Intra- and Interspecific variation of Amblyomma ticks from southern Africa

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In southern Africa, 17 Amblyomma spp. are currently documented. Of these species, Amblyomma hebraeum and Amblyomma variegatum have been well studied due to their wide geographical range and their status as competent vectors of pathogens that are of veterinary and medical importance. Studies on other Amblyomma spp. in southern Africa have been neglected, fostering ongoing debates on the validity of certain species such as Amblyomma pomposum . This study investigated the inter- and intraspecies variation of Amblyomma ticks collected in southern Africa, focusing on resolving the dispute about A. pomposum and A . variegatum as distinct species. Four tick species were collected from Angola, Mozambique, South Africa, Zambia, and Zimbabwe and were identified morphologically as Amblyomma eburneum , A . hebraeum , A . pomposum and A . variegatum using identification keys. Gene amplification was done targeting the 12S and 16S rRNA, cytochrome oxidase I, cytochrome B and internal transcribed spacer-2 genes, and Bayesian inference analyses were performed in MrBayes. These revealed little geographic structuring amongst ticks of the same species from different countries, although intraspecific variation within A. variegatum was high for the 16S and cytB loci. Our study concluds that there is insufficient molecular evidence to differentiate A . pomposum and A . variegatum from each other. We highlight the need for whole mitochondrial genome sequencing of these two species to resolve the ongoing debates. Furthermore, we propose mating and hybrid viability studies between the two species to confirm their reproductive isolation. Tick Diversity Phylogenetic Amblyomma southern Africa Systematics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background The Ixodida order is separated into three families: Argasidae (also referred to as soft ticks), Ixodidae (also referred to as hard ticks) and Nuttalliellidae [ 1 ]. The most recent records collating valid tick names list 707 ixodid and 190 argasid named species, as well as Nuttalliella namaqua , the only tick in the Nuttalliellidae family [ 2 , 3 ]. Out of the 898 recognised species, 206 ixodids, 40 argasids and N . namaqua occur in the Afrotropical region of the world [ 2 ]. Amblyomma , one of the largest genera within the Ixodidae, is found on every continent except Antarctica, and is a major concern in the Afrotropical regions. Amblyomma spp. are known to be aggressive hunters with a vibrant and ornate appearance. The majority of the Amblyomma spp. that have been studied are known to be potential vectors of zoonotic pathogens such as Rickettsia spp.; however, they do not commonly feed on humans. They are vectors of a large variety of pathogens of veterinary importance including, but not limited to, Ehrlichia ruminantium , Theileria mutans and Theileria velifera [ 4 , 5 ]. In the south-eastern parts of Africa, 21 Amblyomma species have been documented [ 5 – 7 ]. Amblyomma variegatum and Amblyomma hebraeum are the most predominant and widespread of the species (Fig. 1 ). Amblyomma hebraeum is located southernmost, where it occurs in the coastal belt of South Africa, the eastern parts of Eswatini, southern Mozambique (below the 22° latitude), eastern Botswana and in south-eastern Zimbabwe [ 8 ]. North of the endemic zone for A . hebraeum [ 9 ], A . variegatum spreads transversely through the continent, from the eastern parts of Angola and northern parts of Botswana, Zimbabwe, and Mozambique to the south from the Sahel transitional zone [ 10 ]. Amblyomma pomposum is distributed in Angola and is documented to share a geographical spread with A . variegatum in western Zambia and southern Democratic Republic of Congo [ 8 ]. Amblyomma eburneum is another species documented to share a geographical distribution with A . variegatum (Smit et al., 2023). Literature on A . eburneum is scarce, however it has been recorded in Somalia, Eritrea, Tanzania, Zimbabwe and northern Ethiopia stretching southwards through Kenya [ 7 ]. Tick taxonomy in southern Africa dates back to 1778 and is focused on the morphological characteristics of adults, geographical distribution and host preference [ 11 – 16 ]. The first phylogenetic representation of ticks was depicted by Hoogstraal and Aeschlimann [ 17 ] but this representation has been revised over the years with increasing information obtained from molecular studies. Several studies have been conducted on the molecular systematics of Amblyomma spp.; however, most studies have included few or no Amblyomma spp. from southern African regions [ 18 – 21 ]. This limited representation of Amblyomma spp. from southern Africa has allowed for several incongruencies to have gone unanswered for decades. The validity of certain Amblyomma spp. classifications has been placed under scrutiny. One noted controversy in southern Africa encompasses the A. variegatum group as described in Dias [ 22 ] and highlighted by Theiler and Salisbury [ 23 ] and Walker and Olwage [ 10 ]. Amblyomma variegatum , as described by Fabricius [ 24 ], is the oldest known species in the A . variegatum group, with a documented geographical spread as mentioned above. Dönitz [ 25 ] described A . pomposum and although it is similar to A . variegatum , sufficient distinguishing phenotypic features are present to separate the two species morphologically. The main distinguishing features included coarse punctation on the conscutum and the fused central and cervical patches. Robinson [ 26 ] described two male specimens collected in southern Rhodesia (now Zimbabwe) as A . variegatum var. nocens. Its distribution was designated to span from Makoni, Umtali (now Mutare) and Melsetter in Zimbabwe to Manica province in Mozambique and is found in bush veldt, at elevations of 2,000 to 3,000 feet [ 23 , 26 ]. He noted that the main morphological difference between A . variegatum and A . variegatum var. nocens was that A . variegatum var. nocens had coarser punctations and was more vibrant in colouration. Robinson concluded in 1926 that the species he named A . variegatum var. nocens was synonymous to with A . pomposum [ 25 ], which was given preference [ 22 ]. Theiler and Salisbury [ 23 ] examined and compared the two males which Robinson [ 26 ] identified as A. variegatum var. nocens with A. pomposum samples and found that the morphologies did not resemble each other. In 1950, Dias described a new species, A. variegatum var. govurensis in Mozambique and compared its morphology to that of A . variegatum and A . pomposum . He described A. variegatum var. govurensis as distinguishable from the type specimens of A . variegatum and A . pomposum by large and coarse punctations, while the patterns were more vibrant and intense. Dias [ 22 ] attempted to revise the A. variegatum group, to include his newly described species A. variegatum var. govurensis; however, upon examination of the samples of Robinson [ 26 ], Dias concluded that his A. variegatum var. govurensis was identical to A . variegatum var. nocens and thus synonymous with A . pomposum . During Dias’s revision of the A . variegatum group, Dias [ 22 ] went on to note that the records of A . pomposum in eastern Africa as described by Dönitz [ 25 ] morphologically resembled the specimens that they found in Mozambique; however, the A . pomposum from western and central Africa did not match the description of the species by Dönitz [ 25 ]. Dias [ 22 ] proposed a new species name, Amblyomma superbum which he illustrated in Dias [ 27 ], for the Amblyomma spp. in central and western Africa. This new nomenclature was rejected and A . pomposum is still used to describe the species occurring in the central and western parts of Africa. In light of these morphological debates, Theiler and Salisbury [ 23 ] examined all the reference material they had access to and advocated for the re-establishment of A . variegatum var. nocens as Amblyomma nocens Robertson 1911, with a geographic distribution ranged confined to latitudes 18–22° S. This change in nomenclature was also rejected [ 28 ]. To date, there is still disagreement regarding the diversity of Amblyomma spp. in Mozambique. It is unclear whether these morphological variations in the Amblyomma spp. found in Mozambique are due to the ticks being different species, or subspecies, or merely due to intraspecific heterogeneity. Although this disagreement on the geographical distribution of A. variegatum and A. pomposum in Mozambique has been ongoing for decades [ 7 , 10 , 22 , 23 ], no molecular evidence has been provided to help resolve this question. Thus, this study investigated the intra- and inter-species variation of A. eburneum, A. hebraeum, A. pomposum and A. variegatum ticks collected in southern Africa using molecular techniques; with the aim to resolve the taxonomic controversy between A . pomposum and A . variegatum in southern Africa. Materials and Methods Sample collection Amblyomma spp. ticks were collected in Angola, Zambia, and Zimbabwe (Fig. 2) by means of hand collections from cattle and goats ( Additional file 1: Dataset S1 ). In Mozambique, Amblyomma spp. ticks were also collected from livestock, and legally hunted wildlife (Fig. 2) ( Additional file 1: Dataset S1 ). Amblyomma spp. ticks from South Africa were obtained from Dlamkile et al. [ 29 ] ( Additional file 1: Dataset S1 ). Collections were performed from February 2021 to April 2021 and again from November 2021 to September 2022. Collected ticks were stored in 70% ethanol and transported to the Department of Veterinary Tropical Diseases, University of Pretoria, Onderstepoort. The ticks were morphologically identified to species level using identification keys obtained from Walker et al. [ 8 ] and Voltzit and Keirans [ 7 ]. Morphological characteristics were documented using the Fujifilm XT-4 camera and images were stacked using Capture One Pro 9 and Zerene Stacker software version 1.04 [ 30 , 31 ]. DNA extraction and amplification DNA from all ticks were individually extracted using the Chelex 100 resin method (total final volume of 100 µl) as described by Smit et al. [ 32 ] and stored at -20˚C for downstream use. Molecular characterization was conducted targeting four mitochondrial [12S rRNA gene, 16S rRNA gene, cytochrome B ( cytB ), cytochrome oxidase 1 ( coi )] genes and the nuclear internal transcriber 2 ( ITS2 ) region. Four male and four female ticks per species per region, if available, were selected for DNA extraction. In a modification of the protocol published by Beati and Keirans [ 33 ], amplification of the 360 bp 12S gene was conducted in a 20 µl reaction consisting of 10 µl Phusion Flash High-Fidelity PCR Master Mix (1X final concentration), 0.5 µl each of the primers T1B and T2A (final concentration of 0.5 µM) (Table 1 ), 8 µl double-distilled water and 1 µl sample DNA. The PCR cycling conditions comprised an initial denaturation at 98°C for 10 s, followed by 10 cycles of denaturation at 94°C for 1 s, annealing at 60°C for 5 s and extension at 72°C for 15 s; then 30 cycles of amplification with denaturation at 94°C for 1 s, annealing at 49°C for 5 s, and extension at 72°C for 15 s. The final extension was performed at 70°C for 1 min. The protocol for amplification of the 16S gene was modified from Black and Piesman [ 34 ]; briefly, a 350 to 450 bp gene was amplified in a 20 µl reaction consisting of 10 µl Phusion Flash High-Fidelity PCR Master Mix (1X final concentration), 1 µl each of primers 16S1 and 16S2 (final concentration of 1 µM) (Table 1 ), 7 µl double-distilled water and 1 µl sample DNA. The PCR cycling conditions started an initial denaturation at 98°C for 10 s, followed by 10 cycles of denaturation at 94°C for 1 s, annealing at 48°C for 5 s and extension at 72°C for 15 s. A further 30 cycles of amplification were conducted with denaturation at 94°C for 1 s, annealing at 54°C for 5 s and extension at 72°C for 15 s. Final extension was conducted at 70°C for 1 min. The amplification of the 500 bp region of cytB was conducted as described by Simon et al. [ 35 ] with modifications. The 20-µl reaction consisted of 10 µl Phusion Flash High-Fidelity PCR Master Mix (1X final concentration), 0.8 µl each of primers CB-N-11367mo and CB-J-10933mo (final concentration of 0.4 µM) (Table 1 ), 6.4 µl double-distilled water and 2 µl sample DNA. The PCR cycling conditions comprised an initial denaturation at 98°C for 10 s, followed by 10 cycles of denaturation at 98°C for 1 s, annealing at 52°C for 5 s and extension at 72°C for 15 s. This was followed by 30 cycles of amplification with denaturation at 98°C for 1 s, annealing at 58°C for 5 s and extension at 72°C for 15 s. Final extension was performed at 70°C for 2 min. In a modification of the protocol published by Beati et al. [ 20 ], the 950 to 1,200 bp ITS2 gene was amplified in a 20 µl reaction consisting of 10 µl Phusion Flash High-Fidelity PCR Master Mix (1X final concentration), 1 µl each of primers ITS2(5.8)F and ITS2(5.8)R (final concentrations of 0.5 µM) (Table 1 ), 7 µl double-distilled water and 1 µl sample DNA. The PCR cycling conditions comprised an initial denaturation at 98°C for 10 s, followed by 35 cycles of denaturation at 98°C for 1 s, annealing at 55°C for 5 s, and extension at 72°C for 15 s. Final extension was performed at 70°C for 3 min. The coi amplification was conducted as described by Smit et al. [ 32 ]. Table 1 Set of primers used in the present study for 12S, 16S, cytB , coi and ITS2 amplification. Target gene Primer Name Primer sequence (5’to3') Reference 12S T1B AAA CTA GGA TTA GAT ACC CT Beati and Keirans [ 33 ] T2A AAT GAG AGC GAC GGG CGA TGT 16S 16S1 CTG CTC AAT GAT TTT TTA AAT TGC TGT GG Black and Piesman [ 34 ] 16S2 TTA CGC TGT TAT CCC TAG AG cytB CB-N-11367mo ATT ACC CCC CCT AAT TTA TTA GGA AT Simon et al. [ 35 ] CB-J-10933mo TAT ATT TTA CCC TGA GGG CAA ATA TC coi LCOI490 GGT CAA CAA ATC ATA AAG ATA TTG G Folmer et al. [ 65 ] HCO2198 TAA ACT TCA GGG TGA CCA AAA AAT CA ITS2 ITS2(5.8)F CCA TCG ATG TGA AYT GCA GGA CA Beati et al. [ 20 ] ITS2(28)R GTG AAT TCT ATG CTT AAA TTC AGG GGG T The PCR products were separated on a 1.5% agarose gel and visualized using the Bio-Rad gel documentation system with assisting visualization programming. All samples that had visible bands were sent to the Central Analytical Facility (CAF), Stellenbosch, South Africa for Sanger sequencing in both directions. Phylogenetic analysis Each gene was analysed separately. Sequences were manually corrected on CLC main workbench version 23.0.2 (developed by CLC Bio, http://www.clcbio.com ), and compared with those available in the GenBank database using BLAST ( https://www.ncbi.nlm.nih.gov/genbank/ ) ( Additional file 2: Dataset S2 ). Contigs were assembled, sequence orientations were confirmed and alignments were generated alongside reference sequences using the online MAFFT version 7 (developed by http://mafft.cbrc.jp/alignment/server/index.html ) with default parameters. The aligned matrices were manually viewed, edited, and truncated using MEGA 11 [ 36 ]. The best fit model was determined for each individual gene using the jModelTest2 [ 37 ] on the CIPRES Science Gateway ( https://www.phylo.org/ ) platform. The file format was changed depending on program requirements using FaBox version 1.61 ( https://users-birc.au.dk/palle/php/fabox/index.php ). Bayesian inference (BI) were performed in MrBayes version 3 [ 38 ]. For the Bayesian analysis, five Monte Carlo Markov Chains (MCMC) were run for 5,000,000 iterations, saving every 1,000th tree with relative burn-in at 25%. Tracer version 1.6 [ 39 ] was used for inspection of estimated sample size (ESS) (> 200) and parameter sampling using graphical plots indicating parameter stabilisation. The resulting tree was visualised and edited in iTOL version 6.8 [ 40 ]. A concatenated file was constructed including only the coi and cytB genes. This was due to the lack of amplification in the 12S and 16S genes for all species. Alignment, model testing and BI analysis was done as described above. Barcode gap analysis Groups based on Amblyomma spp. were created in Mega version 11 and the intra- and inter- species variation of these groups were compared. A pairwise distance matrix (p-distance) was obtained for each gene’s alignment using default parameters with 1,000 bootstrap replicates to calculate the standard error (SE). Barcode gap analysis was conducted using the automatic barcode gap discovery (ABGD) tool on the online web server ( https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html ) [ 41 ]. Analysis was conducted using default parameters (P min = 0.001; P max = 0.1; 10 steps and relative gap width X = 1.5) using the Kimura (K80) TS/TV model. Ethical Considerations This study was approved by the Research Ethics Committee of the University of Pretoria (REC 121 − 20) and approval was also obtained from the Department of Agriculture, Land Reform and Rural Development (DALRRD), South Africa, under Section 20 of the Animal Diseases Act 1984 (Act no. 35 of 84) (12/11/1/1 (1937SS)). Results In total 7,734 adult Amblyomma spp. ticks were obtained from Angola, South Africa, Mozambique, Zambia, and Zimbabwe (Fig. 3 ). These ticks were identified morphologically to species level and described as: A. eburneum , A. hebraeum , A. pomposum and A. variegatum (Fig. 4 ). Amblyomma eburneum was only collected in one location in central Mozambique (18˚S) (Fig. 2). Amblyomma hebraeum was collected in the north-eastern parts of South Africa, southern and central regions in Zimbabwe, and in the southern parts of Mozambique (below 21˚S), with two sampling points extending beyond what is currently documented as the A . hebraeum endemic zone (19°S) (Fig. 2). Amblyomma pomposum was only collected in Angola in the central regions expanding westwards (Fig. 2). Amblyomma variegatum was collected in central northern regions of Mozambique (above 21˚S), northern regions of Zimbabwe, latitudinally across Zambia, and eastern parts of Angola (Fig. 2). Short morphological descriptions Amblyomma eburneum Amblyomma eburneum (Fig. 4 A) males have a brightly ornated conscutum that is red-brown in appearance with symmetrical vibrant beige patterns. The conscutum is smooth in appearance with small shallow punctations. Eyes are present and are slightly convex. The mesial area (central beige patch) is square in shape and is not connected to any of the other enamel patches. The falciform stripe (large “C” shaped enamel patches), beige in colouration, extends anteriorly and is connected indistinctly to the posterior enamel areas. The posterior median stripe is straight and wide, while notches in the posterior enamel areas are large, dividing the enamel area into two separate (but connected) patches. Festoons are rectangular with extensive enamelling (10 out of 11 festoons have varying degrees of enamelling; with the central festoon having no enamel). The lateral median areas of enamel ornamentation are distinctly present and connected to the posterior enamelled areas. They are large ovoid shapes with a beige appearance. Legs are dark in colour with pale rings. Females share several morphological features with the males, including the slightly convex eyes and banded legs. The scutum is red-brown in colouration with a glossy appearance. The mesial area of enamel ornamentation on the scutum is a large circular patch. Lateral areas of enamel ornamentation on the scutum are present forming two well-separated small patches on each lateral side. Two central circular patches are visible on the scutum. The scutum sides are slightly convex and the posterior angle is broad. The scutum appears smooth with small punctations. Amblyomma hebraeum Amblyomma hebraeum (Fig. 4 B) males have a ornate conscutum, that is bright and red-brown in colouration with vibrant and metallic beige-red patterning over most of the surface. The conscutum appears to be smooth with small shallow punctations, situated in each lateral area. Eyes are present and flat. The mesial area of enamel ornamentation on the conscutum is elongate, stretching to just below the capitulum. The falciform stripe is almost triangular in appearance, beige in colouration, extends anteriorly and is clearly connected to the posterior enamel areas. The posterior median stripe is straight and narrow, with short and narrow notches in the posterior enamel areas. Festoons are rectangular with extensive enamelling (enamelling present on 9 out of 11 festoons; with the outermost festoons having no enamel). The lateral median areas of enamel ornamentation are distinctly present, complex and are unconnected to any other enamelled area. Legs are dark in colour with pale rings. Females have flat eyes that are marginalized on the scutum and banded legs. The scutum is red-brown in colouration. The mesial area of enamel ornamentation on the scutum is a large metallic patch covering the central third of the scutum. Lateral areas of enamel ornamentation on the scutum are distinctly present, forming “C”-shaped patches on each lateral side. The scutum sides are slightly convex and the posterior angle is broad. The scutum appears smooth with deep punctations near the eyes; with shallow and small punctations in the scapular and central areas. Amblyomma pomposum Amblyomma pomposum (Fig. 4 C) males have a very bright and ornate conscutum that appears to be more vibrant and metallic in colouration compared to A . variegatum . The conscutum is densely stippled with large punctuations. Eyes are present and are distinctly convex. The mesial area is oval in shape and is not connected to any of the other enamel patches. The falciform stripes resemble two claws, are orange in colouration, extend anteriorly andare connected to the posterior enamel areas. The postmedian stripe is narrow, while the notches in the posterior enamel areas are wide. Enamelling on the festoons are absent. The lateral median areas of enamel ornamentation are distinctly present as large trapezoids with a red appearance. Legs are dark in colour with pale rings. Females share several morphological features with the males including the convex eyes and banded legs. The scutum is dark in colouration with a glossy appearance. The mesial area of enamel ornamentation on the scutum is a small, faint circular patch. Lateral areas of enamel ornamentation on the scutum are present but indistinct. The scutum sides are straight and the posterior angle is narrower than that of A . variegatum . The scutum is densely stippled with large punctations. Amblyomma variegatum Amblyomma variegatum (Fig. 4 D) males have a brightly ornate conscutum that appears to be less vibrant and more muted in colouration compared to A. pomposum males. The conscutum is densely stippled with medium to small punctations. Eyes are present and are distinctly convex. The mesial area has a more squared appearance and is connected to the falciform stripe, which is salmon in colouration, extends anteriorly and is connected to the posterior enamel areas. The posterior median stripe and the notches in the posterior enamel areas are narrow. Festoons have no enamel. The lateral median areas of enamel ornamentation are either absent or present as small circular patches. Legs are dark in colour with pale rings. Females share several morphological features with the males including the convex eyes and banded legs. The scutum is dark in colouration with a dull and more matt appearance. The mesial area of enamel ornamentation on the scutum is a small, clear circular patch. Lateral areas of enamel ornamentation on the scutum are present but indistinct. The scutum sides are slightly convex and the posterior angle is broad. The scutum is densely stippled with small to medium punctations. Phylogenetics Amplification success varied amongst the genes; 12S and coi were the most successful, with amplification and contig assembly achieved with > 50% specimens. The 16S rRNA gene was poorly amplified, with the majority of the sequences excluded due to failure to align forward and reverse reads. CytB was the most successful in producing sequences and contig assembly of these sequences. ITS2 was excluded from further analyses due to low genetic variability only consisting of uninformative single nucleotide mutations preventing any systematic analyses. The number of sequences per species analysed for each gene varied (Table 2 ). All sequences were deposited in GenBank ( Additional file 2: Dataset S2 ). Table 2 The number of sequences analysed for the pairwise distance matrix and for the Automatic Barcode Gap Discovery (ABGD) analysis. Species / Number of sequences analysed 12S 16S coi cytB Concatenated Amblyomma eburneum 12 2 15 6 3 Amblyomma hebraeum 76 50 80 67 27 Amblyomma pomposum 8 4 4 23 2 Amblyomma variegatum 42 33 37 39 8 Outgroup ( Rhipicephalus maculatus ) 1 1 1 1 1 Total 139 90 138 136 41 The average intra-species pairwise distances and the average inter-species pairwise distances are given in Table 3 and Table 4 , respectively. In all inter-species evaluations, A. pomposum and A. variegatum had the least amount of distance between them. The intra-species pairwise distances were on average lower than the average inter-species pairwise distances. The pairwise difference between individual sequences for each gene were also compared ( Additional file 3: Dataset S3 to Additional file 7: Dataset S7 ). Table 3 Estimates of average evolutionary divergence over sequence pairs within groups (intraspecies p-distances). Species / Intraspecies p-distance 12S 16S coi cytB Concatenated Amblyomma eburneum 0.018 0.005 0.021 0.004 0.00 Amblyomma hebraeum 0.005 0.002 0.007 0.005 0.01 Amblyomma pomposum 0.005 0.023 0.007 0.013 0.01 Amblyomma variegatum 0.01 0.078 0.023 0.064 0.03 Table 4 Estimates of evolutionary divergence over sequence pairs between groups (interspecies p-distances). Values in red indicate the intraspecies p-distances. Gene Region Amblyomma eburneum Amblyomma hebraeum Amblyomma pomposum Amblyomma variegatum 12S Amblyomma eburneum 0.18 Amblyomma hebraeum 0.024 0.005 Amblyomma pomposum 0.082 0.085 0.005 Amblyomma variegatum 0.085 0.086 0.009 0.01 outgroup 0.169 0.163 0.168 0.166 16S Amblyomma eburneum 0.005 Amblyomma hebraeum 0.050 0.002 Amblyomma pomposum 0.118 0.111 0.023 Amblyomma variegatum 0.132 0.120 0.058 0.078 outgroup 0.252 0.238 0.228 0.240 coi Amblyomma eburneum 0.021 Amblyomma hebraeum 0.067 0.007 Amblyomma pomposum 0.137 0.133 0.007 Amblyomma variegatum 0.138 0.134 0.026 0.023 outgroup 0.186 0.198 0.190 0.187 cytB Amblyomma eburneum 0.004 Amblyomma hebraeum 0.065 0.005 Amblyomma pomposum 0.132 0.131 0.013 Amblyomma variegatum 0.152 0.151 0.053 0.064 outgroup 0.206 0.205 0.220 0.228 Concatenated Amblyomma eburneum 0.00 Amblyomma hebraeum 0.066 0.01 Amblyomma pomposum 0.137 0.133 0.01 Amblyomma variegatum 0.139 0.136 0.029 0.03 outgroup 0.200 0.202 0.200 0.188 When evaluating the 12S estimations of evolutionary divergence between each of the sequences, low levels of intra-species variation were observed. The intra-species variation of A . eburneum ranged from 0 to 0.074, whereas in A. hebraeum , it ranged from 0 to 0.045. Low levels of intraspecific variation were also observed in A . pomposum , ranging from 0 to 0.024. Amblyomma pomposum and A . variegatum had low levels of inter-species variation amounting to an average of 0.008. The intra-species variation for A . variegatum ranged from 0 to 0.008 for sequences obtained for this study, while nine sequences (four Zambian sequences from this study, and five reference sequences from other countries) had greater variation. The ABGD analysis indicated four operational taxonomic units (OTUs), grouping the majority of A . eburneum with A . hebraeum , and A . pomposum with A . variegatum . One A . eburneum sequence formed its own OTU ( Additional file 8: Fig. S1 ). The 16S pairwise sequence matrix depicts no intraspecific variation between the A . eburneum sequences, although only two sequences were available. The average inter-species variation between A . eburneum and A . hebraeum was 0.050. Low levels of intra-species variation were observed in A . hebraeum , ranging from 0 to 0.009; while for A. pomposum it ranged from 0 to 0.052. High levels of intra-species variation were observed for A . variegatum , ranging from 0 to 0.421, with three sequences (two from Mozambique and one from Uganda) contributing most variation. The ABGD analysis indicated seven OTUs. Amblyomma eburneum and A . hebraeum formed their own OTUs, whereas A . pomposum clustered in the same OTU with the majority of A . variegatum . The three A . variegatum sequences that showed the highest diversity formed their own OTUs ( Additional file 9: Fig. S2 ). The analysis of the coi pairwise sequence matrix illustrated low levels of intraspecific variation for A . eburneum , ranging from 0 to 0.071 with two sequences (both from Mozambique) with higher levels of variation. The intra-species variation in A . hebraeum ranged from 0 to 0.025, while A. pomposum it ranged from 0 to 0.013. High levels of variation in A . variegatum were observed, ranging from 0 to 0.073. The ABGD analysis indicated five OTUs, grouping A . pomposum and A . variegatum together. Amblyomma hebraeum clustered into one main group. Amblyomma eburneum separated into two main groups, one containing the majority of the sequences while the other group consisted of the two variable sequences ( Additional file 10: Fig. S3 ). The cytB pairwise distance matrix depicted low levels of intra-species variation ranging from 0 to 0.006 in A . eburneum . The intra-species variation in A . hebraeum ranged from 0 to 0.037 with one sequence from Mozambique exhibiting high levels of variation. Intra-species variation for A . pomposum ranged from 0 to 0.045 with three sequences that had a higher variation compared to the rest. Amblyomma variegatum had high levels of intra-species variation ranging from 0 to 0.609 with two sequences showing greater variation. The ABGD analysis indicated six OTUs, grouping A . pomposum and A . variegatum together. Amblyomma variegatum clustered into three groups; one main clade and two separate singleton OTUs. Amblyomma eburneum , A . hebraeum and the outgroup clustered in cognate groups ( Additional file 11: Fig. S4 ). For the concatenated alignment, the pairwise distance matrix illustrated low levels of intraspecific variation. For Amblyomma eburneum it ranged from 0.001 to 0.004, while for A . hebraeum ranged from 0 to 0.070. The intraspecific variation for A . pomposum was 0.008; however, only two sequences were compared. Intraspecific variation of A . variegatum ranged from 0.004 to 0.056. The ABGD analysis indicated four OTUs: A . eburneum and A . hebraeum formed their own groups, while A . pomposum clustered with A . variegatum (Fig. 5 ). The BI analysis of the 16S gene illustrated a clear separation between A . eburneum and A . hebraeum , but this was not the case for A . pomposum and A . variegatum , as can be seen by the nesting of A . pomposum within the A . variegatum clades. The BI analysis of the 12S gene also divided A . eburneum and A . hebraeum into well-supported clades; however, while most A . pomposum specimens formed one branch that split from the A . variegatum clade with 0.69 probability, two A . pomposum sequences were not separated from the main A. variegatum clade. The BI analysis of the coi gene resolved all species; A . pomposum branched from the A . variegatum clade with 0.96 probability, and all individuals grouped in the same cluster, although the branch length was very short. This contrasted somewhat with the BI analysis of the cytB gene, in which A . variegatum was positioned as a derived sister group to the A . pomposum lineage with a probability of 0.79. The concatenated BI analysis depicts clear differentiation between all species while A . pomposum branched from the A . variegatum clade with 0.99 probability and all individuals grouped in the same cluster. Three A . variegatum sequences from Mozambique also branched from the main cluster with a probability of 1.00. All BI analyses revealed relatively little intra-species variation among ticks of the same species from different countries, which clustered together without marked geographic structuring, although a clade composed of a subset of A. variegatum from Mozambique was robustly supported in the concatenated analysis. Discussion This study investigated the intra- and inter-species variation of Amblyomma spp. collected in southern Africa. In total 7,734 adult Amblyomma ticks were collected and morphologically identified as A . eburneum , A , hebraeum , A . pomposum and A . variegatum . However, based on our analyses of multiple genes, we cannot conclude that A . pomposum and A . variegatum are distinct species. Amblyomma eburneum is described as an eastern African species with a documented geographical distribution ranging from Somalia, Eritrea and Ethiopia in the north, and south through Kenya, Tanzania and Zimbabwe [ 7 ]. In this study we collected A . eburneum from central Mozambique in the Sofala province from African buffalo. Amblyomma hebraeum , from cattle, was collected in South Africa, Mozambique, and Zimbabwe, corresponding with its documented geographical distribution [ 6 , 8 , 10 , 42 ]. The geographical spread of A . pomposum was described by Robinson [ 43 ], Walker and Olwage [ 10 ], and Petney et al. [ 42 ] to range from Angola to the western regions of Zambia, and northwards to the southern parts of the Democratic Republic of Congo. In the present study, Amblyomma spp. were also collected in these regions, from the western parts of Angola and latitudinally to the eastern parts of Zambia. We found A . pomposum to be restricted to the central-western parts of Angola, whereas Amblyomma specimens collected in eastern Angola were identified as A . variegatum . In this study, A . variegatum was collected in Angola, Mozambique, Zambia and Zimbabwe, corresponding with records of Petney et al. [ 42 ] and Walker and Olwage [ 10 ]. The most predominant collected species was A . hebraeum from South Africa and Mozambique, while the species with the lowest representation was A . pomposum from Angola. The low recovery of A . pomposum , and A . variegatum in Angola, could be ascribed to the collections occurring in March, which is at the end of the adult season (the months when the maximum infestations occur have been documented as November and December [ 42 ]). Amblyomma eburneum was also collected in low numbers, but this can be attributed to the difficulty of collecting ticks from wildlife species. The collections were highly dependent on the amount of wildlife that was purchased and legally hunted in the timeframe of this study. The low prevalence of Amblyomma spp. from Zimbabwe was likely a result of a vigorous campaign by the government to assist livestock farmers in treating their cattle against ticks by encouraging regular acaricde dipping of the animals, as an attempt to control major outbreaks of bovine theileriosis. This study aimed to provide insight to the unresolved debate that has been ongoing for decades as described in the introduction. Both Robinson [ 26 ] and Dias (1950,1953) described morphological variation in the A . variegatum found in Mozambique and suggested new species names A. variegatum var. nocens and A. variegatum var. govurensis , respectively. Dias [ 22 ] also examined what was described as A . pomposum from Angola and concluded that it does not resemble the A . pomposum as described by Dönitz [ 25 ], suggesting a new species description as Amblyomma superbum . In this study, morphological variation in the collected A . variegatum ticks were observed; however, the majority resembled that of the original description by Fabricius [ 24 ]. Morphology is the most important aspect for taxonomy, highlighting the importance of variation in ornamentation. These conflicting observations of morphology in A . variegatum resemble the controversy of the Amblyomma marmoreum complex [ 44 ]. The Amblyomma marmoreum complex encompasses five African species, namely: A . marmoreum sensu stricto, A . sparsum , A . falsomarmoreum , A . nuttalli , and A . paulopunctatum [ 44 ]. As with this study, the A . marmoreum complex was placed under scrutiny due to a lack of genetic and ecological data on its members. Although meticulous descriptions of the five species in the A . marmoreum complex are available, identification remains a challenge and misidentification often occurs, even confusing these species with other Amblyomma spp. outside of the complex [ 23 , 33 , 44 ]. However, Cotes-Perdomo et al. [ 44 ] was able to differentiate between the species with the use of molecular techniques targeting a large part of the mitogenome. Overall, they were able to concatenate and compare 13 protein-coding genes and two ribosomal genes of several Amblyomma spp. In the attempt to provide clarity on the phylogenetic positioning of A . pomposum and A . variegatum , molecular analyses were conducted on these four Amblyomma species with the use of 12S, 16S, coi , cytB and ITS2 molecular markers. However, the ITS2 marker was excluded from further analyses due to uninformative single nucleotide mutations. This research provides additional sequences that have been deposited in the GenBank database for all other markers utilized here, including the first for the 12S and cytB genes of A . eburneum and first for the 12S, coi and cytB genes of A . pomposum . During the course of this study, several challenges with amplification occurred for all genes. The Chelex extraction method has several drawbacks including the rapid degradation of the extracted DNA after two years of storage and the effects of long-term storage on the binding of impurities to DNA. Singh et al. [ 45 ] noted that samples extracted with the Chelex 100 resin method and stored for extended periods of time tended to contain contaminants such as proteins attached to the DNA helix, and these prevented successful PCR reactions. The importance of the impact of storage time on DNA integrity was evident through the successful amplification of cytB , which was conducted with DNA extracted less than one year previously, compared with the less successful amplification of 12S, 16S, coi and ITS2, which were conducted using DNA stored for approximately two years. Additionally, several amplified products did not generate sequences of good quality, and were thus excluded from further analysis, lowering the sample size. The 12S and 16S rRNA gene markers were particularly difficult to amplify in A . pomposum samples and those that did produce bands for these markers did not provide high-quality sequences. For future studies, it would be ideal to use recently extracted DNA when using the Chelex extraction method and DNA should be stored at -80°C, which would maintain the integrity of the DNA for a longer period; or alternative DNA extraction methods should be used that will not compromise the DNA integrity during storage. The ABGD analysis indicated that A . eburneum was divided into two OTUs in the 12S and coi analysis, while only forming one OTU in the 16S, cytB , and concatenated analyses. All the A . eburneum 12S sequences clustered with the A . hebraeum OTU, except for one which formed its own OTU. However, upon investigation, the sample that formed its own OTU was of suboptimal quality and several incongruencies was found between the forward and reverse sequences. The intraspecific variability for A . eburneum was greater for the 12S (0.018) and coi (0.021) genes, while the inter-species variability was sufficient to differentiate between species for 16S, coi , cytB and in the concatenated analyses. The inter-species variability was insufficient for the 12S analysis, as can be seen in the clustering with A . hebraeum during the ABGD analysis, though a clear separation was obtained in the phylogenetic tree. The ABGD analysis indicates that A . hebraeum only formed one OTU in all the single gene and concatenated analyses, although the intra-species variability for A . hebraeum was greater in the concatenated pairwise distance analysis. This supports the clear separation of this species from the other Amblyomma spp. of southern Africa. Strikingly, Amblyomma pomposum clustered with A . variegatum in the ABGD analyses for all the individual genes and in the concatenated analyses. The intraspecific variability in A . pomposum was greatest in the 16S and the cytB analysis. Amblyomma pomposum clustered within A . variegatum for the 12S, 16S, coi and concatenated phylogenetic analyses; however, in the cytB analysis it segregated as the ancestral lineage. The intraspecific variation within A . variegatum was greater than the interspecific variation between A . pomposum and A . variegatum for the 12S, 16S, cytB and concatenated analyses. This may indicate that there is insufficient variation between A . pomposum and A . variegatum to confidently describe these two species as distinct. The ABGD analyses for A . variegatum indicated four OTUs for the 16S and three OTUs for the cytB genes, while the 12S, coi and concatenated analyses indicated only one OTU. Two of the OTUs in the 16S analysis were the result of suboptimal sequences with several incongruencies between the forward and reverse sequences, while the other was a reference sequence from GenBank which covered a larger section of the 16S gene. As with A . pomposum , the intra-species variation for A . variegatum was greater in the 16S and cytB genes. Phylogenetic analysis of the Amblyomma spp. indicates that all genes used in this study were adequate to differentiate between A . eburneum , A . hebraeum and the A . variegatum / A . pomposum complex. The different patterns that emerge between the phylogenetic trees could be a result of the differential mutation rates of each of the molecular markers [ 46 ]. Erster et al. [ 47 ] and Koroiva and Santana [ 48 ] evaluated the marker efficiency of 12S, 16S, coi and cytB , which were also used in the current study, and demonstrated that the mitochondrial markers coi and cytB were most suitable for intra- and inter-species analyses due to their high variability. Furthermore, Vences et al. [ 49 ] reported that cytB was the most variable of the two markers, allowing for clearer separation between closely related species. Norris et al. [ 50 ] found that 16S was the least variable marker when comparing 12S and 16S markers for Ixodes scapularis population genetics. In the current study, cytB proved to be the most efficient single marker to differentiate between southern African Amblyomma spp., while none of the markers in isolation allowed for clear discrimination between samples of the same species from different countries. However, there was strong support for intra-specific population structure in A. variegatum in the concatenated analysis. In this context, a previous analysis of intraspecific variation of A. variegatum molecular markers concluded that genetic diversity was low in West Africa and in introduced Caribbean populations ( i.e ., nucleotide diversity of 0.02–0.25% for 12S), with higher variation in East Africa ( i.e ., 0.65% for 12S) [ 20 ]. Although the study did not apply the more variable markers as used in the present work, the 12S data alone suggests that southern populations of A. variegatum show greater genetic variation (nucleotide diversity of 1%) than elsewhere on the continent. Notably, a population genetic study of A. variegatum in Burkina Faso used microsatellites and concluded that effective population sizes were low at the village level when sampling domestic ruminants [ 51 ]. Similar approaches could also be applied to populations from southern Africa in future studies. Recently, mitochondrial markers were analysed to determine the genetic structure of another African Amblyomma spp., the elephant tick A. tholloni , on host populations in Kenya. The intra-specific variation at the coi locus was found to be low and of similar magnitude to that of A. hebraeum and A. pomposum in the current study (< 1%) [ 52 ]. Population genetic studies of Amblyomma spp. have been more extensive in the New World and offer important lessons for understanding intra-specific variation in the Afrotropical species. There are marked differences between the low intra-specific variation observed in some Neotropical species ( e.g ., Amblyomma triste [ 53 ] and Amblyomma aureolatum [ 54 ], where it was < 1% for mitochondrial markers) compared with others such as Amblyomma ovale [ 55 , 56 ] and Amblyomma mixtum [ 57 ], where pairwise distances for concatenated mitochondrial markers can reach 3–5%. Amblyomma cajennense , which has an extensive distribution across subtropical and tropical regions of the Americas, constitutes a particularly interesting paradigm for the genus. Variation exceeding 8% at the whole mitogenome level between geographically and ecologically distinct populations of A. cajennense formed part of the evidence that was used to designate a species complex and formally describe its members as distinct species [ 58 , 59 ]. The Afrotropical species await similar rigorous analyses using whole mitogenome data across the entirety of their range, in line with the recent study on the Amblyomma marmoreum complex [ 44 ]. While the current study was not designed to address intraspecific population structure in depth, the concatenated phylogenetic analysis depicted clear differentiation between each of the analysed species; albeit A . pomposum branched from the main cluster of A . variegatum with a very short genetic distance, no greater than intra-specific variation within A. variegatum . An important limitation was that the concatenated tree was only constructed with two genes, coi and cytB . This was because not all loci had amplified successfully for each specimen, and also due to Koroiva and Santana [ 48 ]’s conclusion that coi and cytB markers were more suitable for intra- and inter-specific delineation. These were also the only markers that had sufficient representation amongst all the Amblyomma spp. from this study. Thus, based on the phylogenetic analysis of the individual genes, the pairwise distance analyses and the ABGD analyses, we cannot conclude that A . pomposum and A . variegatum are distinct species. Literature on systematic work with A . pomposum is scarce and currently there are no alternative methods to compare differences or similarities between these two species, except for morphological descriptions. No previous studies on the phylogenetic relationship of these two species have been conducted and, as discussed above, there is a scarcity of A . pomposum sequences available in GenBank. Kobayashi et al. [ 60 ] were the first authors who published the only sequences currently available for the A . pomposum on the GenBank database. They collected 15 Amblyomma ticks and morphologically identified them as A . variegatum ( n = 13) and A . pomposum ( n = 2) with the use of Walker et al. [ 8 ]. They then conducted a phylogenetic analysis using the 16S rRNA gene. The A . pomposum that they identified clustered within the A . variegatum clade. This may suggest that the current available sequences are from misidentified A . variegatum ticks. Balinandi et al. [ 61 ] morphologically identified ticks they collected from Uganda as belonging to A . pomposum and A . variegatum ; however, due to a lack of A . pomposum sequences at the time, phylogenetic analyses of the 16S gene clustered A . pomposum with A . variegatum . This discovery led the authors to believe that they had misidentified A . variegatum ticks as A . pomposum . Our 16S phylogenetic analysis depicts A . pomposum dispersed within the A . variegatum cluster. Based on our analysis of several genes, it is possible that Balinandi et al. [ 61 ] correctly identified their ticks as A . pomposum ; however, since they did not upload any sequences nor depicted the morphological discrepancy, no definite conclusion can be made based on this information. On the other hand, a study performed by Barradas et al. [ 62 ] collected 116 ticks from the Huambo province in Angola. With the use of morphological identification using identification keys from Walker et al. [ 8 ] as well as molecular identification targeting the 12S and 16S rDNA genes, they identified their 11 (10%) Amblyomma ticks as A . variegatum . As with the 16S, the 12S also depicts clustering of A . pomposum within A . variegatum . In our study we collected from the same sights in Huambo and identified the Amblyomma spp. circulating in the area as A . pomposum . Our findings are also supported by Sili et al. [ 63 ], who identified all the Amblyomma spp. in the area as A . pomposum . Based on our findings we believe that Barradas et al. [ 62 ] misidentified their Amblyomma spp. as A . variegatum instead of A . pomposum . Although the phylogenetic analyses cannot differentiate between A. variegatum and A. pomposum currently, sufficient morphological features are documented to distinguish these species from each other. The other main factor in defining a species is the ecology and biological habitats [ 64 ]. Mayr [ 64 ] stated that a species can be described as “The segregation of the total genetic variability of nature into discrete packages, so called species, which are separated from each other by reproductive barriers, prevents the production of too great a number of disharmonious, incompatible gene combinations. This is the basic biological meaning of species and this is the reason why there are discontinuities between sympatric species”. A clear parapatric boundary was observed between A . pomposum and A . variegatum ; however, a hypothesis of incipient speciation has yet to be tested. We suggest, alongside whole genome phylogenetic analysis, mating and hybrid viability studies between the two species to confirm their reproductive isolation. Conclusion CytB was the most successful marker in differentiating between A . eburneum , A . hebraeum , A . pomposum , and A . variegatum , closely followed by coi . The concatenated tree distinguished between A . pomposum and A . variegatum ; however, the pairwise distance and ABGD analyses suggest there is insufficient evidence that A . pomposum and A . variegatum are distinct species. In our study, no A . pomposum ticks were collected in any country other than Angola, challenging the current distribution as proposed by Theiler and Salisbury [ 23 ], Walker et al. [ 8 ] and other current literature. Based on our findings, we suggest comparing whole mitochondrial genomes of A . pomposum with those of A . variegatum to determine whether these are distinct species or A . pomposum is a subspecies of A . variegatum . Moreover, further investigations should also be conducted using mating and hybrid viability studies between the two species to determine if they are reproductively isolated. Abbreviations ABGD: Automatic Barcode Gap Discovery; BI: Bayesian inference; CAF: Central Analytical Facility; ESS: Estimates sample size; MCMC: Monte Carlo Markov Chains; OTU’s: operational taxonomic units; SE: Standard error; spp: species Declarations Funding This work was supported by AgriSETA; the Meat industry Trust; and the doctorate research bursary awarded to me by the University of Pretoria. Acknowledgements A special thank you to Delta Safaris, Mungari, who allowed us to collect ticks from the hunted wildlife and hosted us in their camp and to all field technicians and state veterinarians who assisted in the collection of ticks in the represented countries. We would also like to acknowledge Prof Melvyn Quan and Ms Zandile Mkhize, who took all the morphological photos of the ticks that are presented in this manuscript. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. The sequences generated for each tick species for each gene during the current study are available in the GenBank repository, https://www.ncbi.nlm.nih.gov/genbank/. Competing interests The authors declare that they have no competing interests. Author contribution A. Smit : Conceptualization, Sample collection, Methodology, Investigation, Data Curation, Writing - Original Draft, Visualization F.C. Mulandane : Sample collection, Writing - Review & Editing M. Labuschagne : Investigation, Writing - Review & Editing S.H. Wojick : Investigation, Writing - Review & Editing C. Malabwa : Sample collection, Investigation, Writing - Review & Editing G. Sili : Sample collection, Writing - Review & Editing S. Mandara : Sample collection, Writing - Review & Editing Z. Dlamkile : Sample collection, Writing - Review & Editing W.H. Stoltsz : Review & Editing H. Rose Vineer : Visualization, Writing - Review & Editing K. Huber : Review & Editing I. G. Horak : Review & Editing D. Morar-Leather : Writing - Review & Editing, Supervision, Funding acquisition B. L. Makepeace : Writing - Review & Editing, Supervision L. Neves : Conceptualization, Sample Collection, Methodology, Writing - Review & Editing, Supervision, Funding acquisition References Nicholson WL, Sonenshine DE, Noden BH, Brown RN. Ticks (ixodida). Med Vet Entomol: Elsevier; 2019. p. 603–72. Guglielmone A, Robbins R, Apanaskevich D, Petney T, Estrada-Peña A, Horak I. The Hard Ticks of the World (Acari: Ixodida: Ixodidae) Dordrecht, The Netherlands: Springer; 2014. Estrada Pena A, Mangold AJ, Nava S, Venzal JM, Labruna M, Guglielmone AA. A review of the systematics of the tick family Argasidae (Ixodida). Acarologia. 2010;50(3):317–33. Warnecke M, Schein E, Voigt W, Uilenberg G, Young A. Development of Theileria mutans (Theiler, 1906) in the gut and the haemolymph of the tick Amblyomma variegatum (Fabricius, 1794). Z Parasitenkd. 1980;62:119–25. Walker JB. A review of the ixodid ticks (Acari, Ixodidae) occurring in southern Africa. Onderstepoort Journal of Veterinary Research. 1991;58(2):81–105. Horak IG, Heyne H, Williams R, Gallivan GJ, Spickett AM, Bezuidenhout JD, et al. The ixodid ticks (Acari: Ixodidae) of Southern Africa. Cham, Switzerland: Springer; 2018. Voltzit O, Keirans J. A review of African Amblyomma species (Acari, Ixodida, Ixodidae). Acarina. 2003;11(2):135–214. Walker AR, Bouattour A, Camicas JL, Estrada-Pena A, Horak IG, Latif AA, et al. Ticks of domestic animals in Africa: a guide to identification of species: Bioscience Reports Edinburgh; 2003. Bournez L, Cangi N, Stachurski F, Lancelot R, Martinez D, Lefrançois T, et al., editors. Is the distribution of Amblyomma variegatum influenced by interspecific competition with Amblyomma hebraeum ? Preliminary study: distribution range in Mozambique2012: European Society for Vector Ecology. Walker JB, Olwage A. The tick vectors of Cowdria ruminantium (Ixodoidea, Ixodidae, genus Amblyomma ) and their distribution. Onderstepoort Journal of Veterinary Research. 1987;54:353–79. Theiler G, Robinson BN. Tick survey. VIII. Checklists of ticks recorded from the Belgian Congo and Ruanda Urundi, from Angola, and from Northern Rhodesia. Onderstepoort J Vet Res. 1954;26(3). MacLeod J. Tick infestation patterns in the southern province of Zambia. Bull Entom Res. 1970;60(2):253–74. Jack R. Ticks infesting Domestic Animals in S. Rhodesia. Rhod agric j. 1936;33(12). Hoogstraal H, Theiler G. Ticks (Ixodoidea, Ixodidae) parasitizing lower primates in Africa, Zanzibar, and Madagascar. J Parasitol Res. 1959;45(2):217–22. Howard C. A list of the ticks of South Africa. Annals of the Transvaal Museum. 1908;1(2):73–169. Horak IG. A century of tick taxonomy in South Africa. Onderstepoort Journal of Veterinary Research. 2009;76(1):67–74. Hoogstraal H, Aeschlimann A. Tick-host specificity. Bulletin de la Société Entomologique Suisse. 1982;55:5–32. Kushimo O. The tick genus Amblyomma in Africa: phylogeny and mutilocus DNA barcoding: Georgia Southern University; 2013. Pillay A, Nyangiwe N, Mukaratirwa S. Low genetic diversity and population structuring of Amblyomma hebraeum and Rickettsia africae from coastal and inland regions in the Eastern Cape Province of South Africa. Med Vet Entomol. 2022;37:275–85. Beati L, Patel J, Lucas-Williams H, Adakal H, Kanduma EG, Tembo-Mwase E, et al. Phylogeography and demographic history of Amblyomma variegatum (Fabricius)(Acari: Ixodidae), the tropical bont tick. Vector Borne Zoonotic Dis. 2012;12(6):514–25. Kelava S, Mans BJ, Shao R, Moustafa MAM, Matsuno K, Takano A, et al. Phylogenies from mitochondrial genomes of 120 species of ticks: Insights into the evolution of the families of ticks and of the genus Amblyomma . Ticks Tick Borne Dis. 2021;12(1):101577. Dias J. Sobre a posição sistemática de algumas espécies africanas do género Amblyomma CL Koch (Acarina–Ixodoidea). Moçambique. 1953;73:119–39. Theiler G, Salisbury LE. Ticks in the South African zoological survey collection-Part IX-The Amblyomma marmoreum group. Onderstepoort Journal of Veterinary Research. 1959;28(1):47–124. Fabricius JC. Entomologia systematica: impensis CG Proft; 1794. Dönitz. Dber das Zeckengenus Amblyomma . Schrift Ges Nat Freund Berlin. 1909;8: 440–82. Robinson L. New species of ticks ( Haemaphysalis , Amblyomma). Parasitology. 1911;4(4):478–84. Dias JATdS. List of Mozambique ticks and their known hosts. Anais dos Servicos de Veterinaria e Industria Animal. 1950(3):227. Guglielmone AA, Robbins RG, Apanaskevich DA, Petney TN, Estrada-Peña A, Horak IG. Comments on controversial tick (Acari: Ixodida) species names and species described or resurrected from 2003 to 2008. Experimental and Applied Acarology. 2009;48:311–27. Dlamkile Z, Neves L, Morar-Leather D, Brandt C, Pretorius A, Steyn H, et al. Characterization of E . ruminantium Field Isolates from Amblyomma hebraeum Ticks Collected from Cattle in Three South African Provinces Using Multi-Locus Sequence Typing. In: NETWORK, S S R (ed). 2023. Erni S. Capture One Pro 9: Mastering Raw Development, Image Processing, and Asset Management: Rocky Nook, Inc.; 2016. Systems Z. Zerene Stacker, Version 1.04. Zerene Systems Richland, WA; 2018. Smit A, Mulandane FC, Wojcik SH, Horak IG, Makepeace BL, Morar-Leather D, et al. Sympatry of Amblyomma eburneum and Amblyomma variegatum on African buffaloes and prevalence of pathogens in ticks. Ticks and Tick-Borne Diseases. 2023;14(6):102247. Beati L, Keirans JE. Analysis of the systematic relationships among ticks of the genera Rhipicephalus and Boophilus (Acari: Ixodidae) based on mitochondrial 12S ribosomal DNA gene sequences and morphological characters. Journal of Parasitology. 2001;87(1):32–48. Black WC, Piesman J. Phylogeny of hard-and soft-tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences. Proceedings of the National Academy of Sciences. 1994;91(21):10034-8. Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am. 1994;87(6):651–701. Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021;38(7):3022–7. Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods. 2012;9(8):772-. Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003;19(12):1572–4. Rambaut A, Suchard MA, Xie D, Drummond A. Tracer 1.6 2014 [Available from: http://beast.bio.ed.ac.uk/Tracer . Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Research. 2021;49(W1):W293-W6. Puillandre N, Lambert A, Brouillet S, Achaz G. ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Mol Ecol. 2012;21(8):1864–77. Petney T, Horak I, Rechav Y. The ecology of the African vectors of heartwater, with particular reference to Amblyomma hebraeum and Amblyomma variegatum . Onderstepoort J Vet Res. 1987;54:381–95. Robinson LE. The Genus Amblyomma .-Ticks: A Monograph of the Ixodoidea, Part IV. In: Press CU, editor. Ticks: a monograph of the Ixodidae. 2. London1926. p. 99–103. Cotes-Perdomo AP, Sanchez-Vialas A, Thomas R, Jenkins A, Uribe JE. New insights into the systematics of the Afrotropical Amblyomma marmoreum complex (Acari, Ixodidae) and a novel Rickettsia africae strain using morphological and metagenomic approaches. bioRxiv. 2023:2023.08. 18.553479. Singh UA, Kumari M, Iyengar S. Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin. Biol Proced Online. 2018;20(1):1–8. Roth A, Akad F, Zonstein I, King R, Orshan L, Erster O. Molecular characterization of six Hyalomma species using mitochondrial markers. Ticks and Tick-Borne Diseases. 2019;10(4):911–7. Erster O, Roth A, Avni Z, King R, Shkap V. Molecular detection of Rickettsia bellii in Amblyomma rotundatum from imported red-footed tortoise ( Chelonoides carbonaria ). Ticks Tick Borne Dis. 2015;6(4):473–7. Koroiva R, Santana D. Evaluation of partial 12S rRNA, 16S rRNA, COI and Cytb gene sequence datasets for potential single DNA barcode for hylids (Anura: Hylidae) An Acad Bras Cienc. 2022;94. Vences M, Thomas M, Bonett RM, Vieites DR. Deciphering amphibian diversity through DNA barcoding: chances and challenges. Philos Trans R Soc Lond, B, Biol Sci. 2005;360(1462):1859–68. Norris DE, Klompen JSH, Keirans JE, Black IV WC. Population genetics of Ixodes scapularis (Acari: Ixodidae) based on mitochondrial 16S and 12S genes. J Med Entomol. 1996;33(1):78–89. Huber K, Jacquet S, Rivallan R, Adakal H, Vachiéry N, Risterucci A-M, et al. Low effective population sizes in Amblyomma variegatum , the tropical bont tick. Ticks Tick Borne Dis. 2019;10(1):93–9. King'ori EM, Obanda V, Nyamota R, Remesar S, Chiyo PI, Soriguer R, et al. Population genetic structure of the elephant tick Amblyomma tholloni from different elephant populations in Kenya. Ticks and Tick-Borne Diseases. 2022;13(3):101935. Guglielmone AA, Nava S, Mastropaolo M, Mangold AJ. Distribution and genetic variation of Amblyomma triste (Acari: Ixodidae) in Argentina. Ticks and Tick-Borne Diseases. 2013;4(5):386–90. Bitencourth K, Amorim M, Oliveira SVd, Gazêta GS. Amblyomma aureolatum Genetic Diversity and Population Dynamics Are Not Related to Spotted Fever Epidemiological Scenarios in Brazil. Pathogens. 2021;10(9):1146. Fournier GF, Pinter A, Santiago R, Muñoz-Leal S, Martins TF, Lopes MG, et al. A high gene flow in populations of Amblyomma ovale ticks found in distinct fragments of Brazilian Atlantic rainforest. Experimental and Applied Acarology. 2019;77:215–28. Uribe JE, Nava S, Murphy KR, Tarragona EL, Castro LR. Characterization of the complete mitochondrial genome of Amblyomma ovale , comparative analyses and phylogenetic considerations. Experimental and Applied Acarology. 2020;81(3):421–39. Cotes-Perdomo AP, Nava S, Castro LR, Rivera-Paéz FA, Cortés-Vecino JA, Uribe JE. Phylogenetic relationships of the Amblyomma cajennense complex (Acari: Ixodidae) at mitogenomic resolution. Ticks and Tick-Borne Diseases. 2023;14(3):102125. Beati L, Nava S, Burkman EJ, Barros-Battesti DM, Labruna MB, Guglielmone AA, et al. Amblyomma cajennense (Fabricius, 1787)(Acari: Ixodidae), the Cayenne tick: phylogeography and evidence for allopatric speciation. BMC Evolutionary Biology. 2013;13:1–20. Nava S, Beati L, Labruna MB, Cáceres AG, Mangold AJ, Guglielmone AA. Reassessment of the taxonomic status of Amblyomma cajennense () with the description of three new species, Amblyomma tonelliae n. sp., Amblyomma interandinum n. sp. and Amblyomma patinoi n. sp., and reinstatement of Amblyomma mixtum , and Amblyomma sculptum (Ixodida: Ixodidae). Ticks and Tick-Borne Diseases. 2014;5(3):252–76. Kobayashi T, Chatanga E, Qiu Y, Simuunza M, Kajihara M, Hang’ombe BM, et al. Molecular detection and genotyping of Coxiella-Like Endosymbionts in ticks collected from animals and vegetation in Zambia. Pathogens. 2021;10(6):779. Balinandi S, Chitimia-Dobler L, Grandi G, Nakayiki T, Kabasa W, Bbira J, et al. Morphological and molecular identification of ixodid tick species (Acari: Ixodidae) infesting cattle in Uganda. Parasitol Res. 2020;119:2411–20. Barradas PF, Mesquita JR, Ferreira P, Gärtner F, Carvalho M, Inácio E, et al. Molecular identification and characterization of Rickettsia spp. and other tick-borne pathogens in cattle and their ticks from Huambo, Angola. Ticks and Tick-Borne Diseases. 2021;12(1):101583. Sili G, Byaruhanga C, Horak I, Steyn H, Chaisi M, Oosthuizen MC, et al. Ticks and tick-borne pathogens infecting livestock and dogs in Tchicala-Tcholoanga, Huambo Province, Angola. Parasitology Research. 2021;120(3):1097–102. Mayr E. What is a species, and what is not? Philos Sci. 1996;63(2):262–77. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology. 1994;3(5):294–9. Additional Declarations No competing interests reported. Supplementary Files AdditionalFile1TextS1.CollectioncountrieswiththeprovincesmainlocationsandtheGPScoordinatesforthisstudy.docx AdditionalFile2DatasetS2GenBankaccessionnumbersforthesequncesusedandobtainedinthisstudy.xlsx AdditionalFile3DatasetS3Pairwisedistanceanalysisofthe12Sgene.xls AdditionalFile4DatasetS4Pairwisedistanceanalysisofthe16Sgene.xls AdditionalFile5DatasetS5Pairwisedistanceanalysisofthecoigene.xls AdditionalFile6DatasetS6PairwisedistanceanalysisofthecytBgene.xls AdditionalFile7DatasetS7Pairwisedistanceanalysisoftheconcatenatedmatrix.xls AdditionalFile8Fig.S1BITree12S.png AdditionalFile9Fig.S2BITree16S.png AdditionalFile10Fig.S3BITreecoi.png AdditionalFile11Fig.S2BITreecytB.jpg Cite Share Download PDF Status: Published Journal Publication published 28 Aug, 2024 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 03 Mar, 2024 Reviews received at journal 03 Mar, 2024 Reviewers agreed at journal 19 Feb, 2024 Reviewers agreed at journal 24 Jan, 2024 Reviews received at journal 17 Jan, 2024 Reviewers agreed at journal 10 Jan, 2024 Reviewers invited by journal 04 Jan, 2024 Editor assigned by journal 04 Jan, 2024 Submission checks completed at journal 04 Jan, 2024 First submitted to journal 04 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":356501,"visible":true,"origin":"","legend":"\u003cp\u003eMap of Africa showing the current distributions of A. hebraeum (blue), A. pomposum (purple) and A. variegatum (green) on the continent. \u0026nbsp;Blank African map image was obtained from https://worldmapblank.com/blank-map-of-africa/ and modified with distribution information from Walker et al. [8].\u003c/p\u003e","description":"","filename":"Fig.1MapofAfricashowingthecurrentdistributionsofA.hebraeumblueA.pomposumpurpleandA.variegatumgreenonthecontinent.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3833842/v1/cd409a2427a733c8366d996c.jpg"},{"id":49301166,"identity":"62eb601e-e6c0-49cf-b5d9-8bbb940d801d","added_by":"auto","created_at":"2024-01-08 09:38:31","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":400462,"visible":true,"origin":"","legend":"\u003cp\u003eMap of southern Africa, illustrating the collection points during the 2020-2022 sampling period of \u003cem\u003eAmblyomma\u003c/em\u003e spp. The colour of the dot represents the species of \u003cem\u003eAmblyomma\u003c/em\u003e tick collected at the sampling point.\u003c/p\u003e","description":"","filename":"Fig.2MapofsouthernAfricaillustratingthecollectionpointsduringthe20202022samplingperiodofAmblyommaspp.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3833842/v1/1044ae4c2045b1cda6f35fd5.jpeg"},{"id":49301167,"identity":"1b019294-e356-48d7-b1ab-4afa61485253","added_by":"auto","created_at":"2024-01-08 09:38:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63077,"visible":true,"origin":"","legend":"\u003cp\u003eThe number and species of \u003cem\u003eAmblyomma\u003c/em\u003e ticks collected from Angola, Mozambique, South Africa, Zambia, and Zimbabwe over the course of this study.\u003c/p\u003e","description":"","filename":"Fig.3ThenumberandspeciesofAmblyommatickscollectedfromAngolaMozambiqueSouthAfricaZambiaandZimbabweoverthecourseofthisstudy.png","url":"https://assets-eu.researchsquare.com/files/rs-3833842/v1/8330f7a912b81a082270d64d.png"},{"id":49301169,"identity":"5e33b9be-18a2-429a-9a60-a442e15b534e","added_by":"auto","created_at":"2024-01-08 09:38:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":464558,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological comparison of males (indicated by ♂) and females (indicated by ♀) where plate \u003cstrong\u003eA\u003c/strong\u003erepresents \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e, plate \u003cstrong\u003eB\u003c/strong\u003e represents \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e, plate \u003cstrong\u003eC\u003c/strong\u003e represents \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and plate \u003cstrong\u003eD\u003c/strong\u003erepresents \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e. Dorsal and ventral views are illustrated. Photographs were taken by Prof Melvyn Quan and Ms. Zandile Mkhize.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3833842/v1/7143a050aa2c9e2266ad9cbc.png"},{"id":49301172,"identity":"82977c64-3ffb-4c0b-9b6f-f4b3b9a59bc9","added_by":"auto","created_at":"2024-01-08 09:38:31","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":516668,"visible":true,"origin":"","legend":"\u003cp\u003eBayesian inference (BI) analysis of all concatenated genes. The BI analysis used the TPM2uf model with gamma distribution. Five Monte Carlo Markov Chains (MCMC) were employed for 5,000,000 iterations, saving every 1,000th tree. The first 25% of drafted trees were discarded. The resulting tree was visualised and edited in iTOL version 6.8. Posterior probability is indicated at each branch node. Blue, green, pink and purple highlighted regions indicate \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e, respectively. Species names are included with a three-letter country code and the sample name or GenBank accession number. Country codes: AGO – Angola, MOZ – Mozambique, USA – United Stats of America, ZAF – South Africa, ZBM – Zambia and ZWE – Zimbabwe.\u003c/p\u003e","description":"","filename":"Fig.5BayesianinferenceBIanalysisofallconcatenatedgenes.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3833842/v1/ac1f4c5a122962daa3f17c44.jpg"},{"id":63822286,"identity":"cb6f119d-e5c7-4a31-94fb-d9053fd8f57e","added_by":"auto","created_at":"2024-09-02 16:15:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2984878,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3833842/v1/f0168751-363c-414f-b261-8a4e4b0e0b0d.pdf"},{"id":49301646,"identity":"2b6c0750-6b42-46e3-ae1a-f11c55c34a44","added_by":"auto","created_at":"2024-01-08 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09:38:32","extension":"jpg","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":416277,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile11Fig.S2BITreecytB.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3833842/v1/9b062fccc5ae693ef65ec491.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intra- and Interspecific variation of Amblyomma ticks from southern Africa","fulltext":[{"header":"Background","content":"\u003cp\u003eThe Ixodida order is separated into three families: Argasidae (also referred to as soft ticks), Ixodidae (also referred to as hard ticks) and Nuttalliellidae [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The most recent records collating valid tick names list 707 ixodid and 190 argasid named species, as well as \u003cem\u003eNuttalliella namaqua\u003c/em\u003e, the only tick in the Nuttalliellidae family [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Out of the 898 recognised species, 206 ixodids, 40 argasids and \u003cem\u003eN\u003c/em\u003e. \u003cem\u003enamaqua\u003c/em\u003e occur in the Afrotropical region of the world [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eAmblyomma\u003c/em\u003e, one of the largest genera within the Ixodidae, is found on every continent except Antarctica, and is a major concern in the Afrotropical regions.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAmblyomma\u003c/em\u003e spp. are known to be aggressive hunters with a vibrant and ornate appearance. The majority of the \u003cem\u003eAmblyomma\u003c/em\u003e spp. that have been studied are known to be potential vectors of zoonotic pathogens such as \u003cem\u003eRickettsia\u003c/em\u003e spp.; however, they do not commonly feed on humans. They are vectors of a large variety of pathogens of veterinary importance including, but not limited to, \u003cem\u003eEhrlichia ruminantium\u003c/em\u003e, \u003cem\u003eTheileria mutans\u003c/em\u003e and \u003cem\u003eTheileria velifera\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In the south-eastern parts of Africa, 21 \u003cem\u003eAmblyomma\u003c/em\u003e species have been documented [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. \u003cem\u003eAmblyomma variegatum\u003c/em\u003e and \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e are the most predominant and widespread of the species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e is located southernmost, where it occurs in the coastal belt of South Africa, the eastern parts of Eswatini, southern Mozambique (below the 22\u0026deg; latitude), eastern Botswana and in south-eastern Zimbabwe [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. North of the endemic zone for \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e spreads transversely through the continent, from the eastern parts of Angola and northern parts of Botswana, Zimbabwe, and Mozambique to the south from the Sahel transitional zone [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. \u003cem\u003eAmblyomma pomposum\u003c/em\u003e is distributed in Angola and is documented to share a geographical spread with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e in western Zambia and southern Democratic Republic of Congo [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. \u003cem\u003eAmblyomma eburneum\u003c/em\u003e is another species documented to share a geographical distribution with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e (Smit et al., 2023). Literature on \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e is scarce, however it has been recorded in Somalia, Eritrea, Tanzania, Zimbabwe and northern Ethiopia stretching southwards through Kenya [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTick taxonomy in southern Africa dates back to 1778 and is focused on the morphological characteristics of adults, geographical distribution and host preference [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The first phylogenetic representation of ticks was depicted by Hoogstraal and Aeschlimann [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] but this representation has been revised over the years with increasing information obtained from molecular studies. Several studies have been conducted on the molecular systematics of \u003cem\u003eAmblyomma\u003c/em\u003e spp.; however, most studies have included few or no \u003cem\u003eAmblyomma\u003c/em\u003e spp. from southern African regions [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This limited representation of \u003cem\u003eAmblyomma\u003c/em\u003e spp. from southern Africa has allowed for several incongruencies to have gone unanswered for decades.\u003c/p\u003e \u003cp\u003eThe validity of certain \u003cem\u003eAmblyomma\u003c/em\u003e spp. classifications has been placed under scrutiny. One noted controversy in southern Africa encompasses the \u003cem\u003eA. variegatum\u003c/em\u003e group as described in Dias [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and highlighted by Theiler and Salisbury [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and Walker and Olwage [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. \u003cem\u003eAmblyomma variegatum\u003c/em\u003e, as described by Fabricius [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], is the oldest known species in the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e group, with a documented geographical spread as mentioned above. D\u0026ouml;nitz [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] described \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and although it is similar to \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e, sufficient distinguishing phenotypic features are present to separate the two species morphologically. The main distinguishing features included coarse punctation on the conscutum and the fused central and cervical patches.\u003c/p\u003e \u003cp\u003eRobinson [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] described two male specimens collected in southern Rhodesia (now Zimbabwe) as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e var. \u003cem\u003enocens.\u003c/em\u003e Its distribution was designated to span from Makoni, Umtali (now Mutare) and Melsetter in Zimbabwe to Manica province in Mozambique and is found in bush veldt, at elevations of 2,000 to 3,000 feet [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. He noted that the main morphological difference between \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e var. \u003cem\u003enocens\u003c/em\u003e was that \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e var. \u003cem\u003enocens\u003c/em\u003e had coarser punctations and was more vibrant in colouration. Robinson concluded in 1926 that the species he named \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e var. \u003cem\u003enocens\u003c/em\u003e was synonymous to with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], which was given preference [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Theiler and Salisbury [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] examined and compared the two males which Robinson [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] identified as \u003cem\u003eA. variegatum\u003c/em\u003e var. \u003cem\u003enocens\u003c/em\u003e with \u003cem\u003eA. pomposum\u003c/em\u003e samples and found that the morphologies did not resemble each other. In 1950, Dias described a new species, \u003cem\u003eA. variegatum\u003c/em\u003e var. \u003cem\u003egovurensis\u003c/em\u003e in Mozambique and compared its morphology to that of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e. He described \u003cem\u003eA. variegatum\u003c/em\u003e var. \u003cem\u003egovurensis\u003c/em\u003e as distinguishable from the type specimens of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e by large and coarse punctations, while the patterns were more vibrant and intense. Dias [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] attempted to revise the \u003cem\u003eA. variegatum\u003c/em\u003e group, to include his newly described species \u003cem\u003eA. variegatum\u003c/em\u003e var. \u003cem\u003egovurensis;\u003c/em\u003e however, upon examination of the samples of Robinson [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], Dias concluded that his \u003cem\u003eA. variegatum\u003c/em\u003e var. \u003cem\u003egovurensis\u003c/em\u003e was identical to \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e var. \u003cem\u003enocens\u003c/em\u003e and thus synonymous with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eDuring Dias\u0026rsquo;s revision of the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e group, Dias [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] went on to note that the records of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e in eastern Africa as described by D\u0026ouml;nitz [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] morphologically resembled the specimens that they found in Mozambique; however, the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e from western and central Africa did not match the description of the species by D\u0026ouml;nitz [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Dias [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] proposed a new species name, \u003cem\u003eAmblyomma superbum\u003c/em\u003e which he illustrated in Dias [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], for the \u003cem\u003eAmblyomma\u003c/em\u003e spp. in central and western Africa. This new nomenclature was rejected and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e is still used to describe the species occurring in the central and western parts of Africa. In light of these morphological debates, Theiler and Salisbury [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] examined all the reference material they had access to and advocated for the re-establishment of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e var. \u003cem\u003enocens\u003c/em\u003e as \u003cem\u003eAmblyomma nocens\u003c/em\u003e Robertson 1911, with a geographic distribution ranged confined to latitudes 18\u0026ndash;22\u0026deg; S. This change in nomenclature was also rejected [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo date, there is still disagreement regarding the diversity of \u003cem\u003eAmblyomma\u003c/em\u003e spp. in Mozambique. It is unclear whether these morphological variations in the \u003cem\u003eAmblyomma\u003c/em\u003e spp. found in Mozambique are due to the ticks being different species, or subspecies, or merely due to intraspecific heterogeneity. Although this disagreement on the geographical distribution of \u003cem\u003eA. variegatum\u003c/em\u003e and \u003cem\u003eA. pomposum\u003c/em\u003e in Mozambique has been ongoing for decades [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], no molecular evidence has been provided to help resolve this question. Thus, this study investigated the intra- and inter-species variation of \u003cem\u003eA. eburneum, A. hebraeum, A. pomposum\u003c/em\u003e and \u003cem\u003eA. variegatum\u003c/em\u003e ticks collected in southern Africa using molecular techniques; with the aim to resolve the taxonomic controversy between \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e in southern Africa.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAmblyomma\u003c/em\u003e spp. ticks were collected in Angola, Zambia, and Zimbabwe (Fig.\u0026nbsp;2) by means of hand collections from cattle and goats (\u003cb\u003eAdditional file 1: Dataset S1\u003c/b\u003e). In Mozambique, \u003cem\u003eAmblyomma\u003c/em\u003e spp. ticks were also collected from livestock, and legally hunted wildlife (Fig.\u0026nbsp;2) (\u003cb\u003eAdditional file 1: Dataset S1\u003c/b\u003e). \u003cem\u003eAmblyomma\u003c/em\u003e spp. ticks from South Africa were obtained from Dlamkile et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] (\u003cb\u003eAdditional file 1: Dataset S1\u003c/b\u003e). Collections were performed from February 2021 to April 2021 and again from November 2021 to September 2022. Collected ticks were stored in 70% ethanol and transported to the Department of Veterinary Tropical Diseases, University of Pretoria, Onderstepoort. The ticks were morphologically identified to species level using identification keys obtained from Walker et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and Voltzit and Keirans [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Morphological characteristics were documented using the Fujifilm XT-4 camera and images were stacked using Capture One Pro 9 and Zerene Stacker software version 1.04 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and amplification\u003c/h2\u003e \u003cp\u003eDNA from all ticks were individually extracted using the Chelex 100 resin method (total final volume of 100 \u0026micro;l) as described by Smit et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and stored at -20˚C for downstream use. Molecular characterization was conducted targeting four mitochondrial [12S rRNA gene, 16S rRNA gene, cytochrome B (\u003cem\u003ecytB\u003c/em\u003e), cytochrome oxidase 1 (\u003cem\u003ecoi\u003c/em\u003e)] genes and the nuclear internal transcriber 2 (\u003cem\u003eITS2\u003c/em\u003e) region. Four male and four female ticks per species per region, if available, were selected for DNA extraction. In a modification of the protocol published by Beati and Keirans [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], amplification of the 360 bp \u003cem\u003e12S\u003c/em\u003e gene was conducted in a 20 \u0026micro;l reaction consisting of 10 \u0026micro;l Phusion Flash High-Fidelity PCR Master Mix (1X final concentration), 0.5 \u0026micro;l each of the primers T1B and T2A (final concentration of 0.5 \u0026micro;M) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), 8 \u0026micro;l double-distilled water and 1 \u0026micro;l sample DNA. The PCR cycling conditions comprised an initial denaturation at 98\u0026deg;C for 10 s, followed by 10 cycles of denaturation at 94\u0026deg;C for 1 s, annealing at 60\u0026deg;C for 5 s and extension at 72\u0026deg;C for 15 s; then 30 cycles of amplification with denaturation at 94\u0026deg;C for 1 s, annealing at 49\u0026deg;C for 5 s, and extension at 72\u0026deg;C for 15 s. The final extension was performed at 70\u0026deg;C for 1 min. The protocol for amplification of the \u003cem\u003e16S\u003c/em\u003e gene was modified from Black and Piesman [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]; briefly, a 350 to 450 bp gene was amplified in a 20 \u0026micro;l reaction consisting of 10 \u0026micro;l Phusion Flash High-Fidelity PCR Master Mix (1X final concentration), 1 \u0026micro;l each of primers 16S1 and 16S2 (final concentration of 1 \u0026micro;M) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), 7 \u0026micro;l double-distilled water and 1 \u0026micro;l sample DNA. The PCR cycling conditions started an initial denaturation at 98\u0026deg;C for 10 s, followed by 10 cycles of denaturation at 94\u0026deg;C for 1 s, annealing at 48\u0026deg;C for 5 s and extension at 72\u0026deg;C for 15 s. A further 30 cycles of amplification were conducted with denaturation at 94\u0026deg;C for 1 s, annealing at 54\u0026deg;C for 5 s and extension at 72\u0026deg;C for 15 s. Final extension was conducted at 70\u0026deg;C for 1 min. The amplification of the 500 bp region of \u003cem\u003ecytB\u003c/em\u003e was conducted as described by Simon et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] with modifications. The 20-\u0026micro;l reaction consisted of 10 \u0026micro;l Phusion Flash High-Fidelity PCR Master Mix (1X final concentration), 0.8 \u0026micro;l each of primers CB-N-11367mo and CB-J-10933mo (final concentration of 0.4 \u0026micro;M) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), 6.4 \u0026micro;l double-distilled water and 2 \u0026micro;l sample DNA. The PCR cycling conditions comprised an initial denaturation at 98\u0026deg;C for 10 s, followed by 10 cycles of denaturation at 98\u0026deg;C for 1 s, annealing at 52\u0026deg;C for 5 s and extension at 72\u0026deg;C for 15 s. This was followed by 30 cycles of amplification with denaturation at 98\u0026deg;C for 1 s, annealing at 58\u0026deg;C for 5 s and extension at 72\u0026deg;C for 15 s. Final extension was performed at 70\u0026deg;C for 2 min. In a modification of the protocol published by Beati et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], the 950 to 1,200 bp \u003cem\u003eITS2\u003c/em\u003e gene was amplified in a 20 \u0026micro;l reaction consisting of 10 \u0026micro;l Phusion Flash High-Fidelity PCR Master Mix (1X final concentration), 1 \u0026micro;l each of primers ITS2(5.8)F and ITS2(5.8)R (final concentrations of 0.5 \u0026micro;M) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), 7 \u0026micro;l double-distilled water and 1 \u0026micro;l sample DNA. The PCR cycling conditions comprised an initial denaturation at 98\u0026deg;C for 10 s, followed by 35 cycles of denaturation at 98\u0026deg;C for 1 s, annealing at 55\u0026deg;C for 5 s, and extension at 72\u0026deg;C for 15 s. Final extension was performed at 70\u0026deg;C for 3 min. The \u003cem\u003ecoi\u003c/em\u003e amplification was conducted as described by Smit et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSet of primers used in the present study for 12S, 16S, \u003cem\u003ecytB\u003c/em\u003e, \u003cem\u003ecoi\u003c/em\u003e and \u003cem\u003eITS2\u003c/em\u003e amplification.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget gene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrimer sequence (5\u0026rsquo;to3')\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e12S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT1B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAAA CTA GGA TTA GAT ACC CT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBeati and Keirans [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT2A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAAT GAG AGC GAC GGG CGA TGT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e16S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16S1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCTG CTC AAT GAT TTT TTA AAT TGC TGT GG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBlack and Piesman [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16S2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTTA CGC TGT TAT CCC TAG AG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ecytB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCB-N-11367mo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATT ACC CCC CCT AAT TTA TTA GGA AT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSimon et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCB-J-10933mo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTAT ATT TTA CCC TGA GGG CAA ATA TC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ecoi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLCOI490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGT CAA CAA ATC ATA AAG ATA TTG G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFolmer et al. [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCO2198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTAA ACT TCA GGG TGA CCA AAA AAT CA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eITS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eITS2(5.8)F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCA TCG ATG TGA AYT GCA GGA CA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBeati et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eITS2(28)R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGTG AAT TCT ATG CTT AAA TTC AGG GGG T\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe PCR products were separated on a 1.5% agarose gel and visualized using the Bio-Rad gel documentation system with assisting visualization programming. All samples that had visible bands were sent to the Central Analytical Facility (CAF), Stellenbosch, South Africa for Sanger sequencing in both directions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eEach gene was analysed separately. Sequences were manually corrected on CLC main workbench version 23.0.2 (developed by CLC Bio, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.clcbio.com\u003c/span\u003e\u003cspan address=\"http://www.clcbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and compared with those available in the GenBank database using BLAST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/genbank/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/genbank/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (\u003cb\u003eAdditional file 2: Dataset S2\u003c/b\u003e). Contigs were assembled, sequence orientations were confirmed and alignments were generated alongside reference sequences using the online MAFFT version 7 (developed by \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mafft.cbrc.jp/alignment/server/index.html\u003c/span\u003e\u003cspan address=\"http://mafft.cbrc.jp/alignment/server/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with default parameters. The aligned matrices were manually viewed, edited, and truncated using MEGA 11 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The best fit model was determined for each individual gene using the jModelTest2 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] on the CIPRES Science Gateway (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.phylo.org/\u003c/span\u003e\u003cspan address=\"https://www.phylo.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) platform. The file format was changed depending on program requirements using FaBox version 1.61 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://users-birc.au.dk/palle/php/fabox/index.php\u003c/span\u003e\u003cspan address=\"https://users-birc.au.dk/palle/php/fabox/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Bayesian inference (BI) were performed in MrBayes version 3 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. For the Bayesian analysis, five Monte Carlo Markov Chains (MCMC) were run for 5,000,000 iterations, saving every 1,000th tree with relative burn-in at 25%. Tracer version 1.6 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] was used for inspection of estimated sample size (ESS) (\u0026gt;\u0026thinsp;200) and parameter sampling using graphical plots indicating parameter stabilisation. The resulting tree was visualised and edited in iTOL version 6.8 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA concatenated file was constructed including only the \u003cem\u003ecoi\u003c/em\u003e and \u003cem\u003ecytB\u003c/em\u003e genes. This was due to the lack of amplification in the \u003cem\u003e12S\u003c/em\u003e and \u003cem\u003e16S\u003c/em\u003e genes for all species. Alignment, model testing and BI analysis was done as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBarcode gap analysis\u003c/h2\u003e \u003cp\u003eGroups based on \u003cem\u003eAmblyomma\u003c/em\u003e spp. were created in Mega version 11 and the intra- and inter- species variation of these groups were compared. A pairwise distance matrix (p-distance) was obtained for each gene\u0026rsquo;s alignment using default parameters with 1,000 bootstrap replicates to calculate the standard error (SE). Barcode gap analysis was conducted using the automatic barcode gap discovery (ABGD) tool on the online web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html\u003c/span\u003e\u003cspan address=\"https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Analysis was conducted using default parameters (P\u003csub\u003emin\u003c/sub\u003e = 0.001; P\u003csub\u003emax\u003c/sub\u003e = 0.1; 10 steps and relative gap width X\u0026thinsp;=\u0026thinsp;1.5) using the Kimura (K80) TS/TV model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEthical Considerations\u003c/h2\u003e \u003cp\u003eThis study was approved by the Research Ethics Committee of the University of Pretoria (REC 121\u0026thinsp;\u0026minus;\u0026thinsp;20) and approval was also obtained from the Department of Agriculture, Land Reform and Rural Development (DALRRD), South Africa, under Section 20 of the Animal Diseases Act 1984 (Act no. 35 of 84) (12/11/1/1 (1937SS)).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eIn total 7,734 adult \u003cem\u003eAmblyomma\u003c/em\u003e spp. ticks were obtained from Angola, South Africa, Mozambique, Zambia, and Zimbabwe (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These ticks were identified morphologically to species level and described as: \u003cem\u003eA. eburneum\u003c/em\u003e, \u003cem\u003eA. hebraeum\u003c/em\u003e, \u003cem\u003eA. pomposum\u003c/em\u003e and \u003cem\u003eA. variegatum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). \u003cem\u003eAmblyomma eburneum\u003c/em\u003e was only collected in one location in central Mozambique (18˚S) (Fig.\u0026nbsp;2). \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e was collected in the north-eastern parts of South Africa, southern and central regions in Zimbabwe, and in the southern parts of Mozambique (below 21˚S), with two sampling points extending beyond what is currently documented as the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e endemic zone (19\u0026deg;S) (Fig.\u0026nbsp;2). \u003cem\u003eAmblyomma pomposum\u003c/em\u003e was only collected in Angola in the central regions expanding westwards (Fig.\u0026nbsp;2). \u003cem\u003eAmblyomma variegatum\u003c/em\u003e was collected in central northern regions of Mozambique (above 21˚S), northern regions of Zimbabwe, latitudinally across Zambia, and eastern parts of Angola (Fig.\u0026nbsp;2).\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eShort morphological descriptions\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eAmblyomma eburneum\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAmblyomma eburneum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) males have a brightly ornated conscutum that is red-brown in appearance with symmetrical vibrant beige patterns. The conscutum is smooth in appearance with small shallow punctations. Eyes are present and are slightly convex. The mesial area (central beige patch) is square in shape and is not connected to any of the other enamel patches. The falciform stripe (large \u0026ldquo;C\u0026rdquo; shaped enamel patches), beige in colouration, extends anteriorly and is connected indistinctly to the posterior enamel areas. The posterior median stripe is straight and wide, while notches in the posterior enamel areas are large, dividing the enamel area into two separate (but connected) patches. Festoons are rectangular with extensive enamelling (10 out of 11 festoons have varying degrees of enamelling; with the central festoon having no enamel). The lateral median areas of enamel ornamentation are distinctly present and connected to the posterior enamelled areas. They are large ovoid shapes with a beige appearance. Legs are dark in colour with pale rings. Females share several morphological features with the males, including the slightly convex eyes and banded legs. The scutum is red-brown in colouration with a glossy appearance. The mesial area of enamel ornamentation on the scutum is a large circular patch. Lateral areas of enamel ornamentation on the scutum are present forming two well-separated small patches on each lateral side. Two central circular patches are visible on the scutum. The scutum sides are slightly convex and the posterior angle is broad. The scutum appears smooth with small punctations.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAmblyomma hebraeum\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) males have a ornate conscutum, that is bright and red-brown in colouration with vibrant and metallic beige-red patterning over most of the surface. The conscutum appears to be smooth with small shallow punctations, situated in each lateral area. Eyes are present and flat. The mesial area of enamel ornamentation on the conscutum is elongate, stretching to just below the capitulum. The falciform stripe is almost triangular in appearance, beige in colouration, extends anteriorly and is clearly connected to the posterior enamel areas. The posterior median stripe is straight and narrow, with short and narrow notches in the posterior enamel areas. Festoons are rectangular with extensive enamelling (enamelling present on 9 out of 11 festoons; with the outermost festoons having no enamel). The lateral median areas of enamel ornamentation are distinctly present, complex and are unconnected to any other enamelled area. Legs are dark in colour with pale rings. Females have flat eyes that are marginalized on the scutum and banded legs. The scutum is red-brown in colouration. The mesial area of enamel ornamentation on the scutum is a large metallic patch covering the central third of the scutum. Lateral areas of enamel ornamentation on the scutum are distinctly present, forming \u0026ldquo;C\u0026rdquo;-shaped patches on each lateral side. The scutum sides are slightly convex and the posterior angle is broad. The scutum appears smooth with deep punctations near the eyes; with shallow and small punctations in the scapular and central areas.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003eAmblyomma pomposum\u003c/em\u003e\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAmblyomma pomposum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) males have a very bright and ornate conscutum that appears to be more vibrant and metallic in colouration compared to \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e. The conscutum is densely stippled with large punctuations. Eyes are present and are distinctly convex. The mesial area is oval in shape and is not connected to any of the other enamel patches. The falciform stripes resemble two claws, are orange in colouration, extend anteriorly andare connected to the posterior enamel areas. The postmedian stripe is narrow, while the notches in the posterior enamel areas are wide. Enamelling on the festoons are absent. The lateral median areas of enamel ornamentation are distinctly present as large trapezoids with a red appearance. Legs are dark in colour with pale rings. Females share several morphological features with the males including the convex eyes and banded legs. The scutum is dark in colouration with a glossy appearance. The mesial area of enamel ornamentation on the scutum is a small, faint circular patch. Lateral areas of enamel ornamentation on the scutum are present but indistinct. The scutum sides are straight and the posterior angle is narrower than that of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e. The scutum is densely stippled with large punctations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003eAmblyomma variegatum\u003c/em\u003e\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAmblyomma variegatum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) males have a brightly ornate conscutum that appears to be less vibrant and more muted in colouration compared to \u003cem\u003eA. pomposum\u003c/em\u003e males. The conscutum is densely stippled with medium to small punctations. Eyes are present and are distinctly convex. The mesial area has a more squared appearance and is connected to the falciform stripe, which is salmon in colouration, extends anteriorly and is connected to the posterior enamel areas. The posterior median stripe and the notches in the posterior enamel areas are narrow. Festoons have no enamel. The lateral median areas of enamel ornamentation are either absent or present as small circular patches. Legs are dark in colour with pale rings. Females share several morphological features with the males including the convex eyes and banded legs. The scutum is dark in colouration with a dull and more matt appearance. The mesial area of enamel ornamentation on the scutum is a small, clear circular patch. Lateral areas of enamel ornamentation on the scutum are present but indistinct. The scutum sides are slightly convex and the posterior angle is broad. The scutum is densely stippled with small to medium punctations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetics\u003c/h2\u003e \u003cp\u003eAmplification success varied amongst the genes; 12S and \u003cem\u003ecoi\u003c/em\u003e were the most successful, with amplification and contig assembly achieved with \u0026gt;\u0026thinsp;50% specimens. The 16S rRNA gene was poorly amplified, with the majority of the sequences excluded due to failure to align forward and reverse reads. \u003cem\u003eCytB\u003c/em\u003e was the most successful in producing sequences and contig assembly of these sequences. \u003cem\u003eITS2\u003c/em\u003e was excluded from further analyses due to low genetic variability only consisting of uninformative single nucleotide mutations preventing any systematic analyses. The number of sequences per species analysed for each gene varied (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All sequences were deposited in GenBank (\u003cb\u003eAdditional file 2: Dataset S2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe number of sequences analysed for the pairwise distance matrix and for the Automatic Barcode Gap Discovery (ABGD) analysis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies / Number of sequences analysed\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ecoi\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ecytB\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eConcatenated\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma eburneum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma hebraeum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma pomposum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma variegatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOutgroup (\u003cem\u003eRhipicephalus maculatus\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe average intra-species pairwise distances and the average inter-species pairwise distances are given in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, respectively. In all inter-species evaluations, \u003cem\u003eA. pomposum\u003c/em\u003e and \u003cem\u003eA. variegatum\u003c/em\u003e had the least amount of distance between them. The intra-species pairwise distances were on average lower than the average inter-species pairwise distances. The pairwise difference between individual sequences for each gene were also compared (\u003cb\u003eAdditional file 3: Dataset S3 to Additional file 7: Dataset S7\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEstimates of average evolutionary divergence over sequence pairs within groups (intraspecies p-distances).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies / Intraspecies p-distance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ecoi\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ecytB\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eConcatenated\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma eburneum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma hebraeum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma pomposum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma variegatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEstimates of evolutionary divergence over sequence pairs between groups (interspecies p-distances). Values in red indicate the intraspecies p-distances.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene Region\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma eburneum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma hebraeum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma pomposum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma variegatum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e12S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma eburneum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma hebraeum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma pomposum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.082\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.085\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma variegatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.085\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.086\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoutgroup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.166\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e16S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma eburneum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma hebraeum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma pomposum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.111\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma variegatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.058\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.078\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoutgroup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.240\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cem\u003ecoi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma eburneum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma hebraeum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma pomposum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma variegatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.023\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoutgroup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.190\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.187\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cem\u003ecytB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma eburneum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma hebraeum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma pomposum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma variegatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.151\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.064\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoutgroup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.206\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.228\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eConcatenated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma eburneum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma hebraeum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma pomposum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmblyomma variegatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoutgroup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.202\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.188\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhen evaluating the \u003cem\u003e12S\u003c/em\u003e estimations of evolutionary divergence between each of the sequences, low levels of intra-species variation were observed. The intra-species variation of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e ranged from 0 to 0.074, whereas in \u003cem\u003eA. hebraeum\u003c/em\u003e, it ranged from 0 to 0.045. Low levels of intraspecific variation were also observed in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e, ranging from 0 to 0.024. \u003cem\u003eAmblyomma pomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e had low levels of inter-species variation amounting to an average of 0.008. The intra-species variation for \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e ranged from 0 to 0.008 for sequences obtained for this study, while nine sequences (four Zambian sequences from this study, and five reference sequences from other countries) had greater variation. The ABGD analysis indicated four operational taxonomic units (OTUs), grouping the majority of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e. One \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e sequence formed its own OTU (\u003cb\u003eAdditional file 8: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003e16S\u003c/em\u003e pairwise sequence matrix depicts no intraspecific variation between the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e sequences, although only two sequences were available. The average inter-species variation between \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e was 0.050. Low levels of intra-species variation were observed in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e, ranging from 0 to 0.009; while for \u003cem\u003eA. pomposum\u003c/em\u003e it ranged from 0 to 0.052. High levels of intra-species variation were observed for \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e, ranging from 0 to 0.421, with three sequences (two from Mozambique and one from Uganda) contributing most variation. The ABGD analysis indicated seven OTUs. \u003cem\u003eAmblyomma eburneum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e formed their own OTUs, whereas \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e clustered in the same OTU with the majority of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e. The three \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e sequences that showed the highest diversity formed their own OTUs (\u003cb\u003eAdditional file 9: Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe analysis of the \u003cem\u003ecoi\u003c/em\u003e pairwise sequence matrix illustrated low levels of intraspecific variation for \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e, ranging from 0 to 0.071 with two sequences (both from Mozambique) with higher levels of variation. The intra-species variation in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e ranged from 0 to 0.025, while \u003cem\u003eA. pomposum\u003c/em\u003e it ranged from 0 to 0.013. High levels of variation in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e were observed, ranging from 0 to 0.073. The ABGD analysis indicated five OTUs, grouping \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e together. \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e clustered into one main group. \u003cem\u003eAmblyomma eburneum\u003c/em\u003e separated into two main groups, one containing the majority of the sequences while the other group consisted of the two variable sequences (\u003cb\u003eAdditional file 10: Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ecytB\u003c/em\u003e pairwise distance matrix depicted low levels of intra-species variation ranging from 0 to 0.006 in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e. The intra-species variation in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e ranged from 0 to 0.037 with one sequence from Mozambique exhibiting high levels of variation. Intra-species variation for \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e ranged from 0 to 0.045 with three sequences that had a higher variation compared to the rest. \u003cem\u003eAmblyomma variegatum\u003c/em\u003e had high levels of intra-species variation ranging from 0 to 0.609 with two sequences showing greater variation. The ABGD analysis indicated six OTUs, grouping \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e together. \u003cem\u003eAmblyomma variegatum\u003c/em\u003e clustered into three groups; one main clade and two separate singleton OTUs. \u003cem\u003eAmblyomma eburneum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e and the outgroup clustered in cognate groups (\u003cb\u003eAdditional file 11: Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eFor the concatenated alignment, the pairwise distance matrix illustrated low levels of intraspecific variation. For \u003cem\u003eAmblyomma eburneum\u003c/em\u003e it ranged from 0.001 to 0.004, while for \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e ranged from 0 to 0.070. The intraspecific variation for \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e was 0.008; however, only two sequences were compared. Intraspecific variation of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e ranged from 0.004 to 0.056. The ABGD analysis indicated four OTUs: \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e formed their own groups, while \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e clustered with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe BI analysis of the \u003cem\u003e16S\u003c/em\u003e gene illustrated a clear separation between \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e, but this was not the case for \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e, as can be seen by the nesting of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e within the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e clades. The BI analysis of the \u003cem\u003e12S\u003c/em\u003e gene also divided \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e into well-supported clades; however, while most \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e specimens formed one branch that split from the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e clade with 0.69 probability, two \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e sequences were not separated from the main \u003cem\u003eA. variegatum\u003c/em\u003e clade. The BI analysis of the \u003cem\u003ecoi\u003c/em\u003e gene resolved all species; \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e branched from the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e clade with 0.96 probability, and all individuals grouped in the same cluster, although the branch length was very short. This contrasted somewhat with the BI analysis of the \u003cem\u003ecytB\u003c/em\u003e gene, in which \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e was positioned as a derived sister group to the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e lineage with a probability of 0.79. The concatenated BI analysis depicts clear differentiation between all species while \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e branched from the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e clade with 0.99 probability and all individuals grouped in the same cluster. Three \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e sequences from Mozambique also branched from the main cluster with a probability of 1.00. All BI analyses revealed relatively little intra-species variation among ticks of the same species from different countries, which clustered together without marked geographic structuring, although a clade composed of a subset of \u003cem\u003eA. variegatum\u003c/em\u003e from Mozambique was robustly supported in the concatenated analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigated the intra- and inter-species variation of \u003cem\u003eAmblyomma\u003c/em\u003e spp. collected in southern Africa. In total 7,734 adult \u003cem\u003eAmblyomma\u003c/em\u003e ticks were collected and morphologically identified as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e, \u003cem\u003ehebraeum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e. However, based on our analyses of multiple genes, we cannot conclude that \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e are distinct species.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAmblyomma eburneum\u003c/em\u003e is described as an eastern African species with a documented geographical distribution ranging from Somalia, Eritrea and Ethiopia in the north, and south through Kenya, Tanzania and Zimbabwe [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In this study we collected \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e from central Mozambique in the Sofala province from African buffalo. \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e, from cattle, was collected in South Africa, Mozambique, and Zimbabwe, corresponding with its documented geographical distribution [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The geographical spread of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e was described by Robinson [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], Walker and Olwage [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and Petney et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] to range from Angola to the western regions of Zambia, and northwards to the southern parts of the Democratic Republic of Congo. In the present study, \u003cem\u003eAmblyomma\u003c/em\u003e spp. were also collected in these regions, from the western parts of Angola and latitudinally to the eastern parts of Zambia. We found \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e to be restricted to the central-western parts of Angola, whereas \u003cem\u003eAmblyomma\u003c/em\u003e specimens collected in eastern Angola were identified as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e. In this study, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e was collected in Angola, Mozambique, Zambia and Zimbabwe, corresponding with records of Petney et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and Walker and Olwage [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe most predominant collected species was \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e from South Africa and Mozambique, while the species with the lowest representation was \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e from Angola. The low recovery of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e in Angola, could be ascribed to the collections occurring in March, which is at the end of the adult season (the months when the maximum infestations occur have been documented as November and December [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]). \u003cem\u003eAmblyomma eburneum\u003c/em\u003e was also collected in low numbers, but this can be attributed to the difficulty of collecting ticks from wildlife species. The collections were highly dependent on the amount of wildlife that was purchased and legally hunted in the timeframe of this study. The low prevalence of \u003cem\u003eAmblyomma\u003c/em\u003e spp. from Zimbabwe was likely a result of a vigorous campaign by the government to assist livestock farmers in treating their cattle against ticks by encouraging regular acaricde dipping of the animals, as an attempt to control major outbreaks of bovine theileriosis.\u003c/p\u003e \u003cp\u003eThis study aimed to provide insight to the unresolved debate that has been ongoing for decades as described in the introduction. Both Robinson [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and Dias (1950,1953) described morphological variation in the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e found in Mozambique and suggested new species names \u003cem\u003eA. variegatum\u003c/em\u003e var. nocens and \u003cem\u003eA. variegatum\u003c/em\u003e var. \u003cem\u003egovurensis\u003c/em\u003e, respectively. Dias [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] also examined what was described as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e from Angola and concluded that it does not resemble the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e as described by D\u0026ouml;nitz [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], suggesting a new species description as \u003cem\u003eAmblyomma superbum\u003c/em\u003e. In this study, morphological variation in the collected \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e ticks were observed; however, the majority resembled that of the original description by Fabricius [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Morphology is the most important aspect for taxonomy, highlighting the importance of variation in ornamentation. These conflicting observations of morphology in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e resemble the controversy of the \u003cem\u003eAmblyomma marmoreum\u003c/em\u003e complex [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eAmblyomma marmoreum\u003c/em\u003e complex encompasses five African species, namely: \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emarmoreum\u003c/em\u003e sensu stricto, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esparsum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efalsomarmoreum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003enuttalli\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epaulopunctatum\u003c/em\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. As with this study, the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emarmoreum\u003c/em\u003e complex was placed under scrutiny due to a lack of genetic and ecological data on its members. Although meticulous descriptions of the five species in the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emarmoreum\u003c/em\u003e complex are available, identification remains a challenge and misidentification often occurs, even confusing these species with other \u003cem\u003eAmblyomma\u003c/em\u003e spp. outside of the complex [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, Cotes-Perdomo et al. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] was able to differentiate between the species with the use of molecular techniques targeting a large part of the mitogenome. Overall, they were able to concatenate and compare 13 protein-coding genes and two ribosomal genes of several \u003cem\u003eAmblyomma\u003c/em\u003e spp.\u003c/p\u003e \u003cp\u003eIn the attempt to provide clarity on the phylogenetic positioning of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e, molecular analyses were conducted on these four \u003cem\u003eAmblyomma\u003c/em\u003e species with the use of 12S, 16S, \u003cem\u003ecoi\u003c/em\u003e, \u003cem\u003ecytB\u003c/em\u003e and ITS2 molecular markers. However, the ITS2 marker was excluded from further analyses due to uninformative single nucleotide mutations. This research provides additional sequences that have been deposited in the GenBank database for all other markers utilized here, including the first for the 12S and \u003cem\u003ecytB\u003c/em\u003e genes of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e and first for the 12S, \u003cem\u003ecoi\u003c/em\u003e and \u003cem\u003ecytB\u003c/em\u003e genes of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e. During the course of this study, several challenges with amplification occurred for all genes. The Chelex extraction method has several drawbacks including the rapid degradation of the extracted DNA after two years of storage and the effects of long-term storage on the binding of impurities to DNA. Singh et al. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] noted that samples extracted with the Chelex 100 resin method and stored for extended periods of time tended to contain contaminants such as proteins attached to the DNA helix, and these prevented successful PCR reactions. The importance of the impact of storage time on DNA integrity was evident through the successful amplification of \u003cem\u003ecytB\u003c/em\u003e, which was conducted with DNA extracted less than one year previously, compared with the less successful amplification of 12S, 16S, \u003cem\u003ecoi\u003c/em\u003e and ITS2, which were conducted using DNA stored for approximately two years. Additionally, several amplified products did not generate sequences of good quality, and were thus excluded from further analysis, lowering the sample size. The 12S and 16S rRNA gene markers were particularly difficult to amplify in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e samples and those that did produce bands for these markers did not provide high-quality sequences. For future studies, it would be ideal to use recently extracted DNA when using the Chelex extraction method and DNA should be stored at -80\u0026deg;C, which would maintain the integrity of the DNA for a longer period; or alternative DNA extraction methods should be used that will not compromise the DNA integrity during storage.\u003c/p\u003e \u003cp\u003eThe ABGD analysis indicated that \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e was divided into two OTUs in the 12S and \u003cem\u003ecoi\u003c/em\u003e analysis, while only forming one OTU in the 16S, \u003cem\u003ecytB\u003c/em\u003e, and concatenated analyses. All the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e 12S sequences clustered with the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e OTU, except for one which formed its own OTU. However, upon investigation, the sample that formed its own OTU was of suboptimal quality and several incongruencies was found between the forward and reverse sequences. The intraspecific variability for \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e was greater for the 12S (0.018) and \u003cem\u003ecoi\u003c/em\u003e (0.021) genes, while the inter-species variability was sufficient to differentiate between species for 16S, \u003cem\u003ecoi\u003c/em\u003e, \u003cem\u003ecytB\u003c/em\u003e and in the concatenated analyses. The inter-species variability was insufficient for the \u003cem\u003e12S\u003c/em\u003e analysis, as can be seen in the clustering with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e during the ABGD analysis, though a clear separation was obtained in the phylogenetic tree. The ABGD analysis indicates that \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e only formed one OTU in all the single gene and concatenated analyses, although the intra-species variability for \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e was greater in the concatenated pairwise distance analysis. This supports the clear separation of this species from the other \u003cem\u003eAmblyomma\u003c/em\u003e spp. of southern Africa. Strikingly, \u003cem\u003eAmblyomma pomposum\u003c/em\u003e clustered with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e in the ABGD analyses for all the individual genes and in the concatenated analyses. The intraspecific variability in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e was greatest in the 16S and the \u003cem\u003ecytB\u003c/em\u003e analysis. \u003cem\u003eAmblyomma pomposum\u003c/em\u003e clustered within \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e for the 12S, 16S, \u003cem\u003ecoi\u003c/em\u003e and concatenated phylogenetic analyses; however, in the \u003cem\u003ecytB\u003c/em\u003e analysis it segregated as the ancestral lineage. The intraspecific variation within \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e was greater than the interspecific variation between \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e for the 12S, 16S, \u003cem\u003ecytB\u003c/em\u003e and concatenated analyses. This may indicate that there is insufficient variation between \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e to confidently describe these two species as distinct. The ABGD analyses for \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e indicated four OTUs for the 16S and three OTUs for the \u003cem\u003ecytB\u003c/em\u003e genes, while the 12S, \u003cem\u003ecoi\u003c/em\u003e and concatenated analyses indicated only one OTU. Two of the OTUs in the 16S analysis were the result of suboptimal sequences with several incongruencies between the forward and reverse sequences, while the other was a reference sequence from GenBank which covered a larger section of the \u003cem\u003e16S\u003c/em\u003e gene. As with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e, the intra-species variation for \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e was greater in the 16S and \u003cem\u003ecytB\u003c/em\u003e genes.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis of the \u003cem\u003eAmblyomma\u003c/em\u003e spp. indicates that all genes used in this study were adequate to differentiate between \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e and the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e/\u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e complex. The different patterns that emerge between the phylogenetic trees could be a result of the differential mutation rates of each of the molecular markers [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Erster et al. [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and Koroiva and Santana [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] evaluated the marker efficiency of 12S, 16S, \u003cem\u003ecoi\u003c/em\u003e and \u003cem\u003ecytB\u003c/em\u003e, which were also used in the current study, and demonstrated that the mitochondrial markers \u003cem\u003ecoi\u003c/em\u003e and \u003cem\u003ecytB\u003c/em\u003e were most suitable for intra- and inter-species analyses due to their high variability. Furthermore, Vences et al. [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] reported that \u003cem\u003ecytB\u003c/em\u003e was the most variable of the two markers, allowing for clearer separation between closely related species. Norris et al. [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] found that 16S was the least variable marker when comparing 12S and 16S markers for \u003cem\u003eIxodes scapularis\u003c/em\u003e population genetics. In the current study, \u003cem\u003ecytB\u003c/em\u003e proved to be the most efficient single marker to differentiate between southern African \u003cem\u003eAmblyomma\u003c/em\u003e spp., while none of the markers in isolation allowed for clear discrimination between samples of the same species from different countries. However, there was strong support for intra-specific population structure in \u003cem\u003eA. variegatum\u003c/em\u003e in the concatenated analysis.\u003c/p\u003e \u003cp\u003eIn this context, a previous analysis of intraspecific variation of \u003cem\u003eA. variegatum\u003c/em\u003e molecular markers concluded that genetic diversity was low in West Africa and in introduced Caribbean populations (\u003cem\u003ei.e\u003c/em\u003e., nucleotide diversity of 0.02\u0026ndash;0.25% for 12S), with higher variation in East Africa (\u003cem\u003ei.e\u003c/em\u003e., 0.65% for 12S) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Although the study did not apply the more variable markers as used in the present work, the 12S data alone suggests that southern populations of \u003cem\u003eA. variegatum\u003c/em\u003e show greater genetic variation (nucleotide diversity of 1%) than elsewhere on the continent. Notably, a population genetic study of \u003cem\u003eA. variegatum\u003c/em\u003e in Burkina Faso used microsatellites and concluded that effective population sizes were low at the village level when sampling domestic ruminants [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Similar approaches could also be applied to populations from southern Africa in future studies. Recently, mitochondrial markers were analysed to determine the genetic structure of another African \u003cem\u003eAmblyomma\u003c/em\u003e spp., the elephant tick \u003cem\u003eA. tholloni\u003c/em\u003e, on host populations in Kenya. The intra-specific variation at the \u003cem\u003ecoi\u003c/em\u003e locus was found to be low and of similar magnitude to that of \u003cem\u003eA. hebraeum\u003c/em\u003e and \u003cem\u003eA. pomposum\u003c/em\u003e in the current study (\u0026lt;\u0026thinsp;1%) [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePopulation genetic studies of \u003cem\u003eAmblyomma\u003c/em\u003e spp. have been more extensive in the New World and offer important lessons for understanding intra-specific variation in the Afrotropical species. There are marked differences between the low intra-specific variation observed in some Neotropical species (\u003cem\u003ee.g\u003c/em\u003e., \u003cem\u003eAmblyomma triste\u003c/em\u003e [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] and \u003cem\u003eAmblyomma aureolatum\u003c/em\u003e [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], where it was \u0026lt;\u0026thinsp;1% for mitochondrial markers) compared with others such as \u003cem\u003eAmblyomma ovale\u003c/em\u003e [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] and \u003cem\u003eAmblyomma mixtum\u003c/em\u003e [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], where pairwise distances for concatenated mitochondrial markers can reach 3\u0026ndash;5%. \u003cem\u003eAmblyomma cajennense\u003c/em\u003e, which has an extensive distribution across subtropical and tropical regions of the Americas, constitutes a particularly interesting paradigm for the genus. Variation exceeding 8% at the whole mitogenome level between geographically and ecologically distinct populations of \u003cem\u003eA. cajennense\u003c/em\u003e formed part of the evidence that was used to designate a species complex and formally describe its members as distinct species [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The Afrotropical species await similar rigorous analyses using whole mitogenome data across the entirety of their range, in line with the recent study on the \u003cem\u003eAmblyomma marmoreum\u003c/em\u003e complex [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile the current study was not designed to address intraspecific population structure in depth, the concatenated phylogenetic analysis depicted clear differentiation between each of the analysed species; albeit \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e branched from the main cluster of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e with a very short genetic distance, no greater than intra-specific variation within \u003cem\u003eA. variegatum\u003c/em\u003e. An important limitation was that the concatenated tree was only constructed with two genes, \u003cem\u003ecoi\u003c/em\u003e and \u003cem\u003ecytB\u003c/em\u003e. This was because not all loci had amplified successfully for each specimen, and also due to Koroiva and Santana [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u0026rsquo;s conclusion that \u003cem\u003ecoi\u003c/em\u003e and \u003cem\u003ecytB\u003c/em\u003e markers were more suitable for intra- and inter-specific delineation. These were also the only markers that had sufficient representation amongst all the \u003cem\u003eAmblyomma\u003c/em\u003e spp. from this study.\u003c/p\u003e \u003cp\u003eThus, based on the phylogenetic analysis of the individual genes, the pairwise distance analyses and the ABGD analyses, we cannot conclude that \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e are distinct species. Literature on systematic work with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e is scarce and currently there are no alternative methods to compare differences or similarities between these two species, except for morphological descriptions. No previous studies on the phylogenetic relationship of these two species have been conducted and, as discussed above, there is a scarcity of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e sequences available in GenBank. Kobayashi et al. [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] were the first authors who published the only sequences currently available for the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e on the GenBank database. They collected 15 \u003cem\u003eAmblyomma\u003c/em\u003e ticks and morphologically identified them as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13) and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2) with the use of Walker et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. They then conducted a phylogenetic analysis using the 16S rRNA gene. The \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e that they identified clustered within the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e clade. This may suggest that the current available sequences are from misidentified \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e ticks. Balinandi et al. [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] morphologically identified ticks they collected from Uganda as belonging to \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e; however, due to a lack of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e sequences at the time, phylogenetic analyses of the 16S gene clustered \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e. This discovery led the authors to believe that they had misidentified \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e ticks as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e. Our 16S phylogenetic analysis depicts \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e dispersed within the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e cluster. Based on our analysis of several genes, it is possible that Balinandi et al. [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] correctly identified their ticks as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e; however, since they did not upload any sequences nor depicted the morphological discrepancy, no definite conclusion can be made based on this information. On the other hand, a study performed by Barradas et al. [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] collected 116 ticks from the Huambo province in Angola. With the use of morphological identification using identification keys from Walker et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] as well as molecular identification targeting the 12S and 16S rDNA genes, they identified their 11 (10%) \u003cem\u003eAmblyomma\u003c/em\u003e ticks as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e. As with the 16S, the 12S also depicts clustering of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e within \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e. In our study we collected from the same sights in Huambo and identified the \u003cem\u003eAmblyomma\u003c/em\u003e spp. circulating in the area as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e. Our findings are also supported by Sili et al. [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], who identified all the \u003cem\u003eAmblyomma\u003c/em\u003e spp. in the area as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e. Based on our findings we believe that Barradas et al. [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] misidentified their \u003cem\u003eAmblyomma\u003c/em\u003e spp. as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e instead of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAlthough the phylogenetic analyses cannot differentiate between \u003cem\u003eA. variegatum\u003c/em\u003e and \u003cem\u003eA. pomposum\u003c/em\u003e currently, sufficient morphological features are documented to distinguish these species from each other. The other main factor in defining a species is the ecology and biological habitats [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Mayr [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] stated that a species can be described as \u0026ldquo;The segregation of the total genetic variability of nature into discrete packages, so called species, which are separated from each other by reproductive barriers, prevents the production of too great a number of disharmonious, incompatible gene combinations. This is the basic biological meaning of species and this is the reason why there are discontinuities between sympatric species\u0026rdquo;. A clear parapatric boundary was observed between \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e; however, a hypothesis of incipient speciation has yet to be tested. We suggest, alongside whole genome phylogenetic analysis, mating and hybrid viability studies between the two species to confirm their reproductive isolation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cem\u003eCytB\u003c/em\u003e was the most successful marker in differentiating between \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eeburneum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e, closely followed by \u003cem\u003ecoi\u003c/em\u003e. The concatenated tree distinguished between \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e; however, the pairwise distance and ABGD analyses suggest there is insufficient evidence that \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e are distinct species. In our study, no \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e ticks were collected in any country other than Angola, challenging the current distribution as proposed by Theiler and Salisbury [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], Walker et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and other current literature. Based on our findings, we suggest comparing whole mitochondrial genomes of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e with those of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e to determine whether these are distinct species or \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e is a subspecies of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e. Moreover, further investigations should also be conducted using mating and hybrid viability studies between the two species to determine if they are reproductively isolated.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eABGD: Automatic Barcode Gap Discovery; BI: Bayesian inference; CAF: Central Analytical Facility; ESS: Estimates sample size; MCMC: Monte Carlo Markov Chains; OTU\u0026rsquo;s: operational taxonomic units; SE: Standard error; spp: species\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by AgriSETA; the Meat industry Trust; and the doctorate research bursary awarded to me by the University of Pretoria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA special thank you to Delta Safaris, Mungari, who allowed us to collect ticks from the hunted wildlife and hosted us in their camp and to all field technicians and state veterinarians who assisted in the collection of ticks in the represented countries. We would also like to acknowledge Prof Melvyn Quan and Ms\u0026nbsp;Zandile Mkhize, who took all the morphological photos of the ticks that are presented in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. The sequences generated for each tick species for each gene during the current study are available in the GenBank repository, https://www.ncbi.nlm.nih.gov/genbank/.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. Smit\u003c/strong\u003e:\u0026nbsp;Conceptualization, Sample collection, Methodology, Investigation, Data Curation, Writing - Original Draft, Visualization\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.C. Mulandane\u003c/strong\u003e:\u0026nbsp;Sample collection, Writing - Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM. Labuschagne\u003c/strong\u003e:\u0026nbsp;Investigation, Writing - Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS.H. Wojick\u003c/strong\u003e:\u0026nbsp;Investigation, Writing - Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. Malabwa\u003c/strong\u003e:\u0026nbsp;Sample collection, Investigation, Writing - Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG. Sili\u003c/strong\u003e:\u0026nbsp;Sample collection, Writing - Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS. Mandara\u003c/strong\u003e:\u0026nbsp;Sample collection, Writing - Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZ. Dlamkile\u003c/strong\u003e:\u0026nbsp;Sample collection, Writing - Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eW.H. Stoltsz\u003c/strong\u003e:\u0026nbsp;Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH. Rose Vineer\u003c/strong\u003e: Visualization,\u0026nbsp;Writing - Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK. Huber\u003c/strong\u003e:\u0026nbsp;Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI. G. Horak\u003c/strong\u003e: Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. Morar-Leather\u003c/strong\u003e:\u0026nbsp;Writing - Review \u0026amp; Editing, Supervision, Funding acquisition\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. L. Makepeace\u003c/strong\u003e:\u0026nbsp;Writing - Review \u0026amp; Editing, Supervision\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL. Neves\u003c/strong\u003e: Conceptualization, Sample Collection, Methodology, Writing - Review \u0026amp; Editing, Supervision, Funding acquisition\u003cstrong\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNicholson WL, Sonenshine DE, Noden BH, Brown RN. Ticks (ixodida). Med Vet Entomol: Elsevier; 2019. p.\u0026nbsp;603\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuglielmone A, Robbins R, Apanaskevich D, Petney T, Estrada-Pe\u0026ntilde;a A, Horak I. The Hard Ticks of the World (Acari: Ixodida: Ixodidae) Dordrecht, The Netherlands: Springer; 2014.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEstrada Pena A, Mangold AJ, Nava S, Venzal JM, Labruna M, Guglielmone AA. A review of the systematics of the tick family Argasidae (Ixodida). Acarologia. 2010;50(3):317\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarnecke M, Schein E, Voigt W, Uilenberg G, Young A. Development of \u003cem\u003eTheileria mutans\u003c/em\u003e (Theiler, 1906) in the gut and the haemolymph of the tick \u003cem\u003eAmblyomma variegatum\u003c/em\u003e (Fabricius, 1794). Z Parasitenkd. 1980;62:119\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalker JB. A review of the ixodid ticks (Acari, Ixodidae) occurring in southern Africa. Onderstepoort Journal of Veterinary Research. 1991;58(2):81\u0026ndash;105.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHorak IG, Heyne H, Williams R, Gallivan GJ, Spickett AM, Bezuidenhout JD, et al. The ixodid ticks (Acari: Ixodidae) of Southern Africa. Cham, Switzerland: Springer; 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVoltzit O, Keirans J. A review of African \u003cem\u003eAmblyomma\u003c/em\u003e species (Acari, Ixodida, Ixodidae). Acarina. 2003;11(2):135\u0026ndash;214.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalker AR, Bouattour A, Camicas JL, Estrada-Pena A, Horak IG, Latif AA, et al. Ticks of domestic animals in Africa: a guide to identification of species: Bioscience Reports Edinburgh; 2003.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBournez L, Cangi N, Stachurski F, Lancelot R, Martinez D, Lefran\u0026ccedil;ois T, et al., editors. Is the distribution of \u003cem\u003eAmblyomma variegatum\u003c/em\u003e influenced by interspecific competition with \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e? Preliminary study: distribution range in Mozambique2012: European Society for Vector Ecology.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalker JB, Olwage A. The tick vectors of \u003cem\u003eCowdria ruminantium\u003c/em\u003e (Ixodoidea, Ixodidae, genus \u003cem\u003eAmblyomma\u003c/em\u003e) and their distribution. Onderstepoort Journal of Veterinary Research. 1987;54:353\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTheiler G, Robinson BN. Tick survey. VIII. Checklists of ticks recorded from the Belgian Congo and Ruanda Urundi, from Angola, and from Northern Rhodesia. Onderstepoort J Vet Res. 1954;26(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMacLeod J. Tick infestation patterns in the southern province of Zambia. Bull Entom Res. 1970;60(2):253\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJack R. Ticks infesting Domestic Animals in S. Rhodesia. Rhod agric j. 1936;33(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoogstraal H, Theiler G. Ticks (Ixodoidea, Ixodidae) parasitizing lower primates in Africa, Zanzibar, and Madagascar. J Parasitol Res. 1959;45(2):217\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoward C. A list of the ticks of South Africa. Annals of the Transvaal Museum. 1908;1(2):73\u0026ndash;169.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHorak IG. A century of tick taxonomy in South Africa. Onderstepoort Journal of Veterinary Research. 2009;76(1):67\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoogstraal H, Aeschlimann A. Tick-host specificity. Bulletin de la Soci\u0026eacute;t\u0026eacute; Entomologique Suisse. 1982;55:5\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKushimo O. The tick genus \u003cem\u003eAmblyomma\u003c/em\u003e in Africa: phylogeny and mutilocus DNA barcoding: Georgia Southern University; 2013.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePillay A, Nyangiwe N, Mukaratirwa S. Low genetic diversity and population structuring of \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e and \u003cem\u003eRickettsia africae\u003c/em\u003e from coastal and inland regions in the Eastern Cape Province of South Africa. Med Vet Entomol. 2022;37:275\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeati L, Patel J, Lucas-Williams H, Adakal H, Kanduma EG, Tembo-Mwase E, et al. Phylogeography and demographic history of \u003cem\u003eAmblyomma variegatum\u003c/em\u003e (Fabricius)(Acari: Ixodidae), the tropical bont tick. Vector Borne Zoonotic Dis. 2012;12(6):514\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKelava S, Mans BJ, Shao R, Moustafa MAM, Matsuno K, Takano A, et al. Phylogenies from mitochondrial genomes of 120 species of ticks: Insights into the evolution of the families of ticks and of the genus \u003cem\u003eAmblyomma\u003c/em\u003e. Ticks Tick Borne Dis. 2021;12(1):101577.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDias J. Sobre a posi\u0026ccedil;\u0026atilde;o sistem\u0026aacute;tica de algumas esp\u0026eacute;cies africanas do g\u0026eacute;nero \u003cem\u003eAmblyomma\u003c/em\u003e CL Koch (Acarina\u0026ndash;Ixodoidea). Mo\u0026ccedil;ambique. 1953;73:119\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTheiler G, Salisbury LE. Ticks in the South African zoological survey collection-Part IX-The \u003cem\u003eAmblyomma marmoreum\u003c/em\u003e group. Onderstepoort Journal of Veterinary Research. 1959;28(1):47\u0026ndash;124.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFabricius JC. Entomologia systematica: impensis CG Proft; 1794.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD\u0026ouml;nitz. Dber das Zeckengenus \u003cem\u003eAmblyomma\u003c/em\u003e. Schrift Ges Nat Freund Berlin. 1909;8: 440\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobinson L. New species of ticks (\u003cem\u003eHaemaphysalis\u003c/em\u003e, Amblyomma). Parasitology. 1911;4(4):478\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDias JATdS. List of Mozambique ticks and their known hosts. Anais dos Servicos de Veterinaria e Industria Animal. 1950(3):227.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuglielmone AA, Robbins RG, Apanaskevich DA, Petney TN, Estrada-Pe\u0026ntilde;a A, Horak IG. Comments on controversial tick (Acari: Ixodida) species names and species described or resurrected from 2003 to 2008. Experimental and Applied Acarology. 2009;48:311\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDlamkile Z, Neves L, Morar-Leather D, Brandt C, Pretorius A, Steyn H, et al. Characterization of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003eruminantium\u003c/em\u003e Field Isolates from \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e Ticks Collected from Cattle in Three South African Provinces Using Multi-Locus Sequence Typing. In: NETWORK, S S R (ed). 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErni S. Capture One Pro 9: Mastering Raw Development, Image Processing, and Asset Management: Rocky Nook, Inc.; 2016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSystems Z. Zerene Stacker, Version 1.04. Zerene Systems Richland, WA; 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmit A, Mulandane FC, Wojcik SH, Horak IG, Makepeace BL, Morar-Leather D, et al. Sympatry of \u003cem\u003eAmblyomma eburneum\u003c/em\u003e and \u003cem\u003eAmblyomma variegatum\u003c/em\u003e on African buffaloes and prevalence of pathogens in ticks. Ticks and Tick-Borne Diseases. 2023;14(6):102247.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeati L, Keirans JE. Analysis of the systematic relationships among ticks of the genera \u003cem\u003eRhipicephalus\u003c/em\u003e and \u003cem\u003eBoophilus\u003c/em\u003e (Acari: Ixodidae) based on mitochondrial 12S ribosomal DNA gene sequences and morphological characters. Journal of Parasitology. 2001;87(1):32\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlack WC, Piesman J. Phylogeny of hard-and soft-tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences. Proceedings of the National Academy of Sciences. 1994;91(21):10034-8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am. 1994;87(6):651\u0026ndash;701.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021;38(7):3022\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDarriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods. 2012;9(8):772-.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRonquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003;19(12):1572\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRambaut A, Suchard MA, Xie D, Drummond A. Tracer 1.6 2014 [Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://beast.bio.ed.ac.uk/Tracer\u003c/span\u003e\u003cspan address=\"http://beast.bio.ed.ac.uk/Tracer\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLetunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Research. 2021;49(W1):W293-W6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePuillandre N, Lambert A, Brouillet S, Achaz G. ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Mol Ecol. 2012;21(8):1864\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetney T, Horak I, Rechav Y. The ecology of the African vectors of heartwater, with particular reference to \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e and \u003cem\u003eAmblyomma variegatum\u003c/em\u003e. Onderstepoort J Vet Res. 1987;54:381\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobinson LE. The Genus \u003cem\u003eAmblyomma\u003c/em\u003e.-Ticks: A Monograph of the Ixodoidea, Part IV. In: Press CU, editor. Ticks: a monograph of the Ixodidae. 2. London1926. p.\u0026nbsp;99\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCotes-Perdomo AP, Sanchez-Vialas A, Thomas R, Jenkins A, Uribe JE. New insights into the systematics of the Afrotropical \u003cem\u003eAmblyomma marmoreum\u003c/em\u003e complex (Acari, Ixodidae) and a novel \u003cem\u003eRickettsia africae\u003c/em\u003e strain using morphological and metagenomic approaches. bioRxiv. 2023:2023.08. 18.553479.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh UA, Kumari M, Iyengar S. Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin. Biol Proced Online. 2018;20(1):1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoth A, Akad F, Zonstein I, King R, Orshan L, Erster O. Molecular characterization of six \u003cem\u003eHyalomma\u003c/em\u003e species using mitochondrial markers. Ticks and Tick-Borne Diseases. 2019;10(4):911\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErster O, Roth A, Avni Z, King R, Shkap V. Molecular detection of \u003cem\u003eRickettsia bellii\u003c/em\u003e in \u003cem\u003eAmblyomma rotundatum\u003c/em\u003e from imported red-footed tortoise (\u003cem\u003eChelonoides carbonaria\u003c/em\u003e). Ticks Tick Borne Dis. 2015;6(4):473\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoroiva R, Santana D. Evaluation of partial 12S rRNA, 16S rRNA, \u003cem\u003eCOI\u003c/em\u003e and \u003cem\u003eCytb\u003c/em\u003e gene sequence datasets for potential single DNA barcode for hylids (Anura: Hylidae) An Acad Bras Cienc. 2022;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVences M, Thomas M, Bonett RM, Vieites DR. Deciphering amphibian diversity through DNA barcoding: chances and challenges. Philos Trans R Soc Lond, B, Biol Sci. 2005;360(1462):1859\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNorris DE, Klompen JSH, Keirans JE, Black IV WC. Population genetics of Ixodes scapularis (Acari: Ixodidae) based on mitochondrial 16S and 12S genes. J Med Entomol. 1996;33(1):78\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuber K, Jacquet S, Rivallan R, Adakal H, Vachi\u0026eacute;ry N, Risterucci A-M, et al. Low effective population sizes in \u003cem\u003eAmblyomma variegatum\u003c/em\u003e, the tropical bont tick. Ticks Tick Borne Dis. 2019;10(1):93\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKing'ori EM, Obanda V, Nyamota R, Remesar S, Chiyo PI, Soriguer R, et al. Population genetic structure of the elephant tick \u003cem\u003eAmblyomma tholloni\u003c/em\u003e from different elephant populations in Kenya. Ticks and Tick-Borne Diseases. 2022;13(3):101935.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuglielmone AA, Nava S, Mastropaolo M, Mangold AJ. Distribution and genetic variation of \u003cem\u003eAmblyomma triste\u003c/em\u003e (Acari: Ixodidae) in Argentina. Ticks and Tick-Borne Diseases. 2013;4(5):386\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBitencourth K, Amorim M, Oliveira SVd, Gaz\u0026ecirc;ta GS. \u003cem\u003eAmblyomma aureolatum\u003c/em\u003e Genetic Diversity and Population Dynamics Are Not Related to Spotted Fever Epidemiological Scenarios in Brazil. Pathogens. 2021;10(9):1146.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFournier GF, Pinter A, Santiago R, Mu\u0026ntilde;oz-Leal S, Martins TF, Lopes MG, et al. A high gene flow in populations of \u003cem\u003eAmblyomma ovale\u003c/em\u003e ticks found in distinct fragments of Brazilian Atlantic rainforest. Experimental and Applied Acarology. 2019;77:215\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUribe JE, Nava S, Murphy KR, Tarragona EL, Castro LR. Characterization of the complete mitochondrial genome of \u003cem\u003eAmblyomma ovale\u003c/em\u003e, comparative analyses and phylogenetic considerations. Experimental and Applied Acarology. 2020;81(3):421\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCotes-Perdomo AP, Nava S, Castro LR, Rivera-Pa\u0026eacute;z FA, Cort\u0026eacute;s-Vecino JA, Uribe JE. Phylogenetic relationships of the \u003cem\u003eAmblyomma cajennense\u003c/em\u003e complex (Acari: Ixodidae) at mitogenomic resolution. Ticks and Tick-Borne Diseases. 2023;14(3):102125.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeati L, Nava S, Burkman EJ, Barros-Battesti DM, Labruna MB, Guglielmone AA, et al. \u003cem\u003eAmblyomma cajennense\u003c/em\u003e (Fabricius, 1787)(Acari: Ixodidae), the Cayenne tick: phylogeography and evidence for allopatric speciation. BMC Evolutionary Biology. 2013;13:1\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNava S, Beati L, Labruna MB, C\u0026aacute;ceres AG, Mangold AJ, Guglielmone AA. Reassessment of the taxonomic status of \u003cem\u003eAmblyomma cajennense\u003c/em\u003e () with the description of three new species, \u003cem\u003eAmblyomma tonelliae\u003c/em\u003e n. sp., \u003cem\u003eAmblyomma interandinum\u003c/em\u003e n. sp. and \u003cem\u003eAmblyomma patinoi\u003c/em\u003e n. sp., and reinstatement of \u003cem\u003eAmblyomma mixtum\u003c/em\u003e, and \u003cem\u003eAmblyomma sculptum\u003c/em\u003e (Ixodida: Ixodidae). Ticks and Tick-Borne Diseases. 2014;5(3):252\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKobayashi T, Chatanga E, Qiu Y, Simuunza M, Kajihara M, Hang\u0026rsquo;ombe BM, et al. Molecular detection and genotyping of Coxiella-Like Endosymbionts in ticks collected from animals and vegetation in Zambia. Pathogens. 2021;10(6):779.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalinandi S, Chitimia-Dobler L, Grandi G, Nakayiki T, Kabasa W, Bbira J, et al. Morphological and molecular identification of ixodid tick species (Acari: Ixodidae) infesting cattle in Uganda. Parasitol Res. 2020;119:2411\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarradas PF, Mesquita JR, Ferreira P, G\u0026auml;rtner F, Carvalho M, In\u0026aacute;cio E, et al. Molecular identification and characterization of \u003cem\u003eRickettsia\u003c/em\u003e spp. and other tick-borne pathogens in cattle and their ticks from Huambo, Angola. Ticks and Tick-Borne Diseases. 2021;12(1):101583.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSili G, Byaruhanga C, Horak I, Steyn H, Chaisi M, Oosthuizen MC, et al. Ticks and tick-borne pathogens infecting livestock and dogs in Tchicala-Tcholoanga, Huambo Province, Angola. Parasitology Research. 2021;120(3):1097\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMayr E. What is a species, and what is not? Philos Sci. 1996;63(2):262\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFolmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology. 1994;3(5):294\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Tick Diversity, Phylogenetic, Amblyomma, southern Africa, Systematics","lastPublishedDoi":"10.21203/rs.3.rs-3833842/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3833842/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eAmblyomma\u003c/em\u003e spp. ticks, known for their bright ornate appearance and aggressive hunting behaviour, are vectors of a number of important pathogens. In southern Africa, 17 \u003cem\u003eAmblyomma\u003c/em\u003e spp. are currently documented. Of these species, \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e and \u003cem\u003eAmblyomma variegatum\u003c/em\u003e have been well studied due to their wide geographical range and their status as competent vectors of pathogens that are of veterinary and medical importance. Studies on other \u003cem\u003eAmblyomma\u003c/em\u003e spp. in southern Africa have been neglected, fostering ongoing debates on the validity of certain species such as \u003cem\u003eAmblyomma pomposum\u003c/em\u003e. This study investigated the inter- and intraspecies variation of \u003cem\u003eAmblyomma\u003c/em\u003e ticks collected in southern Africa, focusing on resolving the dispute about \u003cem\u003eA. pomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e as distinct species. Four tick species were collected from Angola, Mozambique, South Africa, Zambia, and Zimbabwe and were identified morphologically as \u003cem\u003eAmblyomma eburneum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehebraeum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e using identification keys. Gene amplification was done targeting the 12S and 16S rRNA, cytochrome oxidase I, cytochrome B and internal transcribed spacer-2 genes, and Bayesian inference analyses were performed in MrBayes. These revealed little geographic structuring amongst ticks of the same species from different countries, although intraspecific variation within \u003cem\u003eA. variegatum\u003c/em\u003e was high for the \u003cem\u003e16S\u003c/em\u003e and \u003cem\u003ecytB\u003c/em\u003e loci. Our study concluds that there is insufficient molecular evidence to differentiate \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epomposum\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003evariegatum\u003c/em\u003e from each other. We highlight the need for whole mitochondrial genome sequencing of these two species to resolve the ongoing debates. Furthermore, we propose mating and hybrid viability studies between the two species to confirm their reproductive isolation.\u003c/p\u003e","manuscriptTitle":"Intra- and Interspecific variation of Amblyomma ticks from southern Africa","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-08 09:38:27","doi":"10.21203/rs.3.rs-3833842/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-03T22:32:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-03T11:24:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3306bbd5-780f-4591-8dce-9829bc6debfb","date":"2024-02-19T20:01:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9fa1bfd0-598b-458b-966a-f3035919a364","date":"2024-01-24T06:14:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-01-17T07:36:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4a89ec9c-7939-4572-8d41-f23a1b2b90d2","date":"2024-01-10T06:44:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-04T21:52:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-04T13:24:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-04T13:15:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2024-01-04T06:57:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b1af59d5-3316-4d02-bd45-a56134db7173","owner":[],"postedDate":"January 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-02T16:14:10+00:00","versionOfRecord":{"articleIdentity":"rs-3833842","link":"https://doi.org/10.1186/s13071-024-06394-3","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2024-08-28 15:58:08","publishedOnDateReadable":"August 28th, 2024"},"versionCreatedAt":"2024-01-08 09:38:27","video":"","vorDoi":"10.1186/s13071-024-06394-3","vorDoiUrl":"https://doi.org/10.1186/s13071-024-06394-3","workflowStages":[]},"version":"v1","identity":"rs-3833842","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3833842","identity":"rs-3833842","version":["v1"]},"buildId":"CiT4i_kKBbxQbnFL0ufpk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}