Are there 16 species of brown dog ticks? Phylogenies from 60 entire mitochondrial genomes and 162 cox1 sequences reveal 16 species-level clades in the Rhipicephalus (Rhipicephalus) sanguineus group☆☆

The Rhipicephalus sanguineus group, the brown dog ticks, are cosmopolitan and doubtless the most important ticks of domestic dogs, clinically and economically. Despite four decades of taxonomic enquiry with nucleotide sequences and morphology, the taxonomy of the R. sanguineus group is confused, even chaotic. We provide 13 new mitochondrial (mt) genomes and internal transcribed spacer 2 (ITS2) sequences from nine localities in Australia, Israel and Japan. We inferred phylogenetic trees from 10 mt protein-coding genes (9,514 bp), as well as partial cox1, ITS2, 12S, and 16S rRNA genes, to resolve to common clades the >2,000 nucleotide sequences in GenBank from the R. sanguineus group. Then we applied three species delimitation protocols to 60 entire mt genomes (ca. 15,000 bp) and 162 partial cox1 sequences (472 bp): Automatic Barcode Gap discovery, Assemble Species by Automatic Partitioning, and Poisson Tree Process. We considered pairwise genetic differences and Tamura-Nei genetic distances among 60 entire mt genomes and 162 partial cox1 sequences. We found 16 species-level clades (clades A to P) that we hypothesise represent at least 16 species in the R. sanguineus group. These clades had intra-clade differences of <3.8% (entire mt genomes) and <5.1% (partial cox1) whereas the inter-clade differences were >7.7% (entire mt genomes) and >4.5% (partial cox1). We assigned the species names Rhipicephalus linnaei (Audouin, 1826), Rhipicephalus rutilus (Koch, 1844), Rhipicephalus secundus (Feldman-Muhsam, 1952) and R. sanguineus (Latreille, 1806) to clades A, C, D, and K, respectively. And we hypothesise that the names Rhipicephalus camicasi (Morel, Mouchet & Rodhain, 1976), Rhipicephalus turanicus (Pomerantsev, 1940), Rhipicephalus guilhoni (Morel & Vassilades, 1963), Rhipicephalus sulcatus (Neumann, 1908), Rhipicephalus rossicus (Yakimov & Kol-Yakimova, 1911), Rhipicephalus pumilio (Schulze, 1935) and Rhipicephalus pusillus (Gil Collado, 1936) apply to clades B, E, H, J, M, N and O, respectively. The newly described Rhipicephalus hibericus (Millán, Rodriguez-Pastor & Estrada-Peña, 2024) was genetically indistinguishable from R. sanguineus in clade K and thus is a synonym of R. sanguineus. We could not assign names to clades F (USA, Hungary), I (India, Pakistan), L (Nigeria), G (China, Kazakhstan), and P (Cameroon): some or all of these five clades may be new species in the R. sanguineus group. Our haplotype network of partial mt genes (cox1, cytb and nad2) revealed much genetic similarity among geographically distant populations of R. linnaei. This indicates recent dispersal, likely originating in Africa or the Middle East, since African populations were more genetically diverse than populations in other parts of the world.