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
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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
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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.
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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)
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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.
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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
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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
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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
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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)
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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
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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
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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
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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).
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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,
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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
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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.
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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.
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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.
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