Description of a new tick species, Ixodes collaris n. sp. (Acari: Ixodidae), from bats (Chiroptera: Hipposideridae, Rhinolophidae) in Vietnam

Background In a recent study on ixodid bat ticks from Eurasia, a high genetic difference was found between Ixodes vespertilionis from Europe and Vietnam. Accordingly, it was proposed that I. vespertilionis is a species complex, with at least one additional, hitherto undescribed species. The aim of the present study was to investigate the morphology of bat ticks from Vietnam and to assess their taxonomic status in comparison with those collected in Europe. Findings Ixodid bat ticks (two females and two nymphs) collected from the pomona leaf-nosed bat (Hipposideros pomona) (Hipposideridae) and intermediate horseshoe bat (Rhinolophus affinis) (Rhinolophidae) in Vietnam showed major morphological differences from European isolates of I. vespertilionis, including the shape of the scutum, the enclosure and shape of porose areas, the presence of a caudo-lateral collar-like ridge ventrally on the basis capituli, polytrich coxae with short setae, and grouped (non-linear) arrangement of anterior pit sensillae in Haller’s organ. Conclusions In this study the female and the nymph of an ixodid bat tick species from Vietnam are described for the first time. The genetic and morphological differences between I. vespertilionis Koch, 1844 and these bat ticks from Vietnam justify the status of the latter as a distinct species, Ixodes collaris Hornok n. sp.


Background
In Eurasia, three bat tick species or their genetic variants appear to be geographically widespread: Ixodes vespertilionis Koch, 1844, I. simplex Neumann, 1906 and I. ariadnae Hornok, 2014 [1]. Concerning I. vespertilionis, a high genetic difference was found between specimens collected in Europe and another isolate collected in Vietnam. In particular, in the amplified fragment of their cytochrome c oxidase subunit I (COI) gene the genotypes from Europe and Vietnam differed by 16 %, greatly exceeding the level of the reported average COI sequence divergence between species (i.e. 6.1 %: [2]). Accordingly, it was proposed that I. vespertilionis may be a species complex, with at least one additional, hitherto undescribed species [1]. The aim of the present study was to investigate the morphological differences between ixodid bat ticks collected in Vietnam and Europe, and to verify the existence of a new species in the I. vespertilionis species group.

Methods
Ticks were removed from bats (caught with mist-nets during biodiversity inventory and monitoring projects) and immediately placed into 96 % ethanol. Molecular genetic analysis was performed as previously reported (paratype No. 2 below; see [1]). Pictures were made with a VHX-5000 (Keyence Co., Osaka, Japan) digital microscope.  Etymology: The name of the new species refers to the ventral, caudolateral collar-like ridge on the basis capituli, which appears to be a unique character.

Differential diagnosis
Major morphological differences between females of I. collaris n. sp. and females of I. vespertilionis are the following. The scutum of I. vespertilionis (Fig. 1b) is  . a I. vespertilionis shows lateral flange on basis capituli (blue arrow) and a few, long coxal setae (especially on coxa III: white arrows). Note: V-shaped arrangement of some of these setae is due to reflection. b I. collaris n. sp. with ventral collar on basis capituli (yellow arrow) and multiple, short coxal setae (black arrows) posteriorly tapering, with a shape index exceeding 1.6 [4,5] whereas the scutum of I. collaris n. sp. is posteriorly broad, with a shape index around 1.5 (Fig. 1a). Ventrally, the setae located anteriorly to the genital aperture are shorter than those located posteriorly in I. collaris n. sp. (Fig. 2a), whereas the setae of I.
vespertilionis are similar in length both anteriorly and posteriorly to the genital aperture (Fig. 2b). The porose areas in I. vespertilionis are broader than long, separated by a narrow interval [4,5], and surrounded by an anteriorly converging flank (Fig. 1d). However, the porose areas in I. collaris n. sp. are longer than  broad, separated by a broad interval and surrounded by an initially divergent, then convergent flank (Fig. 1c). The female of I. vespertilionis has a dorsal transverse ridge posteriorly on the basis capituli that continues around the sides as vertical, outwardly directed flange (which is perpendicular to the hypostome) [4]. Adding to this, ventrally on the basis capituli of I. collaris n. sp. a broad, collar-like extension is present behind the palpal base caudolaterally (in a nearly horizontal/longitudinal direction). Furthermore, the coxae of I. vespertilionis female bear few long (100 μm or above) setae posteriorly [5] (Fig. 3a), whereas multipe short setae predominate on the coxae of I. collaris n. sp. (Fig. 3b). The arrangement of anterior pit sensillae in Haller's organ is grouped in the case of I. collaris n. sp., but linear in the case of I. vespertilionis [6]. Regarding nymphs of I. collaris n. sp., the bilateral collars ( Fig. 4b) clearly distinguish the new species from I. vespertilionis (Fig. 4c).
The female of I. collaris n. sp. also differs from the female of I. ariadnae in the shape of the scutum (anteriorly narrow vs anteriorly broad) and porose areas (longer than broad vs broader than long), the palpal shape (elongated vs broad), the presence (vs absence) of collar, the arrangement and length of coxal setae (predominance of short vs long setae) [6]. On the other hand, similarities between I. collaris n. sp. and I. ariadnae include the grouped (non-linear) arrangement of anterior pit sensillae in Haller's organ, as well as the folded surface of nymphal scutum [6].
In contrast to the female of I. collaris n. sp., the female of I. simplex has anteriorly broad scutum, short and broad palps, and short legs with multiple long setae on coxae IV [6].

Discussion
The genetic differences between ixodid bat ticks collected in Europe and Asia were shown to greatly exceed the limit of intraspecific sequence divergence [1]. In line with this, the morphological characteristics of I. collaris n. sp. described above, clearly support its distinct species status. Interestingly, while I. collaris n. sp. was apparently misidentified as I. vespertilionis in several parts of Southeast Asia, based on the analysis of both COI and 16S rDNA genes, this new species is phylogenetically closer to I. ariadnae than to I. vespertilionis [1].
Geographical separation of bat populations may be an important driver of the speciation of their ticks. In line with this, the geographical isolation of lesser horseshoe bats (R. hipposideros) north and south of the Pyrenees [7], was reported to entail high level of genetic difference between their ixodid ticks (I. vespertilionis) [1]. The main host of I. vespertilionis (R. hipposideros) and those of I. collaris n. sp. reported here (R. affinis, H. pomona) show allopatric distribution in Eurasia, i.e. the former occurs west of the Himalayas, whereas the latter two in and east of the Himalayas. This (and possibly other factors in niche segregation) may have acted as selective pressures towards divergent evolution of these bat species (which cluster separately in phylogenetic analyses: [1,8]) and of their ixodid ticks, i.e. I. vespertilionis and I. collaris n. sp.
Ixodes vespertilionis has long been regarded as uncommon in south-east Asia [9], and this may explain why no detailed comparative morphological studies were carried out on this tick species in the region. The description of I. vespertilionis from Japan by Yamaguti et al. [10] indicates several shared features with I. collaris n. sp., e.g. the shape of the scutum, the presence of the ventral collar and the grouping of anterior pit sensillae in the Haller's organ. Therefore, also taking into account the relatively close phylogenetic relationship of bat ticks from Vietnam and Japan [1] it is possible that I. collaris n. sp. also occurs in Japan. In addition, because R. affinis appears to be an important host of the newly described bat tick species (as shown here), and this bat species was reported to be the predominant host of I. vespertilionis in southern China [11], we hypothesise that I. collaris n. sp. has a broad geographical range in south-east Asia.

Conclusion
In this study the female and the nymph of an ixodid bat tick species from Vietnam are described for the first time. The genetic and morphological differences between I. vespertilionis Koch, 1844 and these bat ticks from Vietnam justify the status of the latter as a distinct species I. collaris Hornok n. sp. and Rural Development (Vietnam Administration of Forestry), People's