mtDNA genomes assembly, annotation and analyses

The obtained sequences were carefully examined by checking chromatograms for quality and double peaks following BLASTn analysis to make sure that all amplicons are the desired target sequences. DNA extracted from a single individual of each species was used to infer and annotate its mitogenome, thereby avoiding any intraspecific variation. Eucotylid mtDNA sequences were assembled using DNAstar v.7.1 software [27] and aligned against selected digenean mitogenomes and then against each other using MAFFT 7.149 [36] to determine the relative positions of genes. Boundaries of protein-coding genes (PCGs) were found by searching for open reading frame (ORF; NCBI) and checking alignment against the selected digenean mitogenomes. tRNA sequences and their secondary structure were identified using the MITOS [37] and ARWEN [38] web servers. The two ribosomal RNAs, rrnL and rrnS, were determined via a comparison with the mt genome sequences of selected trematode species and their boundaries were assumed to extend to their adjacent genes. The nucleotide identity (%), nucleotide and amino acid composition, A+T/G+C skewness, codon usage and relative synonymous codon usage (RSCU) for PCGs were determined in PhyloSuite v1.2.1 [39]. The stacked bar chart of amino acids used for the construction of mt PCGs was drawn using ggplot2 [40]. DnaSP v.6 [41] was used to conduct the sliding window analysis: window size of 200 bp and a step size of 20 bp were implemented to estimate the nucleotide divergence (Pi) among PCGs, rRNAs and tRNAs of the three eucotylid mitogenomes. Kimura-2-parameter (K2P) genetic distances of the mt PCGs (substitution included = transitions + transversions) were also calculated using MEGA X [30]. To detect tandem repeats of nucleotides in the two non-coding regions of the complete mt genome of T. zarudnyi, we used Tandem Repeat Finder [42] and mreps [43] software. The circular diagram of the mt genomes was drawn with MTVIZ, an online tool of mitochondrial visualization (available at: (http://pacosy.informatik.uni-leipzig.de/mtviz/).