Mitochondrial 16s rDNA based Analysis of some Hard Ticks Belonging to Genus Hyalomma Koch, 1844 (Acari: Ixodidae)

| A molecular analysis of mitochondrial 16s rDNA sequences of hard ticks belonging to genus Hyalomma from Haryana (India) and those available in genbank database was done so as to resolve inter-relationships between members of genus Hyalomma. For this a total of eighty one 16s rDNA sequences belonging to 16 taxa were subjected to molecular and phylogenetic analysis which was conducted in MEGA6 and Beast 1.8.0 software. Phylogenetic relationships were inferred using the bayesian, maximum likelihood (ML) and neighbor joining (NJ) methods by means of Tamura three parameter model + unequal frequency + gamma distribution (TPMuf+G). The analysis revealed 249 variable sites, 232 conserved sites, and 137 parsimony informative sites in the alignment. Results of the phylogenetic analysis provide support for monophyletic origin of genus Hyalomma. Further our results assert that H. anatolicum, H. excavatum, H. marginatum, H. lusitanicum, H. hussaini and H. brevipunctata represent closely related but rapidly diverging taxa and also substantiate H. asiaticum as a species complex. The molecular clock results exemplify divergence time of subfamily Hyalomminae from the common ancestor of subfamily Rhipicephalinae to be 61.99 mya and the origin of family Ixodidae to be 86.79 mya. A basic phylogenetic relationship tree is also provided to help future studies for phylogenetics of family Ixodidae especially for subfamily Hyalomminae.


MAtErIAl And MEthodS
The hard ticks infesting cattle and buffalo hosts were field collected from different animal farms located in the state of Haryana (India) (Figure 1, Table 1).After initial separation of hard ticks identification up to species level was done by using standard identification keys available for Hyalomma ticks (Kaiser and Hoogstraal, 1964;Geevarghese and Dhanda, 1987;Keirans and Litwak, 1989;Petney and Keirans, 1995;Walker et al., 2003).The ticks were photographed using a trinocular stereo-zoom microscope (Labomed™) and subsequently preserved in 100% ethanol in a -20°C deep freezer (Bluestar).

dna extraCtion
DNA was extracted from individual hard ticks using DNeasy® DNA isolation kit (Qiagen).For this, individual ticks were crushed with sterile glass pestle in liquid nitrogen and subsequently DNA was extracted by following the protocol provided with the kit.The DNA was quantified using Tecan's Infinite® NanoQuant and stored at 4°C.Quality of DNA was checked by resolving on 0.8% Agarose gels using standard procedure.1)

pCr amplifiCation and SequenCing
PCR was performed to amplify 16s rDNA from individual hard tick DNA samples with the following primer pairs: S16S FP (5'-CTGCTCAATGAATAT-TTAAATTGC-3') and S16S RP (5' -CGGTCTAAACT-CAGATCATGTAGG-3') (Tian et al., 2011).PCR reactions were performed in 25µl reaction mixture that had 100ng DNA template and 1.5U of Taq Polymerase (Ge-Nei™) per reaction along with standard reaction ingredients.The PCR cycling conditions set in the program were as follows: initial denaturation at 94°C for 3 min followed by 30 cycles of 94°C for 30 sec (denaturation), 50°C for 40sec (annealing), 72°C for 40 sec (extension) and a final extension step of 72°C for 5 min.PCR products were resolved on 2% Agarose gels and compared with 100bp  DNA standard ladder as the expected product size was in range of 420-440bp.PCR products were purified by using Geneipure™ Quick PCR Purification kit (GeNei™) and sent for commercial DNA sequencing to 1 st base sequencing service (Malaysia).

SequenCe alignment and analySiS
Multiple sequence alignment of eighty one 16s rDNA sequences was generated with Muscle software tool executed in MEGA6 phylogenetic analysis software (Tamura et al., 2013).The alignment was manually edited to remove any alignment errors and exported as mega and fasta format files.The readseq program available at European Bioinformatics Institute (EBI) server link http:// www.ebi.ac.uk/Tools/sfc/readseq/ was used for sequence conversion to nexus format before performing bayesian analysis.The sequences were segregated into groups (Table 2), and analysed for GC% (Figure 2, Supplementary Table 1), maximum likelihood estimate of transition/transversion bias (Table 3), estimates of evolutionary divergence over sequence pairs between groups (Tables 4, 5 and 6), and Tajima's neutrality test (Tajima, 1989) (Table 7) using MEGA6 phylogenetic and sequence analysis software (Tamura et al., 2013).   . Suchard).In all the analysis gaps and missing data were treated as partial deletion with 90% site coverage cut-off.The phylogenetic trees were constructed using the below mentioned parameters for each analysis method: ml analySiS: nucleotide substitution model -Tamura 3-parameter model (Tamura, 1992), test of phylogenybootstrap method (Felsenstein, 1985), 100 replications, rates among sites -gamma distributed, tree inference methodology -nearest-neighbor-interchange (NNI) method, branch swap filter -very strong.

BayeSian analySiS:
A relaxed clock bayesian analysis to estimate the topology and divergence times simultaneously was performed where evolutionary rates along branches followed an uncontrolled log normal distribution and a Yule speciation process was imposed for all analysis.

rESultS
The dataset used in the present analysis includes 81 sequences of 16s rDNA -75 sequences belonging to ten species of genus Hyalomma and 6 sequences from other genera from family Ixodidae (as detailed earlier).All sequences have been aligned and annotated to have a final alignment length of 498bp which when analysed reveal 249 variable sites, 232 conserved sites, 137 parsimony informative sites and 110 singleton sites.

nuCleotide CompoSition
The analysis of nucleotide composition of 16s rDNA sequences demonstrates them to be AT rich having base composition percentage in decreasing order of A>T>G>C except for Amblyomma where it is T>A>G>C (Supplementary Table 1).The average GC% is 22.47, while it is lowest in Hyalomma marginatum of 19.93% and highest in Hyalomma anatolicum of 23.09% (Figure 2).

maximum liKeliHood eStimate of tranSition/ tranSverSion BiaS and SuBStitution matrix
The estimated transition/transversion bias (R) for whole dataset of the genus Hyalomma is estimated to be 0.52 indicating no bias as its very close to expected value of 0.5 but there is marked deviation from the observed and expected ti/tv bias when individual species groups are considered which is When rates of different transitional substitutions (shown in bold in Table 3) and those of transversion substitutions (shown in italics in Table 3) are studied the rate of transition are found to be more than transversion for all base substitutions.A→G and T→C transition are found to have 17.12 probability values while C→T and G→A transition have a probability value of 4.75 only.As for the transversion T→A, A→T, C→A and G→T have a The

taJima'S neutrality teSt
The test results reveal that the value of D for the whole dataset is -1.22 with a p value of 0.06 (Table 6).When the out-groups are removed to test only for the genus Hyalomma the value of D comes out to be -0.39 with p value of 0.04 while for out-groups it comes out to be 0.61 with p value of 0.19.Within genus Hyalomma the D value for the Anatolicum group is -2.17 with p value of 0.018 while the D value for Asiaticum group is -1.51 with p value of 0.003.

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The pHylogenetiC analySiS The analysis of 16s rDNA sequences belonging to hard ticks of genus Hyalomma and outgroups results in a phylogeny with good statistical support using all three approaches having some topology variations but majority of important clades differ only in bootstrap/PP support (Table 7).Ixodes ricinus has been used to root all the trees.The relationships between hard ticks obtained using different approaches are detailed here under:

nJ analySiS
In the evolutionary tree, inferred by using the NJ method based on 1000 bootstrap pseudo-replicates, branches corresponding to partitions reproduced in less than 70% bootstrap pseudo-replicates are collapsed (Figure 4) and bootstrap support values are shown next to the branches.

BayeSian analySiS
In the maximum clade credibility tree the Posterior Probability (PP) values of replicate trees in which the associated taxa clustered together in bayesian analysis is shown above the branches while the node ages are shown against the nodes (Figure 5).There is very strong PP support of 1.0 for the Asiaticum species complex (Hyalomma asiaticum asiaticum+Hyalomma asiaticum kozlovi) and Hyalom

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eStimating divergenCe time
Most recent common ancestor (MRCA) of various nodes has the following divergence times when the molecular clock analysis results are studied: node labelled as A in Figure 5 2 and 6) especially Anatolicum group and Asiaticum species complex.Consequently, when the sequence divergence between groups is studied with the intention of estimating inter-specific variation within genus Hyalomma then it reveals that H. anatolicum, H. excavatum, H. marginatum, H. lusitanicum, H. hussaini and H. brevipunctata are having less than 5% sequence divergence indicating a recent evolutionary split.
In contrast the H. asiaticum is more departed having 7.3% to 13.8% divergence from these species (Table 5).As for H. dromedarii and H. detritum that cluster with H. asiaticum in the phylogenetic trees, H. asiaticum shows 7.65% and 7.84% divergence which is in agreement with between species differences reported earlier for Rhipicephalus (Liu et al., 2013) and Ixodes species (Song et al., 2011;Apanaskevich et al., 2011;Chao et al., 2009).
In this context, a review of literature reveals that there is no explicit study to infer relationships within genus Hyalomma but some of the investigations had a few members of Hyalomma analyzed using various markers.Specifically, H. anatolicum anatolicum and H. anatolicum excavatum were subspecies until Apanaskevich and Horak

relationSHipS witH otHer memBerS of family ixodidae
Filippova (1993) based on the structure of the ventral skeleton of male Ixodid hard ticks suggested that subfamily Hyalomminae be included in the subfamily Rhipicephalinae.Later molecular studies of Black and Piesman (1994), Klompen et al. (1996, 1997), Black et al. (1997), Mangold et al. (1998a, 1998b) further provided evidence that Hyalomma species are found within the monophyletic Rhipicephalinae clade, whereas Dermacentor was placed either basal to Rhipicephalinae or within that clade.In the study of Dobson and Barker (1999)   phylogenetic trees Rhipicephalus (Rhipicephalus) and Rhipicephalus (Boophilus) clade together thus justifying the recent subgeneric status of Boophilus.Although the main aim of our study was to provide a phylogenetic tree as the basis for further comparative studies of inter-relationships within the genus Hyalomma subfamily Hyalomminae rather than a detailed analysis of phylogenetic relationships with other genera within family Ixodidae, still our preliminary results does not support merging the subfamily Hyalomminae within subfamily Rhipicephalinae.This hypothesis needs to be tested using more sequences from other genes covering whole of the metastriate lineage.

origin of tHe SuBfamily Hyalomminae
Estimating the divergence time plays an important role as indicator of speciation events that happened in the past evolutionary history of organisms (Hayakawa et al., 2008;Ricklefs and Outlaw, 2010).Recently Gou et al. ( 2013) have reviewed the divergence times of hard ticks based on three Dominica amber samples estimating that hard ticks began to diverge during the cretaceous approx 86mya (95% HPD 61.11-104-16mya) which is later than earlier estimates (90-120mya) (Mans et al., 2002;Klompen et al., 2005).In our analysis subfamily Hyalomminae seems to have diverged 61.99 mya from the common ancestor of subfamily Rhipicephalinae, while the divergence time of prostriate and metastriate ticks comes out to be 86.79 mya.It can be surmised that all the genera began to form during the early Eocene-late Pleistocene but separation of Hyalomma from the basal stalk happened at the beginning of Oligocene in the warm but cooling climate which coincided with the rapid evolution and diversification of the mammalian hosts of these important ectoparasites.

concluSIonS
The results of the present study have confirmed some of the existing morphological and molecular hypotheses about Hyalomma phylogeny.Furthermore, information about the inter-relationships of taxa not previously included in any phylogenetic study has been provided.In conclusion, the molecular evidence presented here suggests that H. anatolicum, H. excavatum, H. marginatum, H. lusitanicum, H. hussaini and H. brevipunctata represent closely related but rapidly diverging taxa and confirms H. asiaticum as a species complex.The subfamily Hyalomminae diverged from the common ancestor of subfamily Rhipicephalinae approx 62 mya at the beginning of Oligocene.Apart from this, a basic phylogenetic tree is provided (Figure .6) to help any future phylogenetic studies on members of family Ixodidae.Further study covering other members of genus Hyalomma from different geographical regions needs to be done using multiple genes to corroborate findings and shed light on questions raised in the present study.

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Figure 2 :
Figure 2: GC/AT percentage among various species groups of genus Hyalomma and out-groupspHylogenetiC analySiSPhylogenetic analysis was performed using the bayesian, maximum likelihood (ML) and neighbor joining (NJ)

Figure 3 :
Figure 3: Phylogenetic tree (70% consensus) generated by Maximum Likelihood method based on T3PM+G model with 100 bootstrap replicates (values on branches are bootstrap support)

Figure 5 :
Figure 5: Phylogenetic tree generated by Bayesian method based on T3PM+G model executed in Beast (the values on nodes are node ages in million years while the values on bar are Posterior Probability values) based on 18s rDNA the Haemaphysalinae species formed a monophyletic group with high bootstrap (98%) support, and there was also strong support for the clade containing the Rhipicephalus and Hyalomma species (100%) but there was no substantial bootstrap support for the sister group relationship of Dermacentor with the Rhipicephalus + Hyalomma clade.In the analysis of Murrell et al. (2001) the following clade had common ancestor (Dermacentor + ((Hyalomma + Nosomma) + (Boophilus + Rhipicephalus))).Labruna et al. (2009) using 16s and 12s rDNA gene reported Hyalomma and Dermacentor to be paraphyletic and basal to subfamily Rhipicephalinae which itself was a polyphyletic assemblage thus countering the earlier hypothesis.

Figure 6 :
Figure 6: Condensed Phylogenetic tree summarising the results of the present study recommended as a Key for future works In relation to this context, based on the present 16S based phylogeny following points of importance could be inferred viz.subfamily Amblyomminae is most basal in Metastriate after subfamily Haemaphysalinae but the weak support to this node is in lines with the recent contention of Burger et al. (2012) that it is a polyphyletic assemblage.Dermacentor and Rhipicephalus are paraphyletic to Hyalomma with Dermacentor older than the other two genera.In all the three

table 1 :
Collection Details of Four Hyalomma species from Haryana, India sequenced during the present study S.

table 2 :
Taxonomic details of species and groups studied *These groups are not having formal taxonomic status but have been created by authors for analysis based on relationships derived during the present study.

table 4 :
Matrix showing estimates of evolutionary divergence over Sequence Pairs between different genera (divergence is shown in the lower diagonal while the standard error is shown in upper diagonal)

table 5 :
Matrix showing estimates of percentage evolutionary divergence over sequence pairs between groups for genus Hyalomma.(Divergence is shown in the lower diagonal while the standard error values are shown in upper diagonal) (For species codes see Table2) probability value of 11.01 while A→C, T→G, C→G and G→C have a probability value of only 3.06.
dIScuSSIon nuCleotide CompoSition, SequenCe divergenCe, and relationSHipS witHin genuS Hyalomma The nucleotide differences in the 16s rDNA between various members of genus Hyalomma especially ti/tv bias and variation in the tajima's test of neutrality values is indicative of population expansion or a selective sweep.The values are significant for different species groups within genus Hyalomma at 0.05 or 0.01 p values (Tables

table 7 :
Bootstrap support, Posterior Probability, andPosterior age distributions of the major clades of hard ticks using ML, NJ and Bayesian analysis The results of sequence divergence and phylogenetic analysis provides credence to the fact that the Anatolicum group of species in the genus Hyalomma has undergone speciation during the recent evolutionary times which might still be going on.Further, detailed population genetic studies have to be carried out on populations and species from broader geographical localities to better illuminate the taxonomy and population genetic structures of these hard tick species of genus Hyalomma some of which are important vectors in their areas of geographical distribution.
tolicum Koch 1844 and Hyalomma excavatum Koch 1844 based on morphological characters.Based on the present molecular analysis Hyalomma anatolicum and H. excavatum form a single clade, it might be possible to generate molecular discrimination keys for these closely resembling problematic species using multiple genetic markers.Similarly, H. asiaticum was revealed to be a sibling species complex (Apanaskevich and Horak, 2010), while there is ambiguity about the status of H. detritum Schulze, 1919 (recently it has been synonymised with H. scupense(Schulze, 1919;  (Apanaskevich et al., 2010)).Based on our phylogenetic analysis by three different computational approaches (ML, NJ and Bayes) genus Hyalomma appears to be monophyletic with strong bootstrap and PP supports.Further the results reveal strong support for groups of H. anatolicum and related species labelled here as Anatolicum group (Table2) that comprises H. anatolicum, H. excavatum, H. marginatum, H. lusitanicum, H. hussaini and H. brevipunctata taxa and Asiaticum species complex having H. asiaticum asiaticum and H. asiaticum kozlovi.

Journal of Advances in Parasitology April 2016 | Volume 3 | Issue 2 | Page 42
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