# Intra- and interspecific variation of Amblyomma ticks from southern Africa

Andeliza Smit, Fernando Mulandane, Martinet Labuschagne, Stephané Heike

Wójick, Choolwe Malabwa, Gourgelia Sili, Stephen Mandara, Zinathi

Dlamkile, Rebecca Ackermann, Hannah Rose Vineer, et al.

► To cite this version:

Andeliza Smit, Fernando Mulandane, Martinet Labuschagne, Stephané Heike Wójick, Choolwe Malabwa, et al. Intra- and interspecific variation of Amblyomma ticks from southern Africa. Parasites & Vectors, 2024, 17 (1), pp.364. 10.1186/s13071-024-06394-3. hal-04717809

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# Intra- and interspecific variation of *Amblyomma* ticks from southern Africa

Andeliza Smit¹*, Fernando Mulandane², Martinet Labuschagne¹,³, Stephané Heike Wójick¹, Choolwe Malabwa⁴, Gourgelia Sili⁵, Stephen Mandara⁶, Zinathi Dlamkile¹, Rebecca Ackermann¹, Hannah Rose Vineer⁷, Wilhelm Heinrich Stoltsz¹, Karine Huber⁸, Ivan Gerard Horak¹, Darshana Morar-Leather¹, Benjamin Lawrence Makepeace⁷ and Luis Neves¹,²

**Abstract**

**Background** *Amblyomma* spp. ticks, known for their long mouthparts, bright ornate appearance and aggressive hunting behaviour, are vectors of a number of important pathogens. 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 intra-species variation of *Amblyomma* ticks collected in southern Africa, focusing on resolving the dispute about *A. pomposum* and *A. variegatum* being distinct species.

**Methods** Four *Amblyomma* tick species were collected from Angola, Mozambique, South Africa, Zambia and Zimbabwe, and were identified morphologically as *Amblyomma eburneum* (208), *A. hebraeum* (4758), *A. pomposum* (191) and *A. variegatum* (2577) using identification keys. Gene amplification targeting the 12S and 16S rRNA, cytochrome oxidase I, cytochrome B and internal transcribed spacer-2 genes was conducted for 204 ticks, for which varying success was achieved during amplification for each of the markers. Maximum likelihood analyses were performed in IQ-TREE.

**Results** The phylogenetic topologies and ABGD analyses of each individual gene clustered *A. pomposum* within the *A. variegatum* clade, while clearly separating *A. eburneum* and *A. hebraeum* from all other species. None of the genetic markers indicated intraspecific structuring on the basis of geographical origin, despite great distances between sampling sites.

**Conclusion** Our study concludes 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 controversies. Furthermore, we propose mating and hybrid viability studies between the two species to confirm their reproductive isolation.

**Keywords** Tick diversity, Phylogenetic, *Amblyomma*, Southern Africa, Systematics

<footnote>*Correspondence:
Andeliza Smit
u14023190@tuks.co.za
Full list of author information is available at the end of the article</footnote>
## Background

The order Ixodida is divided 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 estimate 750 ixodid and 218 argasid species, as well as *Nuttalliella namaqua*, the only tick in the Nuttalliellidae family, and Deinocrotonidae (monotypic, but extinct) [2–4]. Of the 970 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 of major veterinary and public health concern in the Afrotropical region.

*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 several pathogens of veterinary importance including, but not limited to, *Ehrlichia ruminantium*, *Theileria mutans* and *Theileria velifera* [5, 6]. In the south-eastern parts of Africa, 21 *Amblyomma* species have been documented [6–8]. *Amblyomma variegatum* and *Amblyomma hebraeum* are the most predominant and widespread of the species, while distribution records of *Amblyomma pomposum* and *Amblyomma eburneum* are scant (Fig. 1).

Tick taxonomy in southern Africa dates back to 1778 and is focused on the morphological characteristics of adults, geographical distribution and host preference [9–14]. The first phylogenetic representation of ticks was depicted by Hoogstraal and Aeschlimann [15], but this representation has been revised over the years with increasing information obtained from molecular studies [16, 17]. Several studies have been conducted on the molecular systematics of *Amblyomma* spp.; however, most 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 [24]. *Amblyomma variegatum*, as described by Fabricius [25], is the oldest known species in the *A. variegatum* group, with a documented geographical spread as illustrated in Fig. 1. Dönitz [26] 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 [27] 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 bushveld ecoregions, at elevations of 2000–3000 feet [23, 27]. 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 with *A. pomposum* [26], which was given preference [22]. Theiler and Salisbury [23] examined and compared the two males, which Robinson [27] identified as *A. variegatum* var. *nocens* with *A. pomposum* samples and found that the morphologies did not resemble each other closely enough. Later, Dias described a new species, *A. variegatum* var. *govurensis* in Mozambique and compared its morphology with 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 from Robinson [27], 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, he went on to note that the records of *A. pomposum* in eastern Africa as described by Dönitz [26] 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 [26]. Dias [22] proposed a new species name, *Amblyomma superbum*, which he illustrated in Dias [28], for the *Amblyomma* spp. in central and western Africa. This new nomenclature was rejected [23], and “*A. pomposum*” is still used to describe the species occurring in the central and western parts of Africa [23]. 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 range confined to latitudes 18–22°S. This change in nomenclature was also rejected [29].
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Map of Africa showing the historic distributions of *A. eburneum* (pink), *A. hebraeum* (blue), *A. pomposum* (purple) and *A. variegatum* (green) on the continent. Blank African map image was obtained from https://worldmapblank.com/blank-map-of-africa/ and modified with distribution information from Walker et al. [38] and Voltzit and Keirans [8]</figcaption></figure>

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 [8, 22–24], no molecular evidence has been provided to help resolve this question.

Molecular studies on *Amblyomma* spp. in Africa have been limited to date, focusing on mitochondrial

genetic markers, such as the cytochrome oxidase subunit 1 (coi) gene, 12S rRNA and 16S rRNA genes, and nuclear markers, such as 18S rRNA and 28S rRNA [18, 30, 31]. Phylogenetic analyses with the use of these markers showed varying differentiating capabilities. To increase phylogenetic resolution, whole mitochondrial analyses have been conducted and proven to be highly effective in distinguishing between closely related species [16, 21, 32, 33]. To date, whole mitochondrial genome (mitogenome) sequencing is still considered expensive, limiting the number of mitogenomes available on sequence repositories and limiting its use in
developing countries. Concatenation of several molecular markers into a “super-matrix” is a commonly used tool to differentiate between species, although debates on its accuracy has been long-standing [34–36].

Thus, this study investigated the intra- and inter-
species variation of *A. eburneum*, *A. hebraeum*, *A. pomposum* and *A. variegatum* ticks collected in south-
ern Africa using molecular techniques (concatenation
of four mitochondrial markers), with the aim to resolve
the taxonomic controversy between *A. pomposum* and
*A. variegatum* in southern Africa.

**Methods**

**Sample collection**

*Amblyomma* spp. ticks were collected in Angola, Zambia and Zimbabwe (Fig. 2) by means of hand collections

from cattle and goats. In Mozambique, *Amblyomma*
spp. ticks were also collected from livestock, and legally
hunted wildlife (Fig. 2; Additional file 1: Text S1). Animals were visually inspected for tick infestations before
collections were made. Due to the bright ornate pattern-
ing in *Amblyomma* spp., these ticks can be spotted from
a distance. This prevented the unnecessary restriction
and sampling of animals that would not provide a suffi-
cient number of ticks. Once an infested animal was spot-
ted and restrained, complete tick collections were done
by hand from the animal to ensure optimal species repre-
sentation. Tick samples were taken from the whole body
of the animal, paying particular attention to the predilec-
tion sites such as the dewlap, perineum, axilla, udder, or
interdigital spaces. Collections were performed during
the optimal season for adult ticks in each country: in

<figure><img src="image_4.png" /><figcaption>**Fig. 2** Map of southern Africa, illustrating the collection points during the 2020–2022 sampling period of *Amblyomma* spp. The colour of the dot represents the species of *Amblyomma* tick collected at the sampling point</figcaption></figure>
Angola during March 2022, in Mozambique from February 2021 to April 2021 and again in November 2021, in Zambia during April 2022 and July 2022 and in Zimbabwe from March 2022 to June 2022. *Amblyomma* spp. ticks from South Africa were obtained from Dlamkile et al. [37] (Additional file 1: Text S1). 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 the species level, using identification keys obtained from Walker et al. [38] and Voltzit and Keirans [8]. 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 [39, 40].

## 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. [41] and stored at -20 °C for downstream use. Molecular characterisation was conducted targeting four mitochondrial (12S rRNA gene [42], 16S rRNA gene [43], cytochrome B (*cytB*) [44] and cytochrome oxidase subunit 1 (*coi*) [45]) genes and the nuclear internal transcribed spacer 2 (ITS2) region [20]. These markers were selected on the basis of reference sequence availability on GenBank and as the most popular loci used in the literature. Four male and four female ticks per species per region, if available, were selected for DNA extraction. In total 204 ticks were used: *A. eburneum* (*n*=13), *A. hebraeum* (*n*=92), *A. pomposum* (*n*=26) and *A. variegatum* (*n*=73). Amplification master-mixes are described below. Primer, master-mix and annealing temperatures can be located in Additional file 2: Dataset S1. The general polymerase chain reaction (PCR) cycling conditions for each marker comprised an initial denaturation at 98 °C for 10 s, followed by 10 cycles of denaturation at 98 °C for 1 s, annealing at x °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 y °C for 5 s and extension at 72 °C for 15 s. Final extension was performed at 72 °C for 15 s.

The PCR products were separated on a 1.5% agarose gel and visualised using the Bio-Rad gel documentation system with assisting visualisation 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 the Basic Local Alignment Search Tool (BLAST; https://www.ncbi.nlm.nih.gov/genbank/) [46]. Reference sequences were obtained from GenBank and used for comparison purposes (Additional file 3: Dataset S2). All reference sequences that were available of *A. eburneum* and *A. pomposum*, as well as for *cytB*, were used due to their limited number. *Amblyomma hebraeum* and *A. variegatum* had a larger variety of available sequences; therefore, several sequences were selected that were of similar length to the sequences generated in the study. The outgroup was selected to be *Amblyomma americana num*, as it is sufficiently distant from the species obtained in this study to serve as a well-defined root. 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 frameshift and stop codon detection was conducted for each matrix using MACSE version 2.07 using the invertebrate mitochondrial code [47]. The aligned matrices were manually viewed, edited and truncated using MEGA 11 [48]. The best fit model was determined for each individual gene using ModelFinder [49] on the IQ-TREE version 1 software [50]. Maximum likelihood (ML) analyses were conducted on IQ-TREE version 1 using 1000 bootstrap replicates. The resulting trees were visualised and edited in iTOL version 6.8 [51]. Sequences that were identical to each other were removed from the trees to increase readability. The evaluation criteria for the bootstrap values were as follows: values of 75% and lower were considered nonsignificant, between 75 and 94% were considered moderately significant and values of 95% and higher represented high statistical support [52].

A concatenated file was constructed including 12S, 16S, *coi* and *cytB* genes. Alignments for each of the individual genes were done on MAFFT. The aligned sequences were then concatenated into a super-matrix using MEGA 11. Model selection and ML analysis was done using IQ-TREE. Partitioning was applied to the super-matrix [53] with a 1000 bootstrap replicates using the ultrafast bootstrapping method [54]. The evaluation criteria for the ultrafast bootstrap values were as follows: values of 95% and higher represented high statistical support, while values lower than 95% were considered unsupported [55].

## 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 12S rRNA and 16S rRNA genes, *coi*, *cytB* and the concatenated alignment using
default parameters with 1000 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) [56]. 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.

## Results

In total, 7734 adult *Amblyomma* spp. ticks were obtained from Angola, South Africa, Mozambique, Zambia and Zimbabwe (Fig. 3). These ticks were identified morphologically to the species level; representative specimens of each sex are illustrated in Fig. 4. Four species were observed [*A. eburneum* (*n*=208), *A. hebraeum* (*n*=4758), *A. pomposum* (*n*=191) and *A. variegatum* (*n*=2577)], and the main morphological differences are summarised in Table 1. *Amblyomma eburneum* was only collected in one location in central Mozambique (18°S; Fig. 2). *Amblyomma hebraeum* was collected in the northeastern parts of South Africa, in the 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), in northern regions of Zimbabwe, latitudinally across Zambia and in eastern parts of Angola (Fig. 2).

## Phylogenetics

From the selected 204 *Amblyomma* ticks used for phylogenetic analyses, 24 (11.8%) did not amplify with any of the selected markers and were removed from further analyses. The remaining 180 samples had varying success with amplification and contig construction amongst the genes; success rates were 69.4% (125/180) for 12S rRNA, 57.2% (103/180) for 16S rRNA, 71.1% (128/180) for *coi*, 64.4% (116/180) for *cytB* and 69.4% (125/180) for ITS2. Although the majority of the sequences amplified using ITS2, it was excluded from further analyses due to the genetic variability only consisting of uninformative single nucleotide mutations preventing any systematic analyses. Difficulties arose during the contig construction of the 16S rRNA sequences due to low sequence quality. In contrast, 12S rRNA and *coi* were the most successful in sample amplification and produced sequences of good quality resulting in a high rate of contig assembly of these sequences. The number of sequences per species analysed for each gene varied (Table 2). All sequences were deposited in GenBank (Additional file 3: Dataset S2).

The 12S rRNA gene exhibited an average amplified fragment length of 400 bp, while the final fragment

<figure><img src="image_5.png" /><figcaption>**Fig. 3** The number and species of *Amblyomma* ticks collected from Angola, Mozambique, South Africa, Zambia and Zimbabwe over the course of this study</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 4** Morphological comparison of males (indicated by ♂) and females (indicated by ♀), where plate **A** represents *A. eburneum*, plate **B** represents *A. hebraeum*, plate **C** represents *A. pomposum* and plate **D** represents *A. variegatum*. Dorsal and ventral views are illustrated. Photographs were taken by Prof. Melvyn Quan and Ms. Zandile Mkhize</figcaption></figure>

length for the alignment was 383 bp. The 16S rRNA had the shortest average amplified fragment length at 340 bp, with the final fragment alignment length for the 16S rRNA alignment was 328 bp. The average length of the amplified fragment of *coi* was bp, with the final fragment length in the *coi* alignment being 650 bp. Lastly, the average amplified fragment length for *cytB* was 500 bp, while the final fragment length for the *cytB* alignment was 483 bp.

The average intraspecific pairwise distances and the average interspecific pairwise distances are given in Additional file 4: Dataset S3. In nearly all interspecific evaluations, *A. pomposum* and *A. variegatum* had the least amount of distance between them. The intraspecific pairwise distances were on average lower than the average interspecific pairwise distances. The pairwise difference between individual sequences for each gene were also compared (Additional file 5: Dataset S4 to Additional file 9: Dataset S8).

When evaluating the 12S rRNA estimations of evolutionary divergence between each of the sequences, low levels of intraspecific variation were observed. The

intraspecific variation for *A. eburneum* was the same as the interspecific variation between *A. eburneum* and *A. hebraeum*. *Amblyomma hebraeum* had low intraspecific variation reflecting high levels of co-linearity. The interspecific variation between *A. pomposum* and *A. variegatum* was nearly identical to the intraspecific variation of *A. variegatum*.

The ML phylogenetic topology of 12S rRNA depicted a clear separation between *A. hebraeum* from all other species, forming a single clade. This separation was corroborated by the ABGD analysis, in which *A. hebraeum* clustered in a single operational taxonomic unit (OTU). The *A. eburneum* sequences were observed to be divided into three main clades, separated from all the other species. The first clade (OTU 3) was separated from the *A. variegatum/pomposum* cluster, forming a sister clade with the remaining *A. eburneum* and *A. hebraeum* sequences with high bootstrap support, but contained only two sequences. The main clade of *A. eburneum* sequences (OTU 4) was resolved with strong support, while a third sequence (OTU 2) branched from this main group with moderate support.
<table><caption>**Table 1** Main morphological differences between *Amblyomma eburneum*, *Amblyomma hebraeum*, *Amblyomma pomposum* and *Amblyomma variegatum*</caption><thead><tr><th></th><th>Amblyomma eburneum<br>(Fig. 4A)</th><th>Amblyomma hebraeum<br>(Fig. 4B)</th><th>Amblyomma pomposum<br>(Fig. 4C)</th><th>Amblyomma variegatum<br>(Fig. 4D)</th></tr></thead><tbody><tr><td colspan="5"><strong>Males</strong></td></tr><tr><td>Colouration</td><td>Red-brown with beige patterning</td><td>Red-brown with metallic beige patterning</td><td>Black-brown with red, orange and green patterning</td><td>Black-brown with red, orange and green patterning</td></tr><tr><td>Vibrance</td><td>Vibrant and metallic</td><td>Vibrant and metallic</td><td>Vibrant and metallic</td><td>Muted and metallic</td></tr><tr><td>Eyes</td><td>Slightly convex</td><td>Flat</td><td>Distinctly convex</td><td>Distinctly convex</td></tr><tr><td>Punctations</td><td>Small and shallow</td><td>Small and shallow</td><td>Large and densely scattered</td><td>Medium or small and densely scattered</td></tr><tr><td>Mesial patch</td><td>Square, indistinctly connected to posterior enamel areas</td><td>Elongated, reaching the capitulum</td><td>Oval, unconnected to other enamelled patches</td><td>Square, distinctly connected to falciform stripe</td></tr><tr><td>Falciform stripe</td><td>C-shaped</td><td>Triangular shaped</td><td>Claw shaped</td><td>Horn-like appearance</td></tr><tr><td>Posterior medial stripe</td><td>Straight and wide</td><td>Straight and narrow</td><td>Straight and narrow</td><td>Extremely narrow</td></tr><tr><td>Lateral patches</td><td>Ovoid-shaped, distinctly present</td><td>Complex shape, distinctly present</td><td>Large trapezoid distinctly present</td><td>Generally absent (if present they are indistinct and small)</td></tr><tr><td>Festoon colouration</td><td>10/11 (central festoon is uncoloured)</td><td>9/11 (two outer festoons are uncoloured)</td><td>0/11</td><td>0/11</td></tr><tr><td colspan="5"><strong>Females</strong></td></tr><tr><td>Punctuation</td><td>Small</td><td>Deep near eyes; shallow central and scapular areas</td><td>Large and densely scattered</td><td>Small or medium, densely scattered</td></tr><tr><td>Mesial patch</td><td>Large circular patch with two small central patched</td><td>Large patch covering whole central third</td><td>Small, circular and faint</td><td>Small, circular and clear</td></tr><tr><td>Lateral patch</td><td>Two small unconnected circular patches</td><td>Large c-shaped</td><td>Indistinct but present</td><td>Indistinct but present</td></tr><tr><td>Posterior angle</td><td>Broad</td><td>Broad</td><td>Narrower</td><td>Broad</td></tr></tbody></table>

The *A. pomposum* and *A. variegatum* sequences formed two main clades. The first was a minor clade containing only five *A. variegatum* sequences, all originating from Mozambique. The second, much larger clade exhibited strong support (100 bootstrap value) for a subclade consisting of nine sequences (four from Zambia and five reference sequences from GenBank). Otherwise, phylogenetic evidence for population structure within the larger clade was poor, including for *A. pomposum* as a distinct subclade. In the ABGD analysis, the *A. pomposum* and *A. variegatum* sequences were divided into three OTUs (#5, #7 and #8). While *A. pomposum* sequences segregated together in the tree, they were grouped with a majority of *A. variegatum* sequences into a single OTU (#5). Notably, the minor clade from Mozambique and the subclade described above of nine Zambian and published reference sequences formed a separate OTU (#8). A final, singleton OTU (#7) consisted of an *A. pomposum* sequence located on a long branch, although this was not well supported in the tree. This sequence contained several single nucleotide polymorphisms (SNPs) when compared with others of the same species (Additional file 10: Fig. S1).

The 16S rRNA pairwise sequence matrix revealed low intraspecific variation in both *A. hebraeum* and

*A. pomposum*, while interspecific variation was lower between *A. pomposum* and *A. variegatum* as compared with other species. The ML topology of the 16S rRNA depicted a clear separation between *A. eburneum*, *A. hebraeum* and the *A. variegatum* complex. The *A. hebraeum* sequences formed two distinct clades, sister to an *A. eburneum* clade (OTU 4), with high bootstrap support. However, *A. hebraeum* was classified into a single OTU (#5) in the ABGD analysis. The *A. eburneum* specimens were divided into two main clades, one sister to *A. hebraeum* and the other separated from all other species (OTU 2). The larger clade (OTU 2) contained nine sequences from this study alongside the only reference sequence from GenBank, whereas the other cluster (OTU 4) comprised only two sequences from this study.

The *A. variegatum* and *A. pomposum* cluster was divided into two clades. The first clade consisted of a single *A. variegatum* reference sequence from Sao Tome and Principe, which was supported with a moderate bootstrap value. The second clade comprised the remaining *A. variegatum* and *A. pomposum* sequences, and these were poorly resolved, with a lack of phylogenetic support for population structuring. While most *A. pomposum* sequences were located together in the tree, one reference sequence from Zambia was nestled among *A.*
<table><caption>**Table 2** The number of sequences analysed for the pairwise distance matrix and for the Automatic Barcode Gap Discovery (ABGD) analysis</caption><thead><tr><th>Species/number of sequences analysed</th><th>12S</th><th>16S</th><th>coi</th><th>cytB</th><th>Concatenated</th></tr></thead><tbody><tr><td>Amblyomma eburneum</td><td>12</td><td>12</td><td>15</td><td>12</td><td>11</td></tr><tr><td>Amblyomma hebraeum</td><td>77</td><td>50</td><td>82</td><td>56</td><td>30</td></tr><tr><td>Amblyomma pomposum</td><td>11</td><td>16</td><td>5</td><td>19</td><td>2</td></tr><tr><td>Amblyomma variegatum</td><td>42</td><td>32</td><td>38</td><td>33</td><td>7</td></tr><tr><td>Outgroup (Amblyomma americanum)</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>Total</td><td>143</td><td>111</td><td>141</td><td>121</td><td>51</td></tr></tbody></table>

*variegatum*. Regardless, there was no bootstrap support for the separation of *A. pomposum* from *A. variegatum*, and this was corroborated by the ABGD analysis, which defined a single OTU (#3) encompassing all *A. pomposum* and *A. variegatum* sequences (Additional file 11: Fig. S2).

The analysis of the *coi* pairwise sequence matrix illustrated higher intraspecific variation compared with the other markers, except for *A. hebraeum*, which still exhibited low levels of polymorphism. The ML phylogenetic topology for *coi* depicted a clear separation of *A. hebraeum* from the other *Amblyomma* species. Although *A. hebraeum* was divided into two main clades, supported by a moderate bootstrap value, it formed one OTU (#5) in the ABGD analysis. The first clade consisted of two *A. hebraeum* sequences from Zimbabwe, while the second clade consisted of all other remaining sequences generated during this study alongside reference sequences from GenBank. Similarly, *Amblyomma eburneum* was divided into two well-supported clades. The first contained only two sequences obtained from this study (OTU 3), whereas the other (OTU 2), a sister clade to *A. hebraeum*, consisted of the bulk of the sequences from this study alongside the two reference sequences currently available on GenBank. *Amblyomma pomposum* and *A. variegatum* were segregated into a single clade with no bootstrap support for their separation. Within *A. variegatum*, 14 sequences 7 from Zambia, four from Mozambique, 1 from Senegal and 1 from Guinea-Bissau) formed a well-supported subclade, although they were not classified as a separate OTU. Moreover, within the main *A. pomposum/A. variegatum* clade, one *A. pomposum* sequence (AHB015) branched from the others with high bootstrap support. Nevertheless, the ABGD analysis grouped *A. pomposum* and *A. variegatum* into a single OTU (#4) (Additional file 12: Fig. S3).

The *cytB* pairwise distance matrix depicted low levels of intraspecific variation, especially for *A. eburneum* and *A. hebraeum*. The interspecific variation between *A. pomposum* and *A. variegatum* was much lower than between the other species. The *cytB* ML phylogenetic topology

depict a clear separation between *A. eburneum* and *A. hebraeum* with moderate bootstrap support, and these sister clades were separated into their own OTUs (#2 and #3). Both *A. hebraeum* and *A. eburneum* exhibited evidence of intraspecific population structure, with some branches displaying moderate or strong support. The *A. variegatum* sequences were divided into two main subclades, with high bootstrap support. The first subclade contained representatives from Angola, Mozambique, Zambia and Zimbabwe, while the second consisted of *A. pomposum* sequences alongside some *A. variegatum* specimens from Mozambique and Zambia. These two subclades were grouped into a single OTU (#4) by ABGD analysis, with the exception of one *A. variegatum* sequence from Mozambique that formed its own OTU (#5) on a long branch. When examining this sequence, several SNPs were evident (Additional file 13: Fig. S4).

For the concatenated alignment (12S rRNA and 16S rRNA genes, *coi* and *cytB*), the pairwise distance matrix illustrated low levels of intraspecific variation, especially for *A. hebraeum*. The interspecific variation between *A. pomposum* and *A. variegatum* was nearly five times lower than that between the other species. The ML phylogenetic topology indicated clear separation between all four species with high bootstrap support (Fig. 5). Within the *A. hebraeum* clade, multiple branches were observed with moderate to high bootstrap support; however, only one OTU (#5) was indicated by ABGD analysis. *Amblyomma eburneum* was separated into two clades with moderate to high bootstrap support. The smaller clade contained the two sequences from Mozambique that clustered separately in the individual gene trees, whereas the larger clade comprised the remaining *A. eburneum* sequences. This separation was supported by the ABGD analysis, which divided *A. eburneum* into two OTUs (#1 and #4). Unfortunately, only two *A. pomposum* sequences could be used in the concatenated analysis due to low amplification and sequencing success as described above. These two sequences were separated from *A.*
<figure><img src="image_7.png" /><figcaption>**Fig. 5** Maximum likelihood (ML) analysis of all concatenated genes. The ML analysis used the individual models for each gene: HKY+F+G4 for 12S rRNA, K3Pu+F+I for 16S rRNA and HKY+F+I for both *coi* and *cytB*. Bootstrap support is indicated at each branch node, and values under 70 were removed. Blue, pink, green and purple highlighted regions indicate *A. hebraeum*, *A. eburneum*, *A. variegatum* and *A. pomposum*, 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; ZAF, South Africa; ZM, Zambia; and ZWE, Zimbabwe. GenBank reference strains are marked with +</figcaption></figure>

*variegatum* with high bootstrap support, but the ABGD analysis placed both species in the same OTU (#2).

Discussion

This study investigated the intra- and inter-species vari-
ation of *Amblyomma* spp. collected in southern Africa.
In total 7734 adult *Amblyomma* ticks were collected and
morphologically identified as *A. eburneum*, *A. hebraeum*,
*A. pomposum* and *A. variegatum*. However, on the basis
of our analyses of multiple genes, we cannot conclude
that *A. pomposum* and *A. variegatum* are distinct species.

*Amblyomma eburneum* was only collected in one
location in central Mozambique (18°S) from wildlife
(mainly African buffalo, *Syncerus caffer*). Information

on *A. eburneum* is scant; however, it is described as an
East African species with a documented geographical
distribution as illustrated in Fig. 1 [8, 41]. The sympat-
ric occurrence between *A. eburneum* and *A. variegatum*,
as found in Mozambique, is uncommon for the genus
*Amblyomma* [57]. While no definitive conclusions can be
drawn for this co-occurrence, lack of sexual interference
between the two species is one hypothesis. This sympat-
ric distribution between *A. eburneum* and *A. variegatum*
[41] and the parapatric distribution between *A. hebraeum*
and *A. variegatum* [57] would be an ideal model for fur-
ther investigation into the sexual behaviours of these tick
species and their impact on interspecific competition and
boundary formation.
Amblyomma hebraeum was collected in northeast-
ern parts of South Africa, southern and central regions
of Zimbabwe and in southern areas of Mozambique
(below 21°S), which agrees with the current literature [7,
24, 38, 58]. Amblyomma hebraeum was also collected
at two sampling points extending beyond what is cur-
rently documented as its endemic zone (19°S), indicating
expansion of its geographical distribution. Indeed, it has
been documented that the geographical distribution of
Amblyomma spp. has widened in the past few years [59].
The main culprit for these expansions is always assumed
to be climate change; however, albeit a major factor, it is
not the only cause for these observations [59]. The trans-
portation of livestock within and across countries greatly
impacts the distribution potential of ticks. Regarding our
data, it is more likely that A. hebraeum was introduced
from other parts of Mozambique or from South Africa by
the transportation of infested animals to the new region;
this is supported by the fact that the parapatric bound-
ary between A. hebraeum and A. variegatum in Mozam-
bique has been maintained for almost a century, with no
geographical expansion [57]. Both male and female A.
hebraeum ticks were collected, which could lead to the
establishment of this species in this new geographical
area. It would be of interest to continuously observe this
subpopulation of A. hebraeum in an area where A. varieg-
atum is known to occur and document the competitive
behaviour between the two species.

The geographical spread of *A. pomposum*, as illustrated
in Fig. 1, was described by Robinson [60], Walker and
Olwage [24] and Petney et al. [58] 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
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*. The most prob-
able reason for this discrepancy is the misidentification
of *A. variegatum* as *A. pomposum* in these earlier stud-
ies due to their close morphological appearance. How-
ever, cattle are known to migrate from Angola to Zambia
during drier periods when the marshlands are not flood-
ing; thus, cattle harbouring *A. pomposum* could pos-
sibly be found in Zambia at these times. Yet, if this was
the case, we would expect to see the establishment of *A.
pomposum* in eastern Angola and Zambia, which was
not observed in this study. Since no voucher material of
*A. pomposum* from Zambia is available and the one of
the 16S rRNA sequences for *A. pomposum* on GenBank
appears to be an outlier in our analyses, we maintain
that *A. pomposum* is unlikely to occur in Zambia. In this

study, *A. variegatum* was collected in Angola, Mozambique, Zambia and Zimbabwe, corresponding with the records of Petney et al. [58] and Walker and Olwage [24].

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 season for the adult stage (the months when the maximum infestations occur have been documented as November and December [58]). *Amblyomma eburneum* was also collected in low numbers, but this can be attributed to the difficulty of collecting ticks from wildlife species, as we were highly dependent on the number of wild animals that was legally hunted in the timeframe of this study. The low prevalence of *Amblyomma* spp. from Zimbabwe was likely the result of a vigorous campaign by the government to assist livestock farmers in treating their cattle against ticks by encouraging regular dipping of the animals with acaricides, in an attempt to control major outbreaks of bovine theileriosis.

This study aimed to provide insight into the unresolved
debate that has been ongoing for decades as described
in the introduction. Both Robinson [27] and Dias [22,
28] described morphological variation in *A. variega-
tum* found in Mozambique and suggested the new spe-
cies 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 [26], suggesting a new species
description as *Amblyomma superbum*. In this study, mor-
phological variation in the collected *A. variegatum* ticks
were observed; however, the majority resembled that of
the original description by Fabricius [25]. Morphology is
an important aspect in tick taxonomy, highlighting the
importance of variation in ornamentation. These con-
flicting observations of the morphology in *A. variegatum*
is reminiscent of the controversy regarding the *Ambly-
omma marmoreum* complex [61].

The *Amblyomma marmoreum* complex encompasses five African species, namely: *A. marmoreum* sensu stricto, *A. sparsum*, *A. falsomarmoreum*, *A. nuttalli* and *A. paulopunctatum* [61]. 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, 42, 61]. Cotes-Perdomo et al. [61] was, however, able to differentiate between the species
with the use of molecular techniques targeting a large part of the mitogenome. Overall, they were able to concatenated and compare 13 protein-coding genes and two ribosomal genes of several *Amblyomma* spp.

In an attempt to provide clarity on the phylogenetic positioning of *A. pomposum* and *A. variegatum*, molecular analyses were conducted on four *Amblyomma* spp. of southern Africa using 12S rRNA, 16S rRNA, *coi*, *cytB* and ITS2 molecular markers. The ITS2 marker was, however, excluded from further analyses due to uninformative single nucleotide mutations. Our research provides additional sequences that have been deposited in the GenBank database for all other markers utilised here, including the first for the 12S rRNA and *cytB* genes of *A. eburneum* and first for the 12S rRNA, *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 2 years of storage and the effects of long-term storage on the binding of impurities to DNA. Singh et al. [62] 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 1 year previously, compared with the less successful amplification of 12S rRNA, 16S rRNA, *coi* and ITS2, which were conducted using DNA stored for approximately 2 years. Additionally, several amplified products did not generate sequences of good quality, and were thus excluded from further analysis, lowering the sample size. The *coi* gene marker was 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 ML and ABGD analysis indicated that *A. eburneum* and *A. hebraeum* were sister groups but consistently formed their own OTUs. In the case of *A. hebraeum*, a single OTU was resolved in the single-gene and concatenated analyses with relatively low intraspecific variation. *Amblyomma eburneum* was divided into three OTUs in the 12S rRNA analysis; two OTUs in the 16S rRNA, *coi* and concatenated analyses; and a single OTU in the *cytB* analysis. In all the ML trees, excluding *cytB*,

two *A. eburneum* samples from Mozambique (MSMun 1039 and MSMun 1026) were separated from the main clade with moderate to high bootstrap support, although it is unclear why this is the case. The samples were collected in the same area as all other *A. eburneum*, on the same hosts and over the same timeframe. A possible explanation for both the low polymorphism observed in *A. hebraeum* and the higher genetic diversity observed within *A. eburneum* is the influence of host dispersal on gene flow between generalist and specialist ectoparasites [63]. Matthee [63] documented that *A. hebraeum*, which occurs on hosts with low global range values (i.e. that are located close together), showed the lowest levels of population differentiation and high levels of gene flow. These findings contradicted those of other generalist species studied by Matthee [63]. *Amblyomma hebraeum* was also documented to have the lowest haplotype diversity amongst generalist species [63]. In contrast, *A. eburneum*, is a specialised species that occurs only on wild ungulates, and in Mozambique, is restricted to hosts with a limited geographical range. According to Matthee [63], the low gene flow could results in high population differentiation. We speculate that the two unique *A. eburneum* samples could be from a subpopulation that has undergone more genetic isolation compared with the main clade. This suggests an opportunity to study the intraspecific diversity of *A. eburneum* across its whole latitudinal range, from Ethiopia to Mozambique.

Strikingly, *A. pomposum* clustered with *A. variegatum* in the ABGD analyses for all the individual genes and in the concatenated analyses. Similarly, in the single-gene trees, *A. pomposum* samples were nested within the *A. variegatum* clade, usually as a single cluster with low bootstrap support. However, in the concatenated ML tree, separation of *A. pomposum* from *A. variegatum* was well supported, albeit at a short genetic distance and with limited sequences available for *A. pomposum*. *Amblyomma variegatum* formed a single OTU in all analyses except for the 12S rRNA gene and *cytB*. In the 12S rRNA, *coi* and *cytB* ML topologies, several *A. variegatum* samples from Zambia formed a subclade with moderate to high bootstrap support. Similarly to *A. hebraeum*, *A. variegatum* can be considered a generalist species in which high gene flow is expected. However, Matthee [63] noted that, unlike *A. hebraeum*, many generalist ectoparasite species show high levels of haplotype diversity. There are several potential reasons why species with similar ecology might exhibit different population genetics (especially with regard to mitochondrial markers), such as bottlenecks in evolutionary history, differential effects of migration or even the impact of vertically transmitted bacterial symbionts [64].
Phylogenetic analysis of the *Amblyomma* spp. indicated 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 [65]. Erster et al. [66] (and Koroiva and Santana [67]) evaluated the marker efficiency of 12S rRNA, 16S rRNA, *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. Accordingly, Erster et al. [66] recorded barcode gaps of 12S [0.00 ± 0.03], *coi* [0.03 ± 0.05] and *cytB* [0.04 ± 0.06], while the 16S displayed a negative barcoding gap [-0.01 ± 0.06]. Furthermore, Vences et al. [68] reported that *cytB* was more variable than *coi*, allowing for clearer separation between closely related species of amphibians. Norris et al. [69] found that 16S rRNA was the least variable marker when comparing 12S rRNA and 16S rRNA markers for *Ixodes scapularis* population genetics. The 16S rRNA marker was also shown to be unable to differentiate between closely related species in the *Rhipicephalus sanguineus* group, including *Rhipicephalus turanicus* and *Rhipicephalus sanguineus*, and between *Rhipicephalus guilhoni*, *Rhipicephalus leporis* and *Rhipicephalus camicasi* [70]. The same limitation was observed for *Ixodes ricinus* and *Ixodes inopinatus*, which could not be differentiated between each other using 16S rRNA [71]. In the current study, *cytB* proved to be the most efficient single marker to differentiate between southern African *Amblyomma* spp. (Additional file 4: Dataset S3), while none of the markers in isolation allowed for clear discrimination between samples of the same species from different geographical regions.

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 used in the present work, the 12S data alone suggest 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 [72]. 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* sp., 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%) [73].

Population genetic studies of *Amblyomma* spp. have been more extensive in the New World and offer important lessons for understanding intraspecific variation in the Afrotropical species. There are marked differences between the low intraspecific variation observed in some Neotropical species (e.g., *Amblyomma triste* [74] and *Amblyomma aureolatum* [75], where it was <1% for mitochondrial markers) compared with others such as *Amblyomma ovale* [76, 77] and *Amblyomma mixtum* [33], 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 [78, 79]. The Afrotropical species await similar rigorous analyses using whole mitogenome data across the entirety of their range, as also highlighted in a recent study of *A. sparsum* in the *A. marmoreum* complex [61]. 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. An important limitation was that the concatenated tree only had two representatives from *A. pomposum*. This was because not all loci had amplified successfully for each specimen.

Thus, on the basis of 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. [80] were the first authors who published the only sequences currently available for the *A. pomposum* on the GenBank database. They collected 15 *Amblyomma* spp. ticks and morphologically identified them as *A. variegatum* (*n*=13) and *A. pomposum* (*n*=2) with the use of Walker et al. [38]. They then conducted a phylogenetic analysis using the 16S rRNA gene.
The *A. pomposum* that they identified clustered within the *A. variegatum* clade as in our study. Balinandi et al. [81] 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 rRNA gene clus- tered *A. pomposum* with *A. variegatum*. This discovery led the authors to believe that they had misidentified *A. variegatum* ticks as *A. pomposum*. Our 16S rRNA phylo- genetic analysis also placed *A. pomposum* within the *A. variegatum* cluster. Moreover, based on our analysis of several genes, it is possible that Balinandi et al. [81] cor- rectly identified their ticks as *A. pomposum*; however, since they did not upload any sequences nor depict the morphological discrepancy, no definite conclusion can be made on the basis of this information. Similarly, Barradas et al. [82] collected 116 ticks from the Huambo province in Angola. With the use of morphological identification using the key of Walker et al. [38], as well as molecular identification targeting the 12S rRNA and 16S rRNA genes, they identified 11 (10%) *Amblyomma* ticks as *A. variegatum*. As with the 16S rRNA analyses, the phylo- genetics of 12S rRNA also indicated nesting of *A. pom- posum* within *A. variegatum*. In our study, we collected from the same sites in Huambo and identified the *Ambly- omma* spp. circulating in the area as *A. pomposum*. Our findings are also supported by Sili et al. [83], who identi- fied all the *Amblyomma* spp. in this area as *A. pomposum*. On the basis of our morphological examinations at this geographic location, we believe that Barradas et al. [82] misidentified their *Amblyomma* spp. as *A. variegatum* instead of *A. pomposum*.

Although the phylogenetic analyses cannot differentiate
between *A. variegatum* and *A. pomposum* currently, suf-
ficient morphological features are documented to distin-
guish these species from each other (Table 1; Fig. 4). The
other main factors defining a species are its ecological
niche and reproductive behaviour [84]. Mayr [84] 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 spe-
cies 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 phylo-
genetic analysis, that mating and hybrid viability studies
are performed between the two species to confirm their
reproductive isolation.

Conclusions

In this study, cytB was the most successful marker in dif-
ferentiating between *A. eburneum*, *A. hebraeum*, *A. pom-
posum* 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. [38] and other current literature. On the
basis of our findings, we suggest comparing whole mito-
chondrial genomes and several nuclear loci of *A. pompo-
sum* with those of *A. variegatum* to determine whether
these are distinct species, or whether *A. pomposum* is a
subspecies of *A. variegatum*. Moreover, further investiga-
tions should also be conducted using mating and hybrid
viability studies between the two species to determine
whether they are reproductively isolated.

Abbreviations

<table>
   <tr>
    <td>
     ABGD
    </td>
    <td>
     Automatic Barcode Gap Discovery
    </td>
   </tr>
   <tr>
    <td>
     BI
    </td>
    <td>
     Bayesian inference
    </td>
   </tr>
   <tr>
    <td>
     CAF
    </td>
    <td>
     Central Analytical Facility
    </td>
   </tr>
   <tr>
    <td>
     ESS
    </td>
    <td>
     Estimates sample size
    </td>
   </tr>
   <tr>
    <td>
     ML
    </td>
    <td>
     Maximum likelihood
    </td>
   </tr>
   <tr>
    <td>
     OTUs
    </td>
    <td>
     Operational taxonomic units
    </td>
   </tr>
   <tr>
    <td>
     SE
    </td>
    <td>
     Standard error
    </td>
   </tr>
   <tr>
    <td>
     SNP
    </td>
    <td>
     Single nucleotide polymorphisms
    </td>
   </tr>
  </table>

**Supplementary Information**

The online version contains supplementary material available at https://doi.
org/10.1186/s13071-024-06394-3.

Additional file 1: Text S1. Collection countries with the provinces, main
locations, number of ticks collected, and the GPS co-ordinates for this
stud.

Additional file 2: Table S1: Primer and PCR information. Table contains the primer target region, primer name, primer sequence, the expected product size, PCR master-mix volumes, annealing temperatures and reference article.

Additional file 3: GenBank accession numbers for the sequences used and
obtained in this study.

Additional file 4: Dataset S3: Estimates of evolutionary divergence over sequence pairs between groups (interspecies p-distances). Values in red indicate the intraspecies p-distances.

Additional file 5: Estimates of Evolutionary Divergence between
Sequences. The number of base differences per site from between
sequences are shown. Standard error estimate(s) are shown above the
diagonal. This analysis involved 143 nucleotide sequences. All ambiguous
positions were removed for each sequence pair (pairwise deletion option).
There were a total of 387 positions in the final dataset. Evolutionary analy-
ses were conducted in MEGA11 [1].

Additional file 6: Estimates of Evolutionary Divergence between
Sequences. The number of base differences per site from between
sequences are shown. Standard error estimate(s) are shown above the
diagonal. This analysis involved 111 nucleotide sequences. All ambiguous
positions were removed for each sequence pair (pairwise deletion option).
There were a total of 324 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 [1].

Additional file 7: Estimates of Evolutionary Divergence between Sequences. The number of base differences per site from between sequences are shown. Standard error estimate(s) are shown above the diagonal. This analysis involved 141 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 651 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 [1].

Additional file 8: Estimates of Evolutionary Divergence between Sequences. The number of base differences per site from between sequences are shown. Standard error estimate(s) are shown above the diagonal. This analysis involved 121 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 492 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 [1].

Additional file 9. The number of base differences per site from between sequences are shown. Standard error estimate(s) are shown above the diagonal. This analysis involved 51 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1844 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 [1].

Additional file 10: Fig. S1 Maximum likelihood (ML) analysis of the 12S rRNA gene. The ML analysis used the HKY + F + I model. Bootstrap values are indicated at each branch node; values under 70 were removed. Blue, pink, green and purple highlighted regions indicate *A. hebraeum*, *A. eburneum*, *A. variegatum* and *A. pomposum*, 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; NGA, Nigeria; STP, São Tomé and Príncipe; SWZ, Eswatini; ZAF, South Africa; ZM, Zambia; and ZWE, Zimbabwe. GenBank reference strains are marked with +

Additional file 11: Fig. S2 Maximum likelihood (ML) analysis of the 16S rRNA gene. The ML analysis used the HKY + F + R2 model. Bootstrap values are indicated at each branch node; values under 70 were removed. Blue, pink, green and purple highlighted regions indicate *A. hebraeum*, *A. eburneum*, *A. variegatum*, and *A. pomposum*, respectively. Species names are included with a three-letter country code and the sample name or GenBank accession number. Country codes: AGO, Angola; KEN, Kenya; MOZ, Mozambique; UGA, Uganda; ZAF, South Africa; ZM, Zambia; and ZWE, Zimbabwe. GenBank reference strains are marked with +

Additional file 12: Fig. S3 Maximum likelihood (ML) analysis of the *coi* gene. The ML analysis used the TPM2 + F + G4 model. Bootstrap values are indicated at each branch node; values under 70 were removed. Blue, pink, green and purple highlighted regions indicate *A. hebraeum*, *A. eburneum*, *A. variegatum* and *A. pomposum*, respectively. Species names are included with a three-letter country code and the sample name or GenBank accession number. Country codes: AGO, Angola; CMR, Cameroon; GNB, Guinea-Bissau; KEN, Kenya; MOZ, Mozambique; SEN, Senegal; SWE, Eswatini; ZAF, South Africa; ZMB, Zambi; and ZWE, Zimbabwe. GenBank reference strains are marked with **

Additional file 13: Fig. S4 Maximum likelihood (ML) analysis of the *cytB* gene. The ML analysis used the HKY + F + G4 model. Bootstrap values are indicated at each branch node; values under 70 were removed. Blue, pink, green and purple highlighted regions indicate *A. hebraeum*, *A. eburneum*, *A. variegatum* and *A. pomposum*, respectively. GenBank reference strains are marked with **

## 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 produced the micrographs of the ticks (Fig. 4).

## Author contributions

A. Smit: Conceptualisation, sample collection, methodology, investigation, data curation, writing – original draft and visualisation. F.C. Mulandane: Sample collection and writing – review and editing. M. Labuschagne: Investigation and writing – review and editing. S.H. Wojick: Investigation and writing – review and editing. C. Malabwa: Sample collection, investigation and writing – review and editing. G. Sili: Sample collection and writing – review and editing. S. Mandara: Sample collection and writing – review and editing. Z. Dlamkile: Sample collection and writing – review and editing. R. Ackermann: Visualisation and writing – review and editing. H. Rose Vineer: Visualisation and writing – review and editing. W.H. Stoltsz: Review and editing. K. Huber: Review and editing. I. G. Horak: Review and editing. D. Morar-Leather: Writing – review and editing, supervision and funding acquisition. B. L. Makepeace: Writing – review and editing and supervision. L. Neves: Conceptualisation, sample collection, methodology, writing – review and editing, supervision and funding acquisition.

## Funding

This work was supported by AgriSETA, the Meat Industry Trust and the doctorate research bursary awarded to A. Smit by the University of Pretoria.

## Availability of data and materials

The data supporting the main findings of this study are included in the manuscript and its supplementary files. Sequences generated in this study were deposited in GenBank and the accession numbers are available in Additional file 3: Dataset S2.

## Declarations

### Ethics approval and consent to participate

This study was approved by the Research Ethics Committee of the University of Pretoria (REC 121-20) and 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)).

### Consent for publication

All authors have contributed as described and give consent to publish this article.

### Competing interests

The authors declare that they have no competing interests.

### Author details

¹Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Gauteng, Republic of South Africa. ²Biotechnology Center, Eduardo Mondlane University, Maputo, Mozambique. ³Department of Genetics, Faculty of Natural and Agricultural Sciences, University of Pretoria, Gauteng, Republic of South Africa. ⁴Central Veterinary Research Institute, Lusaka, Zambia. ⁵Department of Basic Science, Faculty of Veterinary Medicine, University Jose Eduardo Dos Santos, Huambo, Angola. ⁶Department of Animal Production Sciences, Marondera University of Agricultural Sciences and Technology, Marondera, Zimbabwe. ⁷Department of Infection Biology and Microbiomes, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, UK. ⁸ASTRE, Univ Montpellier, CIRAD, INRAE, Montpellier, France.

Received: 4 January 2024 Accepted: 5 July 2024

Published online: 28 August 2024

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