Biodiv. Res. Conserv. 42: 9-18, 2016 BRC www.brc.amu.edu.pl DOI 10.1515/biorc-2016-0008 Submitted 29.01.2016, Accepted 30.06.2016 Genetic structure and diversity in Juniperus communis populations in Saxony, Germany Stefanie Reim1, Frank Lochschmidt2, Anke Proft2, Ute Tröber1 & Heino Wolf1 1Public Enterprise Sachsenforst, Wood and Forestry Competence Centre, Forest Genetics and Forest Plant Breeding, Bonnewitzer Str. 34, D-01796 Pirna OT Graupa, Germany 2Green League Osterzgebirge e.V., Große Wassergasse 9, D-01744 Dippoldiswalde, Germany * corresponding author (e-mail: [email protected]) Abstract: In recent years, land use changes led to a rapid decline and fragmentation of J. communis populations in Germany. Population isolation may lead to a restricted gene flow and, further, to negative effects on genetic variation. In this study, ge- netic diversity and population structure in seven fragmented J. communis populations in Saxony, Germany, were investigated using nuclear microsatellites (nSSR) and chloroplast single nucleotide polymorphism (cpSNP). In all Saxony J. communis populations, a high genetic diversity was determined but no population differentiation could be detected whatever method was applied (Bayesian cluster analysis, F-statistics, AMOVA). The same was true for three J. communis out-group samples originating from Italy, Slovakia and Norway, which also showed high genetic diversity and low genetic differences regarding other J. communis populations. Low genetic differentiation among the J. communis populations ascertained with nuclear and chloroplast markers indicated high levels of gene flow by pollen and also by seeds between the sampled locations. Low genetic differentiation may also provide an indicator of Juniper survival during the last glacial maximum (LGM) in Europe. The results of this study serve as a basis for the implementation of appropriate conservation measures in Saxony. Key words: Juniperus communis, nSSR, cpSNP, genetic diversity, population differentiation 1. Introduction and Frankish Alp and in the sandy areas of Lüneburg and Lusatia Heath. In the remaining areas of Germany, Common Juniper (Juniperus communis L.) belongs only very fragmented small groups of J. communis to the Juniperus genus, in the family of Cupressaceae. occur. Therefore, the German Red List of Endangered It is the most widely distributed conifer in the Northern species categorizes J. communis as vulnerable (http:// Hemisphere occurring in North America, Europe, Asia, www.floraweb.de/pflanzenarten/rotelisten.html, state: and parts of North Africa (Thomas et al. 2007). 01.01.2016). The reduction of population numbers J. communis grows on a variety of soil types and tol- and spatial fragmentation reduce connectivity between erates nutrient-poor sites, drought and winter tempera- populations and lead to population isolation. tures as low as -40°C (Thomas et al. 2007). However, Associated with the population size, the fertiliza- Juniper has high light requirements and does not tolerate tion type of J. communis could be a further cause for competitive pressure well (Ellenberg 1996). Despite endangering this species. Juniper is a dioecious species this unpretentiousness, Juniper populations have in- with female and male plants. It is wind-pollinated and creasingly declined in recent decades and have now a seeds are, primarily, dispersed by birds. Compared to threatened status in several regions in Europe (Gruwez hermaphroditic species, the separation of sexes halves et al. 2012). Due to land use changes, typical Juniper the density of potential pollen donors and seed-produc- habitats such as heathlands and calcareous grassland ing individuals. The absence of an appropriate quantity are sharply declining or have already completely dis- of juniper pollen has, in turn, negative effects on the appeared in Germany. At present, a spatially inclusive amount of Juniper cones. In addition, like many other distribution of J. communis populations covering large conifer species, Juniper produces a large proportion of areas can only be found in the Calcareous Alps, Swabian empty seeds (McCartan & Gosling 2013). Seed viability AND PHYLOGENY TAXONOMY VARIABILITY, ©Adam Mickiewicz University in Poznań (Poland), Department of Plant Taxonomy. All rights reserved. 10 Stefanie Reim et al. Genetic structure and diversity in Juniperus communis populations in Saxony, Germany is subject to a remarkable variation depending on plant on J. communis. In order to improve the genetic data set, age, habitat, climate or seed predators (Thomas et al. we also used single nucleotide polymorphism markers of 2007; Vanden-Broeck et al. 2011; Gruwez et al. 2012). the chloroplast genome according to Provan et al. (2009). Juniper seeds are usually deeply dormant and require Based upon the genetic data, we estimated genetic diver- a long stratification time to germinate. On average, sity within the populations and genetic structure among seeds need two years and seven months for germination these populations. Consequences for the implementation (Broome 2003). Limited sexual regeneration, in addition of conservation measures for the Saxony J. communis to the fragmentation of J. communis populations, may populations are discussed. have impact on genetic diversity and genetic structure of the remaining populations. 2. Material and methods Habitat fragmentation leads to a reduction of ran- 2.1. Study site, sampling and DNA extraction dom mating and immigration rates (Michalczyk 2008; Ashley 2010). As a result, genetic diversity decreases The study site was located in the federal state of and genetic differentiation among populations becomes Saxony in South-Eastern Germany (Fig. 1). Saxony more pronounced due to inbreeding and restricted gene comprises the area of 18,413 square kilometers com- flow (Couvet 2002). Genetic diversity limitation may posed, largely, of hills and mountains. Only northern lead to a decline in fitness and, further, to extinction of and western Saxony descends into the great North the species (Severns et al. 2003; Provan et al. 2009). European Plain. Within this area, seven different J. Depending on the mode of pollination (wind or insect communis patches were found and, in total, 404 shrubs pollinated), the extent of negative influences – due were sampled (Table 1). to habitat fragmentation – is more or less distinctive For each shrub originating from Saxony, sex and (reviewed in Ashley 2010; Vranckx 2012). In general, vitality were determined. Additional 29 J. communis wind-pollinators seem to be less influenced by habitat individuals from Slovakia, 27 individuals from Norway fragmentation effects because their pollen is transported and 28 individuals from Northern Italy were included over long distances (Ashley 2010). Recent studies in our analysis as out-group samples. In total, 488 reported high pollen immigration in wind-pollinated J. communis samples were genetically analyzed. populations indicating a random mating over large spa- Fresh needles were collected in 2 ml reaction tubes tial scales. For example, results from a pollen dispersal and dried using silica gel according to a modified pro- investigation in Pinus sylvestris L. showed pollination tocol by Chase et al. (1991). Dried plant material can distances of up to 30 kilometers (Robledo-Arnuncio & be stored at room temperature until DNA isolation. Gil 2005). This suggests that even small isolated frag- DNA extraction and quantification was performed by ments are not necessarily reproductively isolated and the company LGC Genomics (Berlin, Germany). All are able to maintain pollination with more distant trees samples were diluted to 10 ng/µl. or shrubs (Ashley 2010). 2.2. Microsatellite analysis In several studies, J. communis genetic diversity and population differentiation were investigated using dif- For nSSR analysis, 19 nSSR primer pairs developed ferent genetic marker systems (e.g.: allozymes, AFLPs, for J. communis and other Juniperus species (Berube nuclear microsatellite and chloroplast single nucleotide et al. 2003; Michalczyk et al. 2006; Zhang et al. 2008; polymorphisms). In all Juniper populations, a high level Opgenoorth 2009) were tested on 16 J. communis geno- of genetic diversity was found. The degree of population types (Table 2). differentiation varied between moderate to high (Van der Eight primer pairs (Jp01, Jp05, Jt01, Jt02, Jt04, Jt06, Merwe et al. 2000; Provan et al. 2009; Michalczyk et al. y2c12, y2h01) showed no amplification or multibanding 2010) and low (Oostermeijer & De Knegt 2004; Khan- patterns in J. communis, respectively. Four primer pairs temirova & Semerikov 2010; Vanden-Broeck et al. 2011). (Jp04, Jt03, Jt05, y1e10) showed only monomorphic In this study, we used biparentally and uniparentally patterns. For this reason, these twelve primer pairs were inherited markers to estimate the population genetics discarded. For the remaining seven primers, one to two of Juniper samples in the province of Saxony in south- alleles per locus and individual were amplified. In the eastern Germany. For J. communis, only five specific next step, these primers were combined in different microsatellites with scorable polymorphic bands were multiplex reactions. Only five primer pairs developed available (Michalczyk 2008). Therefore, we tested cross- for J. communis by Michalczyk et al. (2006) were suited species applicability of numerous microsatellite mark- for a combination in two multiplexes with two or three ers, which were developed for other Juniperus