High-throughput sequencing data analysis
Mt-genome assembly was conducted with the Geneious 11.1.3 package
(https://www.geneious.com/). First, demulitplexed raw sequence reads
were trimmed and quality-filtered with BBDuk 37.64 of the BBTools
package (https://jgi.doe.gov/data-and-tools/bbtools/) and duplicate
reads were removed with Dedupe 37.64 (BBTools package); for both steps
we applied standard settings. For mt-genome assembly, cleaned reads were
mapped onto the reference mt-genome of L. europaeus (GenBank:
NC_004028.1) using the Geneious assembler with standard settings. Newly
produced mt-genomes were manually checked and then annotated with
Geneious. For phylogenetic tree reconstruction, we added additional
mt-genome sequences from EBHs from Sweden, Poland, Greece, Cyprus and
Turkey available in GenBank and aligned them as described above. Tree
reconstructions based on the maximum-likelihood algorithm and Bayesian
inference were performed as described above using IQ-TREE and MrBayes
software.