Molecular characterisation of the tick Rhipicephalus microplus in Malaysia: new insights into the cryptic diversity and distinct genetic assemblages throughout the world

Background The morphotaxonomy of Rhipicephalus microplus complex has been challenged in the last few years and prompted many biologists to adopt a DNA-based method for distinguishing the members of this group. In the present study, we used a mitochondrial DNA analysis to characterise the genetic assemblages, population structure and dispersal pattern of R. microplus from Southeast Asia, the region where the species originated. Methods A phylogeographic analysis inferred from the 16S rRNA and cytochrome oxidase subunit I (COI) genes was performed with five populations of R. microplus collected from cattle in Malaysia. Malaysian R. microplus sequences were compared with existing COI and 16S rRNA haplotypes reported globally in NCBI GenBank. Results A total of seven and 12 unique haplotypes were recovered by the 16S rRNA and COI genes, respectively. The concatenated sequences of both 16S rRNA and COI revealed 18 haplotypes. Haplotype network and phylogenetic analyses based on COI+16S rRNA sequences revealed four genetically divergent groups among Malaysian R. microplus. The significantly low genetic differentiation and high gene flow among Malaysian R. microplus populations supports the occurrence of genetic admixture. In a broader context, the 16S rRNA phylogenetic tree assigned all isolates of Malaysian R. microplus into the previously described African/the Americas assemblage. However, the COI phylogenetic tree provides higher resolution of R. microplus with the identification of three main assemblages: clade A sensu Burger et al. (2014) comprises ticks from Southeast Asia, the Americas and China; clade B sensu Burger et al. (2014) is restricted to ticks that originated from China; and clade C sensu Low et al. (2015) is a new genetic assemblage discovered in this study comprising ticks from India and Malaysia. Conclusions We conclude that the R. microplus complex consisting of at least five taxa: R. australis, R. annulatus, R. microplus clade A sensu Burger et al. (2014), R. microplus clade B sensu Burger et al. (2014) and the new taxon, R. microplus clade C sensu Low et al. (2015). The use of COI as the standard genetic marker in discerning the genetic assemblages of R. microplus from a broad range of biogeographical regions is proposed. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-0956-5) contains supplementary material, which is available to authorized users.

To date, the ticks of the R. microplus complex consisting of four taxa, namely R. australis, R. annulatus, R. microplus clade A sensu Burger et al. (2014) and R. microplus clade B sensu Burger et al. (2014). Nonetheless, the morphotaxonomy of R. microplus complex has been challenged in the last few years and remained difficult to morphologically differentiate these members [9]. Molecular characterisation is the alternate way to distinguish these closely related taxa, as well as other Rhipicephalus ticks [10].
The phylogeography of R. microplus has been wellstudied in many parts of the world using various molecular approaches, such as random amplified polymorphic DNA [11], restriction fragment length polymorphism [12] and microsatellite analyses [13,14]. In 2009, Labruna et al. [15] used both mitochondrial genes (12S rRNA and 16S rRNA) and microsatellite markers to charactesize the genetic assemblages of R. microplus from Australia, Asia, Africa and the Americas. The results showed three distinct cladesone comprising ticks from the Americas and Africa; another comprising ticks from Australia, Indonesia and New Caledonia; and one comprising ticks from India and Nepal. As a result, R. microplus ticks from Australia, Indonesia and New Caledonia have been reinstated as R. australis based on reproductive isolation and taxonomic evidence [16]. A more recent study discovered a new mitochondrial COI gene lineage that is restricted to ticks from China, suggesting the presence of cryptic species that is more closely related to R. annulatus [10]. Thus far, the current literature has documented two distinct mitochondrial COI gene assemblages of R. microplus, so called clade A sensu Burger et al. There is a gap in knowledge concerning the genetic assemblages and population structure of R. microplus from Southeast Asia. Previous studies have genetically analysed ticks from Australia, the Americas and Africa but sampling was limited, with most countries represented by a single individual. Small sample size provides little support for intraspecific genetic diversity and phylogenetic inferences [17]. Therefore, further studies on the population structure of Southeast Asian R. microplus, including those of Malaysia, are warranted. Given the high resolution of mitochondria-encoded 16S rRNA and COI genes reported in Rhipicephalus ticks [10,15,18], this study attempts to utilize these genes to reveal the hidden intraspecific genetic diversity and dispersal patterns of R. microplus for the first time in Malaysia. To infer the genetic assemblages of Malaysian R. microplus, sequences were compared with existing COI and 16S rRNA haplotypes reported globally in NCBI GenBank.

Ethics statement
All experiments were performed in accordance with relevant guidelines and regulations of the University of Malaya. The research protocols were regulated and approved by the University of Malaya. Prior to the commencement of the sample collection, permission was approved by the Department of Veterinary Services, Ministry of Agriculture and Agro-Based Industry, Malaysia (Reference Number: JPV/PSTT/100-8/1). This study did not involve endangered or protected species.

Tick specimens
Tick collection was conducted on 10 animal farms (each farm with 30-40 individual animals) in seven states in Peninsular Malaysia, namely Kelantan (Farm A, Tanah Merah district), Terengganu (Farm B, Hulu Terengganu district), Pahang (Farm C, Kuantan district and Farm D, Jerantut district), Johore (Farm E, Batu Pahat district), Negeri Sembilan (Farm F, Jelebu district and Farm G, Gemas district), Selangor (Farm H, Serdang district and Farm I, Kuala Langat district) and Kedah (Farm J, Pokok Sena district) (Fig. 1). Cattle were raised at Farm A -Farm E and Farm G -Farm I, whereas sheep, goats and swine were raised at Farm J, Farm F and Farm I, respectively.
Rhipicephalus microplus was preliminary identified and separated from the closely related species R. annulatus by a set of morphological features: (1) short and deeply concave internal margin of the first palp article, (2) presence of a small spur on the second coxa of females, (3) presence of a caudal appendage in males [19]. To differentiate R. microplus from R. australis, the critical morphological features were: (1) dorsal setae are short and slender, and medial alloscutal setae form clusters of 2-3 rows in females, (2) absence of a spur on the ventral surface of first palp article of males, (3) presence of several setae on the lateral margins of the ventral surface of the capitulum in males [16]. The species identity was further confirmed by the 16S rRNA marker which can genetically separate the closely related species R. annulatus and R. australis [10,15,16].
Rhipicephalus microplus ticks were identified from cattle on five farms (Farms B, C, D, G and H) with low to high frequencies (personal observations). The cattle ticks were collected from three imported cattle breeds: Brahman (n = 3), Friesian Sahiwal (n = 4) and Nellore (n = 17); and three local cross breeds of cattle: Kedah Kelantan (n = 27), Mafriwal (n = 1) and Yellow Kedah Kelantan (n = 13). All R. microplus collected at Farm B (n = 13), Farm D (n = 14) and Farm G (n = 6) were used in DNA analyses, whereas randomly selected individuals from a larger sample from Farm C (n = 33, of 106) and Farm H (n = 30, of 249) were analysed. A total of 96 individual ticks from five farms were sequenced and analysed in this study ( Table 1). The voucher specimens were deposited at the Museum of Zoology, University of Malaya, Malaysia.

Polymerase chain reaction (PCR)
Rhipicephalus microplus DNA was extracted from each specimen (n = 96), using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The amplifications of the mitochondrial COI and 16S rRNA genes were performed in a final volume of 50 μL containing 50-100 ng genomic DNA, 25 μL of ExPrime Taq Master Mix (GENETBIO Inc., Daejeon, South Korea) and 10 pmol of each forward and reverse primer. PCR was conducted with an Applied Biosystems Veriti 96-Well Thermal Cycler (Applied Biosystems, Inc., Foster City, CA, USA).
Low amplification success rates (<50 %) were found from three pairs of COI primers [20][21][22], a common problem in the recovery of COI fragment from tick specimens [23]. To solve this issue, the COI of R. microplus was amplified using nested PCR in the present study. The first PCR was performed using the cycling parameters and primer pairs (cox1F and cox1R) from Chitimia et al. [22]. For the nested PCR amplification, 1 μL of the product from the first amplification was used with the primers C1-J-1718 and C1-N-2329 from Shao et al. [21] in a 50 μL reaction mixture. The modified PCR cycling parameters were: 94°C for 5 min, 35 cycles of 94°C for 30 s, 59°C for 1 min and 72°C for 1 min; and 72°C for 10 min. For the 16S rRNA gene, PCR amplification was performed using the primer pairs 16S-F and 16S-R1 and PCR cycling parameters described in Lv et al. [20]. A negative control was included in each PCR run.

DNA sequencing and data analyses
Purified PCR products were sent to a commercial company for DNA sequencing in forward and reverse directions. Sequence data were analysed and edited using Sequence Scanner 1.0 (Applied Biosystems, Foster City, CA, USA) and BioEdit 7.0.9.0 [24]. The partition homogeneity test was conducted using PAUP 4.0b10 [25]. No significant differences were found among separate gene regions (P = 0.88); hence data were concatenated for relevant analyses. The aligned COI and 16S rRNA sequences comprised 625 bp and 399 bp, respectively. The multiple sequences of both COI and 16S rRNA were concatenated to yield a total length of 1024 bp. Sequence alignment of R. microplus haplotypes (H1-H18) based on COI + 16S rRNA genes is shown in Additional file 1: Figure S1. Distinct haplotypes of COI (KM246866-KM246877) and 16S rRNA (KM246878-KM246884) were deposited in NCBI GenBank.
A median-joining analysis implemented in the program SplitsTree4 4.13.1 [26] was used for the intraspecific analysis of the evolutionary relationships among haplotypes. Uncorrected (p) pairwise genetic distances were calculated  [29].
The distinct COI and 16S rRNA haplotypes identified in the Malaysian R. microplus ticks were aligned with all representative sequences of Rhipicephalus taxa available in NCBI GenBank. Hyalomma detritum was used as an outgroup for the construction of phylogenetic trees based on COI and 16S rRNA sequences. A neighbor-joining (NJ) phylogenetic tree [30] was plotted using MEGA5 [31]. The NJ bootstrap values were estimated using 1000 replicates with Kimura's two-parameter model of substitution (K2P distance) [32]. Gaps and missing data were eliminated. A maximum likelihood (ML) analysis was run in PhyML 3.0 [33], using a HKY85 model with parameters estimated by the program.

Haplotype and nucleotide analyses
A total of seven, 12 and 18 unique haplotypes were identified based on the sequence variation of the 16S rRNA, COI and COI + 16S rRNA genes, respectively, from 96 ticks collected from five farms in Peninsular Malaysia. The combined COI + 16S rRNA dataset showed a greater haplotype diversity (0.5875) than COI (0.5123) or 16S rRNA (0.1787) genes alone, whereas the COI gene showed greater nucleotide diversity (0.0124) than the combined COI + 16S rRNA (0.0077) dataset or 16S rRNA (0.0005) alone (Table 1). Haplotype network analysis (COI + 16S rRNA) showed four distinct haplotype clusters among Malaysian populations but revealed a lack of clear separation by tick populations/geographical areas, indicating an overlap of the four genetically divergent groups of R. microplus. Haplotype H1 was the most widespread haplotype (n = 61) in all populations (Figs. 1 and 2). Notably, the H16, H17 and H18 haplotypes, which mainly originated from the Nellore breed, formed a single cluster (clade C), shown in Fig. 2. However, the association of these haplotypes with other cattle breeds was not obvious.

Genetic distance, genetic differentiation and gene flow
In the uncorrected "p" distance matrix, the COI gene indicated stronger resolving power (0.00-9.28 %) compared with 16S rRNA (0.00-0.50 %) ( Table 2). The genetic distance of the representative COI + 16S rRNA haplotypes of R. microplus ranged from 0.10 to 5.76 % (Table 3). Overall, significant genetic differentiation was observed among all populations (P < 0.05). However, a relatively low level of genetic differentiation was found among the five tick populations. The majority of the population pairs showed low genetic differentiation. Moderate genetic differentiation (F ST = 0.15), revealed by the COI and COI + 16S rRNA genes, was found between populations B and D. Additionally, high levels of gene flow were observed among tick populations as shown by the high Nm in the COI (36.92), 16S rRNA (12.47) and COI + 16S rRNA (35.59) genes (Table 4).

Phylogenetic analyses
NJ and ML analyses produced phylogenetic trees with the same topology but with different bootstrap support values (Figs. 2, 3 and 4). Only NJ trees were presented for the sequences of 16S rRNA and COI.
The 18 haplotypes (H1-H18) generated from the concatenated COI and 16S rRNA sequences of Malaysian R. microplus were subjected to phylogenetic analyses. Rhipicephalus microplus generally comprises four main clades: clade A (H4-H7), clade B (H14-H15), clade C (H16-H18) and clade D (H1-H2, H8-H13). There was no bootstrap support for haplotype H3, which formed a single clade (Fig. 2). Similar to the results of the haplotype network analysis, clade C is associated with ticks collected from the Nellore breed. The 16S rRNA phylogenetic tree revealed two R. microplus genetic clades. One well-supported clade comprised R. microplus from China and India and was sister to R. annulatus. Rhipicephalus microplus from China was the basal member, forming a subclade with R. microplus from India. Rhipicephalus australis (formerly identified as R. microplus) from Australia, New Caledonia and Indonesia formed a well-supported clade. Rhipicephalus microplus from Malaysia, Japan, Africa (i.e., Mozambique, South Africa and Tanzania) and the Americas (i.e., Argentina, Bolivia, Brazil, Costa Rica, Paraquay, Peru and Uruguay) formed another clade (clade B) (Fig. 3).
The COI phylogenetic tree revealed three genetic clades of R. microplus.  2014) is more closely related to R. annulatus, with which they formed a sister relationship (Fig. 4).

Discussion
In this study, we delineated the intraspecific genetic diversity and phylogeographical relationships of a major international pest of livestock, R. microplus, using 16S rRNA and COI markers. The results clearly indicate the advantages of using the COI gene for providing sufficient power to resolve the evolutionary relationships of R. microplus. We, like Burger et al. [10] found that the COI sequences were more variable and informative than 16S rRNA sequences. Exceptionally high levels of genetic variability were revealed by the COI gene. The intraspecific genetic distance or haplotype frequency reported here are notably higher than previously described for R. appendiculatus, R. sanguineus sensu lato, R. guilhoni, R. pusillus, R. turanicus, R. muhsamae and R. bursa [18,34,35]. Furthermore, the two, and perhaps three, genetic assemblages inferred from Malaysian R. microplus are more genetically diverse than those reported from other regions of the world (Fig. 4).
Among the five studied populations, R. microplus from Farm G was genetically diverse, as supported by the highest haplotype and nucleotide diversity based on COI, 16S rRNA and COI + 16S rRNA sequences ( Table 1). As one of the farm management strategies in Malaysia, Farm G acts as the 'weaner's park' and supplies weaned calves to other government farms (Farm A -Farm E and Farm G -Farm I) that are owned by the Department of Veterinary Services (DVS), Malaysia. Hence, we suggest that Farm G might be one of the sources for multiple introductions of R. microplus assemblages into other farms in Malaysia.
This study also identified distinct haplotype clusters as shown by a mixture of several genetically divergent groups in the Malaysian tick populations (Fig. 2). The significant genetic differentiation and high gene flow rates among the sampling sites support genetic admixture. The relatively low yet significant genetic differentiation and the widespread genetic admixture detected here were consistent with genetic studies of R. microplus populations from southern Texas [14]. This previous study proposed that southern Texas R. microplus has been introduced on multiple occasions, and these introductions have been associated with two main dispersal mechanisms (i.e., frequent   short-distance dispersal and rare long-distance, humanmediated dispersal), thus promoting genetic admixture among tick populations. Additionally, the highly invasive and widespread movement of R. microplus [14,36,37] may facilitate its occurrence and observed high levels of gene flow. The studied cattle farms owned by the DVS in Malaysia serve as the sources of cattle production for imported and local breeds and eventually distribute the animals to all private farms throughout Malaysia. The natural dispersal ability of ticks might occur in parallel with human-mediated dispersal, causing genetic admixture and high gene flow in Malaysian populations. Morever, this study also attempted to investigate whether haplotypes are distributed according to cattle breed. The specific association of haplotypes with other cattle breeds was not obvious except for some ticks collected from the Nellore breed which was imported from India. A distinct haplotype cluster (H16, H17, H18) was found for R. microplus ticks collected from these cattle. It is unknown whether this indicates a cause-effect association of haplotypes and this cattle breed. However, the prevalence of ticks has been typically associated with cattle breeds [38][39][40].
Over the last decade, a number of molecular approaches have been adopted to infer the phylogeographical relationships of R. microplus populations from different parts of the world [11][12][13][14][15][16]. As far as the molecular approaches are concerned, the mitochondria-encoded 16S rRNA and COI genes have invariably unraveled the distinct genetic assemblages of R. microplus and differentiated its closely related species, R. annulatus and R. australis [10,15,16]. Here, our 16S rRNA data were consistent with previous studies in which two 16S rRNA genetic clades were detected, with the first clade primarily confined to Africa/the Americas and the second clade confined to China/India. In contrast to the results found with the 16S rRNA gene, the COI phylogenetic data provided a new insight into the evolutionary lineages of R. microplus with the identification of three main assemblages: clade A sensu Burger et al. The comparative analysis in this study shows that R. microplus ticks represent at least three distinct genetic assemblages. Additional consideration should be given to the geographical differences between ticks in Malaysia and those from India, China and the Americas. Seasonal variations (e.g., temperature, climate and humidity) among the countries could be factors that shaped the observed patterns of genetic structure. Moreover, the role of anthropogenic disturbances in structuring the current genetic variability of R. microplus cannot be ruled out. This tick has been deemed the 'world's most pesticide-resistant tick' and the evolution of biotypes with resistance to at least 44 active ingredients has been documented worldwide [41]. Hence, the ticks might have experienced selection pressure from pesticides leading to the observed high genetic variability. Additionally, because speciation might be driven by ecological divergence, the cryptic diversity of R. microplus might be underestimated. The recent study of Burger et al. [10] has suggested the presence of cryptic species in R. microplus, complicates the assessment of species status and is a common challenge in the Ixodidae. In this regard, crossbreeding studies from different isolates of R. microplus, including the new taxon R. microplus clade C sensu Low et al. (2015) discovered in the present study should be conducted in the near future.