Calanus glacialis occurs in seas bordering the Arctic Ocean, where it is one of the key zooplankton components. It plays an important role in marine ecosystems, linking ice algae and phytoplankton with other zooplankton species, polar cod and seabirds (Falk-Petersen et al. 2007). Recent environmental changes in the Arctic, such as increasing water temperature and loss of sea ice, affect this species. Its presence and biomass decreases in the Nordic Seas above a critical threshold around 6 °C (Carstensen et al. 2012). At the same time, increasing numbers of its sibling Calanus finmarchicus are observed (Weydmann et al. 2014). The reproduction of these copepods, which need energy from the ice-algal bloom to maximize egg production, may also be affected by the warming and loss of sea-ice (Søreide et al. 2010).

The purpose of this study was to describe novel, variable genetic markers to aid in studies of C. glacialis. These markers are needed for population genetic studies and for species diagnostics, as a contribution to understand the future of Calanus in a warmer Arctic scenario. We also tested the potential for cross-amplification of all the developed microsatellites on the Atlantic C. finmarchicus.

Total RNA was isolated using the RNeasy Kit (Qiagen) from eight C. glacialis copepodites collected from the Barents Sea in June 2009. cDNA was synthesized with the SMARTer™ PCR cDNA synthesis kit (Clontech) and approximately 4 µg of normalized cDNA were sequenced using 454-pyrosequencing technology (Biocant, Cantanhede, Portugal). Cleaned reads were assembled using MIRA v3.0.3. Putative simple sequence repeats (SSRs) were identified using MSATCOMMANDER v0.8.2. Twenty-five primer pairs were designed with Primer3 and further genotyping was conducted on 25 C. glacialis and 24 C. finmarchicus individuals collected in April 2008 from Rijpfjorden (Svalbard), using fluorescent-dye labeled primers. PCR reactions (15 µl) contained ±20 ng of DNA, 0.1 µM of each primer (Table 1), 0.8 mM of dNTPs, 2.0 mM of MgCl2, 3.0 µl of 5× PCR Buffer and 0.4 U of GoTaq Polymerase (Promega Madison, WI). Cycle conditions were as follows: 95 °C for 5 min, 35 cycles (95 °C, 30 s; annealing temperature—Table 1, 30 s; 72 °C, 45 s), 72 °C, 20 min (GeneAmp 9700 thermocycler, Applied Biosystems). Fragments were sized using an ABI PRISM 3130xl DNA analyzer (Applied Biosystems) and allele sizes were scored with STRAND. GENETIX 4.0.5 was used to check allelic richness and expected and observed heterozygosities. Linkage disequilibrium was tested by GENEPOP v.4.1.4 and the frequency of null alleles was estimated using MICRO-CHECKER.

Table 1 Characterization of nine microsatellite loci for Calanus glacialis and cross-amplification with its sibling species C. finmarchicus
Full size table

A total of 9 loci were selected for further studies (Table 1). Allelic richness ranged from 2 to 13 (mean = 7.3) and expected heterozygosity from 0.039 to 0.806. No significant disequilibrium was found between any pair of primers. High frequency of null alleles was likely at two of these loci (Cgla-11, Cgla-14; Table 1).

The test of cross-amplification with C. finmarchicus resulted in four amplified loci, three of which produced polymorphic products (Table 1). These loci showed some species-specific alleles and C. finmarchicus did not amplify for the remaining five of our nine microsatellite loci. Therefore, they are useful to discriminate between C. glacialis and C. finmarchicus, which are often difficult to identify based only on their morphological features. Additionally, the polymorphic locus Cgla-09, with 11 alleles and gene diversity of 0.86, may provide a useful marker for population genetic studies in C. finmarchicus, complementing the markers described in previous reports (Provan et al. 2007).