2.4 Mitochondrial gene amplification and sequencing
A fragment of the mitochondrial gene encoding for the Cytochrome oxidase
subunit I (COI ) was amplified using primers C1-J-2183 Jerry
(Simon et al., 1994) and C1-N-2568 BEN3R (Brady, Gadau, & Ward, 2000).
PCR reactions were performed following Poveda-MartÃnez et al. (2019).
PCR products were checked in 1% agarose gels and purified using the
QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Finally, both
strands of the amplicons were sequenced using Sanger methodology at
Macrogen Inc. (Seoul, South Korea). Additional COI haplotypes
detected in Poveda-MartÃnez et al., (2019) were retrieved from GenBank
(MN013440 - MN013554). All sequences were employed in phylogenetic and
population genetic analyses. New mtDNA haplotypes were deposited in
GenBank under access numbers: MT138921 - MT138931.