With nearly 500 accepted species, Campanula L. is the largest Campanulaceae genus (Roquet et al., 2008). Campanula scheuchzeri Vill. is distributed in meadows and pastures of European mountains and is primarily tetraploid (2n = 68 = 4x; Geslot, 1984; Lauber and Wagner, 2007), although some diploid populations have been identified in the Pyrenees (Geslot, 1984). Campanula rotundifolia L. is a sister species of C. scheuchzeri and is widespread across the northern hemisphere (Roquet et al., 2008; Stevens et al., 2012). In the Alps, both taxa may co-occur on mountain slopes, with C. scheuchzeri found in greatest abundance above c. 1200 m and C. rotundifolia below c. 1200 m (Aeschimann et al., 2004; Frei, 2007). The two species are morphologically similar, phenotypically highly variable, and some populations are difficult to classify (Böcher, 1936; Frei, 2007; Lauber and Wagner, 2007). No nucleotide differences were observed between the two taxa in an 850-bp chloroplast sequence (Roquet et al., 2008; aligning GenBank sequence EF088759 with EF088762). However, typical populations of C. scheuchzeri and C. rotundifolia can be easily distinguished by the number and size of flowers, leaf shape, and hair density (Frei, 2007). In the future, we will use the microsatellite loci developed in this study to evaluate phenotypic and molecular differentiation, gene flow, and local adaptation in C. scheuchzeri and C. rotundifolia in different regions of the Swiss Alps.

METHODS AND RESULTS

We sampled leaf material of C. scheuchzeri individuals in three populations in the Swiss Alps that were separated from one another by at least 60 km: Fondei (canton of Graubünden: 1950 m a.s.l.; N = 20 individuals), Niessen (canton of Bern: 1680 m a.s.l.; N = 20), and Furka (canton of Uri: 2420 m a.s.l.; N = 20). We also sampled leaf material from two lowland populations of C. rotundifolia in Switzerland for cross-amplification: Blauen (canton Basel-Land: 620 m a.s.l.; N = 5) and Bonaduz (canton Graubünden: 660 m a.s.l.; N = 5). Leaf samples were silica-dried, and reference samples (Appendix 1) were stored in the Botanical Institute, University of Basel, Switzerland. Extracted DNA was sent to Ecogenics GmbH molecular marker services (Zurich-Schlieren, Switzerland) to develop microsatellite markers. In brief, Ecogenics used a traditional approach with genomic library enrichment, M13-tailing of the forward primers (Schuelke, 2000; see Table 1), and fluorophore labeling of the M13 primer. ECO500 was used as a size standard in the electropherograms. Additional technical information is described in detail in Kesselring et al. (2013) and Hamann et al. (2014). Ecogenics used a standard PCR program for all loci, with 15-min denaturation at 95°C and PCR start at 95°C for 30 s, 56°C for 45 s, and 72°C for 45 s in 30 cycles followed by eight cycles of 95°C for 30 s, 53°C for 45 s, and 72°C for 45 s. Termination was set to 72°C for 30 min (Kesselring et al., 2013). Each locus was analyzed separately. The library was enriched for tetranucleotide motifs (Table 1). This strategy likely assists in allele scoring, because a maximum of four different allelic peaks in an individual may be stretched over a wide range of base pairs. Ecogenics also performed the allele scoring, which was conducted twice independently. Ten out of 15 polymorphic microsatellite loci were randomly chosen and gave clearly readable electropherograms (Table 1). We used a conservative approach of binning of 1-bp differences due to potential stuttering (Table 1). Sample replicates were processed from the point of DNA extraction for 10% (N = 6) of the C. scheuchzeri samples, and gave identical allele signals to the first run. The polished allelic data of C. scheuchzeri were written in a two-digit code for each allele to calculate average expected heterozygosity (H_e) in the ATETRA software package (version 1.2.a; Van Puyvelde et al., 2010). One thousand Monte Carlo simulations were performed to calculate H_e for each locus in each of the three populations of C. scheuchzeri (Table 2).

Table 1.

Newly developed microsatellite markers in Campanula scheuchzeri.^a

All 10 primer pairs cross-amplified in C. rotundifolia without any PCR dropouts (Table 3), and the allelic range in both species is quite similar. For example, locus Scheuch1 shows alleles between 123 bp and 143 bp in C. scheuchzeri, and between 123 bp and 139 bp in C. rotundifolia (Tables 1, 3). In C. scheuchzeri, between five and 22 alleles were found per locus and population (Table 2). The number of alleles per population in C. rotundifolia was lower (2–9; Table 3), but this is probably a consequence of the lower sample size (N = 20 in C. scheuchzeri, N = 10 in C. rotundifolia). Despite the similarities, some alleles were only found in the 10 individuals of C. rotundifolia (Table 3). The frequency of these C. rotundifolia signals was particularly high (59%) at locus Scheuch7 (Table 3).

Table 2.

Population genetic parameters for three tetraploid populations of Campanula scheuchzeri from the Swiss Alps.

We confirm the tetrapolyploid nature of the study populations of C. scheuchzeri, as up to 60% of all individuals possessed four allelic peaks (locus Scheuch5 and Scheuch7 in the Furka population; Table 2). H_e was high in each locus, ranging from 0.67 to 0.90. Interestingly, we also observed high homozygosity values in some populations (Table 2). High HO₁ values between 0.35 and 0.50 were found in the Fondei population (loci Scheuch3 and Scheuch4) and in the Furka population (locus Scheuch9). We consider four possible explanations for the high HO₁ values: (1) null alleles are present at these loci; (2) increased homozygosity is due to selection of an unknown, linked locus; (3) the observed homozygosity results from autonomous self-fertilization, a scenario that was found in tetraploid individuals of C. rotundifolia (Stevens et al., 2012); or (4) half-sib mating occurred in these populations. Explanations 3 and 4, however, would also have led to higher than the observed HO₁ values at other loci (cf. Table 2). Consequently, we tentatively support either the null-allele scenario or the possibility of selection.

Table 3.

Cross-amplification of 10 microsatellite loci from Campanula scheuchzeri in 10 individuals of two populations of C. rotundifolia.^a

In C. rotundifolia, we also found signals for tetraploidy (see HE₄ in Table 3), which is common in Europe (Böcher, 1936; Hess et al., 1980; Stevens et al., 2012). Notably, Frei (2007), who scored populations of C. rotundifolia from several locations in Switzerland using flow cytometry, observed only tetraploid populations, although some authors reported other ploidy levels (Hess et al., 1980). In the current study, different combinations of one, two, three, or four allelic signals were found in the 10 selected individuals (Table 3).

CONCLUSIONS

Newly developed microsatellite markers confirmed that our focal populations of C. scheuchzeri are tetraploid. All 10 primer pairs cross-amplified in specimens of the widespread sister-species C. rotundifolia. The allelic divergence of C. scheuchzeri and C. rotundifolia at locus Scheuch7 has several possible explanations at this time including genetic drift due to isolation in space and time between the two taxa. However, artificial cross-fertilization was successful in both species, making genetic isolation a weak argument for the observed allelic divergence (Stevens et al., 2012). A clear genetic delimitation of the two species is probably not possible, and at intermediate elevations assigning populations to species may prove difficult due to overlapping variability. Nevertheless, our working hypothesis is therefore that both nominal species evolved under vicariance.