Mitochondrial and Nuclear DNA Analyses of Rhipicephalus microplus from Mizoram, Northeast India: Insights into Genetic Diversity and Endosymbiont

Abstract

Background/Objectives: In this study, we conducted molecular identification of R.microplus and explored the genetic diversity of R. microplus for the first time in Mizoram, a Northeastern Hill (NEH) state of India bordering Myanmar. Methods: To assess genetic variation and evolutionary relationships, we employed phylogenetic analyses, genetic divergence metrics, and haplotype network construction based on mitochondrial (COX1 and 16S rDNA) and nuclear (ITS-2 and 18S rDNA) markers. Additionally, multivariate Principal Coordinate Analysis (PCoA) was used to visualize genetic differentiation among R. microplus populations. Results: Our analyses indicated that populations of R. microplus sensu lato from India, Bangladesh, and Pakistan form a closely related matrilineal lineage distinct from R. microplus sensu stricto, clustering within clade C of the COX1-based phylogeny. Globally, 24 COX1 haplotypes were recovered, with 1 haplotype identified in India. The Mizoram population exhibited a single 16S rDNA haplotype; however, intraspecific divergence was evident across India, with seven matrilineal haplotypes detected and nineteen globally. Further, five haplotypes were identified within R. microplus using the ITS-2 marker, while five haplotypes were observed within the Rhipicephalus genus using the 18S rDNA marker. Moreover, this study revealed the presence of Coxiella-like endosymbionts in 95% of the tick specimens analyzed. Conclusions: This study fills a critical knowledge gap by providing the first molecular documentation of tick diversity in Mizoram, a strategic region along the Indo-Myanmar border, and offers novel insights into the phylogeography and symbiotic associations of R. microplus and related tick taxa.

Keywords: 16S rDNA; 18S rDNA; COX1; ITS-2; Northeast Hills of India; Rhipicephalus microplus; phylogeny.

Figure 1

Digital elevation map showing the sample locations in Mizoram, India: 1. Sihphir, 2. Durtlang, 3. Muthi, 4. Govt. Complex, 5. Bilkhawthlir, 6. Kolasib, 7. Bairabi, 8. Hnahlan, 9. Champhai, 10. Samthang.

Figure 2

(A) Bayesian inference (BI) phylogenetic tree of the mitochondrial COX1 marker in assigning R. microplus as the ingroup while keeping H. doenitzi and Hyalomma anatolicum as outgroups. The posterior probability (PP) support values from the BI tree are given at each branch, and the ultrafast bootstrap (UFB) support for the corresponding branch from the maximum likelihood (ML) inference tree is also given beside the PP values as PP/UFB. (B) Ordination of standardized p-distance (COX1) among the ingroup and outgroup taxa along the principal coordinate (PCo) axes where a total of 61.2% and 29.6% of the variance are captured by PCo1 and PCo2, respectively.

Figure 3

(A) Bayesian inference (BI) phylogenetic tree of the mitochondrial 16S rDNA marker in ixodid ticks with assigning R. microplus and R. annulatus as ingroups while keeping H. bispinosa, Dermacentor and other Rhipicephalus species as outgroups. The posterior probability (PP) support values from the BI tree are given at each branch, and the ultrafast bootstrap (UFB) support for the corresponding branch from the maximum likelihood (ML) inference tree is also given beside the PP values as PP/UFB. The unsupported branching from the ML tree is denoted as dash (PP/–). (B) Ordination of standardized p-distance (16S rDNA) among the ingroup and outgroup taxa along the first and second principal coordinate (PCo) axes, where a total of 70% and 21% of the variance are captured by PCo1 and PCo2, respectively. (C) Ordination of standardized p-distance among the ingroup taxa along the first and second principal coordinate (PCo) axes, where a total of 59% and 29% of the variance are captured by PCo1 and PCo2, respectively.

Figure 4

(A) Ordination of standardized p-distance (ITS-2) among the ingroup and outgroup taxa along the first and second principal coordinate (PCo) axes where a total of 98% and 2% of the variance are captured by PCo1 and PCo2, respectively. (B) Bayesian inference (BI) phylogenetic tree of the nuclear ITS-2 marker in ixodid ticks with Rhipicephalus species assigned as ingroups while keeping Haemophysalis bispinosa as an outgroup taxon. The posterior probability (PP) support values from the BI tree are given at each branch, and the ultrafast bootstrap (UFB) support for the corresponding branch from the maximum likelihood inference tree is also given beside the PP values as PP/UFB.

Figure 5

(A) Bayesian inference (BI) phylogenetic tree of the nuclear 18S rDNA marker in ixodid ticks with the genera Rhipicephalus, Hyalomma, Amblyomma, and Dermacentor assigned as ingroups while keeping A. sphenodonti and the genera Haemophysalis, Ixodes, Argas, Otobius, Ornithodorus, and Demodex as outgroups. The posterior probability (PP) support values from the BI tree are given at each branch, and the ultrafast bootstrap (UFB) support for the corresponding branch from the maximum likelihood inference tree is also given beside the PP values as PP/UFB. (B) Ordination of standardized p-distance (18S rDNA) among the ingroup and outgroup taxa along the first and second principal coordinate (PCo) axes, where a total of 86% and 9% of the variance are captured by PCo1 and PCo2, respectively. (C) Ordination of standardized p-distance among the ingroup taxa along the first and second principal coordinate (PCo) axes, where a total of 87% and 6% of the variance are captured by PCo1 and PCo2, respectively.

Figure 6

Median-joining haplotype networks based on the mitochondrial COX1 and 16S rDNA. Numbers at the branch represent mutational steps found between haplotypes, and black dots at the branch are either inferred missing or unsampled steps. The different color codes denote the different countries where the samples originate from.

Figure 7

Median-joining haplotype networks based on the nuclear 18S rDNA and ITS-2 markers in ixodid ticks. Numbers at the branch represent mutational steps found between haplotypes, and black dots at the branches are either inferred missing or unsampled steps. The different color codes denote the different countries where the samples originate from.

Figure 8

Bayesian inference (BI) phylogenetic tree of the mitochondrial 16S rDNA marker in Coxiella with Legionella pneumophila assigned as the outgroup. The posterior probability (PP) support values from the BI tree are given at each branch, and the ultrafast bootstrap (UFB) support for the corresponding branch from the maximum likelihood (ML) inference tree is also given beside the PP values as PP/UFB. The unsupported branching from the ML tree is denoted as dash (PP/–). The color shading corresponds to the sequences generated in this study.