Flora 207 (2012) 408–413
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Flora
journal homepage: www.elsevier.de/flora
Contrasting diffusion of Quaternary gene pools across Europe: The case of the
arctic–alpine Gentiana nivalis L. (Gentianaceae)
Nadir Alvarez a,∗ , Stéphanie Manel b , Thomas Schmitt c , the IntraBioDiv Consortium1
a
b
c
Department of Ecology and Evolution, Biophore Building, University of Lausanne, 1015 Lausanne, Switzerland
Laboratoire Population Environnement Développement, UMR 151 UP/IRD, Université Aix-Marseille, 3 place Victor Hugo, 13331 Marseille Cedex 03, France
Department of Biogeography, Trier University, Universitätsring 15, D-54286 Trier, Germany
a r t i c l e
i n f o
Article history:
Received 11 October 2011
Accepted 17 March 2012
Keywords:
AFLP
Glacial refugia
High mountain systems
Phylogeography
Range shifts
a b s t r a c t
The fate of European arctic–alpine species during Pleistocene climatic oscillations still remains debated.
Did these cold-adapted species invade much of the continental steppe or did they remain restricted to
warmer slopes of inner mountain massifs? To examine this question, we investigated the phylogeography of Gentiana nivalis, a typical European arctic–alpine plant species. Genome fingerprinting analyses
revealed that four genetic pools are actually unevenly distributed across the continent. One cluster covers
almost all mountain massifs as well as northern areas, and thus coincides with a scenario of past distribution covering a large part of the European glacial steppe. In contrast, the three other lineages are strongly
restricted spatially to western, central, and eastern Alps, respectively, thus arguing towards a scenario of
in situ glacial survival. The coexistence of lineages with such contrasting demographic histories in Europe
challenges our classical view of refugia and corroborates several hypotheses of biogeographers from the
twentieth century.
© 2012 Elsevier GmbH. All rights reserved.
Introduction
Since the formation of the term “phylogeography” twenty-five
years ago (Avise et al., 1987), molecular analyses for a better understanding of the intraspecific differentiation and biogeographical
patterns and history turned out a popular tool in this scientific
field. Especially Europe has become the best studied region, and
numerous studies revealed different repeatable patterns, some of
them even called paradigms (Hewitt, 1999, 2000; Habel et al.,
2005). The maybe clearest differentiation into well distinguished
biogeographical groups on the continental scale is represented
by the Mediterranean, continental and arctic/alpine elements or
chorotypes (“Arealtyp” in German; Frey and Lösch, 2010), which
can be understood as an assemblage of species with similar distribution patterns and thus most probably similar biogeographical
history (Schmitt, 2007). While especially the Mediterranean elements are hitherto relatively well understood (e.g. Taberlet et al.,
1998; Hewitt, 1999; Schmitt, 2007), the continental and the arctic/alpine elements are still in need of considerable further studies
for achieving their comprehensive understanding (Hewitt, 2004;
Schmitt, 2007).
∗ Corresponding author. Tel.: +41 (0)21 692 42 47; fax: +41 (0)21 692 41 65.
E-mail addresses: [email protected] (N. Alvarez),
[email protected] (S. Manel), [email protected] (T. Schmitt).
1
See Electronic Appendix.
0367-2530/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.flora.2012.03.006
The arctic/alpine elements are apparently rather diverse (Varga
and Schmitt, 2008; Schmitt, 2009) and much more complex than
previously thought (e.g. Holdhaus, 1954; De Lattin, 1967). While the
biogeographical history of the classical arctic–alpine species (i.e.
plants with disjunct distributions in the Arctic and the more southern high mountain systems) was interpreted by a simple retreat
into these areas from a wide zonal distribution over the periglacial
steppe areas (De Lattin, 1967), the endemisms in the high mountain systems were interpreted by – at least partly – in situ survival
or survival in near-by areas (Holdhaus, 1954; Varga, 1975; Burnier
et al., 2009).
Recent genetic studies showed that the biogeographical histories of high mountain species (alpine disjunct and arctic–alpine)
are even more complex than previously thought when only
distribution-based analyses were possible (cf. Varga and Schmitt,
2008). Especially the Alps revealed to be genetically more diverse
than previously expected. Thus, several plant species often have
three to four different genetic lineages supporting glacial survival
(and differentiation) in disjunct areas along the southern and southeastern slopes of these mountains (Schönswetter et al., 2005).
While the conditions in the glacial steppes apparently were
unsuitable for many species, maybe due to lack of water supply (cf.
Schmitt and Seitz, 2001), many more widespread mountain elements might have had quite localised distributions during glacial
periods (Schmitt, 2009). In analogy to the Alps, many species survived glacial periods at the foothills of the Pyrenees, Carpathians
and the mountain systems of the Balkan Peninsula (e.g. Kropf et al.,
N. Alvarez et al. / Flora 207 (2012) 408–413
2003; Muster and Berendonk, 2006; Pauls et al., 2006; Schmitt et al.,
2006; Mráz et al., 2007; Kramp et al., 2009; Michl et al., 2010;
Dvořáková et al., 2010; Vila et al., 2011). Other genetic groups could
survive between such mountain systems retreating into both of
them thus showing similar genetic lineages in today highly remote
population groups (e.g. Kropf et al., 2002, 2003; Schönswetter et al.,
2004; Haubrich and Schmitt, 2007).
While these structures are relatively well known for alpine
disjunct species (Schmitt, 2009), the complete phylogeographical
structures of arctic–alpine species are relatively poorly understood.
Therefore, we selected one of these species, Gentiana nivalis L.,
as a study subject, given its well-marked arctic–alpine distribution restricted to the western Palearctic. This species is widely
distributed over the high mountain systems of Europe and the
European Arctic (Hultén and Fries, 1986). We analysed AFLP polymorphisms of populations scattered all over this distribution area
to address the following questions: (i) Does this arctic–alpine plant
species follow the simple pattern of postglacial retreat from one
large continuous periglacial distribution or (ii) does the recent distribution go back to different glacial retreats? If so, we question
for (iii) the geographical location of these retreats and (iv) their
importance for the postglacial recolonisation. (v) By the inclusion
of individuals from Scandinavia and Iceland, we want to reveal their
biogeographical provenance.
Material and methods
Study species
Gentiana nivalis is a diploid (2n = 14) plant (Favarger, 1969)
demonstrating solitary, terminal single flowers on short stems, or
a few flowers clustered in a loose cymose panicle (Kozuharova and
Anchev, 2006). It is the only species in the genus Gentiana along
with G. utriculosa L. (2n = 22; Müller, 1982) showing an annual
habit (Ho and Liu, 2001). It is self-compatible and shows spontaneous self-pollination; visitation by pollinators is rare and does
not seem to substantially account for seed production (Kozuharova
and Anchev, 2002). Dispersal is reported as boleochory – shaking
fruits – (Landolt et al., 2010) and no animals have been recorded
as seed dispersers. Gentiana nivalis is usually found in habitats
on calcareous bedrock or on intermediate wet subalpine grasslands (Kozuharova and Anchev, 2006; Alvarez et al., 2009). It is
distributed in the Alpine arch as well as in surrounding massifs
such as the Jura, the Pyrenees, the Balkan high mountain systems
and the Carpathians including the Tatra (anecdotic records exist
from Scotland and the Massif Central). Its distribution area also
covers the east coast of Canada and continues through Greenland,
Iceland, Scandinavia, and also occurrences in eastern Turkey and
the Caucasus are known (Hultén and Fries, 1986). The species has
been found in late-Pleistocene and early-Holocene palynological
records in several areas where it is absent today, for instance in
western (Abant Lake: S. Bottema, unpublished data) and central
(Ladik lake: S. Bottema, unpublished data) Anatolia, Syria (Ghani:
Lowe, 1978), northern Poland (Niechorze: Ralska-Jasiewiczowa
and Rzetkowska, 1987), the Baltic Sea region (Gotland Island:
Svensson, 1989) and south-eastern Ireland (Arts Lough: Bradshaw
and McGee, 1988), attesting its wider distribution during glacial
periods as proposed for arctic–alpine taxa (Stewart et al., 2010).
409
longitude regular grid cell based on Niklfeld (1971). Three individuals per sampling locality (named hereafter population) were
collected randomly at a distance of at least 10 m, and dried in silica gel. Furthermore, specimens from the Jura, Pyrenees, Norway
and Iceland were added through cooperative sampling with other
researchers/botanists (one or two specimens from one population
in each area). This sampling scheme resulted in 89 sampled populations (from which 85 were sampled by the IntraBioDiv consortium
in the Alps and the Carpathians). Analysing data from only three
individuals per sampling locality was counter-balanced by using a
large number of genetic markers (Nei, 1987) and a large number of
localities. Twenty plants (from 15 populations) were sampled and
extracted twice to test the reproducibility of AFLP fingerprinting
(Bonin et al., 2004). Two samples were used as replicates between
PCR plates, and were replicated more than twice. Voucher specimens are deposited at the herbarium of the University of Neuchâtel.
DNA extraction and AFLP fingerprinting
Total genomic DNA was extracted from similar amounts of
dried tissue (ca. 10 mg) with the DNeasy 96 plant mini kit (Qiagen,
Hilden, Germany) following the manufacturer’s protocol. AFLP profiles were generated following established procedures (Vos et al.,
1995) with minor modifications (Gugerli et al., 2008). After preliminary tests, three primer combinations that resulted in clear
repeatable bands with sufficient variability were chosen: EcoRIACT/MseI-CAC, EcoRI-ATC/MseI-CAC and EcoRI-ATG/MseI-CTG. For
the three AFLP datasets, the procedure followed Gaudeul et al.
(2000). Selective PCR products (1 ␮l labelled products) were mixed
and blended with 10 ␮l HiDi formamide and 0.1 ␮l ROX 500 size
standard. Electrophoresis was carried out on an Applied Biosystems 3100 capillary sequencer (Applied Biosystems, Foster City,
CA, USA). PCR products from each primer combination were run
separately. Blind samples were included to test for contamination
(Bonin et al., 2004). Fragments in the range 100–490 bp were scored
using RawGeno 2.0 (Arrigo et al., 2009). Scores of the primer combinations were further assembled into a binary (presence/absence)
data matrix.
Analysis of AFLP data
Population structure of Gentiana nivalis was analyzed through
Bayesian inference clustering using Structure V2.2 (Pritchard et al.,
Sampling
Gentiana nivalis was sampled across the Alps and Carpathians, following the strategy used by the IntraBioDiv consortium
as described in Gugerli et al. (2008). Leaf samples were collected from at least one locality in every second 12 latitude × 20
Fig. 1. Likelihoods and second derivatives of the Structure analysis. The values of
LnP(D) for each K are summarized using box-plots. The different letters indicate values of likelihood that are significantly different for a risk alpha = 0.05 (post hoc Tukey
honest significant differences test). Values for the second derivative [(slope)] indicate that the optimal K value is reached at K = 4 clusters (arrow) – cf. Evanno et al.
(2005).
410
N. Alvarez et al. / Flora 207 (2012) 408–413
2000; Falush et al., 2007) with an assumption of admixture and
independent allele frequencies, using the recessive alleles option.
Five independent runs were carried out for each value of K (i.e.,
the number of clusters assumed) ranging between 1 and 10, with
parameters and model likelihood estimated over 1,000,000 Monte
Carlo Markov Chains generations following a burn-in period of
200,000 generations. For each value of K, only the run yielding the
best likelihood was considered. We also estimated the allele frequencies at each locus using a Bayesian method with non-uniform
prior distribution of allele frequencies (Zhivotovsky, 1999) using
aflp-surv 1.0 (Vekemans, 2002). Estimates of allele frequencies
were used to calculate the percentage of polymorphic loci to determine the spatial structuring of genetic diversity.
Results
Genetic structure and phylogeny based on AFLP data
The three AFLP primer combinations yielded a total of 220
unambiguously scorable bands for the entire data set comprising
280 specimens with an overall reproducibility rate of 95%. Among
the 220 bands, 46 were generated by the primer EcoRI-ACT/MseICAC, 95 by EcoRI-ATC/MseI-CAC and 79 by EcoRI-ATG/MseI-CTG.
Following Evanno et al. (2005), delta(K) was maximized with K = 4,
which yielded −ln(likelihood) = −6282.67 (Fig. 1). The assignment
probabilities to each cluster (hereafter referred to as lineage) for
each individual at K = 4 are displayed in Fig. 2. Three lineages were
well structured geographically, following the Alpine paradigm proposed by Schönswetter et al. (2005), with western-, central-, and
eastern-Alpine lineages (shown in green, yellow, and blue, respectively; Fig. 2). In contrast, a fourth lineage (shown in red; Fig. 2)
was widespread and little structured geographically, spanning the
Carpathians and the easternmost Alps, the Jura as well as populations close to the Jura in the Western Alps, the Pyrenees, Norway
and Iceland.
Percentage of polymorphic loci ranged from 0.5% to 18.6% in the
different sampled locations, in which more than two specimens
were sampled. Highly diverse populations were mostly found in
the centre of the Alpine Arch, with populations from the edge of
the Alpine distribution as well as those from the Carpathians being
less diverse (Fig. 3).
Discussion
The four well distinguished genetic lineages of G. nivalis in
Europe strongly contradict hypothesis (i) that all populations of
this species have derived from one large and continuous periglacial
distribution during the last ice age and later retraction to the North
and the mountain ranges in the South. Thus, this idea of the biogeographers of the mid-20th century and before (e.g. Holdhaus and
Lindroth, 1939; Holdhaus, 1954; De Lattin, 1967) with respect to
arctic–alpine species cannot be confirmed for this plant species,
although it might apply to one of its lineages (see below). On the
contrary, our alternative hypothesis (ii) of several disjunct glacial
survival centres in Europe is strongly supported, a common feature
of such mountain species, as shown in several recent phylogeographical studies (reviews in Schönswetter et al., 2005; Schmitt,
2009).
The geographic distribution of the four detected lineages is
unequal all over Europe. While all four G. nivalis lineages are present
in the Alps (Fig. 2), the forth lineage, which is the least extended
one in the Alps, is present in all other surveyed regions (Iceland,
Scandinavia, Pyrenees, Jura, Tatras, Carpathians). This phylogeographic pattern is in agreement with three separate, geographically
restricted glacial refugia of the lineages shown (Fig. 2) in green,
yellow and blue in the vicinity of the Alps. Conforming the extent
of the Alpine ice-shield covering larger areas north of the Alps,
but offering suitable areas at the southern margin of the Alps
(Schönswetter et al., 2005), the most likely locations of these three
G. nivalis refugia have to be assumed as having been lined up in
three disjunct regions from the south-western to the eastern margin of the Alps, thus resolving questions (iii) and (iv). Quite similar
pattern of three to four different refugial areas of alpine plants in
these areas are frequently reported in the phylogeographic literature (reviewed in Schönswetter et al., 2005). Similar results of
two or more southern Alpine glacial refugia were also repeatedly
observed in animals (Schmitt et al., 2006; Haubrich and Schmitt,
2007; Schmitt and Haubrich, 2008; Schmitt and Besold, 2010;
Triponez et al., 2011). Thus, G. nivalis is expressing a common feature of arctic–alpine and alpine disjunct taxa referring to its most
likely survival centres at the southern margin of the Alps (Schmitt,
2009). High levels of genetic diversity in the centre of the current Alpine distribution of the plant (Fig. 3) suggest recolonization
by consistently large populations, which kept a large part of the
genetic diversity, which has been maintained in the glacial refugia.
In contrast, the genetic homogeneity of G. nivalis from the Pyrenees, Jura mountains to Tatras and Carpathians including only some
marginal areas of the north-western and easternmost Alps but also
the northern proveniences of samples (Iceland, Scandinavia) is not
as commonly observed in arctic–alpine species. In most cases, at
least one of the mountain ranges of Pyrenees or Carpathians harbours populations with a considerable genetic difference from a
widespread lineage expanding to the North (Kropf et al., 2003;
Muster and Berendonk, 2006; Pauls et al., 2006; Kramp et al., 2009;
Michl et al., 2010). Furthermore, this widespread lineage in most
cases is also common in at least some part of the Alps (Schönswetter
et al., 2004; Muster and Berendonk, 2006; Triponez et al., 2011).
Therefore, these patterns call for large periglacial distributions of
most arctic–alpine species studied so far and postglacial colonisation from this area to the North, (at least some parts of) the Alps
and some other high mountain systems, but also further glacial
distributions south of the Pyrenees, Alps and Carpathians resulting
in additional genetic lineages in at least some of these mountain
systems (Kropf et al., 2003; Muster and Berendonk, 2006).
The pattern found for G. nivalis calls for a somewhat different
biogeographical history. The widespread character of one of its lineages (in red in Fig. 2) makes a large periglacial ice age distribution
between the mountain glaciers in the South and the large northern
glacier the most likely scenario. This is in agreement with palynological records, which demonstrate late-Pleistocene G. nivalis
pollen in the former European Steppe, around the Baltic Sea (Gotland island, northern Poland). However, in contrast to most other
arctic–alpine species (e.g., Ronikier et al., 2008), no additional geographically disjunct retreat seems to have existed south of the
Pyrenees or Carpathians in G. nivalis. Furthermore, the southwards
colonisation from the zonal ice age distribution in Central Europe
through the formerly glaciated areas north of the Alps must have
been relatively slow, so slow that most regions of the deglaciating
Alps already had been colonised by the three southern Alpine lineages when the more northern one reached this region. Therefore,
this lineage only could slightly penetrate two peripheral regions of
the Alps in the extreme northwest and east, as well as the Jura
mountains, located as a mountain range in front of the northwestern Alps.
In contrast to this marginal contribution to the populations in
the Alps, the zonal ice age distribution of G. nivalis in Central Europe
was responsible for all colonisations to the North and even the
individuals analysed from Iceland do not show any remarkable
differentiation against this widespread lineage so that even this
remote Atlantic island at the arctic polar circle has been reached
from this source region during the postglacial period, thus also
N. Alvarez et al. / Flora 207 (2012) 408–413
411
Fig. 2. Geographical origin of the 89 analysed populations of Gentiana nivalis and their grouping according to model-based Structure clustering on the Amplified Fragment
Length Polymorphism (AFLP) dataset. The small frame depicts the Alpine arch. Pie charts represent the proportion of individuals belonging to each of the four detected clusters
(or lineages) using the majority-rule criterion. This proportion is given in thirds, as three specimens were collected in most populations (when one sample did not yield
exploitable AFLP results, the corresponding third of the pie chart is absent). An individual was assigned to a Structure-based cluster, given that its assignment probability
was >0.5 in at least one cluster. Whereas light colour tones (i.e., light green, light blue, light yellow and light red) represent assignment probabilities ranging from 0.5 to 0.75,
dark colour tones (i.e., dark green, dark blue, dark yellow and dark red) indicate a probability >0.75. Blank pies indicate that assignment probabilities were <0.5 for any of
the four clusters.
Fig. 3. Patterns of genetic diversity within Gentiana nivalis in the Alpine arch and the Carpathians, as estimated by the percentage of polymorphic loci per population. Values
are only given in populations where at least three specimens were sampled. Values were categorized into four classes, according to the legend at the bottom.
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N. Alvarez et al. / Flora 207 (2012) 408–413
answering our question (v). Such a colonisation of the northern
regions from one, more southern situated, large source is well in
consensus with the theories erected by the old biogeographers (e.g.
Holdhaus and Lindroth, 1939; Holdhaus, 1954; De Lattin, 1967).
Acknowledgements
We are grateful to Alessia Guggisberg (University of British
Columbia, Canada), to Marc Hämmerli and Philippe Druart (University of Neuchâtel, Switzerland) for collecting samples outside
the Alps and the Carpathians. We also warmly thank Anahí Espíndola and Olivier Broennimann for help in GIS, as well as Philippe
Küpfer for inspiring this study. Nadir Alvarez was funded by
the Swiss National Science Foundation (Ambizione fellowship
PZ00P3 126624) and Stéphanie Manel was supported by the Institut universitaire de France.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.flora.2012.03.006.
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