Genetic similarities between sympatric Ixodes ricinus and Ixodes inopinatus from Tunisia and horizontal gene transfer from their endosymbionts

Ixodes ricinus (Linnaeus, 1758) and Ixodes inopinatus (Estrada-Peña, Nava, and Petney 2014) differ morphologically, yet genetic distinction between these taxa is challenging. Because it is important to understand the biological differences between I. ricinus and I. inopinatus, further clarification of these taxa is needed as the causes of the morphological differences are unknown. Here, we aim to explain the morphological differences between I. ricinus and I. inopinatus. Our hypothesis is that if I. ricinus and I. inopinatus are two separate species or lineages, then I. inopinatus should be genetically distinct from sympatric I. ricinus. An alternative explanation for the differences between I. ricinus and I. inopinatus is alteration of tick morphology by a symbiont. Ticks were collected in Jouza-Amdoun, Northern Tunisia, and morphologically identified as either I. ricinus or I. inopinatus. DNA from six individual ticks was extracted and sequenced using the Oxford Nanopore Technologies platform. The mitochondrial genome and both alleles of three nuclear markers were used to compare I. ricinus and I. inopinatus. Additionally, the presence of tick endosymbiont presence was determined. Phylogenetic analyses were supplemented with sequences of I. ricinus derived from the Netherlands. The morphological identification confirmed the coexistence of I. ricinus and I. inopinatus in Jouza-Amdoun, Tunisia. High quality sequencing data enabled reconstruction of complete mitochondrial genomes and complete separation of both alleles of the TRPA1, TROSPA and calreticulin genes for all six ticks. Additionally, we report the first description of complete bacterial genomes for Candidatus Midichloria mitochondrii, Rickettsia helvetica and R. monacensis from Northern Africa. Surprisingly, we observed stable integration of parts of the Spiroplasma ixodetis and M. mitochondrii genomes into the nuclear genome of these ticks. Phylogenetic analyses of the complete mitochondrial genome, nuclear markers, endosymbionts M. midichondrii, R. helvetica and R. monacensis showed genetic distinction between Tunisian and Dutch ticks, but high genetic similarity between Tunisian I. ricinus and I. inopinatus. Our results provide strong evidence that I. ricinus and I. inopinatus are genetically similar despite morphological differences. There was no correlation between presence of endosymbionts and morphological differences. Our findings highlight next generation sequencing as a powerful tool for research on ticks and tick-borne pathogens.