Rhipicephalus sanguineus

Hekimoğlu, Olcay, 2024, An update on the phylogeny and biogeographical history of Rhipicephalus sanguineus complex, Turkish Journal of Zoology 48, pp. 21-35 : 27-31

3.1. Morphological species identification

Ticks were morphologically classified as R. turanicus , R. rossicus , R. sanguineus sensu lato, R. sanguineus complex, and R. bursa . Among the samples, Rhipicephalus turanicus (74.7%) constituted the majority, while six individuals were identified as R. rossicus and one individual as R. bursa . Thirteen samples ( Rhipicephalus sp. ) could not be identified at the species level and were classified as either R. sanguineus s.l. or R. sanguineus complex ( Table 1 ). The collection patterns based on the hosts were as follows: 36.8% from dogs, 31.7% from sheep, 10.1% from vegetation (flagging), 10.1% from cow, 10.1% from goats, and 1.2% from humans.

3.2. Molecular sequences

The Mt 16S rDNA dataset consisted of 41 sequences. Additional sequences obtained from previous studies conducted by the researchers using samples collected from Türkiye were added to the 16S rDNA dataset ( Hekimoglu et al., 2016; Hekimoglu et al., 2021) ( Table 1 ). Seventeen haplotypes were obtained using Mt 16S rDNA ( Table 2 ). The final dataset, which included GenBank sequences from different localities around the world, consisted of 85 Rhipicephalus sp. sequences with a total length of 389 base pairs. Although T92 + G was determined as the model, TN93 + G was implemented instead since BEAST software does not support T92+G, and TN93+G was chosen as the closest alternative model. The total number of conserved positions in the dataset was 297, with 92 variable positions, out of which 74 were parsimony informative.

After short and unreadable mt 12S rDNA sequences were removed, phylogenetic analysis was conducted with 26 sequences and 16 unique haplotypes were obtained ( Table 3 ). As mentioned previously, some samples were identified molecularly using mt16S rDNA ( Hekimoglu et al., 2021). In this study, mt12S rDNA sequences were generated for these identified samples. The dataset, including downloaded sequences from GenBank, had a length of 342 base pairs and consisted of a total of 60 taxa. The number of conserved sites in the total dataset was 244, the number of variable sites was 98, and 75 of them were parsimony informative. Due to the absence of T92 + G in the BEAST software, TN93 + G was employed.

Using ITS2, a total of 23 sequences were obtained, resulting in 8 unique haplotypes. This dataset comprised 22 taxa and had a length of 255 base pairs ( Table 4 ). The best-fitting model was determined to be T92, and TN93 + G was used. The overall dataset contained 247 conserved sites and 3 variable sites, with 1 of them being parsimony informative.

3.3. Phylogenetic relationships

The phylogenetic tree constructed using mt16S rDNA identified five major clades ( Figure 2 ): R. sanguineus s.s. (temperate lineage), R. turanicus Asian lineage, R. turanicus European lineage, R. sanguineus tropical + R. sanguineus Middle East lineage, and R. rossicus . Rhipicephalus rossicus , which comprised sequences from Türkiye, India, Romania, and China, is distantly located from the other clades (99%). Rhipicephalus sanguineus s.s. comprised samples from Europe, including France, Germany, Spain, Serbia, Croatia, as well as countries from both North ( USA) and South America ( Argentina, Uruguay) (100%). None of the haplotypes from Türkiye grouped within this lineage. Rhipicephalus turanicus Asian lineage seemed to distribute mostly in Middle Eastern countries such as Israel and Türkiye, as well as countries in Asia continent such as Kyrgyzstan, China, Afghanistan, and eastern Siberia (100%). This lineage was closely related to R. turanicus European lineage and R. sanguineus tropical + Middle East lineage (55%). The majority of haplotypes from Türkiye (12/19) grouped within the R. turanicus European lineage and clustered together with sequences from Italy, Croatia, and Greece. The R. sanguineus tropical lineage is separated into two lineages (80%): One lineage involved samples of Middle East ( Egypt, Romania, Northern Cyprus, Türkiye), while the other lineage included sequences from Africa, America, and Australia.

Five clades were identified in the mt 12S rDNA phylogenetic tree ( Figure 3 ): R. turanicus Asian lineage, R. sanguineus tropical + Middle East lineage, R. turanicus European lineage, R. rossicus , and R. sanguineus s.s. Unlike mt 16S rDNA, R. sanguineus s.s. and R. rossicus were sister taxa (65%) according to mt 12S rDNA tree. Rhipicephalus sanguineus s.s. comprised sequences from America continent, such as USA, Uruguay, Argentina, as well as European countries including France and Portugal. Rhipicephalus rossicus consisted of sequences from Türkiye, Romania, and Russia. The majority of haplotypes from Türkiye (9/15) grouped within the R. turanicus European lineage, together with sequences from Greece, Switzerland, and Italy (96%). Rhipicephalus turanicus Asian lineage was closely related to the R. sanguineus tropical + Middle East lineage (77%). The R. turanicus Asian lineage comprised samples from Amasya and Siirt provinces in Türkiye, as well as sequences from Israel, Afghanistan, Kyrgyzstan, and Uzbekistan. R. sanguineus tropical lineage and Middle East lineage were sister taxa (98%). Additionally, two separate lineages were identified in Middle East lineage (100%). Sequences from Romania, Italy, and Türkiye (from Antalya and İstanbul) clustered together, while samples from Egypt and Israel formed a distinct lineage. The R. sanguineus tropical lineage comprised sequences from Australia, America, and Europe.

In contrast to the phylogenetic pattern observed from mitochondrial DNA sequences, ITS2 marker was unable to distinguish species within R. sanguineus complex ( Figure 4 ). In the phylogenetic tree, only R. rossicus was identified as a distinct lineage, whereas other taxa ( R. sanguineus s.s. and tropical lineage, and even R. turanicus ) could not be distinguished. Samples belonging to the Middle Eastern lineage of R. sanguineus (CYP7, CYP9, ANT-Dog1), formed a distinct lineage (96%) like mitochondrial trees, but their clustering with R. turanicus samples makes their identification difficult. The reasons for this observation in the ITS2 marker are discussed in the discussion section.

3.4. Historical biogeography analysis

The S-DIVA and BBM models constructed using the mt 16S rDNA dataset provided different results for certain nodes ( Figure 5 ). According to the S-DIVA analysis, the ancestor of the R. sanguineus complex exhibited a wide geographic distribution. This distribution range (Node 55: B + D = 60.5%) encompassed the entire Europe and the Middle East. However, the BBM model identified Western Europe as the ancestor of the R. sanguineus complex (Node 55: B = 87.5%). Both models indicated that the ancestor of R. pucillus was from Western Europe (Node 54: B = 100%). The ancestor of lineages other than R. pucillus (Node 53) was either the Middle East + Eastern Europe according to the S-DIVA (D = 60%) or either Western Europe (B = 44.7%) or the Middle East + Eastern Europe (D = 38.7%) according to the BBM. The ancestor of R. rossicus , which was included for the first time in ancestral biogeography analyses, was determined as the Middle East + Eastern Europe by the BBM model (Node 52: D = 65%). According to S-DIVA, it was more extensive, including Asia as well (Node 52: D + E = 77%). The ancestor of taxa other than R. rossicus (Node 49) was Europe and the Middle East according to S-DIVA (B + D = 57.6%), while it was either Western Europe or Eastern Europe + the Middle East (B = 43.3%, D = 32.5%) according to the BBM. One of the lineages derived from this clade (Node 34) was R. sanguineus s.s., whose ancestor was Western Europe in both models (S-DIVA: B = 77%, BBM: B = 88.8%). The ancestor of Node 48, which is consisted of Asian and European lineages of R. turanicus , and R. sanguineus Middle Eastern + tropical lineage, was the Middle East + Eastern Europe according to both models (S-DIVA: D = 62%; BBM: D = 85%). Both models suggested that this common ancestor diverged into R. sanguineus tropical lineage in Africa and subsequently spread to America and Australia while also constituting R. sanguineus Middle Eastern lineage in the Middle East + Eastern Europe ( Figure 5 ).

According to the mt 12S rDNA results of S-DIVA, the common ancestor of R. sanguineus complex exhibited ambiguity, and multiple alternatives existed ( Figure 6 ). S-DIVA suggested a widely distributed ancestor encompassing Europe and the Middle East (B + D = 35%), the Middle East + Eastern Europe (D = 33%), or Europe, the Middle East, and Africa (B + C + D = 29%) origin. In contrast, BBM proposed Middle East + Eastern Europe (Node 55: D = 64.9%) origin. Although the ancestor of R. rossicus , R. sanguineus s.s., and R. pucillus (Node 54) was estimated as Europe and the Middle East (B + D = 100%) according to the S-DIVA, BBM indicated that the ancestor originated in the Middle East and Eastern Europe (D = 48.6%). Both models suggested that this ancestor split into two lineages: whereas R. rossicus originated in the Middle East + Eastern Europe (Node 48, S-DIVA: D + E = 100%; BBM: D = 51.7%), the origin of R. sanguineus s.s. and R. pucillus was America and Western Europe (Node 53, S-DIVA: A + B = 84%; BBM: B = 45%). BBM analysis postulated that the ancestor of the R. sanguineus tropical lineage and the R. sanguineus Middle Eastern lineage (Node 46) was the Middle East + Eastern Europe (D = 67%), while S-DIVA placed this taxon to be of Africa + the Middle East and Eastern Europe (C + D = 100%). Both models suggested that one branch diverged from this ancestor (Node 37) and remained in the Middle East + Eastern Europe (D = 100%), while the other lineage (Node 45) separated and migrated to Africa, then to America and Australia (C = 100%). S-DIVA estimated that ancestors of R. turanicus originated in Middle East + Eastern Europe or Middle East + Eastern Europe + Asia (Node 35: D = 46%; D + E = 54%). However, BBM placed the origin of R. turanicus in either Middle East + Eastern Europe or Asia (Node 35: D = 42%, E = 36%). This ancestor diverged into R. turanicus Asian and European lineages ( Figure 6 ).

Figures

Figure 2. Phylogenetic tree of sequences obtained by mt 16S rDNA from this study and sequences of GenBank. Haplotypes obtained from this study are indicated with TRY codes and highlighted in bold.

Figure 3.Phylogenetic tree of sequences obtained by mt 12S rDNA from this study and sequences of GenBank. Haplotypes obtained from this study are indicated with TRY codes and highlighted in bold.

Figure 4. Phylogenetictree of sequences obtained by ITS2 from this study and sequences of GenBank. Haplotypes obtained from this study are indicated with TRY codes and highlighted in bold. Different haplotypes of the same individual are labeled as TRY-1 and TRY-2 on the phylogenetic tree.

Figure 5. The biogeographic analysis of the Rhipicephalus sanguineus complex with S-DIVA and BBM analysis based on mt 16S rDNA.

Figure 6. The biogeographic analysis of the Rhipicephalus sanguineus complex with S-DIVA and BBM analysis based on mt 12S rDN.