The eurybathic isopod species Chelator insignis shows a wide distribution south of Iceland. We analysed 51 specimens from shelf (213–305 m depth), slope (885–891 m and 1380–1390 m depth) and deep−sea habitats (2750 m) south of Iceland with different DNA markers. A fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) was studied for 47 specimens, 16S was studied for 36 specimens, and a fragment for the 18S rRNA gene could be amplified for 11 specimens. For the COI data, specimens clustered into five distinct lineages each separated by ³ 20% uncorrected pairwise distances. Both the mitochondrial 16S and the nuclear 18S sequence data further support this deep divergence, suggesting the presence of overlooked species inside the nominal C. insignis . Populations on the shelf occurring east and west of the Reykjanes Ridge were genetically identical suggesting that this ridge is not a barrier to gene flow. However, populations from different depth ranges differed substantially. Our multi−gene analysis suggests that the newly found species likely have more narrow vertical distribution ranges and highlights a possible role of bathymetry in speciation processes.
Field and laboratory protocols that originally led to the success of published studies have previously been only briefly laid out in the methods sections of scientific publications. For the sake of repeatability, we regard the details of the methodology that allowed broad−range DNA studies on deep−sea isopods too valuable to be neglected. Here, a com− prehensive summary of protocols for the retrieval of the samples, fixation on board research vessels, PCR amplification and cycle sequencing of altogether six loci (three mitochondrial and three nuclear) is provided. These were adapted from previous protocols and developed especially for asellote Isopoda from deep−sea samples but have been successfully used in some other peracarids as well. In total, about 2300 specimens of isopods, 100 amphipods and 300 tanaids were sequenced mainly for COI and 16S and partly for the other markers. Although we did not set up an experimental design, we were able to analyze amplification and sequencing success of different methods on 16S and compare success rates for COI and 16S. The primer pair 16S SF/SR was generally reliable and led to better results than universal primers in all studied Janiroidea, except Munnopsidae and Dendrotionidae. The widely applied universal primers for the barcoding region of COI are problematic to use in deep−sea isopods with a success rate of 45–79% varying with family. To improve this, we recommend the development of taxon−specific primers.