Contrasting Diffusion of Quaternary Gene Pools Across Europe: the Case of The
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Flora 207 (2012) 408–413 Contents lists available at SciVerse ScienceDirect Flora jo urnal homepage: www.elsevier.de/flora Contrasting diffusion of Quaternary gene pools across Europe: The case of the arctic–alpine Gentiana nivalis L. (Gentianaceae) a,∗ b c 1 Nadir Alvarez , Stéphanie Manel , Thomas Schmitt , the IntraBioDiv Consortium a Department of Ecology and Evolution, Biophore Building, University of Lausanne, 1015 Lausanne, Switzerland b Laboratoire Population Environnement Développement, UMR 151 UP/IRD, Université Aix-Marseille, 3 place Victor Hugo, 13331 Marseille Cedex 03, France c Department of Biogeography, Trier University, Universitätsring 15, D-54286 Trier, Germany a r t i c l e i n f o a b s t r a c t Article history: The fate of European arctic–alpine species during Pleistocene climatic oscillations still remains debated. Received 11 October 2011 Did these cold-adapted species invade much of the continental steppe or did they remain restricted to Accepted 17 March 2012 warmer slopes of inner mountain massifs? To examine this question, we investigated the phylogeogra- phy of Gentiana nivalis, a typical European arctic–alpine plant species. Genome fingerprinting analyses Keywords: revealed that four genetic pools are actually unevenly distributed across the continent. One cluster covers AFLP almost all mountain massifs as well as northern areas, and thus coincides with a scenario of past distribu- Glacial refugia tion covering a large part of the European glacial steppe. In contrast, the three other lineages are strongly High mountain systems Phylogeography 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 Range shifts challenges our classical view of refugia and corroborates several hypotheses of biogeographers from the twentieth century. © 2012 Elsevier GmbH. All rights reserved. Introduction The arctic/alpine elements are apparently rather diverse (Varga and Schmitt, 2008; Schmitt, 2009) and much more complex than Since the formation of the term “phylogeography” twenty-five previously thought (e.g. Holdhaus, 1954; De Lattin, 1967). While the years ago (Avise et al., 1987), molecular analyses for a better under- biogeographical history of the classical arctic–alpine species (i.e. standing of the intraspecific differentiation and biogeographical plants with disjunct distributions in the Arctic and the more south- patterns and history turned out a popular tool in this scientific ern high mountain systems) was interpreted by a simple retreat field. Especially Europe has become the best studied region, and into these areas from a wide zonal distribution over the periglacial numerous studies revealed different repeatable patterns, some of steppe areas (De Lattin, 1967), the endemisms in the high moun- them even called paradigms (Hewitt, 1999, 2000; Habel et al., tain systems were interpreted by – at least partly – in situ survival 2005). The maybe clearest differentiation into well distinguished or survival in near-by areas (Holdhaus, 1954; Varga, 1975; Burnier biogeographical groups on the continental scale is represented et al., 2009). by the Mediterranean, continental and arctic/alpine elements or Recent genetic studies showed that the biogeographical histo- chorotypes (“Arealtyp” in German; Frey and Lösch, 2010), which ries of high mountain species (alpine disjunct and arctic–alpine) can be understood as an assemblage of species with similar dis- are even more complex than previously thought when only tribution patterns and thus most probably similar biogeographical distribution-based analyses were possible (cf. Varga and Schmitt, history (Schmitt, 2007). While especially the Mediterranean ele- 2008). Especially the Alps revealed to be genetically more diverse ments are hitherto relatively well understood (e.g. Taberlet et al., than previously expected. Thus, several plant species often have 1998; Hewitt, 1999; Schmitt, 2007), the continental and the arc- three to four different genetic lineages supporting glacial survival tic/alpine elements are still in need of considerable further studies (and differentiation) in disjunct areas along the southern and south- for achieving their comprehensive understanding (Hewitt, 2004; eastern slopes of these mountains (Schönswetter et al., 2005). Schmitt, 2007). 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 ele- ments might have had quite localised distributions during glacial ∗ Corresponding author. Tel.: +41 (0)21 692 42 47; fax: +41 (0)21 692 41 65. periods (Schmitt, 2009). In analogy to the Alps, many species sur- E-mail addresses: [email protected] (N. Alvarez), vived glacial periods at the foothills of the Pyrenees, Carpathians [email protected] (S. Manel), [email protected] (T. Schmitt). 1 and the mountain systems of the Balkan Peninsula (e.g. Kropf et al., 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 N. Alvarez et al. / Flora 207 (2012) 408–413 409 2003; Muster and Berendonk, 2006; Pauls et al., 2006; Schmitt et al., longitude regular grid cell based on Niklfeld (1971). Three indi- 2006; Mráz et al., 2007; Kramp et al., 2009; Michl et al., 2010; viduals per sampling locality (named hereafter population) were Dvorákovᡠet al., 2010; Vila et al., 2011). Other genetic groups could collected randomly at a distance of at least 10 m, and dried in sil- survive between such mountain systems retreating into both of ica gel. Furthermore, specimens from the Jura, Pyrenees, Norway them thus showing similar genetic lineages in today highly remote and Iceland were added through cooperative sampling with other population groups (e.g. Kropf et al., 2002, 2003; Schönswetter et al., researchers/botanists (one or two specimens from one population 2004; Haubrich and Schmitt, 2007). in each area). This sampling scheme resulted in 89 sampled popula- While these structures are relatively well known for alpine tions (from which 85 were sampled by the IntraBioDiv consortium disjunct species (Schmitt, 2009), the complete phylogeographical in the Alps and the Carpathians). Analysing data from only three structures of arctic–alpine species are relatively poorly understood. individuals per sampling locality was counter-balanced by using a Therefore, we selected one of these species, Gentiana nivalis L., large number of genetic markers (Nei, 1987) and a large number of as a study subject, given its well-marked arctic–alpine distribu- localities. Twenty plants (from 15 populations) were sampled and tion restricted to the western Palearctic. This species is widely extracted twice to test the reproducibility of AFLP fingerprinting distributed over the high mountain systems of Europe and the (Bonin et al., 2004). Two samples were used as replicates between European Arctic (Hultén and Fries, 1986). We analysed AFLP poly- PCR plates, and were replicated more than twice. Voucher speci- morphisms of populations scattered all over this distribution area mens are deposited at the herbarium of the University of Neuchâtel. to address the following questions: (i) Does this arctic–alpine plant species follow the simple pattern of postglacial retreat from one DNA extraction and AFLP fingerprinting large continuous periglacial distribution or (ii) does the recent dis- tribution go back to different glacial retreats? If so, we question Total genomic DNA was extracted from similar amounts of for (iii) the geographical location of these retreats and (iv) their dried tissue (ca. 10 mg) with the DNeasy 96 plant mini kit (Qiagen, importance for the postglacial recolonisation. (v) By the inclusion Hilden, Germany) following the manufacturer’s protocol. AFLP pro- of individuals from Scandinavia and Iceland, we want to reveal their files were generated following established procedures (Vos et al., biogeographical provenance. 1995) with minor modifications (Gugerli et al., 2008). After pre- liminary tests, three primer combinations that resulted in clear repeatable bands with sufficient variability were chosen: EcoRI- Material and methods ACT/MseI-CAC, EcoRI-ATC/MseI-CAC and EcoRI-ATG/MseI-CTG. For the three AFLP datasets, the procedure followed Gaudeul et al. Study species (2000). Selective PCR products (1 l labelled products) were mixed and blended with 10 l HiDi formamide and 0.1 l ROX 500 size Gentiana nivalis is a diploid (2n = 14) plant (Favarger, 1969) standard. Electrophoresis was carried out on an Applied Biosys- demonstrating solitary, terminal single flowers on short stems, or tems 3100 capillary sequencer (Applied Biosystems, Foster City, a few flowers clustered in a loose cymose panicle (Kozuharova and CA, USA). PCR products from each primer combination were run Anchev, 2006). It is the only species in the genus Gentiana along separately. Blind samples were included to test for contamination with G. utriculosa L. (2n = 22; Müller, 1982) showing an annual (Bonin et al., 2004). Fragments in the range 100–490 bp were scored habit (Ho and Liu, 2001). It is self-compatible and shows spon- using RawGeno 2.0 (Arrigo et al., 2009). Scores of the primer com- taneous self-pollination; visitation by pollinators is rare and does binations were further assembled into a binary (presence/absence) not seem to substantially