Calculation of intra-specific genetic distance and geographical
distance among populations
For each species, the maximum intra-specific genetic divergence and the
geographical distance between localities were needed for further
analyses (see below the section on statistical tests). For measuring
genetic divergence, we first aligned all the sequences of each species
separately using MUSCLE software (Edgard 2004) as implemented in MEGA 7
(Kumar, Strecher & Tamura 2016; default values). For each separate
species alignment all pairwise genetic distances were calculated using
the Kimura 2-parameter model (K2P%; Kimura 1980) as implemented in MEGA
7 (Kumar et al . 2016); we used K2P% because this is the method
more frequently used in DNA barcoding studies (Bergsten et al .
2012; Gunay, Alten, Simsek, Aldemir & Linton 2015; Shen, Guan, Wang &
Gan et al . 2016). The maximum genetic divergence for each pair of
populations was the maximum genetic distance (K2P%) between any pair of
individuals of those two populations. Pairwise geographical distances
between populations were calculated using QGIS 2.18.9 (QGIS Development
Team 2009) and later corroborated using the cosine-haversine formula
(Robusto 1957).