View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Springer - Publisher Connector Conserv Genet (2010) 11:2169–2177 DOI 10.1007/s10592-010-0103-y RESEARCH ARTICLE Genetic structure of coastal and inland populations of Spergularia media (L.) C. Presl (Caryophyllaceae) in Central Europe Kathleen Prinz • Kurt Weising • Isabell Hensen Received: 1 December 2009 / Accepted: 18 June 2010 / Published online: 2 July 2010 Ó The Author(s) 2010. This article is published with open access at Springerlink.com Abstract In Central Europe, salt-influenced habitats are that showed good correspondence to their geographic restricted mainly to the coastlines of the North Sea and the origins. A Bayesian analysis performed with the program Baltic Sea. The few natural inland salt sites suffer from size STRUCTURE as well as high pairwise UST values supported reduction and loss in biodiversity, while anthropogenic salt the results of the phenetic analyses. The observed patterns sites around potash mining dumps experienced recent and of diversity and differentiation among inland populations massive de novo colonization by numerous halophytes. of S. media are best explained by the isolated nature of Our study aimed to elucidate the general patterns of gene suitable salt habitats, with concomitant reduction of gene flow among coastal and inland salt habitats. We used flow to and among these sites. Our data support the amplified fragment length polymorphisms to assess the hypothesis that the colonization of anthropogenic salt sites genetic diversity and genetic structure of 18 European by S. media originated from natural inland habitats. populations of the halophyte Spergularia media (Caryo- phyllaceae), sampled from inland and coastal salt sites of Keywords AFLP markers Á Halophytes Á Germany, the Netherlands, Denmark, Austria, France and Inland salt sites Á Potash mining dumps Á Genetic structure Á Italy. Estimates of genetic diversity on the population level Spergularia media were generally low, especially within inland salt habitats. Analyses of molecular variance showed comparatively strong differentiation among populations. Multivariate ordination (principal coordinate analysis) and a Neighbor- Introduction Net analysis revealed four distinct groups of populations Under the temperate and humid climatic conditions of Central Europe, salt-influenced habitats are largely Electronic supplementary material The online version of this article (doi:10.1007/s10592-010-0103-y) contains supplementary restricted to a small strip of land along the coastlines of the material, which is available to authorized users. North Sea and the Baltic Sea. These habitats are populated by highly specialized plant species that are able to survive & K. Prinz ( ) under high-salt conditions in the soil, so-called halophytes. Forest Genetics and Forest Tree Breeding, Georg-August- University Go¨ttingen, Bu¨sgen-Insitute, Bu¨sgenweg 2, A few isolated salt sites also exist further inland, e.g., in 37077 Go¨ttingen, Germany the surroundings of natural salty springs. They usually e-mail: [email protected] accommodate a subset of the same halophytic species that are also found along the coasts. Because of their floristic K. Weising Plant Molecular Systematics, University of Kassel, peculiarity, Brandes (1999) referred to these inland salt Heinrich-Plett-Strabe 40, 34109 Kassel, Germany sites as the ‘‘inselbergs of Central Europe’’. However, these remarkable habitats have become seriously endangered by I. Hensen species loss as well as by the ongoing reduction and Institute of Biology/Geobotany and Botanical Garden, Martin-Luther-University Halle-Wittenberg, Am Kirchtor 1, fragmentation of their once larger areas (Westhus et al. 06108 Halle/Saale, Germany 1997; Brandes 1999; Garve and Garve 2000). 123 2170 Conserv Genet (2010) 11:2169–2177 As a consequence of human potash mining activities Saxony, Hesse, Saxony-Anhalt and Thuringia in 1993 since the middle of the nineteenth century, new salt-influ- (Garve and Garve 2000). Presently, S. media is known enced inland habitats have developed in the immediate from about 70% of all anthropogenic inland salt sites in vicinity of the numerous Central European potash mining Central Europe (Garve and Garve 2000). The species is dumps. These purely anthropogenic habitats have been usually pollinated by insects, but is also known to be self- colonized by halophytes only recently, but then at a rapid fertile. Its small, winged seeds are dispersed by wind, water pace (Guder et al. 1998; Garve 1999b; Garve and Garve or by attachment to animals (Hegi 1965; Huiskes et al. 2000; John 2000). Presently, potash mining dumps are 1995). home to large populations of various halophytic species We consider S. media to be a suitable model species for that are highly endangered or even in risk of extinction in examining the biogeography and population genetics of their natural inland salt habitats. Some authors even con- halophytes at natural and anthropogenic inland salt sites of sidered anthropogenic salt sites as potential retreat areas Central Europe. Here we present a genetic analysis based that could serve as stepping stones for the (re)colonization on amplified fragment length polymorphisms (AFLP, Vos of natural inland salt sites (Westhus et al. 1997; Kru¨ger et al. 1995) that addresses the following questions: (1) How et al. 2002; Brock et al. 2007). However, halophyte com- is genetic variation distributed among and within popula- munities at both types of inland salt habitat have probably tions of S. media, and is genetic diversity of inland popu- experienced genetic drift, which is often accompanied by lations lower as compared to that of coastal populations? low levels of genetic diversity (Lienert 2004; Novak and (2) Can putative source populations of the anthropogenic Mack 2005; Prinz et al. 2009). inland salt sites be identified? Floristics and vegetation of inland salt sites in Central Europe have been the subject of numerous publications (e.g., Vollrath and Bo¨nsel 1995; Westhus et al. 1997; Materials and methods Guder et al. 1998; Garve 1999a, b; Garve and Garve 2000). However, still little is known about migration patterns and Plant material and DNA isolation the timing of colonization of these sites by halophytic plants, and there is an ongoing controversial discussion on Leaves of individual S. media plants were collected from these issues. For example, Brandes (1999) postulated a 18 populations of different European coastal and inland continental or Mediterranean origin for Atriplex tatarica, habitats. About 15 plants per population were sampled in Kochia densiflora, Gypsophila perfoliata, G. scorzoneri- Germany and the Netherlands whereas populations from folia and Hymenolobus procumbens, whereas inland pop- Austria, Denmark, France and Italy were represented by ulations of Suaeda maritima, Salicornia ramosissima and three to ten individuals per locality (Fig. 1, Table 1). Spergularia marina were assumed to originate from the Leaves were preserved and stored in NaCl-saturated CTAB coasts of the North Sea and the Baltic Sea. On the contrary, solution (cetyltrimethylammonium bromide; Rogstad Garve (1999a) hypothesized that Suaeda maritima, Gyp- 1992). DNA was extracted by a modified CTAB method sophila scorzonerifolia and other halophytes currently (Doyle and Doyle 1987). Final DNA concentrations were found around mining dumps in Lower Saxony might have determined electrophoretically, with known amounts of immigrated from other inland salt sites further east. Careful phage k-DNA used as standard. population genetic and biogeographic analyses could help to resolve these controversies. However, only a handful of AFLP analysis studies so far addressed issues like genetic diversity, gene flow and genetic structure of coastal and inland halophyte The AFLP protocol followed the method of Ba¨nfer et al. populations by means of molecular markers (e.g., Kru¨ger (2004), with some modifications. Approximately 250 ng of et al. 2002; Brock et al. 2007; Lambracht et al. 2007; Prinz total genomic DNA were double-digested with HindIII and et al. 2009). MseIat37°C for 15 h, and the resulting restriction frag- Spergularia media (Caryophyllaceae) is a small peren- ments were ligated to HindIII and MseI adapters in the nial halophytic herb that grows on saline, mostly wet same reaction. The restriction/ligation assay contained 2.5 substrates in coastal and continental areas of western U each of HindIII and MseI enzyme, 0.1 U T4-DNA ligase, Eurasia (Hegi 1965). It is a common element of coastal salt 0.01 lM HindIII-adapter, 0.1 lM MseI-adapter, 10 mM meadows, but is also present around natural inland salt Tris–HCl (pH 8.4), 10 mM magnesium acetate, 50 mM springs. The species was first recorded from the sur- potassium acetate, 5 mM DTT and 0.2 mM ATP in a final roundings of potash mining dumps in the Alsace in the volume of 30 ll. Five microliters of the restriction-ligation mid-1950s (Simon 1958), followed by records from Baden- mixture were used as template for pre-selective PCR with Wu¨rttemberg in 1978 (Plieninger 1992) and from Lower two different combinations of primers, each containing one 123 Conserv Genet (2010) 11:2169–2177 2171 0.02 U Taq DNA polymerase (Mango-Taq DNA-Poly- merase, Bioline) in a total volume of 50 ll. Pre-selective PCR was performed in a T-Gradient Cycler (Biometra) under the following temperature regime: one cycle at 94°C for 2 min, followed by 19 cycles at 94°C for 30 s, 60°C for 30 s and 72°C for 1 min. Final extension was at 72°C for 7 min. Selective PCRs were then performed with 2.5 llof a 1:10 diluted pre-amplification mixture and various com- binations of an unlabeled MseI ? 3 primer (0.6 lM) and a fluorescence-labeled HindIII ? 3 primer (0.075 lM IRD700, 0.095 lM IRD800; Table 2). Each 10 ll-assay also contained 0.3% Triton X-100, 0.1 mM MgCl2, 0.2 mM of each dNTP, 20 mM Tris–HCl (pH 8.4), 50 mM KCl and 0.025 U Taq DNA polymerase (Mango-Taq DNA- Polymerase, Bioline).