Introduction

The beetle Cucujus cinnaberinus (Scopoli 1763) lives primarily in recently dead, broad-leaved trees in eastern and northern parts of Europe and has status as near threatened at the IUCN redlist (Nieto et al. 2010). The major threat is degradation and loss of habitat, resulting in fragmentation and isolation of populations. In Scandinavia the species is primarily connected to aspen (Populus tremula L.), which occurs scattered in the managed boreal forest (Sverdrup-Thygeson 2008). Given the ephemeral and in many areas scattered nature of the habitat, resource competition may be strong. It is possible that vital resources might be monopolized by the first female to colonize a dead trunk, which would decrease the availability of suitable habitats further. Despite the species’ need for conservation, nothing is known about its genetic structure.

Twenty-six larva or pupa from nine trees were sampled in Pardubice and Jihomoravsky Regions in Czech Republic and 45 larva or pupa from twelve trees were sampled in Telemark and Aust-Agder county in South Norway. Number of individuals from each tree varied from one to eleven. Genomic DNA was extracted using DNeasy™. Tissue Kit (Qiagen). The DNA from six individual samples from each country were pooled and sent to GenoScreen, Lille, France (www.genoscreen.fr). One μg DNA was used for the development of microsatellites libraries through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries as described in Malausa et al. (2011).

Primers were designed for thirteen of the microsatellite motives and tested for amplification and polymorphism using DNA from seven C. cinnaberius from each of the Czech Republic and Norway. Forward primers were labeled with fluorescent, and microsatellite loci were amplified on a GeneAmp PCR System 9700 (Applied Biosystems) in 10 μl reaction mixtures containing 20–40 ng DNA, 3 pmol of each primer, 1× Key buffer (VWR), 0.2 mM dNTP and 0.5 U Taq DNA polymerase (VWR). Thermocycling parameters after denaturation at 95 °C in 2 min were 30 cycles of 95 °C for 30 s, annealing temperature 55 °C for 30 s and 72 °C for 45 s followed by 10 min at 72 °C. The PCR products were electrophoresed using an ABI 3100 sequencer (Applied Biosystems).

Of the thirteen primer sets, ten gave satisfactory amplifications and showed polymorphism, while one did not amplify and two were monomorphic. Linkage disequilibrium and deviations from Hardy–Weinberg equilibrium within countries together with testing for genetic differentiation between countries were determined using Genepop version 4.0.7 (Rousset 2008). We used MICKROCHECKER version 2.2.3 (Van Oosterhout et al. 2004) to test for the presence of null alleles.

All loci were polymorphic in both the Norwegian and Czech samples with number of alleles per locus ranging from two to eleven (Table 1). No loci deviated from Hardy–Weinberg equilibrium after Bonferroni correction (α = 0.05), neither was there any significant linkage disequilibrium among paired loci after correction for multiple tests. Null alleles were not detected. Exact test of genetic differentiation was highly significant between the two samples (P < 0.0001) with significant difference in nine of the ten markers used. In eight out of thirteen trees with more than two beetles sampled, the genotypes in minimum two loci gave evidence that they descended from more than one mating pair. The reason for this was presence of more than four alleles, or more than three alleles together with a homozygote, or more than two alleles together with two different homozygotes. Thus, there is no indication of one female monopolizing a resource with a large number of offspring. These ten microsatellite loci should prove useful in further studies of population structure including mating and dispersal pattern which are important for estimates of efficient population sizes and for making efficient protection plans for this threatened species.

Table 1 Characterization of microsatellites in C. cinnaberius
Full size table