The Ornithodoros capensis group (Acari: Argasidae) - BioOne

Systematic & Applied Acarology 22(1): 28–41 (2017) http://doi.org/10.11158/saa.22.1.5 Article

ISSN 1362-1971 (print) ISSN 2056-6069 (online)

http://zoobank.org/urn:lsid:zoobank.org:pub:D25054A1-C7AA-442C-A576-224D46D9AE4F

The Ornithodoros capensis group (Acari: Argasidae): a morphological diagnosis and molecular characterization of O. capensis sensu stricto from Queimada Grande Island, Brazil SEBASTIÁN MUÑOZ-LEAL1*, RICARDO A. DIAS1, CARLOS R. ABRAHÃO1,2 & MARCELO B. LABRUNA1 1

Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitária, São Paulo, SP, Brazil, 05508-270. *email: [email protected] 2 Centro Nacional de Pesquisa e Conservação de Répteis e Anfíbios, Instituto Chico Mendes de Conservação da Biodiversidade, Ministério do Meio Ambiente, Rua 229, 95, Setor Leste Universitário, Goiânia, GO, Brazil, 74605-090.

Abstract Ornithodoros capensis sensu lato (s. l.) is a worldwide-distributed group of soft ticks that parasitize birds in insular and continental lands. It is currently composed of 11 morphologically closely related species. Several viral and bacterial pathogens, and particularly Coxiella-like endosymbiont organisms have been described coexisting with ticks of this group. Since it last report in 1983, the presence of O. capensis s. l. in Brazil has remained undocumented. By a morphological analysis of larvae and a molecular characterization of ticks and Coxiella genes we describe for the first time O. capensis sensu stricto in Brazil from specimens collected on Queimada Grande Island, in São Paulo state. Key-words: Ornithodoros capensis sensu stricto, Argasidae, Queimada Grande, Brazil

Introduction The Ornithodoros capensis group is currently composed by 11 bird-associated morpho-species distributed in all Zoogeographic Regions of the world with the exception of Antarctica (Kohls et al. 1965; Hoogstraal 1969, 1985; Hoogstraal et al. 1974, 1976, 1979, 1983, 1985; Kitaoka & Suzuki 1973; Clifford et al. 1980; Keirans et al. 1984; Vermeil et al. 1997). Representatives of this group are mainly seabird parasites (Hoogstraal 1985; Keirans et al. 1992; Dietrich et al. 2011), yet some of them also parasitize mainland-bird species and mammals (Hoogstraal et al. 1979), including humans (Estrada-Peña & Jongejan 1999). The type species of the group was originally described by Neumann (1901) as Ornithodoros talaje var. capensis from post-larval stages collected in penguin colonies breeding on islands of the Atlantic coast of South Africa. Reports of O. capensis sensu lato (s. l.) in Brazil were documented by Nuttall et al. (1908) from the examination of specimens collected during the Challenger expedition in 1876, and by Edwards & Lubbock (1983), both at São Pedro and São Paulo Archipelago (refereed as St. Paul’s Rock), located in the North Atlantic Ocean at ≈ 1000 km off Brazilian shores (00º55’ N; 29º20’ W). Since then, the species was subsequently renamed several times, numbering Argas capensis in Bedford (1932) and Carios capensis in Klompen et al. (1996) among its synonyms (Camicas et al. 1998). Although Horak et al. (2002) and Barker & Murrell (2004) included this species within the Carios genus, the last revision of valid tick names (Guglielmone et al. 2010) considered this species as 28

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Ornithodoros capensis. Ticks within this species complex are currently represented by O. capensis sensu stricto (s. s.) Neumann 1901, Ornithodoros amblus Chamberlin 1920, Ornithodoros cheikhi Vermeil, Marjolet & Vermeil 1997, Ornithodoros collocalie Hoogstraal, Kadarsan, Kaiser & Van Peen 1974, Ornithodoros coniceps Canestrini 1890, Ornithodoros denmarki Kohls, Sonenshine & Clifford 1965, Ornithodoros maritimus Vermeil & Marguet 1967, Ornithodoros muesebecki Hoogstraal 1969, Ornithodoros sawaii Kitaoka & Suzuki 1973, Ornithodoros spheniscus Hoogstraal, Wassef, Hays & Keirans 1985, and Ornithodoros yunkeri Keirans, Clifford & Hoogstraal 1984. It has been hypothesized that this group of soft ticks originated in the Neotropical Zoogeographic Region and then spread via migratory birds to its actual distribution (Hoogstraal 1985). In fact, studies have proposed that variability in 16S mitochondrial rDNA haplotypes in populations of O. capensis s. l. from Torishima, Japan (Ushijima et al. 2003) and Cabo Verde Islands (Gómez-Díaz et al. 2012) correspond to seabird-spread ticks from different geographical origins converging in one unique location. Although informative, these molecular studies lack a morphological diagnosis of the analysed ticks, and particularly of larval stage, which has been shown to be taxonomically significant in order to separate the species of the family Argasidae (Hoogstraal 1985; Klompen 1992). In this way, correlating the morphology with a specific genetic array would constitute valuable information in order to separate morphologically closely related species within the O. capensis group. Microorganisms associated with ticks of the O. capensis group include viruses (Hoogstraal 1985) and eubacteria of the genera Borrelia, Coxiella and Rickettsia (Reeves et al. 2006; Duron et al. 2014; Wilkinson et al. 2014; Al-Deeb et al. 2016). There is a particularly high diversity of Coxiella-like bacteria with transstadial and transovarial transmissions that have been molecularly detected in several species of this group and in other argasid ticks worldwide (Almeida et al. 2012, Duron et al. 2015). These bacterial microorganisms have a mutualistic, endosymbiotic relationship with their associated ticks and co-divergence events have been suggested to occur, since closely related Coxiella-like bacteria are often found in closely related tick species (Almeida et al. 2012, Duron et al. 2015). Since the report of unspecified stages by Edwards & Lubbock (1983) 33 years ago, O. capensis s. l. has never been documented again from any part (continental or insular) of the Brazilian territory. In this study, we performed a taxonomic diagnosis of seabird soft ticks collected on an offshore island of São Paulo state using morphological and molecular tools and by analysing its associated Coxiella-like endosymbionts.

Materials and Methods Study site and collection of ticks During May 2015, two nymphs, five females and eight Ornithodoros males were collected under rocks around Sula leucogaster (brown booby, Pelecaniformes: Sulidae) resting places (24º28’39’’S; 46º40’36’’W, 19 m) and one nymph on the higher rocky peak (24º29’21’’S; 46º40’27’’W, 157 m) of Queimada Grande Island (QGI), located at 35 km offshore in the São Paulo state seaboard, Brazil (Fig. 1). Fieldwork was performed under the SISBIO licenses 48445-1 and 11459-1. Morphological identification Ticks were brought alive to laboratory and two cohorts of unfed larvae were obtained from eggs laid by two fertilized females. Ten larvae from each group were clarified in a 20% KOH solution, mounted in semi-permanent slides for optical microscopy examination using Hoyer’s medium. 2017

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Slide-mounted ticks were photographed with an Olympus DP70 camera implemented in an Olympus BX40 optical microscope, and measured with the software Image-Plus Pro v5.1. All measurements are given in millimeters (mm) followed by the mean and standard deviation in parenthesis, and compared with morphometric data of conspecific and highly similar species available in the literature. Morphological identification of Ornithodoros larvae to species level was based on keys by Kohls et al. (1969), Jones & Clifford (1972), and original species descriptions of O. capensis group representatives (Kohls et al. 1965; Hoogstraal 1969, 1985; Hoogstraal et al. 1974, 1976, 1979, 1983, 1985; Kitaoka & Suzuki 1973; Clifford et al. 1980; Keirans et al. 1984; Hoogstraal 1985; Vermeil et al. 1997). Taking into account that the original description of O. capensis s. s. lacks a description of the larval stage (Neumann 1901), in this study we consider as O. capensis s. s. the description of larvae presented by Kohls et al. (1965). These authors obtained larvae from females collected in penguin colonies (Spheniscus demersus L. 1758) from Malagas and Dassen islands, both located on the shores of South Africa, within the geographic area of the type specimens (Neumann 1901). Since there has been no consensus at genus level classification of the Argasidae, we followed the classification of Hoogstraal (1985), which was adopted in the last published list of valid tick species names of the world (Guglielmone et al. 2010).

FIGURE 1. Map with the localization of Queimada Grande Island in the Brazilian coast.

Molecular tools In order to confirm morphological diagnoses, and to detect the presence of Coxiella-like organisms, DNA extraction using the guanidine isothiocyanate technique (Sangioni et al. 2005) was separately performed for a nymph collected at the rocky peak of the island, a male, and one larvae of each laboratory reared group. Two rounds of conventional PCRs were performed. The first using the primers forward 5’-CCGGTCTGAACTCAGATCAAGT-3’ and reverse 5’-GCTCAATGATTTTT TAAATTGCTGT-3’ targeting a ≈460-bp fragment of the tick mitochondrial 16S rDNA gene (Mangold et al. 1998), and the second performed using primers forward 5’-GGGGAAGAAAGTCT CAAGGGTAATATCCTT-3’ and reverse 5’-TGCATCGAATTAAACCACATGCTCCACCGC3’, targeting a ≈500-bp fragment of the Coxiella 16S rDNA gene (Almeida et al. 2012). Expected size products were sequenced in an ABI automated sequencer (Applied Biosystems/ Thermo Fisher Scientific, model ABI 3500 Genetic Analyzer) with the same primers used for the PCRs. Obtained sequences were submitted to BLAST analyses (www.ncbi.nlm.nih.gov/blast) in order to infer relationships between closely related organisms. Phylogenetic analyses Partial sequences of the tick 16S mitochondrial and Coxiella rDNA genes were aligned with sequences of congeneric organisms available in the GenBank database using the program Clustal W (Thompson et al. 1994) implemented in MEGA 6 (Tamura et al. 2013). Only sequences > 400-bp 30

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were considered for the tick mitochondrial 16S rDNA alignment. With both alignments, two phylogenetic trees inferred by the Maximum Likelihood (ML) method were constructed. The tickML tree was based on the Hasegawa-Kishino-Yano model (HKY) and the Coxiella-ML tree using the Kimura 2-parameter model (K2). For both analyses a discrete Gamma (G) distribution was assumed and all codon positions (1st+2nd+3rd+Noncoding) were included. Sequences of Ornithodoros rostratus Aragão 1911 (DQ295780) and Ixodes holocyclus Neumann 1899 (AB051845) were used as outgroups in the tick 16S mitochondrial rDNA tree, and the sequence of Legionella pneumophila (M36024) rooted the Coxiella 16S rDNA phylogenetic tree.

Results Morphological analyses Both groups of 10 larvae were morphologically identified as O. capensis s. s. by the combination of the following characters: dorsal plate pyriform in shape, widest posteriorly, 22 – 25 pairs of dorsal setae, 18 – 21 dorsolateral pairs and 4 central pairs; two pairs of posthypostomal setae; hypostome with a blunt apex, dentition 5/5 in the anterior third, 4/4 near mid length and 2/2 towards the base (Fig. 2A, B, C). Measurements from both groups of laboratory-reared larvae were similar and concordant with the description of O. capensis s. s. summarized by Kohls et al. (1965), at least for the following characters: average size of dorsal and ventral pairs of setae, presence of posteromedian and postcoxal setae, position and size of posthypostomal setae, dentition formula and number of dental rows (except for the fifth row which was 1–5 denticles shorter in the specimens from QGI) (Table 1). Discrepancies were observed for the following characters: body and dorsal plate size, length and width of capitulum and hypostome, palpal and article length. Remarkably, morphological and morphometrical characteristics of the QGI specimens and the O. capensis s. s. larvae reported by Kohls et al. (1965) are also highly similar to O. sawaii (Table 1). Slide-mounted larvae and the other specimens of O. capensis analysed in the present study were deposited in the tick collection “Coleção Nacional de Carrapatos” (CNC) at the Faculty of Veterinary Medicine of the São Paulo University, São Paulo, Brazil, under the following accessing numbers: CNC-3249, -3250, -3251.

FIGURE 2. Larva of Ornithodoros capensis s. s.: (A) Dorsal view; (B) Ventral setae; (C) Hypostome. Scale bar is given in mm.

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TABLE 1. Measurements of Ornithodoros capensis s. s. larvae from QGI, O. capensis s. s. from Kohls et al. (1965), and Ornithodoros sawaii. Characters highlighted in bold were used for the morphological diagnosis. O. capensis s. s.

O. sawaii

This study

Kohls et al. (1965)

Kitaoka & Susuki (1973)

Feeding status

Female a Unengorged

Female b Unengorged

Unengorged

Unengorged

Body length inlcuding capitulum Body width

0.518–0.621 0.329–0.426

0.561–0.616 0.391–0.408

0.635–0.748 0.377–0.440

0.550–0.560 0.360–0.370

Dorsal plate: form

Pyriform

Pyriform

Pyriform

Pyriform

Dorsal plate: length Dorsal plate: width

0.153–0.199 0.122–0.149

0.168–0.195 0.136–0.148

0.205–0.230 0.170–0.190

0.199 – 0.202 0.145 – 0.172

Dorsal setae: total pairs Dorsal setae: dorsolateral pairs Dorsal setae: central pairs

23–25 19–21 4

23–25 19–21 4

22–25 18–21 4

22–25 19–20 3

Dorsal anterolateral setae: average Dorsal posterolateral setae: average Ventral setae (pairs): total Circumanal setae: Ca1 (average) Circumanal setae: Ca2 (average) Circumanal setae: Ca3 (average) Posteromedian setae: Pms Postcoxal setae: Pc Length of capituli Width of basis capituli Posthypostomal setae Ph2 Distance Ph1–Ph1 Distance Ph2–Ph2

0.111 0.087 8 0.062 0.087 0.096 present present 0.212–0.238 0.133–0.166 0.020 0.017 0.085

0.112 0.089 8 0.060 0.087 0.098 present present 0.228–0.249 0.137–0.171 0.020 0.021 0.086

0.114 0.097 8 0.069 0.091 0.101 present present 0.138–0.166 0.177–0.210 0.018 0.018 0.084

0.125 0.098 9 0.065 0.093 0.088 present present ND ND 0.016 0.016 0.067

Palpal lenght Length article I Length article II Length article III Length article IV Hypostome (a) Hypostome: (b) Apex Apical dental formula First third dental formula

0.169–0.182 0.037 0.058 0.060 0.047 0.143–0.153 0.042–0.059 Blunt 5/5 4/4

0.174–0.189 0.039 0.058 0.054 0.045 0.139–0.148 0.042–0.055 Blunt 5/5 4/4

0.205–0.225 0.050 0.068 0.062 0.047 0.169–0.184 0.073–0.081 Blunt 5/5 4/4

0.185 ND ND ND ND 0.127–0.132 0.055 Blunt 5/5 – 6/6 4/4

Basal dental formula Denticles in hypostomal row 1 Denticles in Hypostomal row 2 Denticles in Hypostomal row 3

2/2 16–18 14–15 9–11

2/2 17–18 13 -14 8–10

2/2 13 -17 14–16 9–11

2/2 15 14 8

Denticles in Hypostomal row 4 Denticles in Hypostomal row 5 Tarsus I: length Tarsus I: width

7–8 4–6 0.179–0.196 0.048–0.058

6–8 4–6 0.181–0.206 0.050–0.065

7–10 5a9 0.185–0.210 0.065–0.082

6 5 0.166 –0.171 0.052–0.057

(a) Length measured to the insertion; (b) Width; ND: non-determined.

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Molecular analyses All analysed ticks yielded a 426-bp sequence of the 16S mitochondrial rDNA gene with 100% of nucleotide similarity between them. The morphological diagnosis was confirmed for all tick stages since their sequences were 98.3–100% (418-426/426-bp) similar to O. capensis s. l. available in GenBank (App. 1). Amplification of the Coxiella 16S rDNA gene was positive for all tick-extracted DNA samples, and the sequences were identical to each other and 96.8-100% (445-464/464-bp) identical to Coxiella-like organisms detected in ticks of the O. capensis group (App. 2). Generated sequences of O. capensis and its associated Coxiella-like bacteria were deposited into GenBank under the following accession numbers: KU757069, KU757070. Phylogenetic analyses Both phylogenetic analyses confirmed that ticks collected in QGI belong to the O. capensis group (Fig. 3, 4). In the analysis inferred for the tick mitochondrial 16S rDNA gene, QGI ticks clustered with O. capensis s. l. from Cabo Verde (Atlantic Ocean) and La Reunion Island, France (Indian Ocean), showing 100% nucleotide similarity (App. 1). This clade was included within a major group composed of O. capensis s. l. ticks from Northern Pacific Ocean latitudes and O. sawaii from Japan and Korea. The 16S rDNA partial sequence of the Coxiella-like endosymbiont of O. capensis s. s. from QGI grouped with congeneric organisms detected in O. capensis s. l. from South Carolina (United States), Indian Ocean Basin (Juan de Nova, Europa, La Reunion, Tromelin and Seychelles islands), and also with the Coxiella endosymbionts of O. denmarki, showing 100% of nucleotide similarity with all of them (App. 2).

FIGURE 3. Maximum Likelihood phylogenetic tree of a partial fragment of the tick 16S mitochondrial rDNA gene, constructed with 23 sequences of Ornithodoros capensis s. l. Bootstrap values are indicated for each node. The positions of O. capensis s. s. from Queimada Grande Island and from other two localities are indicated in bold. Abbreviations: I., Ixodes; O., Ornithodoros; s. l., sensu lato; s. s., sensu stricto.

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FIGURE 4. Maximum Likelihood phylogenetic tree of a partial fragment of the Coxiella 16S rDNA gene, constructed with 34 Coxiella spp. sequences. Bootstrap values are indicated for each node and the sequence of Coxiella endosymbionts associated with ticks from Queimada Grande Island is highlighted in bold. Abbreviations: CE, Coxiella endosymbiont; O., Ornithodoros; QGI, Queimada Grande Island; s. l., sensu lato; s. s., sensu stricto.

Discussion Ornithodoros capensis s. l. comprises a morphologically closely related worldwide-distributed group of ticks. However, the larva of the species that morphologically defines the group was not included in the original description of O. capensis s. s. (Neumann 1901). Considering that larval characters are important in taxonomic studies of ticks of the family Argasidae (Hoogstraal 1985; Klompen 1992), in the present study the identification of O. capensis s. s. ticks was based on the original descriptions of the representatives of the group (Kohls et al. 1965; Kitaoka & Suzuki 1973; Hoogstraal et al. 1974, 1976, 1979, 1983, 1985; Keirans et al. 1984; Hoogstraal 1985; Clifford et al. 1980; Vermeil et al. 1997), including the sole detailed description of larval stages for the species summarized by Kohls et al. (1965). Our morphological analysis matched with the larval morphology and morphometry of the later study, though we observed some differences. This interspecific variability in the phenotype of larvae may be explained either by the low number of measured 34


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specimens or by taking into account different geographical origins of the examined material, since larval specimens measured in Kohls et al. (1965) were obtained from females collected in penguin colonies from South African Atlantic shores. Despite the existence of these morphological discrepancies, the species of QGI can still be morphologically classified as O. capensis s. s., since the number of setae and dentition of the hypostome, two characters thought to be the most important for the separation of the species of this group (Keirans et al. 1984), coincide with the description of Kohls et al. (1965). Both tick mitochondrial 16S rDNA and its associated Coxiella-like endosymbiont 16S rDNA sequences clustered together with ticks and Coxiella organisms, respectively, of the O. capensis group. Our phylogenetic analyses showed low bootstrap support for some clades, which can be explained by the shortness of the analysed sequences; however, the topology of the obtained trees was coherent with the results of the morphological identification of ticks. The mitochondrial 16S rDNA partial sequence of QGI ticks was 100% identical to sequences of ticks from Cabo Verde and La Reunion islands. Although a comparison with the complete sequence of the 16S mitochondrial rDNA gene would be more informative, it is highly possible that this 100% matching correspond to the occurrence of O. capensis s. s. in both insular territories. Since QGI, Cabo Verde and La Reunion islands are located in two different ocean basins (Atlantic and Indian, respectively), it is probable that seabirds inhabiting these islands might play an important role dispersing O. capensis s. s. across both oceans. In fact, ticks from QGI were collected near Sula leucogaster (Pelecaniformes: Sulidae), a seabird species distributed among the tropical oceans of the world (Del Hoyo et al. 1992). In Brazil, S. leucogaster breeds on several coastal and oceanic islands along the seaboard of the country (Novelli 1997). Considering this, the distribution of O. capensis s. s. might be much wider in this country. Remarkably, the morphological characters of the O. sawaii larva are almost the same as those of the larval stages of O. capensis s. s., with few exceptions. Ornithodoros sawaii presents 3 instead of 4 pairs of dorsal central setae and 4 instead of 3 pairs of circumanal setae (Kitaoka & Suzuki 1993). However, when comparing the illustrations of O. sawaii given in its original description with the slide-mounted larvae of O. capensis s. s., the alleged differences in dorsal and ventral chaetotaxy are difficult to determine. Firstly, in O. sawaii the number of dorsal central pairs might be underestimated, since the numerous and adjacently disposed posterolateral setae could be masking the position of a fourth central pair, that might be overlooked in the original description. Secondly, in species exhibiting a high quantity of adjacent dorsolateral setae, the last posterolateral setae often results noticeable in a ventral view of the slide-mounted specimens, especially in unengorged ones. With this, the fourth circumanal seta of O. sawaii might be a misinterpretation of the last dorsal posterolateral setae. An analysis of the chaetotaxy in engorged larvae would be informative in order to determine the position of both setae. While morphometric differences between O. sawaii and O. capensis s. s. include slight discrepancies (< 0.017 mm) in the dimensions of dorsal anterolateral, second and third circumanal, and second posthypostomal setae; distances between both pairs of posthypostomal setae; and the length of hypostome and Tarsus I, all of them are not important for separating species of the O. capensis group (Keirans et al. 1984). The high morphological and morphometrical similarity between these soft ticks is also supported by the phylogenetic analysis, since the O. sawaii clade from Japan and Korea clusters with a clade of O. capensis s. l. from the North Pacific Ocean area, including Japan, and then with QGI ticks and O. capensis from Cabo Verde and Reunion Island under high bootstrap support (94%). Furthermore, pairwise comparisons of O. sawaii 16S rDNA sequences show 97.6–97.8% nucleotide identity with O. capensis s. s. sequences (App. 1). Although additional molecular and morphological studies of immature and mature specimens are needed to determine the validity of O. sawaii as a species, in our view, current phenotypic and genetic evidence tackles its treatment as an independent taxon within the Argasidae. 2017


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The Coxiella-like endosymbiont sequences obtained from QGI ticks were genetically identical with O. capensis s. l. microorganisms from several islands in the Indian Ocean and with O. denmarki (App. 2). Since these Coxiella organisms coexist with ticks in a mutualistic endosymbiotic association, which is maternally transmitted, it has been proposed that closely related Coxiella should occur in closely related tick species (Almeida et al. 2012, Duron et al. 2015). Although in the present study the vertical transmission of these microorganisms was not completely proven, we demonstrated that Coxiella endosymbionts associated with O. capensis s. s. ticks from QGI are closely related with other Coxiella coexisting with ticks of the same species group. While a major molecular characterization of these endosymbionts is necessary, our results indicate that sequences of Coxiella from QGI ticks correspond to the endosymbiont organisms associated with O. capensis s. s., O. denmarki, and other species of the group of unknown identity. It is noteworthy that several Coxiella endosymbionts of O. capensis s. l. have been recently associated with particular species of the group, such as O. amblus, O. denmarki, O. maritimus, O. muesebecki and O. spheniscus (Duron et al. 2014, 2015; Al-Deeb et al. 2016). Considering that species level identification of O. capensis group representatives should include an examination of larval stages, these tick-Coxiella associations should also be revaluated. Furthermore, the identification of O. capensis s. l. ticks relying on the examination of free-living post-larval stages collected in the historically described localities for the different species of the group should be also carefully assessed, since the distribution of some species may overlap. Finally, this study demonstrates the presence of O. capensis s. s. in QGI, which corresponds to the first confirmation of this soft tick by the examination of larval stages in Brazil, after more than 30 years since its last collection by Edwards & Lubbock (1983) at São Pedro and São Paulo Archipelago. Moreover, we demonstrate that the combination of the genetic characterization of associated Coxiella-like endosymbionts with the morphological study of larval stages provides a useful taxonomical tool in order to support specific diagnoses. Morphological differentiation between O. capensis s. l. ticks is a difficult task, mainly because of the high phenotypic similarity exhibited by immature and mature stages. In this sense, the collection of specimens at the type localities with further molecular and morphological characterization should be a priority in order to accurately determine the phylogenetic and morphological relationships of this worldwide-distributed group of soft ticks.

Acknowledgments The authors acknowledge Carlos Azevedo (ARIE Ilhas da Queimada Pequena e Queimada Grande, ICMBio), Airton Lourenço in memorian (CEVAP/UNESP), Rhaiza Esteves (Instituto Vital Brasil) for their valuable help during field-work and logistic support in the expedition to Queimada Grande Island. This work received financial support by CAPES, Brazil. SML was funded by CONICYT Programa de Formación de Capital Humano Avanzado, Beca Chile Nº 72140079.

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