RESULTS: The phylogenetic inference revealed five highly divergent clades (genetic distances among clades: 4.4-13.9%) that are morphologically indistinguishable, supporting the assumption that this presumed nominal species may represent a cryptic species complex. The species group may have originated in the humid subtropical plains of Nepal or in southern adjacent regions in the Early Miocene. The major cladogenetic events leading to the fives clades occurred successively from the Early Miocene to the Early Pleistocene, coinciding with major periods of monsoonal intensification associated with major regional paleogeographic events in the Miocene and repeated climate changes due to the Plio-Pleistocene climatic oscillations. Our coverage of the Indo-Australian Archipelago (IAA) highlights the presence of a single clade there. Contrary to expectations, an AMOVA did not reveal any population genetic structure among islands or along a widely recognised zoogeographical regional barrier, suggesting a recent colonisation independent of natural biogeographical constraints. Neutrality tests and mismatch distributions suggested a sudden demographic and spatial population expansion that could have occurred naturally in the Pleistocene or may possibly result of a modern colonisation triggered by anthropogenic activities.
CONCLUSIONS: Even though Indoplanorbis is the main focus of this study, our findings may also have important implications for fully understanding its role in hosting digenetic trematodes. The existence of a cryptic species complex, the historical phylogeographical patterns and the recent range expansion in the IAA provide meaningful insights to the understanding and monitoring of the parasites potential spread. It brings a substantial contribution to veterinary and public health issues.
METHODS: Forty full-length pktrap sequences from clinical isolates of Malaysia along with the reference H-strain were downloaded from published databases. Genetic diversity, polymorphism, haplotype and natural selection were determined using DnaSP 5.10 software. McDonald-Kreitman test was conducted using P. vivax and Plasmodium coatneyi as ortholog sequence in DnaSP 5.10 software. Population genetic differentiation index (FST) of parasite populations was determined using Arlequin v3.5. Phylogenetic relationships between trap ortholog genes were determined using MEGA 5.0 software.
RESULTS: Comparison of 40 full-length pktrap sequences along with the H-strain identified 74 SNPs (53 non-synonymous and 21 synonymous substitutions) resulting in 29 haplotypes. Analysis of the full-length gene showed that the nucleotide diversity was lower compared to its nearest ortholog pvtrap. Domain-wise analysis indicated that the proline/asparagine rich region had higher nucleotide diversity compared to the von Willebrand factor domain and the thrombospondin-type-1 domain. McDonald-Kreitman test identified that the ratio of the number of nonsynonymous to synonymous polymorphic sites within P. knowlesi was significantly higher than that of the number of nonsynonymous to synonymous fixed sites between P. knowlesi and P. vivax. The von Willebrand factor domain also indicated balancing selection using MK test, however, it did not give significant results when tested with P. coatneyi as an outgroup. Phylogenetic analysis of full-length genes identified three distinct sub-clusters of P. knowlesi, one originating from Peninsular Malaysia and two originating from Malaysian Borneo. High population differentiation values was observed within samples from Peninsular Malaysia and Malaysian Borneo.
CONCLUSIONS: This study is the first to report on the genetic diversity and natural selection of full-length pktrap. Low level of genetic diversity was found across the full-length gene of pktrap. Balancing selection of the von Willebrand factor domain indicated that TRAP could be a target in inducing immune response against P. knowlesi infections. However, higher number of samples would be necessary to further confirm the findings.