Genomic DNA was extracted from the sampled tissue from each individual following the protocol described in Martín-Torrijos et al. [39 (link)]. Mitochondrial 16S and COI genes were used as markers in this study for detecting intraspecific genetic variation in crayfish. The primers pair used to amplify the mitochondrial 16S gene, 1472 [54 (link)] and Tor12sc [55 (link)], amplified a fragment that included partial sequences of the 12S rRNA, the 16S and the val-tRNA. The primer pairs used to amplify the mitochondrial COI gene was C/N 2769 [56 (link)] and LCO1490 [57 ]. Both primers pairs were used in a single round PCR following the protocols in Matallanas et al. [38 (link)], including negative and positive controls. Three microliters of aliquots of the amplification products were analyzed to check positive amplicons in 1% agarose TAE gels stained with SBYR1Safe (Thermo Fisher Scientific, Waltham, MA, USA). The amplified products were purified using a QIAquick PCR Purification Kit (Qiagen, Germany). Double-stranded PCR products were sequenced using an automated sequencer (Macrogen, Spain). Sequences were assembled and edited using the program Geneious v6.14 8 [58 (link)], and a BLAST search was run on each sequence to confirm they belonged to WCC species.
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