3.1 Patterns of genetic diversity, numts and the presence of a
duplicated region
We obtained an average of 192 sequences per marker (length range
307-1323 bp; Table 1). Mitochondrial data produced 148 polymorphic sites
yielding 150 haplotypes, while nuclear data exhibited a total of 111
variable sites and 150 alleles (see Table 1 for summary of polymorphic
sites, haplotypes and diversities per marker). Mitochondrial markers
were twice as variable as nuclear markers, though nuclear haplotype and
nucleotide diversity in βfib reached a level similar to
mitochondrial haplotype diversity (Table 1).
None of the coding markers (cox1 , cytb and pax )
presented any insertion, deletion, nonsense or stop-codon following
translation (see Supplementary Material 1). Double peaks on Sanger
chromatograms were however detected for each of the three mtDNA markers.
While all double peaks at cox1 were removed by the exonuclease
treatment, 60 CR sequences (33%) still showed double-peaks at 73
positions, as well as 37 positions for 22 individuals (10%) forcytb . Double-peaks were not specific to any population or sex,
and were not linked to the position of the individuals in the sequencing
plate (see Supplementary Material 3). Only 12 (5%) individuals showed
double-peaks both at CR and cytb , so the presence of double peaks
seemed unlinked between the two markers. Replicating DNA extractions,
PCR and sequencing confirmed these results, making laboratory
contamination unlikely. Contamination in the field was also unlikely
since new sampling supplies were used for every sample. Given that only
10% of the cytb sequences presented such ambiguities (which may
be due to heteroplasmy, Torres et al 2018), we removed such sequences
for further analyses expecting little impact on the analyses. However,
for the CR, since a third of the total sequences were involved, we kept
all CR data in further analyses, considering two haplotypic phases for
MrBayes and *BEAST analyses (although this is a violation of the
assumption that mixed sequences to be phased are under Hardy-Weinberg
equilibrium; see discussion below).