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;

ela.winkler@univie.ac.at

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.