Analyses of mitochondrial genes reveal two sympatric but genetically divergent lineages of Rhipicephalus appendiculatus in Kenya

Abstract

Background: The ixodid tick Rhipicephalus appendiculatus transmits the apicomplexan protozoan parasite Theileria parva, which causes East coast fever (ECF), the most economically important cattle disease in eastern and southern Africa. Recent analysis of micro- and minisatellite markers showed an absence of geographical and host-associated genetic sub-structuring amongst field populations of R. appendiculatus in Kenya. To assess further the phylogenetic relationships between field and laboratory R. appendiculatus tick isolates, this study examined sequence variations at two mitochondrial genes, cytochrome c oxidase subunit I (COI) and 12S ribosomal RNA (rRNA), and the nuclear encoded ribosomal internal transcribed spacer 2 (ITS2) of the rRNA gene, respectively.

Results: The analysis of 332 COI sequences revealed 30 polymorphic sites, which defined 28 haplotypes that were separated into two distinct haplogroups (A and B). Inclusion of previously published haplotypes in our analysis revealed a high degree of phylogenetic complexity never reported before in haplogroup A. Neither haplogroup however, showed any clustering pattern related to either the geographical sampling location, the type of tick sampled (laboratory stocks vs field populations) or the mammalian host species. This finding was supported by the results obtained from the analysis of 12S rDNA sequences. Analysis of molecular variance (AMOVA) indicated that 90.8 % of the total genetic variation was explained by the two haplogroups, providing further support for their genetic divergence. These results were, however, not replicated by the nuclear transcribed ITS2 sequences likely because of recombination between the nuclear genomes maintaining a high level of genetic sequence conservation.

Conclusions: COI and 12S rDNA are better markers than ITS2 for studying intraspecific diversity. Based on these genes, two major genetic groups of R. appendiculatus that have gone through a demographic expansion exist in Kenya. The two groups show no phylogeographic structure or correlation with the type of host species from which the ticks were collected, nor to the evolutionary and breeding history of the species. The two lineages may have a wide geographic distribution range in eastern and southern Africa. The findings of this study may have implications for the spread and control of R. appendiculatus, and indirectly, on the transmission dynamics of ECF.

Keywords: 12S rRNA; COI; East coast fever; Genetic differentiation; Genetic markers; ITS2; Phylogeography; Population genetics; Ticks.

Fig. 1

Phylogenetic tree showing the relationships between the 28 Rhipicephalus appendiculatus COI haplotypes and a reference sequence from GenBank (AF132833 [RA]). The 28 haplotypes are represented by Hap 1–28. Percent bootstrap values above 75 % (1000 replications) are shown. COI sequence of R. turanicus (JQ737086) from the GenBank database and another from a Kenya tick confirmed to be Rhipicephalus evertsi were included as the outgroup

Fig. 2

Median-Joining network of 28 COI haplotypes observed in 332 Rhipicephalus appendiculatus ticks. The network was based on the polymorphic sites in the 558 bp COI gene segment. Each circle represents a haplotype and the area of the circle is proportional to the haplotype frequency. Numbers represent nucleotide position. Colours represent a group of tick populations classified on the basis of the origin of the sequences: blue, laboratory stocks; yellow, populations sampled from pastures grazed by wildlife; red, populations sampled from pastures grazed by both cattle and wildlife; green, populations sampled from cattle pastures. Median vectors are represented by “mv”

Fig. 3

Tree showing the phylogenetic relationships between the Kenyan COI haplotypes and sequences generated by Mtambo et al. [17]. Eleven sequences from GenBank were included in the analysis. Five were from eastern Zambia [accession number DQ859261 (E-ZAM1); DQ859263 (E-ZAM2); DQ859264 (E-ZAM3); DQ859265 (E-ZAM4) and DQ859266 (E-ZAM5)], one from southern Zambia [DQ859262 (S-ZAM1)], three from Rwanda [DQ901360 (RWDA1), DQ901362 (RWDA2), DQ901363 (RWDA3)], one from Comoros Island [DQ901357 (COMS)] and one from Kenya [DQ901358 (KE-Mug)]. Another R. appendiculatus sequence [AF132833 (RA)] was included in the analysis as a reference while a sequence from R. turanicus [JQ737086 (R. turan)] was used as the outgroup. Percent bootstrap values above 75 % are shown

Fig. 4

(a) shows the overall mismatch distribution pattern for the 22 R. appendiculatus populations analysed. (b) and (c) depict the distribution profiles of 10 field and 12 laboratory populations respectively. (d) and (e) shows the distribution patterns of ticks in haplogroup A and B respectively