Molecules and morphology: evidence for cryptic hybridizationin African Hyalomma (Acari: Ixodidae)

David J. Rees,a,* Maurizio Dioli,b and Lawrence R. Kirkendalla

a Department of Zoology, University of Bergen, All�eegaten 41, 5007 Bergen, Norwayb Royal Veterinary College, London, UK

Received 24 May 2002; revised 7 September 2002

Abstract

The role of natural hybridization and introgression as part of the evolutionary process is of increasing interest to zoologists,

particularly as more examples of gene exchange among species are identified. We present mitochondrial and nuclear sequence data

for Hyalomma dromedarii, Hyalomma truncatum, and Hyalomma marginatum rufipes (Acari: Ixodidae) collected from one-humped

camels in Ethiopia. These species are well differentiated morphologically and genetically; sequence data from the mitochondrial

DNA (mtDNA) cytochrome oxidase I gene indicates 10–14% divergence between the species. However, incongruence between

morphology and the mtDNA phylogeny was observed, with multiple individuals of H. dromedarii and H. truncatum present on the

same mtDNA lineage as H. marginatum rufipes. Thus, individuals with morphology of H. dromedarii and H. truncatum are in-

distinguishable from H. marginatum rufipes on the basis of mtDNA. Multiple copies of ITS-2 were subsequently cloned and se-

quenced for a subset of individuals from the mtDNA phylogeny, representing both �normal� and �putative hybrid� individuals. Verylow sequence divergence (0.3%) was observed within �normal� individuals of both H. dromedarii and H. truncatum relative to the�putative hybrid� individuals (6 and 2.7%, respectively). The pattern of intra-individual variation in ITS-2 within �putative hybrid�individuals, particularly in H. dromedarii, strongly suggests that gene flow has occurred among these Hyalomma species, but no

indication of this is given by the morphology of the individuals.

� 2002 Elsevier Science (USA). All rights reserved.

Keywords: Hyalomma; Ixodidae; mtDNA; ITS-2; Hybridization; Phylogeny

1. Introduction

The significance of hybridization and introgression as

part of the evolutionary process has long been recog-

nized by botanists but has traditionally received less

attention from zoologists (Dowling and Secor, 1997).

Natural hybridization has been hypothesized as acting

as an evolutionary stimulus, with blocks of genestransferred between differentially adapted systems, re-

sulting in potential for adaptive shifts and evolutionary

diversification (Anderson and Stebbins, 1954). Reluc-

tance to assign an important evolutionary role to hy-

bridization and introgression has arisen in part from a

perceived rarity of animal hybrids, although it is

emerging that gene exchange among animal species has

been more common than previously believed (Dowling

and Secor, 1997). Two factors that may contribute to

the apparent rarity of animal hybrids are: (a) the tra-

ditional view that hybridization disrupts coadapted gene

complexes, resulting in less fit offspring that are then

selected against (but see Barton, 2001), and (b) the dif-

ficulty of detecting natural hybrids, particularly as they

may not often be looked for.The tick genus Hyalomma Koch is represented in

Africa, southern Europe, and Asia and inhabits regions

with a long dry season (Matthysse and Colbo, 1987).

The genus currently comprises 30 species and subspecies

and has a complex history of synonymization (Camicas

et al., 1998; Hoogstraal, 1956; Matthysse and Colbo,

1987; Pegram and Higgins, 1992). Ticks have adapted

remarkably well to human domestication of livestock(Hoogstraal, 1978; Hoogstraal and Aeschlimann, 1982)

and are of considerable economic importance through-

Molecular Phylogenetics and Evolution 27 (2003) 131–142

www.elsevier.com/locate/ympev

MOLECULARPHYLOGENETICSANDEVOLUTION

* Corresponding author. Fax: +47-555-89675.

E-mail address: david.rees@zoo.uib.no (D.J. Rees).

1055-7903/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved.

doi:10.1016/S1055-7903(02)00374-3

out Africa due to both the direct effects of feeding onlivestock and tick-borne diseases such as Theileria equi,

T. annulata, and Babesia caballi (De Kok et al., 1993;

Hoogstraal, 1956; Pegram et al., 1981). Species of Hy-

alomma are also known as potential vectors of many

viral, bacterial, and protozoan pathogens that are of

direct concern to humans. For example, H. marginatum

marginatum is known as a potential vector of Cri-

mean–Congo haemorrhagic fever (CCHF), tick-borneencephalitis, and of the rikketsia, Coxiella burnetti (Q-

fever) (De Kok et al., 1993; Hoogstraal, 1956).

Ten species of Hyalomma have been recorded in

collections from livestock (cattle, camels, sheep, and

goats) in Ethiopia (Pegram et al., 1981). While the ma-

jority of these species are localized, three are relatively

widespread: Hyalomma dromedarii, H. truncatum, and

H. marginatum rufipes. For this study, these species werecollected from a herd of one-humped camels (Camelus

dromedarius) in south-east Ethiopia. H. dromedarii is

considered mainly a North African and Asian tick with

a small population in East Africa (Hoogstraal, 1956)

and the distribution of this species coincides with that

of the most common host, the one-humped camel

(Hoogstraal, 1956; van Straten and Jongejan, 1993).

H. dromedarii has been described as the most completelydesert-adapted ixodid tick of Africa (Pegram et al.,

1981). Hyalomma marginatum rufipes and H. truncatum

are most commonly found on domestic cattle but also

utilize other domestic and wild hosts (Cumming, 1998;

Hoogstraal, 1956). Both species are widespread

throughout Africa (Matthysse and Colbo, 1987) but

absent from Eurasia and Arabia (Pegram and Higgins,

1992).Several studies involving collections of ticks from

camels have noted a predilection in H. dromedarii for

attachment in the nostrils, a specialized attachment site

not utilized by other species of Hyalomma (e.g., Dioli,

1992; Dolan et al., 1983; Rutagwenda, 1985). The most

common attachment sites for H. marginatum rufipes and

H. truncatum on both camels and cattle are the perineal/

inguinal area and the base of the tail (Baker et al., 1989;Dioli, 1992; Dolan et al., 1983; Mohammed, 1977; Ru-

tagwenda, 1985). The initial impetus for a molecular

analysis of this group of Hyalomma species came from

the observation of a considerable number of ticks, typ-

ical of H. dromedarii morphology, utilizing attachment

sites considered unusual for this species, primarily the

anogenital region (M. Dioli, pers. obs.). Our original

goal was to ascertain whether the H. dromedarii-likeindividuals from the anus could represent a cryptic

species utilizing very different attachment sites than

those generally associated with H. dromedarii. Mater-

nally inherited mitochondrial DNA (mtDNA) sequences

for the cytochrome oxidase I (COI) gene have previously

been used in phylogenetic studies and have clearly sep-

arated Hyalomma species (Murrell et al., 2000) and this

gene was therefore selected for initial use. Following theresults of the mtDNA study, a nuclear gene (the internal

transcribed spacer ITS-2) was chosen to investigate

possible hybridization among H. dromedarii, H. mar-

ginatum rufipes, and H. truncatum. ITS-2 has been used

in a number of tick phylogenetic studies at a range of

scales (e.g., McLain et al., 1995; Wesson et al., 1993;

Zahler et al., 1997) and the relatively high level of var-

iability associated with this marker (including intra-in-dividual variation, e.g., Rich et al., 1997) led to its

selection over other candidates such as 28S, 16S, and

12S rDNA.

2. Materials and methods

2.1. Sample collection

Ticks were collected by the second author from a

domestic herd of one-humped camels near the town of

Gode in south-east Ethiopia (5�550N, 43�340E) in Oc-tober 1999 and June 2000. Hyalomma were also col-

lected from camels near Isiolo in eastern Kenya (0�210N,37�350E) (May 1998). Only male specimens were used inphylogenetic analyses because females of species in thisgenus can be difficult to distinguish. Samples in this

study consisted of H. dromedarii collected from the nose

and anus, and H. marginatum rufipes and H. truncatum

collected from the anus only. Extensive collection from

the nose yielded no specimens of H. marginatum rufipes

or H. truncatum. No other Hyalomma species were

present in collections. All specimens were stored in 70%

EtOH, examined under a stereomicroscope and identi-fied using available taxonomic keys (Hoogstraal, 1956;

Matthysse and Colbo, 1987). Our species determinations

were confirmed, for a voucher set of individuals used in

the study, by J.L. Camicas of the Laboratoire d�Epide-miologie des Maladies a Vecteurs, Montpellier, France

(pers. comm.). All specimens used in this study are re-

tained in the collection of M. Dioli.

2.2. DNA extraction, PCR amplification, and sequencing

of COI

Prior to DNA extraction, each sample was immersed

in liquid nitrogen for 20min. Entire individuals were

then digested for 4 h and DNA subsequently column

purified using reagents from a QIAamp DNA Mini Kit,

following manufacturer�s recommendations. Individualticks remained morphologically intact at the end of this

DNA extraction process; 12 individuals were subse-

quently used to prepare scanning electron micrographs

(see Fig. 4). The primers used for amplification of COI

were TY-J-1449 50-AATTTACAGTTTATCGCCT-30

(Murrell et al., 2000) and C1-N-2312 50-CATACAATAAAGCCTAATA-30 (Murrell et al., 2000). Together

132 D.J. Rees et al. / Molecular Phylogenetics and Evolution 27 (2003) 131–142

these primers amplify a fragment of 863 bp. Polymerasechain reactions (PCRs) were performed in 25-ll volumesincluding 0.75 ll of each 10 lM PCR primer, 0.5U of

AmpliTaq DNA polymerase (Perkin–Elmer) and with a

MgCl2 concentration of 2mM. Two microlitres of DNA

extract were used for amplification. Each of 40 PCR

cycles comprised denaturation at 94 �C for 30 s, an-

nealing at 45 �C for 1min and extension at 72 �C for

1min. For some samples, annealing temperatures be-tween 42 �C and 47 �C were necessary to achieve optimalamplification results. PCR products were column puri-

fied using a QIAquick PCR Purification Kit following

manufacturer�s recommendations. Sequencing reactionsusing the PCR primers and the Perkin–Elmer BigDye

terminator reaction mix were run on a Perkin–Elmer

ABI automated DNA sequencer. The COI fragment was

sequenced in both directions for all individuals and se-quences were aligned by eye against the GenBank se-

quence of Dermacentor variabilis (Murrell et al., 2000).

2.3. PCR amplification, cloning, and sequencing of ITS-2

On the basis of the results of the COI sequencing, a

subset of the samples from the mtDNA phylogeny was

included in a study utilizing the nuclear marker ITS-2.Amplification of a portion of ITS-2 was performed us-

ing the primers of Zahler et al. (1997); RIB-8 50-GTCGTAGTCCGCCGTC-30 and a modified RIB-1150-GAGTACGACGCCCTACC-30 (this primer was

modified by removal of the XbaI linker present in the

original). Both primers are located in a variable region

at the 30 end of ITS-2 and are conserved between Der-macentor spp. and Rhipicephalus sanguineus (Zahler etal., 1997). Together, these primers amplify a fragment of

approximately 300 bp. PCRs were again performed in a

25-ll volume including 0.75 ll of each 10 lM PCR pri-

mer, 2 ll of DNA and 2mM MgCl2. AmpliTaq Gold

DNA polymerase (Perkin–Elmer) was used in place of

AmpliTaq to allow Hot Start PCR with minimal mod-

ification of reaction conditions. The PCR profile con-

sisted of an initial enzyme activation step at 95 �C for10min, followed by 40 cycles of denaturation at 94 �Cfor 30 s and annealing/extension at 60 �C for 1min, anda final step of 72 �C for 10min. Fresh (less than 1-day-old), unpurified PCR products were used in cloning re-

actions and transformation of chemically competent

Escherichia coli (TOPO TA Cloning Kit for Sequencing,

Invitrogen) following manufacturer�s recommendations.Transformation mixtures were then plated overnight at37 �C on LB plates containing 100 lgml�1 ampicillin.Colonies were then picked and transferred to 5ml LB

containing 50 lgml�1 ampicillin for overnight culture.Plasmids were then column purified using the Promega

Wizard Plus SV minipreps DNA purification system

following manufacturers recommendations. Sequencing

reactions were performed as described for COI, using

the M13 forward and reverse primers supplied with theTOPO TA Cloning Kit (Invitrogen).

2.4. Phylogenetic analyses

Published COI sequences for several species of Hya-

lomma were obtained from GenBank and included in

our dataset. These were H. dromedarii (laboratory

strain, Egypt), H. truncatum (South Africa), H. mar-ginatum rufipes (Zimbabwe), and H. aegyptium (labo-

ratory strain, Belgium) (all from Murrell et al., 2000).

Aligned COI sequence data were analyzed using PAUP*

(Swofford, 1998). Maximum-parsimony (MP) analysis

was performed using D. variabilis as an outgroup. ITS-2

sequence alignment was performed with the Multalin

package (Corpet, 1988; available online via http://

prodes.toulouse.inra.fr/multalin/multalin.html) using theDNA comparison table, a gap weight of 5 and a gap

length weight of 0. Haplotype networks were con-

structed from the aligned ITS-2 sequence data using the

program TCS (Clement et al., 2000). This program al-

lows the estimation of genealogical relationships from

DNA sequences using the statistical parsimony method

of Templeton et al. (1992). This method involves cal-

culation of a pairwise distance matrix for haplotypesand calculation of the probability of parsimony for all

pairwise comparisons until a cut-off point of 0.95 is

exceeded. The maximum number of mutational differ-

ences allowed between pairs of sequences under the

parsimony criterion is determined by the number of

differences associated with the probability just before the

95% cut-off. This is the basis for connections made be-

tween haplotypes and the resulting graphical outputrepresents the plausible connections made between

haplotypes, including missing intermediates.

DNA sequences have been deposited in the EMBL

Nucleotide Sequence Database under Accession Nos.

AJ437061–AJ437101 (COI) and AJ437360–AJ437402

(ITS-2).

3. Results

3.1. mtDNA analysis

Aligned COI sequences, including those of Murrell

et al. (2000) obtained from GenBank, consisted of

793 bp with variability at 29.9% of sites. Of the variable

sites, 71.3% were parsimony-informative. Maximum-parsimony analysis resulted in nine equally parsimoni-

ous trees that differed only in minor rearrangements of

terminal branches. The maximum-parsimony phylogeny

(Fig. 1) consists of three major mtDNA lineages. One

lineage (A) comprises H. dromedarii from Kenya,

the Egyptian laboratory strain, and several Ethiopian

samples from both nose and anus attachment sites.

D.J. Rees et al. / Molecular Phylogenetics and Evolution 27 (2003) 131–142 133

Maximum divergence within this clade is 0.5% (uncor-

rected). A second lineage (B) contains samples of H.

truncatum from Ethiopia and the South African Gen-Bank sequence. Divergence among the Ethiopian sam-

ples is 1.1% while between Ethiopia and South Africa,

the maximum divergence is 10.3%. A third mtDNA

lineage (C) comprises all H. marginatum rufipes (Ethio-

pia plus the GenBank sequence from Zimbabwe) and

also three individuals of H. truncatum and six H.

dromedarii collected from the anus. Identical haplotypes

are shared by H. marginatum rufipes, H. dromedarii, and

H. truncatum. Maximum divergence within this clade is

3.8%. Minimum COI divergences between these lineages

are 14.3% (A–B), 12.2% (A–C), and 10.0% (B–C).

3.2. ITS-2 data

On the basis of the mtDNA phylogeny, both �normal�(morphology +mtDNA) and �putative hybrid� (normalmorphology but H. marginatum rufipes-type mtDNA)

individuals of H. dromedarii and H. truncatum were se-

quenced for multiple clones of ITS-2 along with indi-

A

B

C

Fig. 1. One of the nine equally parsimonious trees for species of Hyalomma using mitochondrial COI sequence data. Branch lengths are proportional

to substitutional change. Bootstrap values indicate nodes gaining more than 70% support (heuristic search, 500 replicates). Major clades are indicated

by the letters A, B, and C. For Ethiopian H. dromedarii, samples collected from camels noses and anogenital region are indicated by N and A,

respectively. Individuals subsequently used for cloning and sequencing of ITS-2 are indicated by an asterisk (*).

134 D.J. Rees et al. / Molecular Phylogenetics and Evolution 27 (2003) 131–142

viduals of H. m. rufipes. For both H. dromedarii and H.truncatum, two �normal� individuals were cloned andsequenced, one for eight clones, and a second individual

for two clones. Two individuals of H. marginatum rufi-

pes were sequenced, one for five clones, and a second for

two. One �putative hybrid� H. dromedarii was sequencedfor seven clones, and two �putative hybrid� H. truncatumwere sequenced; one for seven clones and a second for

two clones. The aligned ITS-2 sequences are shown inFig. 2. Construction of a haplotype network for the ITS-

2 sequence data using TCS resulted in two distinct net-

works (Fig. 3). These two networks differ by a minimum

of 11 substitutions, two one-base and one three-base

insertion/deletion (see Figs. 2 and 3). One network (a)

comprised only H. dromedarii clones. The sequencesobtained from seven clones from one �normal� H.dromedarii (HY01) were identical (termed �H. dromedariitype� in Fig. 3 for simplicity), while the eighth differedfrom the majority by only one substitution. Two clones

from a second �normal� individual (HY43) were alsoidentical to this majority type. Of the seven clones se-

quenced for the �putative hybrid� H. dromedarii indi-vidual (HY37), two were identical to the majority H.dromedarii sequence type; the remainder were not con-

nected to this network.

The second network (b) generated by the TCS anal-

ysis consisted of all clones from H. truncatum and H.

marginatum rufipes individuals and also the remaining

Fig. 2. Alignment of polymorphic sites among 43 ITS-2 clones for Hyalomma individuals. Nucleotide positions relate to those following sequence

alignment using Multalin. Sequence labels indicate individual code, clone number, and species, respectively (D, H. dromedarii; M, H. marginatum

rufipes; T, H. truncatum). Dashes represent alignment gaps and dots indicate sequence identity to H. dromedarii individual HY01, clone 1.

D.J. Rees et al. / Molecular Phylogenetics and Evolution 27 (2003) 131–142 135

five clones from the �putative hybrid� H. dromedarii(HY37). Two groupings are apparent in this second

network, relating to the majority sequence types seen in

H. marginatum rufipes and H. truncatum. These two

groups are differentiated by two substitutions (Figs. 2

and 3). The sequences for three clones from one indi-

vidual H. marginatum rufipes (HY28) were identical and

matched by the two clones from a second individual(HY52) (this sequence type has been termed �H. mar-ginatum rufipes type� in Fig. 3). Of the two remaining H.marginatum rufipes clones, both differed from the ma-

jority type by two substitutions, but one (HY28 clone

10) was of identical sequence to the ITS-2 sequence

observed in the majority of �normal� H. truncatumclones. Four of seven clones from the �putative hybrid�

H. dromedarii (HY37) were of identical sequence to the

majority H. marginatum rufipes type.

Greater sequence variation was observed among H.

truncatum clones than in H. dromedarii or H. margina-

tum rufipes. For �normal� H. truncatum, seven clones hadidentical sequences and the eighth differed by one sub-

stitution (HY58). One clone from a second individual

(HY33) was again identical to this majority �H. trunca-tum type� ITS-2 sequence, and a second differed by onesubstitution. One clone from the �putative hybrid� H.dromedarii (HY37) was identical to the majority ITS-2

sequence type observed in H. truncatum. For the �puta-tive hybrid� H. truncatum, of seven clones from one in-

dividual (HY57), three were identical to the majority

H. truncatum type, three differed from this by two

Fig. 3. Haplotype network resulting from the analysis of ITS-2 sequence data using TCS. Branches connecting haplotypes represent one-step

mutations and small circles indicate missing haplotypes. Haplotypes with the highest outgroup probability are displayed as squares and other

haplotypes are displayed as ovals. Where a haplotype comprises more than one sequence, the morphological species, individual code, and number of

clones involved are listed (connected to the haplotype by a broken line). For unique haplotypes, the individual and clone number is shown within the

oval (e.g., clone 2 from individual HY57 appears as HY57-2). Clones from individuals considered �normal� and �putative hybrid� on the basis ofmtDNA are indicated by filled circles and crosses, respectively.

136 D.J. Rees et al. / Molecular Phylogenetics and Evolution 27 (2003) 131–142

substitutions, and one was closer to the H. marginatumrufipes majority type (differing by two substitutions).

One clone from a second �putative hybrid� H. truncatumindividual (HY54) was identical to the majority H.

truncatum type, and a second clone differed from the H.

marginatum rufipes ITS-2 type by one substitution.

4. Discussion

4.1. Incongruence between morphology and mtDNA

On the basis of mtDNA sequence data, none of the

three Hyalomma species included in this study form a

monophyletic group (Fig. 1). Multiple samples of both

H. truncatum and H. dromedarii collected from the anus

are indistinguishable from H. marginatum rufipes interms of mtDNA sequence. In the mtDNA phylogeny,

the main H. dromedarii lineage (Fig. 1, lineage A)

comprises the GenBank reference sequence (Murrell et

al., 2000) and samples from Kenya, as well as all Ethi-

opian �nose� samples and multiple samples collected

from the anus. The H. truncatum lineage (Fig. 1, lineage

B) comprises the GenBank reference sequence from

South Africa (Murrell et al., 2000) and two of five in-dividuals from Ethiopian camel herds, with significant

COI divergence between our Ethiopian samples and the

published GenBank sequence for this species (10.3%).

The H. marginatum rufipes lineage (Fig. 1, lineage C)

contains the GenBank reference sample from Zimbabwe

(Murrell et al., 2000) and all Ethiopian H. marginatum

rufipes samples. In addition, this lineage contains mul-

tiple individuals of both H. dromedarii (anus only nonefrom the nose) and H. truncatum. These individuals are

morphologically typical of their respective species but

are indistinguishable from H. marginatum rufipes on the

basis of mtDNA.

Given that many different gene trees comprise a

species tree (see, e.g., Avise, 2000; Doyle, 1997; Page and

Charleston, 1997; Page and Holmes, 1998), several

processes may be invoked to explain apparent paraphylyof a species in a phylogeny constructed from a single

locus. These include: (i) convergent evolution, (ii) re-

tention of ancestral genetic diversity, (iii) amplification

of pseudogenes, and (iv) introgressive hybridization.

The presence of individuals with H. dromedarii and

H. truncatum morphology on the H. marginatum rufipes

mtDNA lineage is unlikely to be explained by conver-

gent evolution of these two morphotypes. Specimens ofH. dromedarii and H. truncatum possessing the

H. marginatum rufipes mtDNA type (those on lineage C

in Fig. 1) do not just �tend towards� these species mor-phologically, they are indistinguishable from �normal�specimens (see Fig. 4). Persistence of an ancestral allelic

polymorphism within a species can lead to incongruence

between gene and species trees, particularly if the di-

vergence time between species is short (Pamilo and Nei,1988). Several studies have invoked incomplete sorting

of alleles to explain the presence of a species on multiple

mtDNA lineages (e.g., Juan et al., 1996; Rees et al.,

2001; Sperling et al., 1999; Vogler and DeSalle, 1993).

For Hyalomma, however, incomplete sorting of mtDNA

alleles is not a compelling explanation due to the depth

of divergence between these Hyalomma species as a

whole (10–14.3% COI) indicating that these species arenot of recent origin.

Nuclear copies of mitochondrial sequences have been

detected in a variety of organisms (reviewed by Ben-

sasson et al., 2001; http://www.pseudogene.net). These

nuclear mitochondrial pseudogenes (Numts) may be

inadvertently amplified by PCR and may lead to in-

correct phylogenetic reconstruction. Other than the

unexpected placement of H. dromedarii and H. trunca-tum individuals on the H. marginatum rufipes mtDNA

lineage, no symptoms associated with Numts (Bensas-

son et al., 2001) were observed in Hyalomma.

An alternative explanation that would account for

the observed variation in both the mtDNA and ITS-2

data involves introgressive hybridization, that is, the

incorporation of the genes of one species into the gene

pool of another. Past or ongoing hybridization betweenmales of H. dromedarii and H. truncatum and female H.

marginatum rufipes could account for the observed

mtDNA phylogeny. This possibility was further inves-

tigated by cloning and sequencing multiple copies of an

additional nuclear marker (ITS-2) for both �normal� andputative hybrid individuals.

4.2. Intra-individual and interspecific variation in ITS-2

Significant intra-individual sequence variation among

cloned copies of ITS-2 (which, being located in ribo-

somal DNA, occurs in multiple copies in eukaryote ge-

nomes) has previously been reported for ticks of the

genus Ixodes (Rich et al., 1997). In that case, eight

copies of ITS-2 (�300 bp) were cloned and sequencedfrom each of two individuals, revealing 3.5–4.5% nu-cleotide polymorphism within individuals. This con-

trasts with other organisms where lower intra-individual

ITS-2 variability has been found (e.g., mosquitoes, less

than 2%: Wesson et al., 1992; Onyabe and Conn, 1999).

In the Hyalomma individuals in this study, within-indi-

vidual heterogeneity of ITS-2 copies is clearly not uni-

versal. In �normal� individuals of both H. dromedarii(HY01) andH. truncatum (HY58) the level of nucleotidepolymorphism was 0.3% (1 substitution in 302 bp, eight

clones from each individual). Far greater levels of se-

quence diversity were observed in the �putative hybrid�individuals; 6% in H. dromedarii (HY37; 18 nucleotide

polymorphisms, seven clones) and 2.7% in H. truncatum

(HY57; eight polymorphisms, seven clones). The pattern

of intra-individual variation in ITS-2 sequence appears

D.J. Rees et al. / Molecular Phylogenetics and Evolution 27 (2003) 131–142 137

to support the existence of gene flow among Hyalomma

species. Clones from �normal� H. dromedarii show verylittle intra-individual sequence variation (one substitu-

tion in a total of 10 clones). In contrast, clones from the

�putative hybrid� H. dromedarii individual (HY37) dis-played significant sequence variation, with clones iden-tical to the majority types found within H. marginatum

rufipes and H. truncatum as well as �normal� H. drome-darii (involving 10–12 substitutions, one three-base and

three one-base indels between clones; Fig. 2). Although

other factors could account for the presence of multiple,

divergent copies of ITS-2 within an individual (e.g., re-

tention of ancestral polymorphic copies despite con-

certed evolution of rDNA), the fact that intra-individualvariation was only found in the �putative hybrid� indi-vidual, and that the divergent copies were identical to

those found in other species, makes interspecific hy-

bridization a more plausible explanation.

The pattern of intraspecific variation in ITS-2 ob-

served in H. dromedarii was also evident in H. trunca-

tum. For �normal� H. truncatum, low intra-individual

divergence was observed (one substitution in eightclones from one individual, and two substitutions in one

of two clones from a second individual). For �putativehybrid� H. truncatum, three clones from one individual

(HY57) matched the sequence predominant in �normal�H. truncatum, while three others differed from this se-

quence by two substitutions. A seventh clone differed

from the majority sequence of H. marginatum rufipes by

two substitutions (and by four from H. truncatum). Fortwo clones from a second �putative hybrid� H. truncatumindividual (HY54), one clone matched the majority

Fig. 4. Scanning electron micrographs of Hyalomma individuals included in the molecular study (prepared after DNA extraction). Dorsal view,

ventral view, and spiracular plates, respectively, are shown for �normal� (morphology and mtDNA) individuals of H. dromedarii (a, b, c: all fromindividual HY09),H. truncatum (d, e, f: all from HY33), andH. marginatum rufipes (g, h, i: all from HY52). Morphology of �putative hybrid� (normalmorphology but H. marginatum rufipes type mtDNA) individuals is also shown: H. dromedarii (j, k, l: HY42 dorsal, ventral; HY44 spiracular plate)

and H. truncatum (m, n, o: HY54 dorsal; HY57 ventral, spiracular plate).

138 D.J. Rees et al. / Molecular Phylogenetics and Evolution 27 (2003) 131–142

H. truncatum sequence and the second differed from the

H. marginatum rufipes sequence by one substitution. No

clones from �putative hybrid� H. truncatum were identi-cal to the majority H. marginatum rufipes type. This mayreflect sequence divergence since a past hybridization

event between these species, or alternatively, it may re-

flect the presence of greater diversity of ITS-2 types

within H. marginatum rufipes. Among the H. margina-

tum rufipes clones, five are identical, one differs from this

sequence by two substitutions, and one is identical to the

majority H. truncatum sequence (Figs. 2 and 3). The

presence of H. truncatum-type ITS within individual H.marginatum rufipes could be accounted for in two ways,

(a) retention of ancestral ITS within H. marginatum

rufipes or (b) introgression of H. truncatum nuclear

genes into the H. marginatum rufipes genome as a result

of hybridization. Either of these is possible but the latter

is perhaps more compelling, given the other indications

of hybridization among these Hyalomma species.

4.3. Natural hybridization in Hyalomma

Because of the absence of individuals of intermediate

morphology, hybridization among the Hyalomma spe-

cies in this study was not initially suspected, however,

the genetic data suggest that natural hybridization has

indeed occurred. Potential for hybridization among

species of Hyalomma has been noted by previous

workers (e.g., Matthysse and Colbo, 1987), and experi-

mental interspecific crosses occasionally produce juve-

nile or adult offspring in Hyalomma (Cwilich andHadani, 1963; Pervomaisky, 1954) and in the closely

related genus Rhipicephalus (Pegram et al., 1987; Zi-

vkovic et al., 1986). Significantly, in two such studies

(Pervomaisky, 1954; Zivkovic et al., 1986), F1 progeny

usually inherited the morphology of one of the parental

species (rather than showing intermediate morphology).

Fig. 5 illustrates two possible pathways to hybrid

individuals that account for both the observed mor-phology and genetic data in this study. We assume that

the majority-type ITS-2 sequence observed in H. trunc-

atum and H. marginatum rufipes indicates gene flow

between these species and does not merely represent the

retention of ancestral polymorphism. We attempt to

account for the presence of the following classes of in-

dividuals revealed by this study: (a) H. truncatum mor-

phology, H. marginatum rufipes mtDNA, ITS-2 fromboth species, (b) H. dromedarii morphology, H. mar-

ginatum rufipes mtDNA, ITS-2 from H. dromedarii, H.

marginatum rufipes, and H. truncatum, (c) H. margina-

tum rufipes morphology, H. marginatum rufipes mtDNA

and ITS-2 from both H. marginatum rufipes and H.

truncatum. The mtDNA (matrilineal) phylogeny indi-

cates the involvement of males of H. dromedarii and

Fig. 4. (continued)

D.J. Rees et al. / Molecular Phylogenetics and Evolution 27 (2003) 131–142 139

Fig. 5. Simplest routes to hybrid individuals observed in this study. The three types of hybrid individuals suggested by mtDNA and ITS-2 sequence data are accounted by direct inheritance of

paternal morphology in hybrid offspring (1) and by hybridization followed by backcrossing (2).

140

D.J.Rees

etal./Molecu

larPhylogenetics

andEvolution27(2003)131–142

H. truncatum and female H. marginatum rufipes. In thecase of direct inheritance of paternal morphology (Fig.

5.1), the classes of individuals (a)–(c) described above

can therefore be accounted for by the following se-

quence of events: an initial hybridization event between

male H. truncatum and female H. marginatum rufipes

would result in male hybrid offspring with H. truncatum

morphology, H. marginatum rufipes mtDNA, and ITS-2

characteristic of both species (a). Secondary hybridiza-tion events between female progeny from this initial

event and males of either H. dromedarii or H. margin-

atum rufipes would result in individuals with (b)

H. dromedarii morphology, H. marginatum rufipes

mtDNA, and ITS-2 from H. dromedarii, H. margina-

tumrufipes, and H. truncatum, and (c) H. marginatum

rufipes morphology, H. marginatum rufipes mtDNA and

ITS-2 from both H. marginatum rufipes and H. trunca-tum. An alternative pathway to the observed individu-

als, involving backcrossing, is presented in Fig. 5.2. In

this scenario, an initial hybridization event between

male H. truncatum and female H. marginatum rufipes is

followed by repeated backcrossing of female progeny to

males of each of the three species, for as many genera-

tions as necessary to re-establish �typical� morphology.Problems exist with both of these scenarios, and at thepresent time we are unable to determine which process,

if either, has led to the existence of the individuals we

have observed in this study. Further field and laboratory

studies are required to determine the extent and fre-

quency of hybridization in Hyalomma as well as the

mechanisms leading to maintenance or re-establishment

of typical morphology.

4.4. Concluding remarks

If intermediate individuals are observed in the field,

then the possibility of interspecific hybridization is evi-

dent. However, the indications from experimental

crosses (Pervomaisky, 1954; Zivkovic et al., 1986) and

our study involving a natural population are that, in

some cases, morphology alone may not indicate thehybrid origin of individuals. In the case of Hyalomma,

hybridization was not suspected among the three species

and hybrid individuals were only identified by molecular

analysis involving multiple, independent markers. It is

disconcerting to consider that the pattern evident in

these Hyalomma may be repeated in other species and

genera. Inferences from gene trees concerning relation-

ships among populations or species may be incorrect ifthe gene tree does not accurately reflect organismal

phylogeny. The effects of introgression on levels of ge-

netic variability within individuals and populations, and

among populations and species, may be extremely dif-

ficult to assess, and may go unnoticed if a study involves

only one species among several which are or have been

hybridizing. Concerning the importance of identifying

introgression, as Moore (1995) reminds us, ‘‘if hybrid-ization is apparent, its effects can be taken into account

and its occurrence incorporated into the historical nar-

rative of the group—as it should be.’’ As a cautionary

measure, particularly in cases where hybridization is a

possibility, we emphasize that molecular phylogenetic

studies should not rely on sequence data from a single

individual as being representative of a species. Addi-

tionally, in analyses of nuclear genetic diversity amongpopulations or species, it is wise to make an initial as-

sessment of the level of intra-individual variability in

several individuals. Clearly, different interpretations

would have resulted from assessment of either �normal�(0.3% ITS-2 variability) or �putative hybrid� H. drome-darii (6% ITS-2 variability), although both individuals

would be treated the same on the basis of morphology.

A number of questions are raised by our findings. AreHyalomma hybrids fertile? If so, what are the fitness

consequences of hybridization? By what mechanism is

species-specific morphology maintained? Does hybrid-

ization affect the role of these species as disease vectors?

Are other species of Hyalomma involved? How wide-

spread and how frequent is hybridization in Hyalomma

and does it occur in other genera? Is hybridization

linked to host use? These and further challenges await.

Acknowledgments

We express our thanks to Dr. M.T. Fox, Department

of Pathology and Infectious Diseases, Royal Veterinary

College, London and Paul Hylliard and Janet Beccaloni

of the Natural History Museum, London, for advice on

Hyalomma and loan of type specimens. Thanks go to

Prof. J.L. Camicas, Laboratoire d�Epidemiologie desMaladies a Vecteurs, Montpellier, France for confir-

mation identification of samples. We thank Dr. SileshiMekonnen, Dr. Ibrahim Hussein, Dr. Nigist Mekonnen,

and Mr. Abebe Mekonnen of the Acarology and En-

tomology Team at the Sabeta National Animal Health

Center, Ethiopia, for help and advice on collecting. We

also thank Eigil Erichsen, Laboratory for Electron Mi-

croscopy, University of Bergen for invaluable assistance

with the scanning microscope. Helpful comments on the

manuscript were provided by Brent Emerson and EndreWillassen, Rob DeSalle, and an anonymous referee.

Funding was provided by the Norwegian Research

Council (NFR), Project No. 128388/420, ‘‘Applications

of Molecular Techniques in Systematic Biology.’’

References

Anderson, E., Stebbins Jr., G.L., 1954. Hybridization as an evolu-

tionary stimulus. Evolution 8, 378–388.

Avise, J.C., 2000. Phylogeography: The History and Formation of

Species. Harvard University Press, Cambridge, MA.

D.J. Rees et al. / Molecular Phylogenetics and Evolution 27 (2003) 131–142 141

Baker, M.K., Ducasse, F.B.W., Sutherst, R.W., Maywald, G.F., 1989.

The seasonal tick populations on traditional and commercial cattle

grazed at four altitudes in Natal. J. S. Afr. Vet. Assoc. 60, 95–101.

Barton, N.H., 2001. The role of hybridization in evolution. Mol. Ecol.

10, 551–568.

Bensasson, D., Zhang, D.-X., Hartl, D.L., Hewitt, G.M., 2001.

Mitochondrial pseudogenes: evolution�s misplaced witnesses.

Trends Ecol. Evol. 16, 314–321.

Camicas, J.-L., Hervy, J.-P., Adam, F., Morel, P.C., 1998. The Ticks

of the World (Acarida, Ixodida). �EEditions de l�Orstom, Paris.Clement, M.D., Posada, D., Crandall, K.A., 2000. TCS: a computer

program to estimate gene genealogies. Mol. Ecol. 9, 1657–1660.

Corpet, F., 1988. Multiple sequence alignment with hierarchical

clustering. Nucleic Acids Res. 16, 10881–10890.

Cumming, G.S., 1998. Host preference in African ticks (Acari:

Ixodida): a quantitative data set. Bull. Ent. Res. 88, 379–406.

Cwilich, R., Hadani, A., 1963. Inter-specific hybridization of ticks of

the genus Hyalomma. Acta Trop. 20, 178–180.

De Kok, J.B., d�Oliveira, C., Jongejan, F., 1993. Detection of theprotozooan parasite Theileria annulata in Hyalomma ticks by the

polymerase chain reaction. Exp. Appl. Acarol. 7, 846–849.

Dioli, M., 1992. A study of tick infestation in four nomadic camel

herds in Northern Kenya. In: Schwartz, H.J., Dioli, M. (Eds.), The

One-humped Camel (C. dromedarius) in Eastern Africa: A Pictorial

Guide to Disease, Health Care, and Management. Margraf,

Weikersheim, pp. 263–267.

Dolan, R., Wilson, A.J., Schwartz, H.J., Nelson, R.M., Field, C.R.,

1983. Camel production in Kenya and its constraints. II. Tick

infestation. Trop. Anim. Health Prod. 15, 179–185.

Dowling, T.E., Secor, C.E., 1997. The role of hybridization and

introgression in the diversification of animals. Ann. Rev. Ecol.

Syst. 28, 593–619.

Doyle, J.J., 1997. Trees within trees: genes and species, molecules and

morphology. Syst. Biol. 46, 537–553.

Hoogstraal, H., 1956. ‘‘African Ixodoidea. I. Ticks of the Sudan’’ Bur.

Med. Surg., Dept. Navy. US Gov. Print. Off. (USGPO), Wash-

ington.

Hoogstraal, H., 1978. Biology of ticks. In: Wilde, J.K.H. (Ed.),

Tickborne Diseases and their Vectors. Centre for Tropical Veter-

inary Medicine, Edinburgh, pp. 3–14.

Hoogstraal, H., Aeschlimann, A., 1982. Tick-host specificity. Bull.

Soc. Ent. Suisse 55, 5–32.

Juan, C., Ibrahim, K.M., Orom�ıı, P., Hewitt, G.M., 1996. Mitochon-

drial DNA sequence variation and phylogeography of Pimelia

darkling beetles on the island of Tenerife (Canary Islands).

Heredity 77, 589–598.

McLain, D.K., Wesson, D.M., Oliver Jr., J.H., Collins, F.H., 1995.

Variation in ribosomal DNA internal transcribed spacers 1 among

eastern populations of Ixodes scapularis (Acari: Ixodidae). J. Med.

Entomol. 32, 353–360.

Matthysse, J.G., Colbo, M.H., 1987. The Ixodid Ticks of Uganda.

Entomological Society of America, Maryland.

Mohammed, A.N., 1977. The seasonal incidence of ixodid ticks of cattle

in northern Nigeria. Bull. Anim. Health Prod. Afr. 25, 273–293.

Moore, W.S., 1995. Inferring phylogenies from mtDNA variation:

mitochondrial-gene trees versus nuclear-gene trees. Evolution 49,

718–726.

Murrell, A., Campbell, N.J.H., Barker, S.C., 2000. Phylogenetic

analyses of the Rhipicephaline ticks indicate that the genus is

paraphyletic. Mol. Phylogenet. Evol. 16, 1–7.

Onyabe, D.Y., Conn, J.E., 1999. Intragenomic heterogeneity of a

ribosomal DNA spacer (ITS2) varies regionally in the neotropical

malaria vector Anopheles nuneztovari (Diptera: Culicidae). Insect

Mol. Biol. 8, 435–442.

Page, R.D.M., Charleston, M.A., 1997. From gene tree to organismal

phylogeny: reconciled trees and the gene tree/species tree problem.

Mol. Phylogenet. Evol. 7, 231–240.

Pamilo, P., Nei, M., 1988. Relationships between gene trees and

species trees. Mol. Biol. Evol. 5, 568–583.

Pegram, R.G., Higgins, A.J., 1992. Camel ectoparasites: a review. In:

Allen, W.R., Higgins, A.J., Mayhew, I.G., Snow, D.H., Wade, J.F.

(Eds.), Proc. 1st Int. Camel Conf., pp. 69–78.

Pegram, R.G., Hoogstraal, H., Wassef, H.Y., 1981. Ticks (Acari:

Ixodoidea) of Ethiopia. I. Distribution, ecology, and host

relationships of species infesting livestock. Bull. Ent. Res. 71,

339–359.

Pegram, R.G., Walker, J.B., Clifford, C.M., Keirans, J.E., 1987.

Comparison of populations of the Rhipicephalus simus group: R.

simus, R. praetextatus, and R. muhsamae (Acari: Ixodidae). J. Med.

Entomol. 24 (6), 665–682.

Pervomaisky, G.S., 1954. Variation in pasture ticks (Acarina, Ixodi-

dae) and its significance for systematics. Trud. Vsesoyuz. Ent.

Obshch. 44, 62–201.

Rees, D.J., Emerson, B.C., Orom�ii, P., Hewitt, G.M., 2001. Reconcil-

ing gene trees with organism history: the mtDNA phylogeography

of three Nesotes species (Coleoptera: Tenebrionidae) on the western

Canary Islands. J. Evol. Biol. 14, 139–147.

Rich, S.M., Rosenthal, B.M., Telford III, S.R., Spielman, A., Hartl,

D.L., Ayala, F.J., 1997. Heterogeneity of the internal transcribed

spacer (ITS-2) region within individual deer ticks. Insect Mol. Biol.

6, 123–129.

Rutagwenda, T., 1985. The control of important camel diseases in the

IPAL study area. In: IPAL Technical Report No. E-7. UNESCO,

Nairobi, Kenya, pp. 10–75.

Sperling, F.A.H., Raske, A.G., Otvos, I.S., 1999. Mitochondrial DNA

sequence variation among populations and host races of Lambdina

fiscellaria (Gn.) (Lepidoptera: Geometridae). Insect Mol. Biol. 8,

97–106.

Straten, M. van, Jongejan, F., 1993. Ticks (Acari: Ixodidae) infesting

the Arabian Camel (Camelus dromedarius) in the Sinai, Egypt with

a note on the acaricidal efficacy of Ivermectin. Exp. Appl. Acarol.

17, 605–616.

Swofford, D.L., 1998. PAUP*. Phylogenetic Analysis Using Parsi-

mony (* and Other Methods), Version 4. Sinauer Associates,

Sunderland, MA.

Templeton, A.R., Crandall, K.A., Singh, C.F., 1992. A cladistic

analysis of phenotypic associations with haplotypes inferred from

restriction endonuclease mapping and DNA sequence data. III.

Cladogram estimation. Genetics 132, 619–633.

Vogler, A.P., DeSalle, R., 1993. Mitochondrial DNA evolution and

the application of the phylogenetic species concept in the Cicindella

dorsalis complex (Coleoptera: Cicindelidae). In: Desender, K.

(Ed.), Carabid Beetles: Ecology and Evolution. Kluwer Academic

Publishers, Dordrecht, The Netherlands, pp. 79–85.

Wesson, D.M., McLain, D.K., Oliver, J.H., Piesman, J., Collins, F.H.,

1993. Investigation of the validity of species status of Ixodes

dammini (Acari: Ixodidae) using rDNA. Proc. Natl. Acad. Sci.

USA 90, 10221–10225.

Wesson, D.M., Porter, C.H., Collins, F.H., 1992. Sequence and

secondary structure comparisons of ITS rDNA in mosquitoes

(Diptera: Culicidae). Mol. Phylogenet. Evol. 1, 253–269.

Zahler, M., Fillipova, N.A., Morel, P.C., Gothe, R., Rinder, H., 1997.

Relationships between species of the Rhipicephalus sanguineus

group: a molecular approach. J. Parasitol. 83, 302–306.

Zivkovic, D., Pegram, R.G., Jongejan, F., Mwase, E.T., 1986. Biology

of Rhipicephalus appendiculatus and R. zambeziensis and produc-

tion of a fertile hybrid under laboratory condtions. Exp. Appl.

Acarol. 2, 285–298.

142 D.J. Rees et al. / Molecular Phylogenetics and Evolution 27 (2003) 131–142