especially Central Asia
and (south-)eastern European mountain ranges. As for
substantial parts of the Eurasian alpine flora (Kadereit
et al. 2008), Central Asian mountain ranges have been
suggested to be the species’ place of origin (Abbott et al.
2000). Owing to the difficulties in obtaining samples
from these, however, this hypothesis has not been tested
yet. In (south-)eastern European mountain ranges, plas-
tid DNA haplotypes falling into the North American
Clade of S. oppositifolia previously unknown from
western Eurasia have been found in S. retusa Gouan
(M. Winkler, A. Tribsch, G.M. Schneeweiss, S. Brodbeck,
F. Gugerli, R. Holderegger, P. Schonswetter, unpubl.
data), a close relative of S. oppositifolia (Kaplan 1995). As
these two species frequently share haplotypes in areas
of co-occurrence in the Alps (M. Winkler, A. Tribsch,
G.M. Schneeweiss, S. Brodbeck, F. Gugerli, R. Holderegger,
P. Schonswetter, unpubl. data), this may also be the
case for the (south-)eastern European mountain ranges,
but this assertion has not been tested yet, either.
Consequently, our aims were to test (i) whether S. op-
positifolia originated in Central Asia; (ii) whether plastid
DNA haplotypes of the North American Clade also
found in (south-)eastern European S. retusa occur in so
far unstudied mountain ranges (e.g. Carpathians, Balkan
Peninsula). These questions were tackled against the
background of (iii) a re-evaluation of the species’ large-
scale range dynamics employing a recently developed
Bayesian approach (Lemey et al. 2009) applied to a com-
prehensive data set of plastid sequences. The maternal
inheritance of plastid genomes (for Saxifragaceae: Soltis
et al. 1990) makes them ideally suited for tracing migra-
tion histories (Petit et al. 2002). Furthermore, we added a
more restricted data set of AFLP markers, which are
biparentally inherited (Bussell et al. 2005) and allow for
the reconstruction of reticulation events.
Material and methods
Study species
Saxifraga oppositifolia has a wide circumpolar distribu-
tion that extends southwards into the North American
Cordilleras and many Eurasian mountain ranges, all of
which except for the Himalayas are included here
(Fig. 1). The present study covers S. oppositifolia s. str. as
� 2012 Blackwell Publishing Ltd
(a) (b)
(c)
(d)
Fig. 1 Sampled populations (numbered 1–62; Table S1, Supporting Information) and patterns of plastid DNA (psbA-trnH, trnTF) and
AFLP variation in Saxifraga oppositifolia. (a) Statistical parsimony network of plastid DNA haplotypes. Small black dots represent not
sampled haplotypes. Haplotypes of the Europe-centred Clade (EC-Clade) are shown in shades of blue, those of the Asia-centred
Clade (AC-Clade) in red and yellow. (b, c) Distribution of sampling sites and plastid haplotypes in the northern hemisphere (b) and
the Alps (c). Colour coding as in (a), haplotypes sampled only once are indicated by their number. For underlined populations, AFLP
data are not available. Distribution of ice cover (white) and tundra (dark grey) in the northern hemisphere at the last glacial maxi-
mum in (b) are modified from Frenzel (1968), Frenzel et al. (1992) and Ehlers et al. (2011). Margins of exposed continental shelves at
the last glacial maximum are indicated by dotted lines. (d) NeighborNet diagram of AFLP data. Splits with weight <0.1 were omitted
to aid legibility. Numbers along branches are bootstrap values based on a neighbour-joining analysis (given for major branches and
>50% only). The red bootstrap value results from a separate analysis without three admixed individuals from populations 5 and 21.
Colour coding of the circles represents cpDNA haplotypes and numbers in the circles are population numbers.
PHYLOGEOGRAPHY OF SA XIFRAGA OPPOSITIFOLIA 3
well as S. oppositifolia ‘murithiana’ from the Western Alps,
subsp. speciosa (Dorfler & Hayek) Engler & Irmscher
from the Apennines (Italy), subsp. paradoxa D.A. Webb
from the Pyrenees and subsp. smalliana (Engler &
Irmscher) Hulten from Beringia (nomenclature follows
� 2012 Blackwell Publishing Ltd
McGregor & Harding 1998), all of which have a doubt-
ful taxonomic status (e.g. Annotated Checklist of the
Panarctic Flora; Elven 2011). In turn, our sampling did
not include the genetically (M. Winkler, A. Tribsch, G.M.
Schneeweiss, S. Brodbeck, F. Gugerli, R. Holderegger,
4 M. WINKLER ET AL.
P. Schonswetter, unpubl. data) and morphologically
clearly distinct (Fischer et al. 2008) S. biflora Allioni,
S. oppositifolia subsp. blepharophylla (A. Kerner ex Hay-
ek) Engler & Irmscher and subsp. rudolphiana (Horns-
chuch) Engler & Irmscher from the Alps. As not all
available samples of S. oppositifolia had a sufficiently
high quality for the production of reliable AFLP finger-
prints, some areas covered by plastid DNA data are not
or only poorly covered by AFLP data, that is, northern
Greenland, arctic Canada and the Beringian region.
Plant material, DNA isolation, plastid DNAsequencing and AFLP fingerprinting
Leaf material of one to three individuals per sampling
site was collected and immediately stored in silica gel
(Table S1, Supporting Information, also including vou-
cher numbers). Total genomic DNA was extracted from
about 10 mg of dried tissue with the DNeasy 96 plant
mini kit (Qiagen, Hilden, Germany) following the man-
ufacturer’s protocol. The psbA-trnH intergenic spacer
was amplified and sequenced as described in Holdereg-
ger & Abbott (2003). The plastid trnTUGU-trnLUAA-
trnFGAA intergenic spacers including the trnLUAA intron
(from here on referred to as trnTF) were amplified
using the specific primers Sax-a (5¢-ACCTACCGGGAT
CGTAGCTATT) and Sax-f (5¢-TTTTTGCTCGGATCCTT
TTG), which were developed based on the primer pair
a and f from Taberlet et al. (1991). The PCR reaction
mix contained 9 lL of ReadyMix (Sigma-Aldrich, Stein-
heim, Germany), 13 lL water, 1 lL BSA (1 mg ⁄ mL;
Promega, Madison, WI), 0.5 lL of each primer (10 lM),
0.5 lL of MgCl2 (25 mM), and 0.5–1 lL of total genomic
DNA. We used the following PCR conditions: 95� C for
5 min, followed by 35 cycles of 94� C for 30 s, 60� C for
1 min and 65� C for 4 min, followed by a final exten-
sion period of 65� C for 10 min. Purification of PCR
products and cycle sequencing were performed as
described in Surina et al. (2011) except that ClAP was
replaced with FastAP (Thermosensitive Alkaline Phos-
phatase, Fermentas). For sequencing, primers Sax-a and
Sax-f as well as internal primers Sax-c (5¢-CGAAAT
TGGTAGACGCTACG) and ‘b’ and ‘d’ from Taberlet
et al. (1991) were used.
The AFLP procedure followed Vos et al. (1995) with
the modifications described by Schonswetter et al.
(2009). To test the reproducibility of AFLP fragments
and to allow an estimation of the error rate, 13 samples
were replicated from the restriction ⁄ ligation step
onwards. An initial screening of selective primers using
twelve primer combinations was performed. The three
final primer combinations for the selective PCR (fluores-
cent dyes in brackets) were EcoRI (6-FAM)-ACA ⁄ MseI-
CAC, EcoRI (VIC)-AGG ⁄ MseI-CTC, EcoRI (NED)-ACC ⁄
MseI-CAG. The selective PCR products were purified
and subjected to electrophoresis as described in Schons-
wetter et al. (2009).
Data analyses
For plastid DNA data, a statistical parsimony network
was constructed from the concatenated sequence data
using TCS 1.21 (Clement et al. 2000) treating sequence
gaps as fifth character state after re-coding inser-
tions ⁄ deletions (indels) longer than 1 bp as single base
pair indels and excluding polymorphic mononucleotide
repeats. For all other analyses the unmodified alignment
was used. Haplotype diversity was estimated using p,
the mean number of pairwise nucleotide differences (Taj-
ima 1983), calculated with ARLEQUIN 3.11 (Excoffier et al.
2005). Phylogeographic analyses of the plastid data set
were conducted in BEAST 1.6 (Drummond & Rambaut
2007). Model-fit of nucleotide substitution models was
assessed via the Bayesian Information Criterion (BIC) as
implemented in JMODELTEST 0.1.1 (Posada 2008). As the
set of models with cumulative BIC weights of at least
0.95 contained three medium-complex models (F81,
HKY, F81 + Gamma), we finally used an HKY model
with rate heterogeneity modelled by a gamma distribu-
tion (with six rate categories). As prior for the transition–
transversion ratio j, we used a normal distribution with
mean 1 (derived from the model-averaged value for this
parameter determined via BIC) and a deliberately wide
standard deviation of 1.0. Rate evolution was modelled
in a strict clock framework, because a relaxed clock
model had an only slightly better marginal log-likelihood
()2839.72 versus )2841.03, respectively) and the coeffi-
cient of rate variation had its highest posterior density
around zero (data not shown). As prior on the substitu-
tion rate, we used a truncated (at 1 · 10)4) normal prior
with mean and standard deviation of 2.8 · 10)3 and
4 · 10)3 substitutions per site per million years, respectively.
This ensured a modal value of the distribution around
4 · 10)3 substitutions per site per million years in line with
previously suggested values (Smith et al. 2008; Yamane et al.
2003). As population model, we used the Bayesian skyline
plot (Drummond et al. 2005) with a group interval m = 5.
Stationarity of the Markov chain was determined using
TRACER 1.4 (http://tree.bio.ed.ac.uk/software/tracer/).
Spatial distribution through time was inferred employ-
ing a discrete model of geographic diffusion, where rates
of diffusion between a priori defined discrete locations
are estimated using a continuous-time Markov chain
model starting from the unobserved location at the root
of the tree derived from a uniform distribution over all
sampled locations (Lemey et al. 2009). This geospatial
model may be reversible, that is, the diffusion rate
between regions is identical in both directions, or nonre-
� 2012 Blackwell Publishing Ltd
PHYLOGEOGRAPHY OF SA XIFRAGA OPPOSITIFOLIA 5
versible, that is, the diffusion rate in one direction can
differ from that in the reverse direction. Although
geographically distinct sampling localities constitute
intuitive discrete geographical units, the enormous num-
ber of possible rate parameters renders their use impossi-
ble. To reach sufficient geographic resolution with as few
groups as possible, we finally delimited nine geographic
regions (for details on used criteria see Text S1, Support-
ing Information): Pyrenees; Alps; Carpathians; southern
European mountains (Apennines and mountains of the
Balkan Peninsula); Central Asian mountains (Tien Shan
and Altai); western Atlantic Arctic (Greenland and
neighbouring islands); eastern Atlantic Arctic (Scandina-
via and European Russian Arctic, Svalbard to Franz
Joseph Land); northern Russian Arctic (Taymyr and Lena
Delta); Beringia (including a single population from the
American Cordillera). To achieve statistical efficiency,
dispersal rates were allowed to be zero with some proba-
bility (determined by a truncated Poisson distribution) in
the framework of Bayesian stochastic search variable
selection (BSSVS). Following Lemey et al. (2009), for the
reversible model the truncated Poisson prior had a mean
of 0.693 (i.e. ln2) and an offset of 8 (the number of rates
necessary to minimally connect all regions, that is, num-
ber of regions minus 1). For the nonreversible model, the
Poisson prior was parameterized with a mean of 8 (num-
ber of regions minus 1) and an offset of 0 (P. Lemey, pers.
comm.). Sensitivity analyses with different prior means
(0.693, 5, 8) supported previous findings (Escobar Garcıa
et al. 2012) that, in contrast to the reversible model, the
nonreversible model is very sensitive to prior choice
yielding partly nonsensible results with low prior means
(data not shown). Therefore, we only present the results
from analyses with the default prior settings. Owing to
the presence of a deep basal phylogeographic split (see
Results), the two main clades were also analysed sepa-
rately using the same geographic units and prior settings
adjusted for the lower number of geographic regions per
clade (six instead of nine regions corresponding to the
clades’ actual occurrences). To assess the effects of
uneven sampling densities in different geographic
regions, in particular the high number of investigated
populations in the Alps, we repeated the analyses for the
whole data set and for the EC-Clade only (see Results)
including only five populations from the Alps spread
over the Alpine distribution area (hereinafter called
reduced data sets). In all cases, we used equal expecta-
tions for all rates, that is, the prior on the diffusion rates is
not informed by the geographic distances among geo-
graphic units. Two runs per parameterization were con-
ducted, each for 108 generations with sampling every
5000th generation. As both runs converged on the same sta-
tionary distribution and effective sample sizes (ESS) safely
exceeded 200, they were combined after removal of the first
� 2012 Blackwell Publishing Ltd
10% of sampled generations as burn-in. All parameter esti-
mates were based on these two runs combined (36 000
sampling points). Identification of well-supported rates (i.e.
those with Bayes Factor support of at least three) was done
using the program SPREAD 1.0.4 (Bielejec et al. 2011).
Raw AFLP data were collected, aligned with Gene-
Scan 500 ROX (Applied Biosystems, Foster City, USA)
internal size standard and scored using DAx (Van Mier-
lo Software Consultancy, Eindhoven) as described in
Bendiksby et al. (2011). Thirteen samples were repeated
and the error rate was calculated as the number of mis-
matches (i.e. 0 ⁄ 1 or 1 ⁄ 0) divided by the number of
matches (i.e. 0 ⁄ 0 and 1 ⁄ 1) in each pair of replicates
(Bonin et al. 2004). Fragments with mismatches in more
than one replicate pair were omitted from the analysis.
Using SPLITSTREE 4.8 (Huson & Bryant 2006), a Neigh-
borNet diagram was produced from Nei–Li distances
(Nei & Li 1979). Node support was estimated in a
neighbour-joining analysis based on Nei-Li distances
and 1000 bootstrap pseudo-replicates.
The genetic covariance structure among geographic
regions as defined for the phylogeographic analyses of
the plastid sequence data was modelled for the AFLP
data set within a graph theoretic framework (popula-
tion graphs: Dyer & Nason 2004) using POPGRAPHS
(http://dyerlab.bio.vcu.edu/software/). A network is
constructed where regions, which constitute the nodes,
are connected by edges only if there is significant
genetic covariance between the regions after removing
the co-variation each region has with the remaining
regions in the dataset. Stability of edges among geo-
graphic regions was assessed using a bootstrap
approach with 200 bootstrap replicates. Pseudoreplicate
data sets were generated using seqboot from the PHYLIP
package (Felsenstein 1989) and analysed like the origi-
nal data set. The proportion of replicates where a cer-
tain edge is found constitutes its bootstrap support.
To infer genetic ties between pairs of geographical
regions based on the AFLP data set, the number of
shared fragments among regions divided by the num-
ber of fragments present in a region was calculated. To
account for differing sample sizes among regions, ten
samples per region were randomly selected with
replacement and the percentage of shared fragments
calculated using R 2.13.1 (R Development Core Team
2011). This process was repeated 100 times and the
results were averaged.
Results
Plastid DNA
The lengths of the psbA-trnH intergenic spacer and the
trnTF intergenic spacers in S. oppositifolia were 212 and
6 M. WINKLER ET AL.
1633 bp, respectively. The combined alignment was
1845 bp long and comprised 43 variable characters, 33
of which were nucleotide substitutions and ten were
indels (2.33% variability). Excluding six polymorphic
mononucleotide repeats gave a total of 27 haplotypes in
114 individuals analysed. The original alignment is
available on datadryad.org; GenBank accession num-
bers are provided in Table S1 (Supporting Information).
The statistical parsimony network (Fig. 1) revealed
two lineages, hereinafter referred to as Europe-centred
Clade (EC-Clade) and Asia-centred Clade (AC-Clade).
The EC-Clade was distributed in the Pyrenees, the
Alps, the Western Carpathians and the Atlantic Arctic,
whereas the AC-Clade occurred in the Apennines, the
Eastern and Southern Carpathians, mountains of the
Balkan Peninsula, Central and Northern Asia, Beringia
and Northern Canada, and North Greenland. The
mean number of pairwise nucleotide differences (p) in
these clades amounted to 2.22 ± 1.20 and 2.13 ± 1.20,
respectively.
Results of the Bayesian phylogeographic analysis were
sensitive to the sampling density in the Alps, strongly
affecting asymmetries in diffusion rates and ancestral
location probabilities of the Alps (Fig. 2: reduced data-
set, Fig. S2, Supporting Information complete dataset
including all Alpine populations). This is likely due to
the prevalent haplotype sharing between the Alps and
the European Arctic. For one, haplotypes from the less
represented region will often coalesce with haplotypes
from the more frequently represented region resulting
in frequent intermixing of populations from these
regions (Fig. S1, Supporting Information) and a bias in
diffusion rates from the more frequently to the less fre-
quently represented region. Furthermore, the (back-
wards in time) latest coalescence events will be
dominated by the more frequently represented region,
which will receive higher ancestral location probabili-
ties. Therefore, we only present results from the reduced
data set (Fig. 2), where sampling is more even.
For the whole dataset (i.e. comprising all populations
of the reduced dataset), the reversible model (Fig 2a)
identified seven significant connections, the Central
Asian mountains remaining unconnected (i.e. receiving
BF support <3). The number of significant connections
inferred from the nonreversible model (Fig. 2b) was
higher (six unidirectional and six bidirectional ones),
but this set included all rates identified with the revers-
ible model. Among the connections exclusively found
under the nonreversible model was the sole connection
involving the Central Asian mountains. Connections
and directionalities inferred from the whole data set
were usually also found in separate analyses of the
AC-Clade (Fig. 2c,d) and the EC-Clade (Fig. 2e,f).
Differences between the analyses of the whole and the
separate datasets concerned only a few weakly sup-
ported rates (BF <4.5) and the connection between the
western and the eastern Atlantic Arctic not identified
from the analysis of the whole dataset with the revers-
ible model. Whereas in the EC-Clade connections were
mostly latitudinal, that is, between a temperate moun-
tain range and an Arctic region, connections were
mostly longitudinal in the AC-Clade.
For the whole dataset (Fig. 2a,b), ancestral location
probabilities under both models ranged from 0.10 to
0.12, thus merely reflecting the prior probability of one-
ninth (0.11). For the EC-Clade, the Alps and the eastern
European Arctic had the highest posterior probabilities
of being the ancestral location (0.27 and 0.26 for the
Alps and 0.29 and 0.18 for the eastern European Arctic
under the reversible and nonreversible models, respec-
tively). Under the reversible model, the set of regions
with cumulative posterior probability of at least 0.8
comprised the Alps, the eastern Atlantic Arctic, the Py-
renees, and the northern Russian Arctic, whereas under
the nonreversible model it included all regions harbour-
ing this clade. Similar results were obtained from sepa-
rate analysis of the EC-Clade (Fig. 2e,f): Although the
posterior probabilities for the Alps and the eastern
European Arctic being the ancestral location dropped
(0.23 and 0.22 for the Alps and 0.26 and 0.18 for the
eastern European Arctic under the reversible and non-
reversible models, respectively), they remained highest
and above the prior probability of 0.17. For the AC-
Clade (Fig. 2a,b), the Central Asian mountains had the
highest posterior probability of being the ancestral loca-
tion (0.20 and 0.26 under the reversible and nonrevers-
ible models, respectively), the set of regions with
cumulative posterior probability of at least 0.8 including
all regions harbouring this clade. Similar results were
obtained from separate analysis of the AC-Clade
(Fig. 2c,d), where the posterior probabilities slightly
increased for the Central Asian mountains being the
ancestral location (0.24 and 0.29 under the reversible
and nonreversible models, respectively) and thus
remained above the prior probability of 0.17.
AFLPs
A total of 490 reproducible AFLP bands were scored for
76 individuals. Eleven bands found in all or all but one
individual were excluded from further analyses. Fifty
singular markers were retained because they were
present in more than one individual with respect to a
dataset including close relatives of S. oppositifolia
(M. Winkler, A. Tribsch, G.M. Schneeweiss, S. Brodbeck,
F. Gugerli, R. Holderegger, P. Schonswetter, unpubl.
data). The error rate was 1.6%. Eighteen nonreproduc-
ible fragments were removed from the AFLP matrix.
� 2012 Blackwell Publishing Ltd
(a) (b)
(c) (d)
(e) (f)
Fig. 2 Range connectivity and ancestral location probabilities among nine discrete geographical regions (hatched lines) in Saxifraga
oppositifolia inferred using reversible (i.e. diffusion rates are identical in both directions; a, c, e) and nonreversible (i.e. the diffusion
rate in one direction can differ from that in the reverse direction; b, d, f) models of geographic diffusion of (a, b) the whole plastid
DNA data set (including only five populations from the Alps: 24, 34, 37, 40, 48; see text for details), (c, d) the Asia-centred Clade,
and (e, f) the Europe-centred Clade. The thickness of the connections is proportional to their support by Bayes factors (only connec-
tions receiving BF support >3 are shown). The posterior probability of a region to be the ancestral area is indicated by the size of
white and black dots for the Asia-centred and the Europe-centred Clade, respectively. Dots are overlaid in the analyses of the whole
data set (a, b).
PHYLOGEOGRAPHY OF SA XIFRAGA OPPOSITIFOLIA 7
The NeighborNet diagram (Fig. 1d) revealed differen-
tiation into two groups, hereinafter referred to, in analogy
to plastid DNA data, as the Europe-centred Group
(EC-Group) and the Asia-centred Group (AC-Group).
The EC-Group contained populations from the Pyrenees,
the Alps and the Atlantic Arctic (Newfoundland to north-
ern Urals), the AC-Group included populations from
the Apennines, the Carpathians, the Balkan Peninsula,
Central and Northern Asia as well as Beringia. Three
� 2012 Blackwell Publishing Ltd
accessions from northern Greenland and the Taymyr
Peninsula (northern Siberia) shared similarly weighted
splits with both main groups. The EC-Group largely
lacked internal structure; only accessions from the Atlan-
tic Arctic were weakly separated from those from the
Alps and the Pyrenees. The AC-Group had a stronger
internal structure, with samples from (i) the Balkans and
the Carpathians; (ii) the Apennines; and (iii) Central and
Northern Asia forming three distinct groups.
8 M. WINKLER ET AL.
Results from the POPGRAPHS network (Fig. 3a) were
largely congruent with those from the NeighborNet
analysis. This concerned the separation of the EC- from
the AC-Group, which remained unconnected, as well as
the distinctness of the European mountain ranges from
regions elsewhere within the AC-Group, whose connec-
tions received insufficient support. Connections
between temperate and Arctic regions involved the
Alps and the eastern Atlantic Arctic in the EC-Group as
well as Central Asian mountains and Beringia in the
AC-Group. Some connections between disjunct geo-
graphical regions revealed by the plastid DNA data
(e.g. between the Pyrenees and the Northern Russian
Arctic) were not supported in the POPGRAPHS network
based on AFLP data; those between the Central Asian
mountains and the Northern Russian Arctic received
only weak support.
The Alps harboured by far most AFLP fragments. All
regions shared more than 60% of their fragments with
the Alps, especially the western and eastern Atlantic Arc-
tic and the Pyrenees (Fig 3b). The eastern Atlantic Arctic
and northern Russian Arctic were rich in fragments as
compared to Beringia, the western Atlantic Arctic, and
Central Asia. The western Atlantic Arctic shared 78.7%
of its fragments with the eastern Atlantic Arctic. Beringia
shared more than 60% of its fragments with the other
arctic regions (e.g. 80.6% with the northern Russian Arc-
tic), Central Asia and the Alps, but no region shared
more than a third of its fragments with Beringia.
Discussion
Despite intensive phylogeographical research on north-
ern hemisphere cold-adapted biota during the past two
(a) (b
Fig. 3 Connectivity among nine discrete geographical regions (hatche
Population Graphs network illustrating the genetic covariance struc
strap support >10% are shown. (b) Numbers of AFLP fragments w
among regions. The thickness of arrows is proportional to the percen
present in the region to which the arrows point, with values betwee
shares 80.4% of its fragments with the Northern Russian Arctic). No
by two samples in a single population located on Wrangel Island in t
decades, we still hold surprisingly limited information
on the spatiotemporal diversification of plants and ani-
mals that constitute this biota. Consequently, our
understanding of how this biota formed and evolved,
particularly in regard to its establishment and spread in
the Arctic and across northern hemisphere mountain
ranges, remains limited. Even for those species previ-
ously subjected to phylogeographic analysis, it is likely
that restricted sampling and molecular analysis
revealed only part of their history, and thus, more com-
prehensive analysis is required for a more complete
understanding of their spatiotemporal diversification in
the past. Indeed, this has emerged from our analysis of
the arctic-alpine model species Saxifraga oppositifolia,
where the inclusion of populations from several so far
unstudied temperate mountain ranges and use of state-
of-the-art phylogeographic analyses of plastid DNA and
AFLP data sets resulted in a refined and in parts
revised interpretation of this species’ phylogeographic
history. Major changes concerned the circumscription of
the geographical distribution of the two plastid DNA
clades previously identified (Abbott et al. 2000) and
patterns of haplotype diversity, which affect the infer-
ence of range dynamics within the Arctic and between
the Arctic and temperate mountain ranges of the north-
ern hemisphere.
The ‘North American Clade’ extends to the southernEuropean mountains
The distribution of the Asia-centred Clade (AC-Clade,
corresponding to the North American Clade of Abbott
et al. 2000) was considerably extended to range not only
from northern Greenland over the Rocky Mountains
)
d lines) in Saxifraga oppositifolia based on AFLP fingerprints. (a)
ture among regions, connections between regions with a boot-
ithin regions and mean percentage of shared AFLP fragments
tage of shared fragments in relation to the number of fragments
n 60% and the maximum value of 80.4% shown (e.g. Beringia
te that the Beringian region (given in grey) is only represented
he north-eastern Russian Arctic.
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PHYLOGEOGRAPHY OF SA XIFRAGA OPPOSITIFOLIA 9
and the Beringian region to the Taymyr Peninsula
(Abbott et al. 2000), but also to Central Asian (Tien Shan
and Altai) as well as to southern and south-eastern
European mountain ranges (Apennines, Southern Car-
pathians, mountain ranges of the Balkan Peninsula;
Fig. 1). Plants from the latter areas have eglandular
sepals, precluding that this clade is congruent with
subsp. glandulisepala as suggested previously (Abbott &
Comes 2004). The distribution area of the Europe-
centred Clade (EC-Clade, corresponding to the Eurasian
Clade of Abbott et al. 2000) ranged from Newfoundland
and Greenland throughout the Atlantic Arctic to the
Taymyr Peninsula in northernmost Siberia in the Arctic
and, in the south, from the Pyrenees over the Alps to
the Tatra Mountains in the Western Carpathians. The
latter occurrence represented an extension of this
clade’s distribution that corroborated phylogeographic
links between the Alps and Carpathians previously
detected in other plant species with arctic-alpine distri-
butions (Schonswetter et al. 2006a,b; Skrede et al. 2006).
Good correspondence between the two plastid DNA
clades (EC- and AC-Clade) and the two main AFLP
groups (EC- and AC-Group), only blurred by a few
admixed individuals (Fig. 1; see next paragraph), sug-
gests a long and independent vicariant history. Both
clades started to diversify roughly at the same time
long after their initial separation (Fig. S1, Supporting
Information). This supports the hypothesis of an old
disjunction between Europe (based on AFLP data prob-
ably the Alps; Fig 3b) and Central Asia, the most likely,
albeit only weakly supported ancestral areas of the two
clades (Fig. 2). This deep divergence is most probably
responsible for the failure, with our data, to infer the
place of origin of S. oppositifolia as a whole. A phyloge-
netic study in a taxonomically much broader context
will be necessary to rigorously test the hypothesis of an
Asian origin of S. oppositifolia. Such an approach will
additionally enable testing whether the deep divergence
truly reflects an early diversification within S. oppositifo-
lia or whether it is the result of an ancient chloroplast
capture from related Saxifraga species, as suggested
by haplotype similarity with other European Saxifraga
species of sect. Porphyrion Tausch (M. Winkler,
A. Tribsch, G.M. Schneeweiss, S. Brodbeck, F. Gugerli,
R. Holderegger, P. Schonswetter, unpubl. data).
An obvious contact zone identified as the single area
of incongruence between plastid and AFLP datasets was
located in the Tatra Mountains (western Carpathians).
While both sampled populations exhibited haplotypes
of the EC-Clade, their AFLP profiles unambiguously
clustered with other Carpathian and Balkan populations,
and thus with the AC-Group. Corroborating our results,
the Tatra Mountains have repeatedly been shown to be
a meeting ground of major phylogeographic lineages
� 2012 Blackwell Publishing Ltd
(reviewed in Ronikier 2011). Two additional contact
zones between the main lineages were encountered in
the Arctic in accordance with Abbott et al. (2000), that
is, in Northern Greenland and on the Taymyr Peninsula.
The admixed state of AFLP fingerprints from both areas
(albeit based on limited sampling: Greenland, popula-
tion 5; Taymyr, population 21; Fig. 1) was not compati-
ble with an interpretation of Taymyr as a region of
primary arctic occurrence of both clades of S. oppositifo-
lia (Abbott et al. 2000). The data instead suggest that
populations from both Northern Greenland and Taymyr
underwent similar histories, shaped by recent contact of
differentiated lineages colonizing the Arctic from south-
ern Europe and central and ⁄ or eastern Eurasia including
Beringia (Fig. 2), respectively. However, only dedicated
sampling may reveal the true origins of S. oppositifolia in
these regions.
Contrasting range dynamics in western and easternEurasia
The AC- and the EC-Clades differed with respect to
their range dynamics as inferred by the comparison of
latitudinal vs. longitudinal gene flow. Latitudinal gene
flow was obviously important in the history of the
EC-Clade for shaping the genetic ties between disjunct
areas, as illustrated by the Alps and the eastern Atlantic
Arctic (Fig. 2). This was further supported by the distri-
bution of plastid DNA haplotypes (three frequent hapl-
otypes were shared; Fig. 1), probably causing
ambiguities in reconstructing ancestral location proba-
bilities with the reversible phylogeographic model
(Fig. 2), as well as by the shallow AFLP divergence
between both areas (Fig. 1d) and the strong connection
in the Population Graphs network (Fig 3a). The propor-
tion of shared AFLP fragments along a richness gradi-
ent (Fig. 3b) strongly suggested a prevalence of south
to north directed gene flow contrary to previous find-
ings (Vargas 2003). Latitudinal gene flow has likely
been fostered by the ample availability of suitable habi-
tats at intermittent latitudes during glacial periods
(Birks & Willis 2008), as evident from macrofossils
found in the lowlands between the Alps and the Scan-
dinavian ice sheet (Birks 1994; Burga & Perret 1998).
Latitudinal gene flow together with the presence of
long-term refugia in southern Europe may also have
prevented a loss of genetic diversity in the EC-Clade,
which based on our estimates of the mean number of
pairwise nucleotide differences (p) and contrary to pre-
vious findings (Abbott et al. 2000) is actually as variable
as the AC-Clade. A counterintuitive connection, yet in
line with the prevalence of latitudinal connections in
the EC-Clade, was suggested by the plastid data, where
haplotype h17 from the Taymyr Peninsula was derived
10 M. WINKLER ET AL.
(by a substitution at alignment position 1262) from h16
restricted to the Pyrenees (Fig. 1, Fig. S1, Supporting
Information). This may indicate gene flow from the
southwestern to the northeastern edges of the distribu-
tion of the EC-Clade (Fig. 2), a connection lacking sup-
port in the AFLP data (Fig. 3).
In contrast to the EC-Clade, latitudinal gene flow
appears to have played a minor role in the range
dynamics of the AC-Clade. This was evident from the
lack of a clear signal for connections between any of
the temperate mountain ranges and arctic regions in
the plastid data (support for the only exception involv-
ing Central Asian mountains and the western Atlantic
Arctic is low; Fig. 2). Additionally, the more pro-
nounced AFLP-structure (Fig. 1d) indicates a stronger
role of allopatric differentiation as compared to the
EC-Clade. In contrast, connectivity among the arctic
regions and between the Carpathians and the southern
European mountains was much stronger (Figs 2 and
3), suggesting that range expansion into the Arctic by
the AC-Clade happened more rarely than in the EC-
Clade. Based on haplotype distribution and diversity
(Fig. 1), the northern Russian Arctic likely represented
the primary entry point into the Arctic for the AC-
Clade, as suggested previously for the entire species
by Abbott et al. (2000). The scarcity of gene flow
between temperate and arctic regions may also be
responsible for the unexpectedly low genetic diversity
in Beringia: haplotype diversity in this area was zero
and the number of AFLP fragments (albeit based on
only one population in Wrangel Island, north-east Rus-
sia) was the lowest of all investigated geographic
regions (Fig. 3b). This corroborates results based on
the psbA-trnH spacer only (Holderegger & Abbott
2003), but strongly contrasts with previous plastid
RFLP data, which suggested that Beringia represents a
hotspot of genetic diversity (Abbott et al. 2000; Abbott
& Comes 2004). As the number of mutational steps
separating the two major plastid DNA clades identi-
fied here matches those found in previous studies, an
artefact related to different levels of resolution appears
highly unlikely. Lack of congruence in the pattern of
diversity resolved by RFLP vs. sequence analysis sug-
gests that sequencing of the entire plastid genome
may be required to establish firmly how diverse this
species’ plastid genome is in Beringia. As macrofossils
dating to the Last Glacial Maximum (from ca. 21 500
BP, Goetcheus & Birks 2001) suggest the continuous
persistence of S. oppositifolia in Beringia, the low diver-
sity found in our study is unexpected unless the spe-
cies passed through a strong bottleneck owing to a
dramatic decrease in resident individuals caused by
temporally restricted habitat availability in the rela-
tively recent past.
Both plastid and AFLP data (Figs 2 and 3) may sug-
gest a southern migration corridor connecting Central
Asia with south-eastern Europe. As S. oppositifolia does
not and, based on the lack of any supporting (sub)fossil
evidence, probably never did occur in any of the moun-
tain ranges between the Carpathians and the Tien Shan
(Hulten & Fries 1986; Losina-Losinskaja 1939), this
would require a long-distance dispersal event to span
this roughly 4000 km disjunction. A northern migration
route in lowlands, whose traces were eradicated by
advances of the Scandinavian and Kara ice shields,
would provide an alternative explanation. A previously
wider arctic distribution of the AC-Clade would be in
line with the strongly disjunct occurrence of haplotype
h18 in the Tien Shan and on the northern coast of
Greenland (a back-mutation from haplotype h19, sam-
pled in the same population and differing in a point
mutation at alignment position 1173, appears unlikely).
As macrofossils from this part of North Greenland were
dated to the Pliocene ⁄ Pleistocene transition (Bennike &
Bocher 1990; Matthews & Ovenden 1990), in situ sur-
vival of pre-Pleistocene immigrants has to be taken into
account. Whereas glacial survival in formerly strongly
glaciated areas of the Arctic, especially Scandinavia,
was initially ruled out (Gabrielsen et al. 1997), recent
evidence from species with a western arctic distribution
(Westergaard et al. 2011; Parducci et al. 2012) renders
nunatak survival a plausible alternative.
Our study underlines the importance of southern
mountain ranges for the evolution and range formation
of arctic-alpine biota. Specifically, the present-day,
essentially continuous circum-Arctic distribution of
S. oppositifolia was achieved via independent coloniza-
tion from long-term divergent populations in European
and Central Asian mountain ranges, respectively. Major
range expansions also affected the species’ distribution
in temperate mountain ranges as is evident from a rela-
tively recent westwards range expansion resulting in an
unexpectedly close relationship of Central Asian and
southeastern European populations. This indicates that
a connection between both areas – as also evidenced by
the highly disjunct distribution of, for example, Sibiraea
altaiensis s.l. (Rosaceae, distributed in Central Asian
mountains and disjunctly in southeastern Europe) –
was indeed relevant, even if the exact migration routes
and range dynamics remain intractable given present-
day knowledge. An important message emerging for
future studies is that adequate sampling of the ‘rear
edge’ (Hampe & Petit 2005), that is, the southern refu-
gial populations, is crucial for understanding the range
dynamics not only of temperate trees and shrubs, but
also of arctic-alpine species. Finally, as global warming
imposes the highest risk of extirpation in the southern
parts of arctic-alpine distribution ranges (Thuiller et al.
� 2012 Blackwell Publishing Ltd
PHYLOGEOGRAPHY OF SAX IFRAGA OPPOSITIFOLIA 11
2005; Alsos et al. 2012), eradication of southern lineages
may significantly reduce the evolutionary potential of
arctic-alpine species. In conclusion, the specific case of
S. oppositifolia serves as an example for how arctic-
alpine plant species with often far-ranging dispersal
potential were – and possibly will be – able to cope
with ever changing habitat availability in response to
past and future climate oscillations.
Acknowledgements
We thank Victoria Sork and three anonymous reviewers for
their helpful comments. I. G. Alsos, A. Brysting, R. Elven,
S. Ertl, B. Frajman, O. Gilg, A. Hilpold, M. & A. Ronikier,
C. Schmiderer, H. Solstad, C. Thiel-Egenter, K. Westergaard,
M. Wiedermann and others (see Abbott et al. 2000) helped
collect samples of S. oppositifolia. We thank O. Paun and
M. Ronikier for accompanying P.S. in the Romanian Carpathi-
ans and the Tatra Mountains, D. Ehrich and M. Kapralov for
accompanying A.T. in the Urals, and S. Smirnov, F. Essl for
accompanying A.T. in the Altai Mountains and I. Kunzle for
company to F.G. in the Tien Shan. M. Affenzeller and M. Eder
are thanked for technical assistance in obtaining plastid DNA
sequences from several samples. C. Brochmann allowed us to
use samples stored at the DNA-bank of the National Centre
for Biodiversity (NCB), University of Oslo.
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P.S., R.H., F.G. and A.T. designed the research, M.W., S.B., P.S.
and A.T. performed the research, M.W., G.M.S., A.T. and P.S.
analysed the data, P.S., M.W., G.M.S. and R.J.A. wrote the
paper and all authors have commented on several drafts of the
manuscript.
Data accessibility
DNA sequences have been deposited in GenBank under acces-
sion numbers JX131382 – JX131609. Details regarding individ-
ual samples are available in Table S1 (Supporting information).
Phylogenetic data (original plastid DNA alignment used for
� 2012 Blackwell Publishing Ltd
the BEAST analysis, separate alignments for psbA-trnH and
trnT-trnF sequences including GenBank accession numbers),
AFLP data matrix (excluding not repeatable, monomorphic
and single fragments), and detailed results of the geographic
diffusion model (Bayes factors and ancestral area probabilities)
are available at Dryad: doi: 10.5061/dryad.gf3qp.
Supporting information
Table S1 Population numbers, sampling locations and their
coordinates, number of individuals analysed for AFLP (NAFLP)
and plastid DNA (Ncp) variation, encountered plastid haplo-
types, GenBank accession numbers, herbarium and number of
voucher specimens and collectors of 62 populations of Saxifraga
oppositifolia used in the present study.
Fig. S1 Maximum clade credibility tree from strict clock Bayes-
ian analysis of plastid DNA (psbA-trnH, trnTF) haplotypes of
Saxifraga oppositifolia with the software BEAST.
Fig. S2 Range connectivity and ancestral location probabilities
among nine discrete geographical regions in Saxifraga oppositifo-
lia inferred using models of geographic diffusion applied to
the complete data set (i.e. including all sampled populations
from the Alps).
Fig. S3 Bayesian Skyline Plot of Saxifraga oppositifolia showing
changes in effective population size over time.
Text S1 Information regarding the definition of geographic
regions.