Phylogenetic analysis of Haemaphysalis erinacei Pavesi, 1884 (Acari: Ixodidae) from China, Turkey, Italy and Romania

Haemaphysalis erinacei is one of the few ixodid tick species for which valid names of subspecies exist. Despite their disputed taxonomic status in the literature, these subspecies have not yet been compared with molecular methods. The aim of the present study was to investigate the phylogenetic relationships of H. erinacei subspecies, in the context of the first finding of this tick species in Romania. After morphological identification, DNA was extracted from five adults of H. e. taurica (from Romania and Turkey), four adults of H. e. erinacei (from Italy) and 17 adults of H. e. turanica (from China). From these samples fragments of the cytochrome c oxidase subunit 1 (cox1) and 16S rRNA genes were amplified via PCR and sequenced. Results showed that cox1 and 16S rRNA gene sequence divergences between H. e. taurica from Romania and H. e. erinacei from Italy were below 2%. However, the sequence divergences between H. e. taurica from Romania and H. e. turanica from China were high (up to 7.3% difference for the 16S rRNA gene), exceeding the reported level of sequence divergence between closely related tick species. At the same time, two adults of H. e. taurica from Turkey had higher 16S rRNA gene similarity to H. e. turanica from China (up to 97.5%) than to H. e. taurica from Romania (96.3%), but phylogenetically clustered more closely to H. e. taurica than to H. e. turanica. This is the first finding of H. erinacei in Romania, and the first (although preliminary) phylogenetic comparison of H. erinacei subspecies. Phylogenetic analyses did not support that the three H. erinacei subspecies evaluated here are of equal taxonomic rank, because the genetic divergence between H. e. turanica from China and H. e. taurica from Romania exceeded the usual level of sequence divergence between closely related tick species, suggesting that they might represent different species. Therefore, the taxonomic status of the subspecies of H. erinacei needs to be revised based on a larger number of specimens collected throughout its geographical range.

Haemaphysalis erinacei is one of the few ixodid tick species for which valid names of subspecies exist. Subspecies are conspecific taxa, representatives of which show differences in morphology and geographical range from each other, but can naturally interbreed. Accordingly, until now H. erinacei subspecies were described on the basis of different morphology and geographical range, but this resulted in a controversy in their taxonomy. Camicas et al. [11] (Fig. 1).
Despite this taxonomic controversy, no studies have attempted molecular phylogenetic comparison of H. erinacei subspecies. Based on the above literature data on their morphology and geographical range, we hypothesized that phylogenetic analyses would support H. e. turanica as a separate species from H. e. taurica and H. e. erinacei. Therefore, in the present study the phylogenetic relationships of H. erinacei subspecies (collected in four countries) were investigated, in the context of the first finding of this tick species in Romania.
Cytochrome c oxidase subunit 1 (cox1) and 16S rDNA genes are well-established barcoding genes for molecular identification and phylogenetic analyses of ticks [13][14][15][16]. Therefore analysis of these two genes was chosen to investigate the phylogenetic relationships of H. e. taurica, H. e. erinacei and H. e. turanica in the present study.

Molecular analysis
DNA was extracted with QIAamp DNA Mini Kit (QIA-GEN, Hilden Germany) as described [18], including an overnight digestion step at 56°C in tissue lysis buffer and 6.6% proteinase-K (provided by the manufacturer). Two mitochondrial markers were amplified from selected samples: an approx. 710 bp long fragment of the cytochrome c oxidase subunit 1 (cox1) gene using the primers HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3') and LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) [19], and an approx. 460 bp fragment of the 16S rDNA gene using the primers 16S + 1 (5′-CTG CTC AAT GAT TTT TTA AAT TGC TGT GG-3′) and 16S-1 (5′-CCG GTC TGA ACT CAG ATC AAG T-3′) [13]. Reaction conditions were set as reported [20]. Concerning samples collected in China, another set of primers (designed by Primer Premier 5.0 software) was used for the cox1 gene (forward: 5′-ATT TAC AGT TTA TCG CCT-3′; reverse: 5′-CAT ACA ATA AAG CCT AAT A-3′), and PCR conditions were different (preheating at 94°C for 4 min,  Co. (Shanghai, China). The newlygenerated sequences were manually edited, aligned and compared to reference GenBank sequences by nucleotide BLASTN program (https://blast.ncbi.nlm.nih.gov). Representative sequences were submitted to the GenBank database ( Table 1). The automatic MEGA model selection method (analysis: Maximum Likelihood model selection, substitution type: nucleotide) was applied to choose the appropriate model for phylogenetic analyses. The dataset was resampled 1000 times to generate bootstrap values. Phylogenetic analyses were conducted with the Maximum Likelihood method (HKY model [21]) by using MEGA version 6.0 [22]. Outgroups of phylogenetic trees were selected from GenBank (from ixodid genera other than Haemaphysalis), and are referenced according to accession numbers.

Results and discussion
The cox1 nucleotide sequence of H. e. taurica from Romania was 100% identical with the sequence for the same subspecies from Turkey (Tokat province), and 99.4% identical with H. e. erinacei from Italy, but had only 94. 6 Taken together, the cox1 and 16S rDNA gene sequence divergences between H. e. taurica from Romania and Turkey (Tokat province) and H. e. erinacei from Italy were low (below 2%). This may be consistent with allopatric separation of these two subspecies (Fig. 1). Similar magnitudes of intraspecific genetic (i.e. 1.2%) variation in the 16S rRNA target region have been recorded for other ixodid species, such as I. scapularis, over large geographical distances [15]. However, the sequence divergence between H. e. taurica from Romania and H. e. turanica from Central-Asia was high (up to 5.4% for the cox1 gene, and up to 7.3% for the 16S rRNA gene; Table 1), i.e. exceeding the expected (average) level of sequence divergence between closely related tick species [14]. For comparison, the 16S rRNA gene sequence similarity between I. inopinatus (KM211790) and I. ricinus (GU074592) is 98.2% (383/390 bp), amounting to 1.8% difference [16]. When species boundaries were evaluated for several tick species [14], the sequence divergence delineating tick species was reported to be 5.3% for the 16S rRNA gene, i.e. much lower than the 7.3% shown here for H. e. taurica and H. e. turanica.
Interestingly, two females from Turkey (Sivas province), which were morphologically identified as adults of H. e. taurica (Fig. 2) had higher 16S rRNA gene similarity to isolates of H. e. turanica from China (maximum 396/406 bp = 97.5%) than to H. e. taurica from Romania (96.3%) ( Table 1). However, these two samples clustered phylogenetically more closely to H. e. taurica than to H. e. turanica (Fig. 4), indicating the existence of different genetic lineages within H. e. taurica. Fig. 2 Morphology of genetically divergent H. e. taurica female from Turkey (Sivas Province) identified according to Filippova [17]. a Caudolateral setae on coxa IV are much longer than the spur (arrow). b The pulvillus (arrow) almost reaches the ends of claws The number/percentage of nucleotide differences between H. e. taurica, H. e. erinacei and H. e. turanica are well reflected by the topology of cox1 phylogenetic tree, with H. e. taurica and H. e. erinacei clustering close to each other, but separately from H. e. turanica (Fig. 3). This separation was supported by a high probability (94%), and chronologically (based on branch lengths) preceded the separation of H. e. taurica and H. e. erinacei (Fig. 3). The phylogenetic analysis of 16S rRNA gene sequences confirmed these relationships, i.e. all genotypes of H. e. turanica clustered in one clade, as a sister group to all H. e. erinacei and H. e. taurica isolates (Fig. 4).
In a geochronological context, the divergence of H. punctata and H. flava was estimated to have taken place approx. 40 million years ago [23]. Relative to this event, as inferred from the branch lengths in the 16S rRNA gene phylogenetic tree (Fig. 4), the divergence of H. e. turanica from H. e. taurica/erinacei might have occurred much more recently.
Several factors may have contributed to this divergence and its maintenance. Southern peninsulas of Europe acted as major refugia during ice age(s), from which genetically distinct clades of animal species emerged [24], as also exemplified by H. e. erinacei and H. e. taurica. Similarly, glacial surfaces confluent with the Caspian Sea [25] Table 1 for details). The vertical red, yellow and blue lines mark the H. e. taurica, H. e. erinacei and H. e. turanica clades, respectively. Branch lengths represent the number of substitutions per site inferred according to the scale shown In addition, based on an extensive collection material, while the hosts of H. e. taurica and H. e. turanica are common, these two tick subspecies exhibit biotope isolation in overlapping parts of their geographical range [17], which most likely reduced further the chances of gene flow between their populations.

Conclusions
This is the first finding of H. erinacei in Romania, and the first (although preliminary) phylogenetic comparison of H. erinacei subspecies. Phylogenetic analyses do not support that the three H. erinacei subspecies evaluated here are of equal taxonomic rank. In particular, the genetic divergence between H. e. turanica from China and H. e. taurica from Romania exceeded the usual level of sequence divergence between closely related tick species, suggesting (especially if formerly reported morphological differences are also taken into account) that they might represent different species. Therefore, the taxonomic status of the subspecies of H. erinacei needs to be revised based on a larger number of specimens collected throughout its geographical range.  Table 1 for details). The vertical red, yellow and blue lines mark the H. e. taurica, H. e. erinacei and H. e. turanica clades, respectively. Branch lengths represent the number of substitutions per site inferred according to the scale shown