Experimental and Applied Acarology

, Volume 75, Issue 1, pp 107–128 | Cite as

Phylogenetic insights on Mediterranean and Afrotropical Rhipicephalus species (Acari: Ixodida) based on mitochondrial DNA

  • Maria João Coimbra-Dores
  • Mariana Maia-Silva
  • Wilson Marques
  • Ana Cristina Oliveira
  • Fernanda Rosa
  • Deodália Dias


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.


Rhipicephalus duttoni Rhipicephalus senegalensis Cryptic species Mitochondrial genes Ticks Phylogeny 



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.


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.

Supplementary material

10493_2018_254_MOESM1_ESM.xlsx (37 kb)
Supplementary material 1 (XLSX 37 kb)
10493_2018_254_MOESM2_ESM.xlsx (28 kb)
Supplementary material 2 (XLSX 28 kb)
10493_2018_254_MOESM3_ESM.xlsx (14 kb)
Supplementary material 3 (XLSX 14 kb)


  1. Alexandre N, Santos AS, Núncio MS et al (2009) Detection of Ehrlichia canis by polymerase chain reaction in dogs from Portugal. Vet J 181:343–344. CrossRefPubMedGoogle Scholar
  2. Alexandre N, Santos AS, Bacellar F et al (2011) Detection of Rickettsia conorii strains in Portuguese dogs (Canis familiaris). Ticks Tick Borne Dis 2:119–122. CrossRefPubMedGoogle Scholar
  3. Almeida C, Simões R, Coimbra-Dores MJ et al (2017) Mitochondrial DNA analysis of Rhipicephalus sanguineus s.l. from the western Iberian peninsula. Med Vet Entomol 31:167–177. CrossRefPubMedGoogle Scholar
  4. Anderson JF (2002) The natural history of ticks. Med Clin North Am 86:205–218. CrossRefPubMedGoogle Scholar
  5. Bacellar F, Regnery RL, Nuncio MS, Filipe AR (1995) Genotypic evaluation of rickettsial isolates recovered from various species of ticks in Portugal. Epidemiol Infect 114:169–178CrossRefPubMedPubMedCentralGoogle Scholar
  6. Baptista S, Quaresma A, Aires T et al (2004) Lyme borreliosis spirochetes in questing ticks from mainland Portugal. Int J Med Microbiol Suppl 293:109–116Google Scholar
  7. Barker SC (1998) Distinguishing species and populations of rhipicephaline ticks with its 2 ribosomal RNA. J Parasitol 84:887–892CrossRefPubMedGoogle Scholar
  8. Barker SC, Murrell A (2004) Systematics and evolution of ticks with a list of valid genus and species names. Parasitology. PubMedGoogle Scholar
  9. Beati L, Keirans JE (2001) Analysis of the systematic relationships among ticks of the genera Rhipicephalus and Boophilus (Acari: Ixodidae) based on mitochondrial 12S ribosomal DNA gene sequences and morphological characters. J Parasitol 87:32–48CrossRefPubMedGoogle Scholar
  10. Black WC IV, Piesman J (1994) Phylogeny of hard- and soft-tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences. Proc Natl Acad Sci USA 91:10034–10038CrossRefPubMedPubMedCentralGoogle Scholar
  11. Bowman AS, Nuttall PA (2008) Ticks: biology, disease and control. Cambridge University Press, CambridgeCrossRefGoogle Scholar
  12. Burger TD, Shao R, Barker SC (2014) Phylogenetic analysis of mitochondrial genome sequences indicates that the cattle tick, Rhipicephalus (Boophilus) microplus, contains a cryptic species. Mol Phylogenet Evol 76:241–253. CrossRefPubMedGoogle Scholar
  13. Camicas J-L, Hervy J-P, Adam F, Morel P-C (1998) The ticks of the world: nomenclature, described stages, hosts, distribution. Éditions de Orstom, ParisGoogle Scholar
  14. Chitimia L, Lin RQ, Cosoroaba I et al (2010) Genetic characterization of ticks from southwestern Romania by sequences of mitochondrial cox1 and nad5 genes. Exp Appl Acarol 52:305–311. CrossRefPubMedGoogle Scholar
  15. Chitimia-Dobler L, Langguth J, Pfeffer M et al (2017) Genetic analysis of Rhipicephalus sanguineus sensu lato ticks parasites of dogs in Africa north of the Sahara based on mitochondrial DNA sequences. Vet Parasitol 239:1–6. CrossRefPubMedGoogle Scholar
  16. Coimbra-Dores MJ, Nunes T, Dias D, Rosa F (2016) Rhipicephalus sanguineus (Acari: Ixodidae) species complex: morphometric and ultrastructural analyses. Exp Appl Acarol 70:455–468. CrossRefPubMedGoogle Scholar
  17. Dantas-Torres F, Latrofa M, Annoscia G et al (2013) Morphological and genetic diversity of Rhipicephalus sanguineus sensu lato from the New and Old Worlds. Parasites Vectors 6:213. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Dantas-Torres F, Maia C, Latrofa MS et al (2017) Genetic characterization of Rhipicephalus sanguineus (sensu lato) ticks from dogs in Portugal. Parasites Vectors 10:133. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Dias VAS (1950) Subsídios para o estudo dos ixodídeos de Angola. Pecuaria Anais dos Serviços de Veterinária e Indústria Animal 1947–1948(2):3–154Google Scholar
  21. Dias JATS (1994) As carraças (Acarina-Ixodoidea) da Península Ibérica: Algumas considerações sobre a sua biogeografia e relacionamento com a ixodofauna Afropaleárctica e Afrotropical. Instituto de Investigação Científica Tropical. Estudos, ensaios e documentos, LisboaGoogle Scholar
  22. ECDC (2016) Crimean–Congo hemorrhagic fever in Spain, 8 Sept 2016, StockholmGoogle Scholar
  23. Ehounoud CB, Yao KP, Dahmani M et al (2016) Multiple pathogens including potential new species in tick vectors in Côte d’Ivoire. PLoS Negl Trop Dis 10:1–18. Google Scholar
  24. Estrada-Peña A, Sánchez C (1988) Morfología comparada de Rhipicephalus sanguineus y R. turanicus (Acarina: Ixodidae). Rev Ibérica Parasitol 48:51–62Google Scholar
  25. Estrada-Pena A, Estrada-Sanchez A, Estrada-Sanchez D (2012) Occurrence patterns of afrotropical ticks (Acari: Ixodidae) in the climate space are not correlated with their taxonomic relationships. PLoS ONE 7:e36976. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Estrada-Pena A, Pfaffle M, Baneth G et al (2017) Ixodoidea of the Western Palaearctic: a review of available literature for identification of species. Ticks Tick Borne Dis. Google Scholar
  27. Estrada-Peña A, Bouattour A, Camicas J, Walker A (2004) Ticks of domestic animals in the Mediterranean region: a guide to identification of species. University of Zaragoza, ZaragozaGoogle Scholar
  28. Estrada-Peña A, Venzal JM, Nava S et al (2012) Reinstatement of Rhipicephalus (Boophilus) australis (Acari: Ixodidae) with redescription of the adult and larval stages. J Med Entomol 49:794–802. CrossRefPubMedGoogle Scholar
  29. Ferrolho J, Antunes S, Santos AS et al (2016) Detection and phylogenetic characterization of Theileria spp. and Anaplasma marginale in Rhipicephalus bursa in Portugal. Ticks Tick Borne Dis 7:443–448. CrossRefPubMedGoogle Scholar
  30. Folmer O, Black M, Hoeh W et al (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299. PubMedGoogle Scholar
  31. Gray JS, Dautel H, Estrada-Pena A et al (2009) Effects of climate change on ticks and tick-borne diseases in Europe. Interdiscip Perspect Infect Dis 2009:12. CrossRefGoogle Scholar
  32. Guglielmone AA, Nava S (2014) Names for ixodidae (Acari: Ixodoidea): valid, synonyms, incertae sedis, nomina dubia, nomina nuda, lapsus, incorrect and suppressed names—with notes on confusions and misidentifications. Zootaxa 3767:001–256. CrossRefGoogle Scholar
  33. Guglielmone AA, Robbins RG, Apanaskevich DA et al (2010) The Argasidae, Ixodidae and Nuttalliellidae (Acari: Ixodida) of the world: a list of valid species. Zootaxa 2528:1–28Google Scholar
  34. Hall T (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  35. Hekimoğlu O, Sağlam İK, Özer N, Estrada-Peña A (2016) New molecular data shed light on the global phylogeny and species limits of the Rhipicephalus sanguineus complex. Ticks Tick Borne Dis. PubMedGoogle Scholar
  36. Hornok S, Sándor AD, Tomanović S et al (2017) East and west separation of Rhipicephalus sanguineus mitochondrial lineages in the Mediterranean Basin. Parasites Vectors 10:39. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Huelsenbeck JP, Ronquist F (2001) MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755. CrossRefPubMedGoogle Scholar
  38. Jeyaprakash A, Hoy MA (2009) First divergence time estimate of spiders, scorpions, mites and ticks (subphylum: Chelicerata) inferred from mitochondrial phylogeny. Exp Appl Acarol 47:1–18. CrossRefPubMedGoogle Scholar
  39. Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol Biol Evol 30:772–780. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Kumar S, Nei M, Dudley J, Tamura K (2008) MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform 9:299–306. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Labruna MB, Naranjo V, Mangold AJ et al (2009) Allopatric speciation in ticks: genetic and reproductive divergence between geographic strains of Rhipicephalus (Boophilus) microplus. BMC Evol Biol 9:1–12. CrossRefGoogle Scholar
  42. Labruna MB, Gerardi M, Krawczak FS, Moraes-Filho J (2017) Comparative biology of the tropical and temperate species of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) under different laboratory conditions. Ticks Tick Borne Dis 8:146–156. CrossRefPubMedGoogle Scholar
  43. Latrofa MS, Dantas-Torres F, Giannelli A, Otranto D (2014) Molecular detection of tick-borne pathogens in Rhipicephalus sanguineus group ticks. Ticks Tick Borne Dis 5:943–946. CrossRefPubMedGoogle Scholar
  44. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452. CrossRefPubMedGoogle Scholar
  45. Low VL, Tay ST, Kho KL et al (2015) Molecular characterisation of the tick Rhipicephalus microplus in Malaysia: new insights into the cryptic diversity and distinct genetic assemblages throughout the world. Parasites Vectors 8:341. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Mans BJ, De Klerk D, Pienaar R, Latif AA (2011) Nuttalliella namaqua: a living fossil and closest relative to the ancestral tick lineage: implications for the evolution of blood-feeding in ticks. PLoS ONE. PubMedPubMedCentralGoogle Scholar
  47. Mans BJ, de Klerk D, Pienaar R et al (2012) The mitochondrial genomes of Nuttalliella namaqua (Ixodoidea: Nuttalliellidae) and Argas africolumbae (Ixodoidae: Argasidae): estimation of divergence dates for the major tick lineages and reconstruction of ancestral blood-feeding characters. PLoS ONE 7:1–12. CrossRefGoogle Scholar
  48. Mediannikov O, Davoust B, Socolovschi C et al (2012) Spotted fever group rickettsiae in ticks and fleas from the Democratic Republic of the Congo. Ticks Tick Borne Dis 3:371–373. CrossRefPubMedGoogle Scholar
  49. Millán J, Ruiz-fons F, Márquez FJ et al (2007) Ectoparasites of the endangered Iberian lynx Lynx parinus and sympatric wild and domestic carnivores in Spain Ectoparasites of the endangered Iberian lynx Lynx pardinus and sympatric wild and domestic carnivores in Spain. Med Vet Entomol 21:248–254. CrossRefPubMedGoogle Scholar
  50. Moraes-Filho J, Marcili A, Nieri-Bastos FA et al (2011) Genetic analysis of ticks belonging to the Rhipicephalus sanguineus group in Latin America. Acta Trop 117:51–55. CrossRefPubMedGoogle Scholar
  51. Moraes-Filho J, Krawczak FS, Costa FB et al (2015) Comparative evaluation of the vector competence of four South American populations of the rhipicephalus sanguineus group for the bacterium Ehrlichia canis, the agent of canine monocytic ehrlichiosis. PLoS ONE. PubMedPubMedCentralGoogle Scholar
  52. Mtambo J, Madder M, Van Bortel W et al (2007a) Genetic variation in Rhipicephalus appendiculatus (Acari: Ixodidae) from Zambia: correlating genetic and ecological variation with Rhipicephalus appendiculatus from eastern and southern Africa. J Vector Ecol 32:168–175.[168:gviraa];2 CrossRefPubMedGoogle Scholar
  53. Mtambo J, Madder M, Van Bortel W et al (2007b) Further evidence for geographic differentiation in R. appendiculatus (Acari: Ixodidae) from Eastern and Southern provinces of Zambia. Exp Appl Acarol 41:129–138. CrossRefPubMedGoogle Scholar
  54. Murrell A, Barker SC (2003) Synonymy of Boophilus Curtice, 1891 with Rhipicephalus Koch, 1844 (Acari: Ixodidae). Syst Parasitol 56:169–172CrossRefPubMedGoogle Scholar
  55. Murrell A, Campbell NJH, Barker SC (2000) Phylogenetic analyses of the rhipicephaline ticks indicate that the genus Rhipicephalus is paraphyletic. Mol Phylogenet Evol 16:1–7. CrossRefPubMedGoogle Scholar
  56. Murrell A, Campbell NJ, Barker SC (2001a) A total-evidence phylogeny of ticks provides insights into the evolution of life cycles and biogeography. Mol Phylogenet Evol 21:244–258. CrossRefPubMedGoogle Scholar
  57. Murrell A, Campbell NJH, Barker SC (2001b) Recurrent gains and losses of large (84–109 bp) repeats in the rDNA internal transcribed spacer 2 (ITS2) of rhipicephaline ticks. Insect Mol Biol 10:587–596CrossRefPubMedGoogle Scholar
  58. Nava S, Guglielmone AA (2012) A meta-analysis of host specificity in Neotropical hard ticks (Acari: Ixodidae). Bull Entomol Res 103:216–224. CrossRefPubMedGoogle Scholar
  59. Nava S, Estrada-Peña A, Petney T et al (2015) The taxonomic status of Rhipicephalus sanguineus (Latreille, 1806). Vet Parasitol 208:2–8. CrossRefPubMedGoogle Scholar
  60. Ogden NH, Lindsay LR (2016) Effects of climate and climate change on vectors and vector-borne diseases: ticks are different. Trends Parasitol. PubMedGoogle Scholar
  61. Olson DM, Dinerstein E, Wikramanayake ED et al (2001) Terrestrial ecoregions of the world: a new map of life on earth. Bioscience 51:933.[0933:TEOTWA]2.0.CO;2 CrossRefGoogle Scholar
  62. Olwoch JM, van Jaarsveld AS, Scholtz CH, Horak IG (2007) Climate change and the genus Rhipicephalus (Acari: Ixodidae) in Africa. Onderstepoort J Vet Res 74:45–72. CrossRefPubMedGoogle Scholar
  63. Papadopoulos B, Núncio M, Filipe A (1992) The occurrence of Rhipicephalus turanicus Pomerantzev, Matikashvily & Lototsky, 1940, a species of R. sanguineus group, in Portugal. Acarologia 33:331–333Google Scholar
  64. Parola P, Raoult D (2001) Ticks and tickborne bacterial diseases in humans: an emerging infectious threat. Clin Infect Dis 32:897–928. CrossRefPubMedGoogle Scholar
  65. Pegram RG, Clifford CM, Walker JB, Keirans JE (1987a) Clarification of the Rhipicephalus sanguineus group (Acari, Ixodoidea, Ixodidae). I. R. sulcatus Neumann, 1908 and R. turanicus Pomerantsev, 1936. Syst Parasitol 10:3–26. CrossRefGoogle Scholar
  66. Pegram RG, Keirans JE, Clifford CM, Walker JB (1987b) Clarification of the Rhipicephalus sanguineus group (Acari, Ixodoidea, Ixodidae). II. R. sanguineus (Latreille, 1806) and related species. Syst Parasitol 10:27–44. CrossRefGoogle Scholar
  67. Pina-Martins F, Paulo OS (2008) Concatenator: sequence data matrices handling made easy. Mol Ecol Resour 8:1254–1255. CrossRefPubMedGoogle Scholar
  68. Rambaut A (2014) FigTree v1. 4.2. A graphical viewer of phylogenetic trees. Institute of Evolutionary Biology University of EdinburghGoogle Scholar
  69. Rambaut A, Surchard MA, Xie D, Drummond AJ (2014) Tracer v1.6. Accessed 21 Jan 2017
  70. Rosa F, Crespo M, Ferreirinha D et al (2006) Ticks on dogs and its role as vectors/intermediate hosts in the Center-West of Portugal. In: 11th international congress of parasitology—ICOPA XI pp 567–570Google Scholar
  71. Santos-Silva MM (2010) Portuguese Ixodids (Acari, Ixodidae). Systematics, Geographical Distribution and Host-Vector Relationships. PhD dissertation, Universidade Tecnica de LisboaGoogle Scholar
  72. Santos-Silva MM, Beati L, Santos AS et al (2011) The hard-tick fauna of mainland Portugal (Acari: Ixodidae): an update on geographical distribution and known associations with hosts and pathogens. Exp Appl Acarol 55:85–121. CrossRefPubMedGoogle Scholar
  73. Shubber HWK, Al-Hassani NAW, Mohammad MK (2014) Ixodid ticks diversity in the middle and south of Iraq. Int J Recent Sci Res 5:1518–1523Google Scholar
  74. Skoracka A, Magalhães S, Rector BG, Kuczyński L (2015) Cryptic speciation in the Acari: a function of species lifestyles or our ability to separate species? Exp Appl Acarol 67:165–182. CrossRefPubMedPubMedCentralGoogle Scholar
  75. Sobrino R, Millán J, Oleaga Á et al (2012) Ecological preferences of exophilic and endophilic ticks (Acari: Ixodidae) parasitizing wild carnivores in the Iberian Peninsula. Vet Parasitol 184:248–257. CrossRefPubMedGoogle Scholar
  76. Speybroeck N, Madder M, Thulke HH et al (2004) Variation in body size in the tick complex Rhipicephalus appendiculatus/Rhipicephalus zambeziensis. J Vector Ecol 29:347–354PubMedGoogle Scholar
  77. Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312–1313. CrossRefPubMedPubMedCentralGoogle Scholar
  78. Sugiura N (1978) Further analysts of the data by akaike’s information criterion and the finite corrections. Commun Stat Theory Methods 7:13–26. CrossRefGoogle Scholar
  79. Szabó MPJ, Mangold AJ, João CF et al (2005) Biological and DNA evidence of two dissimilar populations of the Rhipicephalus sanguineus tick group (Acari: Ixodidae) in South America. Vet Parasitol 130:131–140. CrossRefPubMedGoogle Scholar
  80. Tendeiro JL (1956) Estudos de Ixodologia. Notas sobre o Rhipicephalus simus simus C. L. Koch 1844 e o Rhipicephalus simus senegalensis C. L. Koch 1844 na Guiné Portuguesa. BolCult Guiné Port 42:99–109Google Scholar
  81. Tendeiro JL (1959) Sur quelques ixodidés du Mozambique et de la Guinée portugaise. BolCult Guiné Port 14:21–95Google Scholar
  82. Vilhena H, Martinez-Díaz VL, Cardoso L et al (2013) Feline vector-borne pathogens in the north and centre of Portugal. Parasites Vectors 6:99. CrossRefPubMedPubMedCentralGoogle Scholar
  83. Walker JB, Keirans JE, Horak IG (2000) The genus Rhipicephalus (Acari, Ixodidae): a guide to the brown ticks of the world, 1st edn. Cambridge University Press, New YorkCrossRefGoogle Scholar
  84. Walker A, Bouattour A, Camicas J et al (2003) Ticks of domestic animals in Africa: a guide to identification of species, 1st edn. Bioscience Reports, EdinburghGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Animal Biology, Faculty of SciencesUniversity of LisbonLisbonPortugal
  2. 2.Faculty of Sciences, Centre for Environmental and Marine Studies (CESAM)University of LisbonLisbonPortugal
  3. 3.Casa dos Animais Veterinary ClinicLuandaAngola
  4. 4.Instituto Superior de AgronomiaUniversity of LisbonLisbonPortugal

Personalised recommendations