Laboratory procedures

Total genomic DNA was extracted from tube feet and spine muscles using the DNeasy® Blood and Tissue Kit (QIAGEN) following the manufacturer’s instructions. PCR amplifications with the TopTaq DNA Polymerase (QIAGEN) were conducted using 1 μl of extracted genomic DNA (approximately 10-15 ng). Reaction conditions using primers CR15fwd and CR08rev were 3 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 57 °C and 60 s at 72 °C, ending with a final extension step of 10 min at 72 °C. PCR products were visualized on a 1.5% agarose gel, purified using ExoSAP-IT (Affymetrix) and sent for sequencing to Microsynth GmbH (Vienna, Austria) using the PCR primers. In cases of weak amplifications (amplicons of the expected size showing only as faint bands on an agarose gel), target selected re-amplifications were performed following the methods of Bjourson and Cooper [49] using the same primers and reaction conditions as above. For this purpose, the respective PCR fragments were visualised on an agarose gel under UV light and templates for the re-amplification were transferred from the gel to the new PCR tubes using a sterile needle.

Despite yielding a product at the expected length, all amplicons failed to sequence through using the external PCR primers, with the sequencing signal dropping ca. half way through the expected amplicon length (see results for details). Consequently, the internal primers CR_int_fwd and CR_int_rev were used to generate a two-way sequencing of each amplicon (before and after the break-off). The sequences determined in the present study were deposited in GenBank under the accession numbers: {"type":"entrez-nucleotide-range","attrs":{"text":"MG198122-MG198170","start_term":"MG198122","end_term":"MG198170","start_term_id":"1386678519","end_term_id":"1386678567"}}MG198122-MG198170.