DNA barcoding reveals novel biodiversity in ticks (Acari: Ixodida: Ixodidae) of Ethiopia

Identification of hard ticks (Acari: Ixodida: Ixodidae) is problematic due to inconsistent diagnostic characters, sexual dimorphism, individual variation, and vast morphological differences among life stages, as well as distortion through blood feeding. DNA barcoding has proven useful in delineating tick species, but available genetic databases are littered with misleading mis-identifications, hindering tick-borne disease mitigation strategies. Efforts are needed to generate high quality reference sequences from expertly identified and morphologically vouchered samples. To date, 56 species of hard ticks are reported from Ethiopia. Here, we subsampled and e-vouchered exemplars of 22 expert-validated hard tick species from 25,343 total ticks collected on a range of livestock from five regions across Ethiopia. We successfully sequenced the barcoding region of the mitochondrial cytochrome c oxidase subunit I (COI) for 208 individual ticks, representing 21 of these target taxa (The COI gene of one tick species, Haemaphysalis parmata, was not able to be amplified). Our data suggest the presence of 29 species-level groupings among the 21 morphologically identified taxa successfully sequenced, an increase of 38%, reiterating the gaps and challenges of accurate tick identification in Ethiopia, as well as the greater East Africa region. Here we report novel hidden diversity in Amblyomma variegatum, Hyalomma truncatum, Rhipicephalus lunulatus, and Rh. praetextatus in Ethiopia, and provide the first expertly-verified mtDNA COI barcodes for Hae. aciculifer and Rh. bergeoni. Molecular confirmation of the presence of Hy. marginatum in Ethiopia is documented for the first time. Dorsal and ventral photographic vouchers are provided for examples of all known and newly discovered COI barcodes sequenced in this study, enhancing accuracy in future taxonomic and ecologic studies of ticks in Ethiopia and across East Africa. This validated barcoding resource improves tick-borne disease surveillance across East Africa, and provides a framework for broader vector-borne disease biosurveillance efforts.