Eight microsatellite loci characterised in the European blackbird, Turdus merula

Abstract

Although the European blackbird, Turdus merula, is one of the most abundant and conspicuous songbirds of the Western Palaearctic and, as such, has been subject of numerous behavioural and ecological studies, there is to date no specific, PCR-based marker system for this species, and information on the applicability of genetic markers from other species or genera is scant. Here, we report the successful amplification of eight microsatellite loci in the European blackbird. We compared levels of polymorphism between groups of individuals sampled during the breeding season at different geographic localities (Heligoland Island, North Sea and Radolfzell, south-western Germany). We found high levels of polymorphisms, which enabled us to ascertain population membership of individuals. The properties of the tested microsatellite markers make them suitable for population genetic studies as well as for kinship analyses.

Introduction

Being able to distinguish between individuals of different origin is central to most ornithological studies, but often this cannot be achieved through ringing and observation alone. The application of molecular genetic markers can help establish population connectivity (Webster et al. 2002; Wink 2006) and has surely revolutionised our view of avian mating systems (Griffith et al. 2002). However, for the majority of species, including the European blackbird, Turdus merula, it seems that the dawn of the molecular age has only just begun.

The European blackbird is one of the most abundant and conspicuous songbirds of the Western Palearctic (Cramp 1988)—and one of the most successful, judged by its ability to adjust to a wide range of environments in many parts of the world (see Kentish et al. 1995; Sol et al. 2002). Since the beginning of the twentieth century, it has rapidly extended its breeding distribution to northern and eastern Europe, colonising habitats along the entire rural–urban gradient (Luniak et al. 1990; Luniak 2004; Stephan 1999). The blackbird has been the focus of several studies concerning the mechanisms that drive behavioural and life-history changes with increasing urbanisation (e.g. Partecke et al. 2004, 2006). But information about the extent of genetic exchange between populations distributed along the rural–urban gradient is still scarce. This information, however, can be decisive in resolving whether or not different ecotypes of the blackbird represent independent genetic entities resulting from local adaptation.

Microsatellites—simple, non-coding sequence repeats within nuclear DNA—have become the genetic marker of choice for answering questions at the level of individuals and populations, mainly because of their high mutation rate and polymorphism (Webster et al. 2002; Schlötterer 2004). So far, no specific marker system has been developed for the European blackbird, and information about the applicability of genetic markers from other species or genera is scant. In this short communication, we report the successful amplification of eight microsatellite loci in the European blackbird.

Materials and methods

Genetic variability was examined in a total of 147 blackbirds from two study sites. The first was situated in the surroundings of Radolfzell (47°44′N, 08°58′E) in south-western Germany. The second was the island of Heligoland (54°12′N, 07°56′E) in the North Sea about 50 km from the German mainland.

DNA was extracted from blackbird feathers or blood samples, using a silica-based column method (Qiagen, Hilden, Germany) following the manufacturer’s protocol. PCR amplifications were performed in 10 μl reactions in a Perkin–Elmer thermal cycler. Each reaction mix contained 1 μl DNA extraction, 2.5 mM MgCl₂, 25 mM Tris–HCl (pH 8.0), 35 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.5% (v/v) Tween-20, 0.5% IGEPAL CA-630, 0.2 mM of each nucleotide, 5 pmol of each primer) and 0.5 units Taq polymerase (Eppendorf HotmasterTaq; Eppendorf, Hamburg, Germany). PCR profiles consisted of 2 min denaturation at 94°C, 35 cycles of 30 s denaturation at 94°C, 30 s annealing at the specified temperature and 30 s extension at 68°C with a final 5 min 68°C step. PCR fragments were resolved by electrophoresis on an ABI 377 sequence analyser.

Allele frequencies and estimates of within-population diversity (observed number of alleles, heterozygosity) and between-population divergence were calculated using FSTAT 2.9.3 (Goudet 1995) and CERVUS (Marshall et al. 1998).

Results and discussion

High levels of variability could be detected in the analysed loci, with the number of alleles ranging from 4 to 21 per locus (Table 1). Genetic differentiation between the two study sites was high (F _ST = 0.135). The number of alleles was lower in the island than in the mainland population, even after correcting for the considerable difference in sample size (Table 2).

Table 1 Characterisation of eight polymorphic microsatellite loci for the blackbird Turdus merula, originally isolated from other passerine species. T _a Annealing temperature, n number of individuals tested, A number of alleles, H _O observed heterozygosity, H _E expected heterozygosity

Table 2 Genetic diversity of two blackbird populations from Radolfzell and Heligoland (Germany). AR Allelic richness

Observed and expected heterozygosity differed significantly in four microsatellite loci (Cuμ 28, Mjg1, Pdoμ4, Pdoμ5). However, our samples from Radolfzell comprised a number of siblings collected from the same nests, which may explain these differences; when analysing whole families we found possible null alleles only in locus Pdoμ5.

Our preliminary analysis shows that the eight tested microsatellite loci are generally suitable for analysing genetic divergence among blackbird populations. We tested a number of other passerine loci, but these did not amplify any PCR product (Lox7, Piertney et al. 1998; Ls1, Mundy and Woodruff 1996; Phtr3, Fridolfsson et al. 1997; Gf5B, Petren 1998; Pca3, Pca9, Dawson et al. 2000) or were monomorphic (Cuμ4, Gibbs et al. 1999; Ltr7, Lillandt et al. 2002; Vecr2, Vecr8, Stenzler et al. 2004) in the blackbird. The set of eight polymorphic genetic markers could, for example, be used to estimate the rate of gene flow between migratory and sedentary blackbird populations (Sacher et al. 2006) or to determine the extent to which urban blackbird populations represent distinct and independent genetic entities (see Luniak 2004; Partecke et al. 2006). The presented marker system may also be used for pedigree analyses and paternity testing. We eagerly await the first biological results from its application.

Zusammenfassung

Acht Mikrosatelliten-Loci für die Amsel, Turdus merula

Obwohl die Amsel, Turdus merula, einer der häufigsten und auffälligsten Singvögel der westlichen Paläarktis darstellt und somit bereits Gegenstand zahlreicher, verhaltensökologischer Untersuchungen war, lag bislang für diese Art kein spezifisches, molekulargenetisches Marker-System vor, und Information über die Anwendbarkeit von an anderen Arten entwickelten Markern fehlte weitgehend. Hier berichten wir von der erfolgreichen Amplifizierung von acht variablen Mikrosatelliten-Loci bei der Amsel und vergleichen Ergebnisse zwischen Individuen, die zur Brutzeit in unterschiedlichen Gebieten beprobt wurden (Helgoland, Nordsee und Radolfzell, SW-Deutschland). Wir fanden einen hohen Polymorphismusgrad, der uns ermöglichte, die Populationszugehörigkeit von Individuen eindeutig zu bestimmen. Die getesteten Mikrosatelliten-Marker eignen sich demzufolge sowohl für populationsgenetische Studien als auch für Verwandtschaftsanalysen.