Molecular characterisation of Amblyomma integrum circulating in southern India.

Amblyomma integrum is a large gooseberry sized longirostrate tick (when fully repleted) found in India and Sri Lanka. In Kerala (India), this tick is commonly found in the forest and its fringe areas frequently infesting deer and hence it is locally known as "maan chellu / maanunny" (deer tick). In the present study, molecular characterisation and phylogenetic analysis of A. integrum collected from the area grazed by the sambar deer (Rusa unicolor) of Kerala, south India was performed using three molecular markers viz., the mitochondrial cytochrome c oxidase subunit 1 (COI), mitochondrial 16S ribosomal RNA, and nuclear 18S ribosomal RNA genes. Cytochrome c oxidase subunit 1 (COI) gene showed better resolving ability for elucidating the evolutionary relationship of A. integrum and identified two distinct clades, viz., A and B. The Tamil Nadu isolates of south India and Marayoor isolate 1 (from Idukki district of Kerala bordering with Tamil Nadu) belonged to clade A. Majority of Wayanad isolates from Kerala, occupied clade B. The intraspecific genetic distance among the A. integrum species ranged from 0.00 to 13.34%. Between clades A and B, the genetic distance observed was 11.49%. The clade B isolates were genetically close to A. geoemydae (GD: 1.22%). Morphological variations between the clades included darker exoskeletal coloration in clade A and distinct differences in the shape of basis capitulum. Further analysis using Assemble Species by Automatic Partitioning (ASAP) and Generalized Mixed Yule Coalescent (GMYC) provided additional insights. Assemble Species by Automatic Partitioning (ASAP) identified 26 Molecular Operational Taxonomic Units (MOTUs) at a threshold distance of 5.38%, supporting the species partition of A. integrum clade B. Generalized Mixed Yule Coalescent (GMYC) analysis retained the same species complex (A. integrum-geoemydae Complex) inferred from the ASAP analyses. It could be inferred from the present study that the A. integrum clades A and B could be two different putative pseudocryptic species.