Exp Appl Acarol https://doi.org/10.1007/s10493-018-0254-y Phylogenetic insights on Mediterranean and Afrotropical Rhipicephalus species (Acari: Ixodida) based on mitochondrial DNA Maria João Coimbra‑Dores1,2 · Mariana Maia‑Silva1 · Wilson Marques1 · Ana Cristina Oliveira3 · Fernanda Rosa4 · Deodália Dias1,2 Received: 11 February 2018 / Accepted: 24 March 2018 © Springer International Publishing AG, part of Springer Nature 2018 Abstract A multigene phylogeny including 24 Rhipicephalus species from the Afrotropical and Mediterranean regions, based on mitochondrial DNA genes (COI, 12S and 16S), was constructed based on Bayesian inference and maximum likelihood estimations. The phylogenetic reconstruction revealed 31 Rhipicephalus clades, which include the first molecular records of Rhipicephalus duttoni (Neumann), and Rhipicephalus senegalensis (Koch). Our results support the R. pulchellus, R. evertsi and R. pravus complexes as more phylogenetically close to Rhipicephalus (Boophilus) than to the remaining Rhipicephalus clades, suggesting two main monophyletic groups within the genus. Additionally, the phenotypic resembling R. sanguineus s.l. and Rhipicephalus turanicus (Pomerantsev) are here represented by nine clades, of which none of the R. turanicus assemblages appeared as distributed in the Iberian Peninsula. These results not only indicate that both species include more cryptic diversity than the already reported, but also suggest that R. turanicus distribution is less extended than previously anticipated. This analysis allowed to improve species identification by exposing cryptic species and reinforced mtDNA markers suitability for intra/inter-species clarification analyses. Incorporating new species molecular records to improve phylogenetic clarification can significantly improve ticks’ identification methods which will have epidemiologic implications on public health. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1049 3-018-0254-y) contains supplementary material, which is available to authorized users. * Maria João Coimbra-Dores mjcdores@gmail.com 1 Department of Animal Biology, Faculty of Sciences, University of Lisbon, Campo Grande, 1749-016 Lisbon, Portugal 2 Faculty of Sciences, Centre for Environmental and Marine Studies (CESAM), University of Lisbon, Campo Grande, 1749-016 Lisbon, Portugal 3 Casa dos Animais Veterinary Clinic, Luanda, Angola 4 Instituto Superior de Agronomia, University of Lisbon, Tapada da Ajuda, 1349-017 Lisbon, Portugal 13 Exp Appl Acarol Keywords Rhipicephalus duttoni · Rhipicephalus senegalensis · Cryptic species · Mitochondrial genes · Ticks · Phylogeny Introduction Ticks (Acari: Ixodida) are hematophagous arthropods implicated in the (re)emerging of infectious diseases such as Lyme borreliosis, Q fever, tularemia, anaplasmosis, rickettsioses, and some arboviruses (Bacellar et al. 1995; Parola and Raoult 2001; Baptista et al. 2004; Alexandre et al. 2009, 2011; Santos-Silva et al. 2011; Vilhena et al. 2013; ECDC 2016; Ferrolho et al. 2016). Due to the increasing number of studies related to tick-borne diseases, an accurate determination of ixodids phylogenetic relationships and species delimitation are of extreme importance considering that closely related species, or even different populations of the same species, could present a different ability to transmit pathogens (Barker 1998; Anderson 2002). Members of the Rhipicephalus genus are a great example of taxonomic units that are often clustered within species complexes, due to exhibit a high number of morphologically similar and cryptic species (Skoracka et al. 2015). Recent divergence evidenced between some of its species (Jeyaprakash and Hoy 2009) could explain why it is hard to place these entities within the species definition, based on the continuous nature of evolution. Because it is still unclear how these genus species are related to each other, it is common that different authors/specialists do not agree to include some Rhipicephalus species in the same complex (Pegram et al. 1987a, b; Camicas et al. 1998; Walker et al. 2000; Murrell et al. 2001a; Estrada-Pena et al. 2017). Since most authors apply the species complexes proposed by Walker et al. (2000), at least one-quarter of Rhipicephalus species can be comprehended in eight species complexes accordingly to their morphological features similarities (see Table 1). These complexes were compiled based on similarities between phenotypic characters, such as spiracle plate and capituli shapes, posterior grooves and caudal appendage forms and lengths. Among 14 genera known for the Ixodidae family, Rhipicephalus genus is one of the most widespread, being present in almost all continents except the Antarctic (Walker et al. 2000). As a vector of several zoonoses, these blood-feeding arthropods are considered of Public Health importance. Regardless that status, there is still much disarray around its taxonomic classification, and its biosystematics status still needs further clarification. This genus, based on current data, is mainly distributed in the African continent, the geographical area where is also suggested by some evolutionary studies that ticks first appeared (Walker et al. 2000; Jeyaprakash and Hoy 2009; Guglielmone et al. 2010; Mans et al. 2011, 2012). Nevertheless, several Rhipicephalus species such as all R. follis species complex members, most of the R. pravus and R. simus complexes elements, and even some species of the R. appendiculatus complex (sensu Walker et al. 2000), remain until now genetically uncharacterised. Rhipicephalus genus diversity clarification is then of great importance since it is known that climate changes will have severe implications in the distribution range change not only of tick-vectors but also their transmitted pathogens (Olwoch et al. 2007; Gray et al. 2009; Estrada-Pena et al. 2012). With the aim to contribute to the phylogenetic clarification of this tangled genus, a phylogenetic approach using three mitochondrial genes (cytochrome oxidase subunit I or COI, 12S rDNA and 16S rDNA) was applied. DNA sequences of ticks collected in the Afrotropical and Palearctic Mediterranean regions were used, which 13 Exp Appl Acarol Table 1 List of currently accepted Rhipicephalus species complexes (Camicas et al. 1998; Walker et al. 2000; Murrell et al. 2001a) Rhipicephalus complex Species list R. appendiculatus R. appendiculatus, R. armatusc,d, R. carnivoralis, R. duttoni, R. humeralisd, R. maculatusd,e, R muehlensi, R. nitens, R. pulchellusd,e, R. sculptus, R. zambeziensis R. capensisa R. evertsi R. follisa R. capensis, R. compositus, R. longus, R. pseudolongus R. bursa, R. evertsi, R. glabroscutatum R. follisd, R. gertrudaed, R. hurtid, R. jeannelid, R. lounsburyid, R. lunulatusd, R. neumannid, R. tricuspisd R. haemaphysaloidesd, R. pilansd, R. ramachandraid R. arnoldid, R. exophthalmos, R. kochi, R. oculatus, R. pravus, R. warburtoni R. camicasi, R. guilhoni, R. leporis, R. pumilio, R. pusillus, R. rossicus, R. sanguineus s.l., R. schulzei, R. sulcatus, R. turanicus R. distinctusd, R. muhsamae, R. planusd, R. praetextatus, R. senegalensis, R. simpsoni, R. simus, R. zumpti R. haemaphysaloidesb R. pravus R. sanguineus R. simusa a Included species are difficult to be morphologically identified and grouped. Needs revision b Show morphological disparities between different life stages. Needs revision c Grouping this species within this or in another complex may be regarded as questionable d Included in another complex by Camicas et al. (1998) e Included in R. pulchellus complex by Murrell et al. (2001a, b) are Rhipicephalus’ key study areas due to be well-known diversity hotspots of ticks, their hosts, and associated tick-borne pathogens (Walker et al. 2000; Estrada-Peña et al. 2004). For this study, new molecular records of Rhipicephalus senegalensis (Koch) and Rhipicephalus duttoni (Neumann) morphologically identified species, collected in Guinea-Bissau and Angola respectively, were obtained. Materials and methods Samples Our study included 62 mature specimens morphologically identified as Rhipicephalus species, which were obtained in the Afrotropical and Mediterranean regions between 1950 and 2017. Twenty-three Rhipicephalus ticks collected between 1950 and 2001 in Angola, Cape Verde, Guinea Bissau and Mozambique, were not successfully sequenced. Remaining 39 ticks were successfully sequenced, which were collected in Angola, Cape Verde Islands, Guinea-Bissau, Portugal and South Africa (see Table 2). Fieldwork sampling was performed and supervised by a veterinarian in accordance with animal manipulation international rules and carried out with appropriate local authorities’ permissions. Rhipicephalus species are considered a widespread genus; then they are not considered an endangered or protected species in the International Union for Conservation of Nature (IUCN) lists (available at http://www.iucnredlist.org/). Specimens used for DNA extraction were preserved in 70% ethanol and stored at room temperature. 13 13 Table 2 List of morphologically identified taxa, geographical region, source, samples IDs and GenBank accession numbers by molecular marker used in this study Taxon Rhipicephalus duttoni (Neumann) Rhipicephalus evertsi mimeticus (Donitz) Rhipicephalus geigyi (Aeschlimann and Morel) Rhipicephalus pusillus (Gil Collado) Rhipicephalus sanguineus s.l. (Nava et al.) Geographical origin Angola: Lubango Source Capra hircus Angola: Lubango Guinea-Bissau: Cufada Lagoons Natural Park Portugal: Caldas da Rainha Angola: Luanda Capra hircus Bos taurus Canis familiaris Canis familiaris Cape Verde: Santiago Praia Canis familiaris Portugal: Caldas da Rainha Canis familiaris Portugal: Samora Correia Portugal: Santarém Canis familiaris Canis familiaris Sample ID GenBank acc. no. 16S rDNA COI A3009 MF425964 MF425974 MF425989 A3012 A3010 GB3052 CR1563 A3013 A3014 A3015 A3021 A3022 A3045 CV3041 CV3042 CR1551 CR1553 CR1568 CR1570 CR1575 SC3005 S29 S32 S33 S37 S48 MF425966 MF425965 MF425937 MF425936 MF425967 MF425968 MF425969 MF425970 MF425971 MF425934 MF425958 MF425959 MF425935 – – – – MF425945 – – – – – MF425976 MF425975 MF425948 MF425983 MF425977 MF425978 MF425979 MF425980 MF425981 MF425947 MF425961 MF425962 MF425982 – – – – MF425956 – – – – – MF425991 MF425990 – MF425999 MF425992 MF425993 MF425994 MF425995 MF425996 – – MF426003 MF425997 MF425998 MF426000 MF426001 MF426002 MF425987 MF426007 MF426008 MF426009 MF426013 MF426015 Exp Appl Acarol 12S rDNA Taxon Geographical origin Source Rhipicephalus senegalensis (Koch) Portugal: São Facundo Guinea-Bissau: Cufada Lagoons Natural Park Vegetation Vegetation Rhipicephalus simus (Koch) South Africa: Mpumalanga Panthera leo Sample ID S52 S56 S58 S337 S344 S358 S475 S827 S943 S1060 SF3003 GB3058 GB3059 GB3060 RAS3062 GenBank acc. no. 12S rDNA 16S rDNA COI – – – MF425940 MF425941 MF425942 MF425943 – MF425944 MF425939 MF425946 MF425960 MF425972 MF425973 MF425938 – – – MF425951 MF425952 MF425953 MF425954 – MF425955 MF425950 MF425957 MF425963 MF425984 MF425985 MF425949 MF426016 MF426017 MF426018 MF426010 MF426011 MF426012 MF426014 MF426019 MF426020 MF426006 MF425988 – MF426004 MF426005 MF425986 Exp Appl Acarol Table 2 (continued) 13 Exp Appl Acarol Morphological identification Rhipicephalus species morphological examination was carried out based on standard keys and descriptions (Dias 1950, 1994; Papadopoulos et al. 1992; Walker et al. 2000, 2003). Also, recent Rhipicephalus sanguineus s.l. (Nava et al. 2015) morphological evaluations (Dantas-Torres et al. 2013; Coimbra-Dores et al. 2016) was also taken into account. Identification of R. senegalensis and R. duttoni was based on males’ conscutum caudal appendage and grooves, adanal, accessory and spiracular plates; and on females’ genitalia aperture and sclerites shapes (Tendeiro 1956, 1959; Walker et al. 2000). Key morphological traits were examined and photographed using a Leitz Laborlux K light microscope and a Leica M165C stereomicroscope, coupled to a Leica DFC420 digital microscope camera. DNA extraction, PCR and sequencing Entire specimens were used for DNA extraction. After 48 h of enzymatic digestion, exoskeletons were recovered for further analyses. Genomic DNA was extracted using E.Z.N.A.® Tissue DNA Isolation Kit (Omega Bio-tek, Norcross, GA, USA) accordingly to manufacturer’s instructions. Fragments between 600 and 800 bp were obtained by polymerase chain reaction (PCR) amplification of the mitochondrial gene cytochrome oxidase subunit I (COI), using two pair of primers: LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA ATC A-3′) (Folmer et al. 1994), and the pair Cox1F (5′-GGA ACA ATA TAT TTA ATT TTT GG-3′) and Cox1R (5′-ATC TAT CCC TAC TGT AAA TAT ATG-3′) (Chitimia et al. 2010). The mitochondrial 12S ribosomal DNA (rDNA) and 16S rDNA regions were also amplified, producing fragments of 250–400 and 350–450 bp, respectively. The following primers were used: 12SrDNA-F (5′-AAA CTA GGA TTA GAT ACC CTA TTA TTT TAG-3′) and 12SrDNA-R (5′-CTA TGT AAC GAC TTA TCT TAA TAA AGA GTG-3′) (Szabó et al. 2005), and the pair 16S + 1 (5′-CTG CTC AAT GAT TTT TTA AAT TGC TGT GG-3′) and 16S-2 (5′-TTA CGC TGT TAT CCC TAG AG-3′) (Black and Piesman 1994). PCR reactions were performed in 25 µL volume, including 1 × PCR buffer, 2 mM MgCl2, 100 µM dNTPs, 0.4 µM of each primer, 0.16 µg/µL BSA, 0.5U Taq polymerase (NzyTech, Lisbon, Portugal) and approximately 20 ng of genomic DNA. Cycling conditions used for COI mitochondrial DNA (mtDNA) amplification were: 95 °C for 5 min (initial denaturation), followed by 40 cycles of 95 °C for 45 s (denaturation), 45 °C for 1 min (annealing), 72 °C for 1 min (extension), and a final extension of 72 °C for 5 min. For the 12S and 16S rDNA genes, the used conditions were: 94 °C for 2 min (initial denaturation), followed by 40 cycles of 94 °C for 1 m (denaturation), 50 °C for 1 min (annealing), 72 °C for 1 min (extension), and a final extension of 72 °C for 7 min. Each PCR product was evaluated by 2% agarose gel electrophoresis to validate amplification efficiency. All PCR products were purified with SureClean Plus Kit (Bioline Reagents, London, UK) following the manufacturer’s protocol, and sequenced with both primers at STABVIDA (Monte da Caparica, Portugal). New generated sequences were submitted to GenBank database, provided by the National Center for Biotechnology Information (https:// www.ncbi.nlm.nih.gov/). Taxa, geographical origin, source, samples IDs and GenBank accession numbers are provided in Table 2. 13 Exp Appl Acarol Molecular analysis and phylogenetic reconstruction Due to rarity and long-conservation time of some samples, 87 sequences were retrieved from 39 specimens for this study. With the aim to improve statistical and phylogenetic support in our analyses, 374 additional sequences were retrieved from the GenBank and included in the phylogenetic analysis. Hyalomma species were used as an outgroup in all phylogenetic analyses. Retrieve sequences GenBank accession numbers, geographical origin, as well as associated taxa, are available in the Online Resource Table S1. These sequences were chosen based on previously published work regarding Rhipicephalus mitochondrial lineages, their taxa, sequence quality, and our geographic region of interest. Collection locations, which include mainly the Palearctic and Afrotropical regions, are represented in Fig. 1 (except Iran, Russia, and Turkmenistan, that are on the Asian continent). Remaining countries (namely China, India and Pakistan) belong to a different geographical region (Oriental ecozone), out of our regions of interest. Since Rhipicephalus genus comprehends multiple species and not all regions are genetically represented on the GenBank, the isolates collected out of our interest region were here included for comparison purposes. Since sometimes more than one clade was formed under the same Rhipicephalus species morphologic identification, it became necessary to adopt differential nominal references to distinguish them. The designations “R. sanguineus tropical” and “temperate” are two well-known references regarding differentiated R. sanguineus s.l. mitochondrial lineages (Moraes-Filho et al. 2011; Latrofa et al. 2014; Coimbra-Dores et al. 2016; Hekimoğlu et al. 2016; Hornok et al. 2017; Almeida et al. 2017). Recent studies based on morphological and phylogenetic analyses also referred the existence of at least four more operational taxonomic units, denominated as “Rhipicephalus species I–IV” (Dantas-Torres et al. 2013; Hornok et al. 2017), which were also adopted here. Concerning the remaining clades, we adopted the denominations “R. turanicus I–III” since the sequence-authors described the specimens as presenting R. turanicus-like morphologies. R. turanicus s.s. is referent to the lineage sensu Filippova (1997) described in Dantas-Torres et al. (2013). All sequences were verified and edited using the software SEQUENCHER v.4.0.5 (Gene Codes, Ann Arbor, Michigan, USA) and BIOEDIT v.7.2.5 (Hall 1999), aligned using MAFFT v.7.304 (Katoh and Standley 2013), converted and concatenated to appropriate formats with CONCATENATOR v.1.1.0 (Pina-Martins and Paulo 2008). Phylogenetic analyses were performed for individual and concatenated mtDNA datasets (12S rDNA, 16S rDNA and COI), using the Bayesian inference (BI) and maximum likelihood (ML) methods, in MRBAYES v.3.1.2 (Huelsenbeck and Ronquist 2001) and RAxML v.8.2.4 (Stamatakis 2014), respectively. Two runs with four chains for 300 thousand generations with sampling every 1000th generations were applied. For the concatenated dataset, unlinked partitions corresponding to each DNA region were used. Trace files obtained in the BI analysis were checked using TRACER v.1.6 (Rambaut et al. 2014) since it is standard to ensure that chains had reached convergence, and 25% of trees were discarded as burn-in. Branch support was estimated by performing 1000 bootstrap replicates. The software jMODELTEST v.2.1.10 (Darriba et al. 2012) was used to select the best-fit substitution model for each mitochondrial marker, using the corrected Akaike Information Criterion or AICc (Sugiura 1978). In the ML analyses, the GTR model was selected as the best fit available in RAxML for the 16S rDNA and COI mtDNA single-genes datasets, and the GTR + G was the model obtained for the 12S rDNA partition. For the BI analyses, the selected models were the HKY + G for 12S rDNA fragment and JC + G for the 16S rDNA and COI mtDNA partitions. Bipartitions and consensus tree files obtained as outputs were 13 Exp Appl Acarol Fig. 1 Rhipicephalus species sampling locations situated in the (A) Afrotropical and (P) Palearctic geographical regions (Olson et al. 2001). Newly collected samples are represented by a dark circle, and GenBank retrieved samples are represented by a white circle. AG Angola, BF Burkina Faso, BO Botswana, CO Comoros, CV Cape Verde, EG Egypt, ET Ethiopia, FR France, GB Guinea-Bissau, GH Ghana, GR Greece, IC Ivory Coast, IQ Iraq, IS Israel, IT Italy, KE Kenya, MA Malta, ML Mali, MZ Mozambique, NA Namibia, NI Nigeria, PT Portugal, RE Reunion, RO Romania, RW Rwanda, SA South Africa, SE Senegal, SP Spain, ZA Zambia, ZI Zimbabwe 13 Exp Appl Acarol visualised and edited using the software FIGTREE v.1.4.2 (Rambaut 2014). For both ML and BI analyses, single-gene trees were previously analysed to evaluate if sequences concatenation would be consensual considering phylogenetic relationships obtained. Although some differences were observed, they do not show to be significant. Final figures were obtained using the Inkscape software (available at https://inkscape.org). DnaSP v.5.10.01 (Librado and Rozas 2009) software was used to compute parsimonyinformative, segregating and conserved sites for each dataset. Divergence values were obtained using MEGA v.7.0.21 (Kumar et al. 2008), by the use of p-distances. These results are available as Online Resources (Tables S2, S3). Results Regarding the molecular analysed ticks, 30 specimens were genetically identified as Rhipicephalus sanguineus s.l., three as R. senegalensis, two as R. duttoni, one Rhipicephalus evertsi mimeticus (Donitz), one Rhipicephalus geigyi (Aeschlimann and Morel), one Rhipicephalus pusillus (Gil Collado), and one Rhipicephalus simus (Koch). These identifications were based on the amplicons obtained for the cytochrome c oxidase subunit I (COI), 12S ribosomal DNA (rDNA) and 16S rDNA mitochondrial genes (see Fig. 2). Except for the ones identified as R. sanguineus tropical lineage (PP = 0.770, BS = 53), all our obtained sequences grouped in well-supported clades (PP = 1, BS = 100). Identification of five specimens referred as R. senegalensis and R. duttoni was based on differential morphological traits (Tendeiro 1956, 1959; Walker et al. 2000) since additional sequences were not available at any molecular database for comparison. Morphological traits are shown in Figs. 3 and 4. As expected, the R. senegalensis sequences grouped within the R. simus complex (PP = 0.998, BS = 60; Table 1), a result that supports the morphological identification. The same not happen with R. duttoni, that shown to be genetically more closely related to R. pulchellus (PP = 1, BS = 59) than to the R. appendiculatus complex. Based on that result, we grouped both species within the R. pulchellus complex, a species group suggested by Murrell et al. (2001a, b). Molecularly identified R. sanguineus s.l. specimens, corresponding to R. sanguineus tropical and temperate mitochondrial lineages (Moraes-Filho et al. 2011), were morphologically identified as R. sanguineus-like (22/30) and R. turanicus-like (8/30) specimens by comparison with reference phenotypes (as referred in Coimbra-Dores et al. 2016). All R. turanicus-like individuals grouped in the R. sanguineus temperate lineage. Rhipicephalus sanguineus s.l. and Rhipicephalus turanicus (Pomerantsev) were split into several clades. R. sanguineus s.l. gave origin to three distinct clades presenting predominantly R. sanguineus-like specimens, suggesting a division in conformity with their geographic distributions: R. sanguineus tropical (PP = 0.770, BS = 53) and temperate (PP = 1, BS = 100) lineages, which present a mainly tropical (Afrotropical ecoregions, but also Southern France and Iraq) and temperate-climate (Mediterranean, but also Senegal) areas of distribution, respectively; and Rhipicephalus sp. I (PP = 0.574, BS = 65) distributed across the Central and South-east Mediterranean ecoregion. Although only the “temperate” clade was well-supported in our phylogeny, divergence values between these clades (between 7.8 and 10.3%) support their clustering in distinct groups. Rhipicephalus sanguineus temperate and tropical lineages presented the highest divergence values obtained within the complex (10.3%), suggesting these two lineages had already diverged at a 13 Exp Appl Acarol Fig. 2 a Rhipicephalus species sampling locations situated in the (A) Afrotropical and (P) Palearctic geographical regions (Olson et al. 2001) (for more details, see Fig. 1); b maximum likelihood phylogenetic tree based on three concatenated mitochondrial genes (12S rDNA, 16s rDNA, and COI) (865 bp). Species were grouped accordingly to the current morphological classification of Rhipicephalus genus (grey boxes). Numbers above branches correspond to Bayesian posterior probability and Maximum Likelihood bootstrap values (PP/BS) (some values, PP < 50 and BS < 50%, are not shown). Collapsed branches correspond to partitions that were reproduced in less than 50% of bootstrap replicates. Sequences obtained for this study are in bold. Asterisk indicate that only a reference sequence is represented (see the Online Resource Table S1 for further details) 13 Exp Appl Acarol Fig. 3 Morphological traits of Rhipicephalus duttoni a–c male, d–f female specimens collected in Capra hircus hosts, in Angola (isolate numbers: A3009, A3012). The male presents highly distinct morphological characters, such as more elongated spiracles and absence of accessory adanal plates (detailed morphological descriptions are available at Tendeiro 1956, 1959; Walker et al. 2000). a Conscutum, presenting a rectangle-shaped caudal appendage and elongated posterior grooves; b adanal plates, that are triangleshaped and narrow anteriorly; c, f spiracular areas; d scutum; e mounted genital aperture, presenting triangular-shaped sclerites that taper anteriorly. Scale-bars lengths: a 500 µm, b, d 200 µm, c 50 µm, e 20 µm, f 100 µm considerable time despite their shared phenotype. This divergence could also be directly correlated with different geographic distribution presented by both lineages. On the other hand, R. turanicus-like specimens were divided into six distinct clades, suggesting an association between clades and their geographic distribution again. Rhipicephalus turanicus I (PP = 0.634, BS = 91) presenting a South-east African distribution; R. turanicus II (PP = 0.993, BS = 99) that is mainly present in the 13 Exp Appl Acarol Fig. 4 Morphological traits of Rhipicephalus senegalensis a–c male, d–f female specimens collected in Guinea-Bissau (isolate numbers: GB3058, GB3060). The male shows a distinct adanal plates shape, and the female a typical R. senegalensis genitalia (detailed morphological descriptions are available at Walker et al. 2000). a Conscutum, presenting the “simus pattern” punctuations, with indistinct posterior grooves and absent caudal appendage; b adanal plates are broadly sickle-shaped, and accessory adanal plates with bluntly-rounded points; c, f broad spiracular areas; d scutum, with a smooth posterior margin, large porose areas visible on the capitulum; e mounted genital aperture, with rectangular-shaped sclerites. Scale-bars lengths: a, b 500 µm, c, f 100 µm, d 200 µm, e 20 µm East Mediterranean ecoregion; R. turanicus s.s. (PP = 1, BS = 100) distributed through Central and East Mediterranean; Rhipicephalus sp. III (PP = 1, BS = 100) that is distributed in the East Mediterranean border with the Oriental ecozone; R. turanicus III (PP = 0.571, BS = 55) distributed through the South-east Mediterranean; and Rhipicephalus sp. IV (PP = 1, BS = 100) that presents a West African distribution. Among 13 Exp Appl Acarol these R. turanicus clades, only the R. turanicus III is not well supported. However, due to divergence values obtained between clades (4.5–9.9%), the groups formation seems supported. Both Rhipicephalus appendiculatus (Neumann) and Rhipicephalus microplus (Canestrini), species comprehended in the R. appendiculatus complex and Rhipicephalus (Boophilus) (Murrell and Barker 2003), also comprehended two well-supported clades each (PP = 0.979–1; BS = 95–99; PP = 1, BS = 100; respectively) under the same morphological species classification. Rhipicephalus microplus species clustering is corroborated by differential geographical origins (Oriental and African). Moreover, within the R. evertsi complex, the Rhipicephalus evertsi (Neumann) species also seems to suggest the existence of two well-supported clades (PP = 1, BS = 77). These mitochondrial lineages distinction is in accordance with the two morphological differentiated subspecies taxonomic classification of R. evertsi, namely R. evertsi evertsi and R. evertsi mimeticus. In addition, the divergence between the two clades is of 6.2%, what also supports the existence of two differentiated clades. By last, two basal clades were formed (PP = 1, BS = 100), what suggests a division within the Rhipicephalus genus. One is composed by Rhipicephalus (Boophilus), R. evertsi complex, R. pravus complex and R. pulchellus complex; and another including the remaining three complexes, namely the R. appendiculatus, R. simus and R. sanguineus complexes. As observed before, each clade is suggesting a division that seems to conform with their geographic distributions. The most dispersed is the R. sanguineus tropical lineage since it is present in the Mediterranean Palearctic ecoregion (Southern France, Iraq) and in all Afrotropical ecoregions (from Cape Verde to Reunion and South Africa, as represented in Fig. 5). Rhipicephalus leporis (Pomerantsev) specimens, which were included in the R. sanguineus tropical clade, are suggested as closely related to four R. sanguineus s.l. sequences (9, 14, 16, 17). Altogether, this group is evidenced to be distributed through the West, East and South African ecoregions (Senegal, Burkina Faso, Ivory Coast, Ghana, Kenya, South Africa); and through the East Mediterranean ecoregion (Iraq). A second group is composed of specimens collected in Cape Verde Islands (CV3041 and CV3042) and Senegal (R. sanguineus s.l. 13), and thus distributed in the West African ecoregion. The last closely related group of sequences included in R. sanguineus tropical lineage were found in the Central-Western Mediterranean (Southern France) and East, West and South African ecoregions (Senegal, Angola, Kenya, Botswana and South Africa). Concerning the R. sanguineus temperate lineage, it is shown to be distributed through the Central (Italy and Malta) and West (Iberian Peninsula) Mediterranean ecoregion. However, it is also found in both West (Senegal) and South (South Africa) African ecoregions. Clades suggested to be delimited to the Afrotropical ecozone, based on our dataset collection sites, include Rhipicephalus camicasi (Morel, Mouchet and Rodhain) (Ethiopia) and R. turanicus I (Zambia and Zimbabwe) in the East African ecoregion; Rhipicephalus guilhoni (Morel and Vassiliades) (Nigeria and Zambia) and Rhipicephalus sulcatus (Neumann) (Guinea-Bissau and Zambia) in both East and West African ecoregions; and Rhipicephalus sp. IV (Guinea-Bissau and Nigeria) in the West African ecoregion. Restricted to the Palearctic ecozone are the R. turanicus s.s. clade, more specifically in Central-East Mediterranean and Siberian (Turkmenistan) ecoregions; Rhipicephalus sp. I, found through Central, East and South Mediterranean (Italy, Greece, Iran, Israel, Egypt); R. turanicus III, distributed in South and East Mediterranean (Egypt, Iraq, and Israel); R. pusillus, that shown to be distributed in West and Central Mediterranean (Portugal, France, and Italy) ecoregions; Rhipicephalus sp. III in far East Mediterranean (Pakistan), located 13 Exp Appl Acarol Fig. 5 Geographical distribution of Rhipicephalus sanguineus s.l. complex phylogenetic clades in the Afrotropical and Palearctic ecozones (Olson et al. 2001). a—R. sanguineus tropical lineage, a1—specimens collected in Angola (A3013–3015, A3021–3022, A3045) and closely related sequences (R. camicasi 1, 3; R. sanguineus s.l. 8, 10–12, 15, 18–19), a2—specimens collected in Cape Verde Islands (CV3041 and CV3042) and Senegal (R. sanguineus s.l. 13), a3—R. leporis and closely related sequences (R. sanguineus s.l. 9, 14, 16, 17), b—R. sanguineus temperate lineage, c—R. turanicus s.s., c1—R. turanicus 1–27, c2—R. turanicus 28–29 (Turkmenistan), d—Rhipicephalus sp. I, e—R. turanicus III, f—R. camicasi, g—R. guilhoni, h—R. pusillus, i—R. turanicus I, j—Rhipicephalus sp. III, kvR. pumilio and R. rossicus, l—R. sulcatus, m—Rhipicephalus sp. IV, n—R. turanicus II near the Oriental ecozone border; the R. pumilio and R. rossicus clade, distributed through the European Palearctic ecoregion (Russia and Romania); and R. turanicus II was only found in East Mediterranean (Israel). 13 Exp Appl Acarol Discussion Overall, this study supports that not all Rhipicephalus clades can be assigned to previously morphologically defined operational complexes and species. One example is the re-use of the R. pulchellus complex as a valid operational cluster, a deviation to the morphologically defined complexes by Walker (2000). This operational group was proposed by Murrell et al. (2001a, b) based on molecular and biosystematics results, but included only the ornate species Rhipicephalus maculatus (Neumann) and Rhipicephalus pulchellus (Gerstacker). Based on the inclusion of two new Rhipicephalus molecular records in the phylogenetic analysis, R. duttoni is here proposed as an additional member to the complex due to be evidenced as a sister species of R. pulchellus (Fig. 2). Remaining incongruities regarding the morphological based classification and this phylogenetic analysis results include R. appendiculatus and R. microplus species, which comprehended two isolated clades each; the clade formed by Rhipicephalus pumilio (Schulze) and Rhipicephalus rossicus (Yakimov and Kol-Yakimova) sequences; and R. sanguineus s.l. and its morphologically similar species R. turanicus. Rhipicephalus appendiculatus clades were already described in detail elsewhere (Mtambo et al. 2007a, b), based molecularly on one nuclear (internal transcribed spacer 2 or ITS2) and two mitochondrial (cytochrome oxidase subunit I or COI, and 12S ribosomal DNA or 12S rDNA) regions, and also in ecological differences. Although their mitochondrial based results also support the existence of two lineages, denominated Southern (South Africa, Zimbabwe, Kenya and Comoros) and East (Rwanda and Zambia) African groups, R. appendiculatus is regarded as a single species based on the nuclear markerbased analysis. Nevertheless, distinction between these lineages allowed to support that the East African lineage can have a higher vectorial competent regarding the transmission of Theileria parva (etiologic agent of East Coast Fever) which could be the reason of significant epidemiolocal differences of this disease expression between the southern and eastern African provinces (Speybroeck et al. 2004; Mtambo et al. 2007b). Equivalently, R. microplus species also comprehended two clades. Nonetheless, the difference to the previous case resides on the fact that former works already reported the existence of cryptic diversity under this species name (Labruna et al. 2009; Estrada-Peña et al. 2012; Burger et al. 2014; Low et al. 2015). Moreover, it was even proposed that the two mitochondrial lineages of R. microplus here represented (R. microplus s.s. collected in Yunnan, China; and R. microplus lineage obtained in Africa), in addition to its genetically closely-related Rhipicephalus annulatus (Say) and to the recently reinstated Rhipicephalus australis (Fuller) species, should be included within the denominated R. microplus complex (Estrada-Peña et al. 2012; Burger et al. 2014). Although R. australis and R. annulatus were shown to present morphological differences regarding the R. microplus complex members (Walker et al. 2003; Estrada-Peña et al. 2012), more studies will be needed to clarify the morphologic and taxonomic status of the R. microplus s.l. lineages. Interestingly, R. decoloratus from India in our phylogeny cluster within the members of the R. microplus complex, although without support, showing as more closely-related to R. microplus s.l. and R. annulatus lineages than to the R. geigyi as proposed by previous works (Burger et al. 2014). Regarding R. sanguineus complex, R. pumilio and R. rossicus labelled sequences were grouped in the same phylogroup, what is an indicator that they are easily misidentified. This result led us to suggest that both species need further morphologic and genetic clarification in future studies to elucidate their status and genetic relationship further. Moreover, 13 Exp Appl Acarol and likewise to previous works (Chitimia-Dobler et al. 2017), R. leporis and R. camicasi grouped with R. sanguineus tropical lineage, what do not support its’ differential morphological-based classification either. Complicating the matter further, R. sanguineus temperate lineage evidenced in this study to accommodate at least two type-morphologies, the R. sanguineus-like and R. turanicus-like phenotypes (described in detail in Coimbra-Dores et al. 2016). Unlike our results seems to suggest, R. guilhoni was also included within this clade in a previous analysis (Chitimia-Dobler et al. 2017), result which needs to be clarified further in future research. Based on the morphological differences that were shown to exist between these entities (Pegram et al. 1987b; Coimbra-Dores et al. 2016; Hornok et al. 2017), at least four type-morphologies could be represented within this mitochondrial lineage, what again support the expression of several or hypervariable phenotypes within the clade. The same is suggested to occur in the R. turanicus III clade. These results evidence that both clades present phenotypic plasticity, probably occurring due to hybridization processes, which explain why some authors morphologically identified R. turanicus as present in the Iberian Peninsula (Estrada-Peña and Sánchez 1988; Rosa et al. 2006; Millán et al. 2007; Santos-Silva 2010; Sobrino et al. 2012; Coimbra-Dores et al. 2016) whereas molecular based results support otherwise (Santos-Silva et al. 2011; Dantas-Torres et al. 2017). Furthermore, our study confirmed the existence of several clades under the R. sanguineus s.l. and R. turanicus species. Some were previously described (Moraes-Filho et al. 2011; Dantas-Torres et al. 2013; Hekimoğlu et al. 2016; Hornok et al. 2017; Almeida et al. 2017), and some new clades were here identified, namely R. turanicus I–III. Previous studies also support that R. sanguineus species complex comprehend more than one cryptic species (Skoracka et al. 2015; Hekimoğlu et al. 2016; Hornok et al. 2017; Almeida et al. 2017), and again here is suggested it includes more diversity than formerly reported. Even taking into account all R. turanicus clades in the obtained phylogeny, their distribution seems to be more restricted than previously anticipated (Estrada-Peña et al. 2004) since, as can be seen in Fig. 5, it does not include the Western Mediterranean ecoregion (Iberian Peninsula). In that ecoregion are in fact R. sanguineus temperate lineage specimens which present an identical phenotype to R. turanicus (Santos-Silva et al. 2011; Coimbra-Dores et al. 2016; Dantas-Torres et al. 2017). Because R. turanicus presents high phenotypic resemblances to several genetic clades, which could present differentiated vectorial competences regarding several pathogenic agents, R. turanicus morphological-like species should be referred as R. turanicus s.l. previous to molecular identification. Unlike R. sanguineus-like morphology, R. turanicus s.l. present a lectotype sensu Filippova 1997 (Dantas-Torres et al. 2013), referred here as R. turanicus s.s. clade, which in addition to the R. turanicus II–III, seem to be the R. turanicus clades distributed in the Mediterranean ecoregion (Fig. 5; Italy, Greece, Israel, Iraq, Egypt, Turkmenistan). R. turanicus I clade was found in East Africa ecoregion, that with Rhipicephalus sp. IV might be the R. turanicus lineages reported to be represented in the Afrotropical region (Fig. 5) (Walker et al. 2000). Nevertheless, these three R. turanicus lineages (I–III) were not so far evidenced as putative species. Moreover, it is of note that although only the R. turanicus s.s. lineage is morphologically and phenotypically well defined (Guglielmone and Nava 2014; Chitimia-Dobler et al. 2017), several works suggested that the R. turanicus-like morphology seem to be identified in samples belonging to other mitochondrial lineages (Pegram et al. 1987a; Beati and Keirans 2001; Mtambo et al. 2007b; Dantas-Torres et al. 2013; Shubber et al. 2014; Coimbra-Dores et al. 2016) based on both Walker et al. (2000) and Filippova et al. (1997) descriptions. Further studies will be needed 13 Exp Appl Acarol to clarify their species status based on their morphological and ecological characteristics, geographical distribution and possible distinct vectorial competences. Concerning our low supported clade Rhipicephalus sp. I, a recent phylogenetic analysis supports not only the mitochondrial lineage, as well as suggests its distribution as being in the northeast African region (Egypt) and south-eastern Europe (Turkey, Greece, Romania) (Chitimia-Dobler et al. 2017), in addition to its’ central Mediterranean distribution (Italy) (Dantas-Torres et al. 2013). With the R. sanguineus tropical lineage clade, both of these R. sanguineus s.l. clades should be found in the over-Saharan African region (ChitimiaDobler et al. 2017). Additionally, our phylogeny also suggests the formation of two basal monophyletic clades within the Rhipicephalus genus: one comprehending the Rhipicephalus (Boophilus) (Murrell and Barker 2003), R. evertsi complex, R. pravus complex, and R. pulchellus complex; and the second including the remaining three complexes, namely the R. appendiculatus, R. simus and R. sanguineus complexes. Earlier studies corroborate Rhipicephalus (Boophilus) as a closely related clade to Rhipicephalus (Digineus) (Pomerantsev) (here represented as R. evertsi complex) and R. pravus (Murrell et al. 2001a, b; Murrell and Barker 2003). Here, we also suggest the inclusion of R. pulchellus complex in the group. Morphological and ecological evidence also support these findings, namely: (1) two species included in R. pulchellus complex (R. maculatus and R. pulchellus) are part of a group of only four Rhipicephalus species that present ornate scuta (Walker et al. 2000; Barker and Murrell 2004); (2) Rhipicephalus (Boophilus) and R. (Digineus) evertsi complex comprehend the most morphological dissimilar species of Rhipicephalus genus (Walker et al. 2000; Bowman and Nuttall 2008), and (3) both this subgenus are the only two presenting species with a reduced lifecycle (one and two-host ticks, respectively) (Murrell et al. 2000; Walker et al. 2000). Regarding the second clade, R. sanguineus complex was already suggested as a sister clade of R. simus complex (Murrell et al. 2001a, b; Murrell and Barker 2003). Their relationship with R. appendiculatus complex could not be here unambiguously resolved due to lack of support obtained in the correspondent basal branches (Fig. 2). More studies will be needed to clarify this phylogenetic relationship. All this high cryptic diversity reported within the Rhipicephalus genus can be associated to several factors, such as geographic segregation (Mtambo et al. 2007b; Low et al. 2015), host density, different climates adaptation (Estrada-Pena et al. 2012; Ogden and Lindsay 2016), selective pressure from pesticides (Low et al. 2015), or could even be an adaptation strategy based on lack of direct food sources competition between species and no hostspecificity (Nava and Guglielmone 2012; Skoracka et al. 2015). Despite these hypotheses, no definite association has been found so far. Nonetheless, since the Rhipicephalus ticks’ speciation might be driven by different ecological pressures, the overall diversity of these ixodids might be underestimated. The importance of clarifying these species and associated genetic lineages, particularly in sympatric areas, arise with their involvement and their different rates of transmission regarding major epidemiological pathogens (Walker et al. 2000; Mediannikov et al. 2012; Ehounoud et al. 2016). Recent phylogenetic works already evidenced different vectorial competences shown by two morphological similar phylogroups within the R. sanguineus s.l. taxon regarding the transmission of zoonotic pathogen Ehrlichia canis to dogs (MoraesFilho et al. 2015; Nava et al. 2015; Labruna et al. 2017). Subsequently, it is imperative not only to identify different phylogroups within the genus but also to study their vectorial and transmission capacity concerning several tick-borne pathogens. 13 Exp Appl Acarol Concerning the use of mitochondrial markers in phylogenetic analyses, which presents its limitations, it is of note that it might have cause results over-resolution due to the use of short sequences. Taking these limitations into account, our results suggest several possibilities that should be studied further to be validated. Nevertheless, the availability of new molecular records for ixodids species provides a more comprehensive mitochondrial database that will improve the ticks’ identification accuracy for future scientific studies. Conclusions Tick complexes include several closely related species, whereas genetic history is tangled due to a probably high rate of both hybridisation and gene flow events between its members, which is supported by the emergence of several cryptic species. Some geneflow between R. sanguineus complex lineages are likely to occur since most of them appear to have some sympatric distributions, as it is the case of the East Mediterranean ecoregion (Egypt and Israel) regarding R. turanicus s.l. Our results allowed to trace the mitochondrial genetic profile of two Afrotropical species, R. duttoni and R. senegalensis, and to clarify their phylogenetic relationships further, what will be useful to elucidate their status as pathogenic-vectors in future studies. Evidence support that R. duttoni and R. pulchellus belong to the same clade which is here proposed to be the R. pulchellus complex. In addition, this species-group and the R. evertsi and R. pravus complexes are here supported to be more closely related to the Rhipicephalus (Boophilus) (Murrell and Barker 2003) that to the remaining members of Rhipicephalus genus. These results suggest a more shared evolutive history by these clades than previously anticipated. Furthermore, our analysis based on three molecular markers allowed to emphasise hidden genetic clades and presumptive cryptic species. These mitochondrial based results also suggested at least nine distinct genetic clades exist under the highly phenotypically resembling R. sanguineus s.l. and R. turanicus, which need to be studied further to clarify their species status. Finally, our molecular evidence strongly suggests R. turanicus lineages, which should be referred as R. turanicus s.l. due to its high cryptic diversity, are not distributed through the Iberian Peninsula, although a very similar type-morphology among R. sanguineus temperate lineage seems to suggest otherwise. Moreover, it is supported that some Rhipicephalus clades show phenotypic plasticity, which probably occurs as a result of ticks’ adaptation processes. Nonetheless, the causes remain unexplained, what make ixodids good study models of adaptation. Based on these results, additional molecular studies are still needed to clarify further this genetic tangled genus. As each tick species lineage has its specific, or even unique, epidemiological, pathogenic or host specific traits, some ecoregions are probably preferred instead of others, and a comprehensible determination of these species neglected diversity, and specific geographic distribution, can eventually lead to the identification of tick-borne diseases transmission risk areas. Acknowledgements We are grateful to the colleagues Sara Ema Silva and Ana Sofia Rodrigues (Computational Biology and Population Genomics Group (CoBiG2), Centre for Ecology, Evolution and Environmental Changes (cE3c), Department of Animal Biology, Faculty of Sciences, University of Lisbon, Portugal) for revising the final draft and to the CoBiG2 group for the help provided relating software handling. We are also thankful to volunteer students for fieldwork assistance and to the anonymous reviewers for their constructive comments that helped us to improve our work. 13 Exp Appl Acarol Funding This study was funded by Fundação para a Ciência e a Tecnologia (FCT) of the Portuguese Government (Grant No. PD/BD/109408/2015) to MJCD, and CESAM RU from FCT/MEC financial support (UID/AMB/50017) to DD through national funds. Calouste Gulbenkian Foundation (2001), Luso-American Development Foundation (2007), and Fundação Portugal-África (2008) provided field sampling funds to FR. Compliance with ethical standards Conflict of interest All authors declare that they have no conflict of interest. References Alexandre N, Santos AS, Núncio MS et al (2009) Detection of Ehrlichia canis by polymerase chain reaction in dogs from Portugal. 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