YMPEV 4854
No. of Pages 13, Model 5G
2 April 2014 Molecular Phylogenetics and Evolution xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev 6 7
Phylogenetic analysis of mitochondrial genome sequences indicates that the cattle tick, Rhipicephalus (Boophilus) microplus, contains a cryptic species
3 4 5 8
Q1
a
9 10
b
12 11 13
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia GeneCology Research Centre, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore DC, QLD 4556, Australia
a r t i c l e
1 2 5 8 16 17 18 19 20 21 22 23 24 25 26 27
Thomas D. Burger a, Renfu Shao b,⇑, Stephen C. Barker a,⇑
i n f o
Article history: Received 29 July 2013 Revised 11 March 2014 Accepted 17 March 2014 Available online xxxx
Q2
Keywords: Cattle ticks Rhipicephalus (Boophilus) microplus Cryptic species Mitochondrial genomes Phylogenetics
a b s t r a c t Cattle ticks of the subgenus Rhipicephalus (Boophilus) are major agricultural pests worldwide, causing billions of dollars in losses annually. Rhipicephalus (Boophilus) annulatus and R. microplus are the most wellknown and widespread species, and a third species, R. australis, was recently reinstated for ‘R. microplus’ from Australia and parts of Southeast Asia. We use mitochondrial genome sequences to address the phylogenetic relationships among the species of the subgenus Boophilus. We sequenced the complete or partial mitochondrial genomes of R. annulatus, R. australis, R. kohlsi, R. geigyi, and of three geographically disparate specimens of R. microplus from Brazil, Cambodia and China. Phylogenetic analyses of mitochondrial genomes, as well as cox1 and 16S rRNA sequences, reveals a species complex of R. annulatus, R. australis, and two clades of R. microplus, which we call the R. microplus complex. We show that cattle ticks morphologically identified as R. microplus from Southern China and Northern India (R. microplus clade B) are more closely related to R. annulatus than other specimens of R. microplus s.s. from Asia, South America and Africa (R. microplus clade A). Our analysis suggests that ticks reported as R. microplus from Southern China and Northern India are a cryptic species. This highlights the need for further molecular, morphological and crossbreeding studies of the R. microplus complex, with emphasis on specimens from China and India. We found that cox1 and, to a lesser extent, 16S rRNA were far more successful in resolving the phylogenetic relationships within the R. microplus complex than 12S rRNA or the nuclear marker ITS2. We suggest that future molecular studies of the R. microplus complex should focus on cox1, supplemented by 16S rRNA, and develop nuclear markers alternative to ITS2 to complement the mitochondrial data. Ó 2014 Elsevier Inc. All rights reserved.
29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
51 52 53 54 55 56 57 58 59 60 61 62 63
1. Introduction Cattle ticks of the subgenus Rhipicephalus (Boophilus) are important, globally distributed, parasites of livestock. Tick infestations, in addition to transmitting disease-causing organisms such as Babesia and Anaplasma, have a direct impact on cattle production by causing weight and blood loss, as well as damage to hides (de Castro, 1997). Hence, considerable resources are required to treat tick infestations and control the spread of ticks; the cost of ticks and tick-borne diseases in the cattle industry globally has been estimated to be between USD 13.9–18.7 billion annually (de Castro, 1997). Rhipicephalus (Boophilus) species were formerly in the genus Boophilus Curtice, 1891, due to several distinctive morphological
⇑ Corresponding authors. E-mail addresses: [email protected] (R. Shao), [email protected] (S.C. Barker).
features which distinguish them from the genus Rhipicephalus Koch, 1844 (e.g. small in size, compact mouthparts, small eyes, lacking festoons). However, modern molecular analysis, as well as combined molecular and morphological analysis, revealed that the genus Rhipicephalus was paraphyletic with respect to the genus Boophilus (Murrell et al., 2000, 2001; Beati and Keirans, 2001). Hence, the Boophilus species were transferred to the genus Rhipicephalus, though Boophilus was retained as a subgenus ‘‘in order to preserve the name Boophilus’’ (Murrell et al., 2003, p. 170). There are currently six valid species in the subgenus Rhipicephalus (Boophilus). Rhipicephalus (Boophilus) annulatus (Say, 1821) and Rhipicephalus (Boophilus) microplus (Canestrini, 1888) are the most widely distributed species in the subgenus Boophilus, and have long been thought to be sister species on the basis of morphology (Feldman-Muhsam and Shechter, 1970; Graham et al., 1972) and molecular markers (Murrell et al., 2000). Rhipicephalus annulatus is found across Southern Europe, Western and Central Asia,
http://dx.doi.org/10.1016/j.ympev.2014.03.017 1055-7903/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: Burger, T.D., et al. Phylogenetic analysis of mitochondrial genome sequences indicates that the cattle tick, Rhipicephalus (Boophilus) microplus, contains a cryptic species. Mol. Phylogenet. Evol. (2014), http://dx.doi.org/10.1016/j.ympev.2014.03.017
64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80
YMPEV 4854
No. of Pages 13, Model 5G
2 April 2014 2 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146
T.D. Burger et al. / Molecular Phylogenetics and Evolution xxx (2014) xxx–xxx
Northern and tropical sub-Saharan Africa (Fig. S1). After a long and costly eradication campaign in the first half of the 20th century, the North American distribution of R. annulatus is now limited to Mexico and the border regions of Texas. Rhipicephalus microplus is found in South, East and Southeast Asia, Central and South America (including Mexico), and Eastern and Southern Africa (Fig. S1; Kolonin, 2009). Recently, cattle ticks from Australia previously known as R. microplus were reinstated as a separate species, Rhipicephalus (Boophilus) australis (Fuller, 1899), and this species was found to be present in Australia, the Philippines, Cambodia, Tahiti, New Caledonia, and the islands of Borneo, Sumatra, Java, and New Guinea (Estrada-Peña et al., 2012). The distributions of two further Rhipicephalus (Boophilus) species are limited to Africa (Fig. S2): Rhipicephalus (Boophilus) decoloratus (Koch, 1844) is found across sub-Saharan Africa, whereas Rhipicephalus (Boophilus) geigyi (Aeschlimann and Morel, 1965) is limited to the tropical region of sub-Saharan Africa (Estrada-Peña et al., 2006). Rhipicephalus (Boophilus) kohlsi (Hoogstraal and Kaiser, 1960) is found in the Middle East and Western Asia (Fig. S2); this species has a preference for sheep and goats rather than cattle. Indeed, we note that most species of the subgenus Boophilus have been reported from sheep, goats, deer, antelope, and horses as well as domestic cattle (Yeruham et al., 1999; Jubb and Campbell, 2002; Kolonin, 2009; de Meeûs et al., 2010; Pound et al., 2010; Madder et al., 2011). The geographic distributions of R. annulatus, R. australis and R. microplus (Fig. S1) have been greatly affected by the trade of live cattle. Though the spread of R. microplus is not well documented, it is thought to have originated in India (Hoogstraal, 1986) and then spread to Madagascar and Southern Africa. There is little evidence for how or when R. microplus spread to the Americas (Labruna et al., 2009), but it was likely on cattle from India or Africa in the 16th and 17th centuries (Barré and Uilenberg, 2010). Rhipicephalus microplus has also recently been reported as an introduced species in Western Africa, where it appears to be replacing the indigenous Rhipicephalus (Boophilus) species, including R. annulatus (Madder et al., 2007, 2011, 2012). Rhipicephalus annulatus (common in Southern Europe) was likely introduced into Mexico by Spanish colonists (Hoogstraal, 1986) and then spread into the United States, some time prior to the first recorded outbreak of cattle fever in 1796 (Angus, 1998). Rhipicephalus australis was apparently introduced into the Northern Territory of Australia on cattle or buffalo from Indonesia in 1829 (Angus, 1996). Cattle from Australia subsequently carried R. australis to New Caledonia in 1942 (De Meeûs et al., 2010). Mating experiments between R. microplus and R. annulatus have established that these two species are reproductively isolated (Graham et al., 1972; Thompson et al., 1981; Davey et al., 1983). A study of the distributions of R. microplus and R. annulatus along the Texas-Mexico border revealed a stable parapatric boundary between the two species, which also suggests the two species are reproductively isolated (Lohmeyer et al., 2011). Rhipicephalus microplus and R. australis have also been shown to be reproductively isolated; the mating of R. microplus from South Africa and R. australis from Australia produced infertile offspring (Spickett and Malan, 1978). More recently, geographically disparate R. microplus strains from Mozambique and Argentina were found to produce fertile crosses, but crosses between either strain and R. australis from Australia were infertile (Labruna et al., 2009). The phylogenetic relationships among Boophilus species are not well studied, and most studies investigating these relationships have relied on partial 12S rRNA and 16S rRNA sequences (Murrell et al., 2000, 2001; Beati and Keirans, 2001; Labruna et al., 2009; Estrada-Peña et al., 2012). However, molecular analysis (Beati and Keirans, 2001) has confirmed the sister species relationship between R. annulatus and R. microplus suggested by morphology (Feldman-Muhsam and Shechter, 1970). The total-evidence phylogeny of Murrell et al. (2001) strongly supported monophyly
of the Boophilus clade, within the genus Rhipicephalus, but could not resolve relationships among Boophilus species, however this study did not include R. microplus s.s.; the ‘R. microplus’ specimen used in the study was from Australia, and thus actually R. australis. Later studies with wide geographic sampling of R. microplus have also supported monophyly of the subgenus Rhipicephalus (Boophilus), and the reinstatement of R. australis (Labruna et al., 2009; Estrada-Peña et al., 2012). Intriguingly, two strains of R. microplus from India and Nepal were shown to be highly divergent from R. microplus from the Americas and Africa (Labruna et al., 2009). However, the phylogenetic placement of R. microplus from India and Nepal was not strongly resolved, though R. microplus from India clustered with R. annulatus in the 16S rRNA analysis (Labruna et al., 2009). Mitochondrial genomes are a rich source of data for investigating phylogenetic relationships (Shao and Barker, 2007), and we Q3 have demonstrated the use of mitochondrial genomes in resolving the phylogentic reltionships among tick lineages (Burger et al., 2012, 2013). To date, the mt genomes of only two rhipicephaline ticks have been sequenced: the entire mt genome of R. sanguineus (Black and Roehrdanz, 1998), and 13 gene boundary fragments of the mt genome of R. australis (Campbell and Barker, 1999). We present complete mitochondrial (mt) genome sequences from Rhipicephalus (Boophilus) australis, Rhipicephalus (Boophilus) geigyi, Dermacentor nitens, and three geographically disparate specimens of Rhipicephalus (Boophilus) microplus from Cambodia, China and Brazil, as well as partial (ca. 70%) mt genome sequences of Rhipicephalus (Boophilus) kohlsi, Rhipicephalus (Boophilus) annulatus, and Rhipicephalus appendiculatus. We use these mt genome sequences, as well as the cox1, 12S rRNA, 16S rRNA, and ITS2 markers, to address the phylogenetic relationships among the species of the subgenus Boophilus. We also compare four common molecular markers, cox1, 12S rRNA, 16S rRNA, and ITS2, to determine which of these markers have the most resolving power to determine relationships among the species of the subgenus Boophilus. We also sequenced partial 18S and 28S nuclear rRNA of R. appendiculatus, R. australis, R. geigyi, R. kohlsi, R. microplus, and D. nitens which we reported in a separate study (Burger et al., 2014); these sequences had very high pairwise identities (99–100%) and were not informative for the species-level analysis in this study.
147
2. Materials and methods
187
2.1. Specimens and DNA extraction
188
Specimens sequenced in the present study are listed in Table 1, along with accession numbers for sequences deposited in GenBank. Specimens of R. microplus and R. annulatus were identified according to Walker et al. (2003). Of the R. microplus SCB collected in China only three specimens were male. All three males had a caudal appendage and long spurs on coxa I, as per R. microplus; R. annulatus males lack a caudal appendage and have short spurs on coxa I (Walker et al., 2003). However, we noted that the male R. microplus from China had distinct (or sharp) adanal plate spurs. Rhipicephalus microplus and R. annulatus have indistinct (or blunt) adanal plates spurs (Walker et al., 2003), as does R. australis (Estrada-Peña et al., 2012), however this feature may be variable among populations (Estrada-Peña et al., 2012). The mouthparts of most of the female specimens of R. microplus from China were either damaged or obscured by host tissue. Thus, we relied on the coxal spurs for identification of females: distinct spurs on coxa I and spurs present on coxae II–III in R. microplus; indistinct spurs on coxa I and spurs absent on coxae II–III, in R. annulatus (Walker et al., 2003). DNA was extracted from tick specimens using the DNeasy Tissue Extraction Kit (QIAGEN). Individual ticks were cut in half
189
Please cite this article in press as: Burger, T.D., et al. Phylogenetic analysis of mitochondrial genome sequences indicates that the cattle tick, Rhipicephalus (Boophilus) microplus, contains a cryptic species. Mol. Phylogenet. Evol. (2014), http://dx.doi.org/10.1016/j.ympev.2014.03.017
148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186
190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208
YMPEV 4854
No. of Pages 13, Model 5G
2 April 2014 3
T.D. Burger et al. / Molecular Phylogenetics and Evolution xxx (2014) xxx–xxx Table 1 Collection data for the tick mitochondrial genomes sequenced in this study. Species
Collection location
Collector
GenBank ID mt genome
GenBank ID ITS2
Rhipicephalus (B.) microplus Rhipicephalus (B.) microplus Rhipicephalus (B.) microplus Rhipicephalus (B.) annulatus Rhipicephalus (B.) australis Rhipicephalus (B.) kohlsi Rhipicephalus (B.) geigyi Rhipicephalus appendiculatus Dermacentor nitens
Cambodia Guiyang, Guizhou, China Pantanal Research Station, MS, Brazil Tulcea County, Romania Bunya, QLD, Australia Salalah, Oman CIRDES colony strain, Burkina Faso Lake Chivero National Park, Zimbabwe Campo Grande, MS, Brazil
John Kopinski Stephen C Barker Stephen C Barker Lidia Chitimia Philip Vitale Stephen C Barker Hassan Adakel William Mazhowu Stephen C Barker
KC503260 KC503259 KC503261 KC503256 KC503255 KC503262 KC503263 KC503257 KC503258
KC503272 KC503274 KC503273 KC503267 KC503268 KC503271 KC503270 KC503269 KC503275
215 216 217
220 221
1
R. (B.) microplus Brazil 14,905 bp R. (B.) microplus Cambodia 14,903 bp R. (B.) microplus China 14,864 bp R. (B.) australis 14,891 bp R. (B.) geigyi 14,948 bp R. (B.) annulatus* 9,756 bp R. (B.) kohlsi* 9,678 bp R. appendiculatus* 9,569 bp Dermacentor nitens 14,839 bp
3
H
ATP8
K D
ATP6
222
P
2
219
Y
COX
218
L1
D6
NA
T
W
X1 CO
214
4L
2
X3
CO
Short (ca. 400–700 bp) regions of the cox1, cytb and 12S rRNA genes were first amplified and sequenced using universal-arthropod primers (Table S1; Simon et al., 1994; Kambhampati and Smith, 1995; Shao et al., 2005). Species-specific primers were then designed for each species from these three regions, and used in conjunction with universal-tick primers in the 12S rRNA and cytb genes, designed from all known tick 12S rRNA and cytb sequences (Table S1). Entire mitochondrial genomes were then amplified in three overlapping fragments, from cytb to cox1, cox1 to 12S rRNA, and 12S rRNA to cytb (Fig. 1). ITS2 regions were amplified using primers in the 5.8S and 28S rRNA genes, designed from a consensus of tick sequences available on GenBank, and sequenced with these PCR primers and species-specific internal sequencing primers (Table S1). Expand Long Range dNTPack kits (Roche) were used to amplify long mitochondrial PCR products (>1000 bp). TaKaRa Ex Taq DNA polymerase kits (Takara Biotechnology) were used to amplify short mitochondrial gene fragments (
No. of Pages 13, Model 5G
2 April 2014 Molecular Phylogenetics and Evolution xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev 6 7
Phylogenetic analysis of mitochondrial genome sequences indicates that the cattle tick, Rhipicephalus (Boophilus) microplus, contains a cryptic species
3 4 5 8
Q1
a
9 10
b
12 11 13
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia GeneCology Research Centre, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore DC, QLD 4556, Australia
a r t i c l e
1 2 5 8 16 17 18 19 20 21 22 23 24 25 26 27
Thomas D. Burger a, Renfu Shao b,⇑, Stephen C. Barker a,⇑
i n f o
Article history: Received 29 July 2013 Revised 11 March 2014 Accepted 17 March 2014 Available online xxxx
Q2
Keywords: Cattle ticks Rhipicephalus (Boophilus) microplus Cryptic species Mitochondrial genomes Phylogenetics
a b s t r a c t Cattle ticks of the subgenus Rhipicephalus (Boophilus) are major agricultural pests worldwide, causing billions of dollars in losses annually. Rhipicephalus (Boophilus) annulatus and R. microplus are the most wellknown and widespread species, and a third species, R. australis, was recently reinstated for ‘R. microplus’ from Australia and parts of Southeast Asia. We use mitochondrial genome sequences to address the phylogenetic relationships among the species of the subgenus Boophilus. We sequenced the complete or partial mitochondrial genomes of R. annulatus, R. australis, R. kohlsi, R. geigyi, and of three geographically disparate specimens of R. microplus from Brazil, Cambodia and China. Phylogenetic analyses of mitochondrial genomes, as well as cox1 and 16S rRNA sequences, reveals a species complex of R. annulatus, R. australis, and two clades of R. microplus, which we call the R. microplus complex. We show that cattle ticks morphologically identified as R. microplus from Southern China and Northern India (R. microplus clade B) are more closely related to R. annulatus than other specimens of R. microplus s.s. from Asia, South America and Africa (R. microplus clade A). Our analysis suggests that ticks reported as R. microplus from Southern China and Northern India are a cryptic species. This highlights the need for further molecular, morphological and crossbreeding studies of the R. microplus complex, with emphasis on specimens from China and India. We found that cox1 and, to a lesser extent, 16S rRNA were far more successful in resolving the phylogenetic relationships within the R. microplus complex than 12S rRNA or the nuclear marker ITS2. We suggest that future molecular studies of the R. microplus complex should focus on cox1, supplemented by 16S rRNA, and develop nuclear markers alternative to ITS2 to complement the mitochondrial data. Ó 2014 Elsevier Inc. All rights reserved.
29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
51 52 53 54 55 56 57 58 59 60 61 62 63
1. Introduction Cattle ticks of the subgenus Rhipicephalus (Boophilus) are important, globally distributed, parasites of livestock. Tick infestations, in addition to transmitting disease-causing organisms such as Babesia and Anaplasma, have a direct impact on cattle production by causing weight and blood loss, as well as damage to hides (de Castro, 1997). Hence, considerable resources are required to treat tick infestations and control the spread of ticks; the cost of ticks and tick-borne diseases in the cattle industry globally has been estimated to be between USD 13.9–18.7 billion annually (de Castro, 1997). Rhipicephalus (Boophilus) species were formerly in the genus Boophilus Curtice, 1891, due to several distinctive morphological
⇑ Corresponding authors. E-mail addresses: [email protected] (R. Shao), [email protected] (S.C. Barker).
features which distinguish them from the genus Rhipicephalus Koch, 1844 (e.g. small in size, compact mouthparts, small eyes, lacking festoons). However, modern molecular analysis, as well as combined molecular and morphological analysis, revealed that the genus Rhipicephalus was paraphyletic with respect to the genus Boophilus (Murrell et al., 2000, 2001; Beati and Keirans, 2001). Hence, the Boophilus species were transferred to the genus Rhipicephalus, though Boophilus was retained as a subgenus ‘‘in order to preserve the name Boophilus’’ (Murrell et al., 2003, p. 170). There are currently six valid species in the subgenus Rhipicephalus (Boophilus). Rhipicephalus (Boophilus) annulatus (Say, 1821) and Rhipicephalus (Boophilus) microplus (Canestrini, 1888) are the most widely distributed species in the subgenus Boophilus, and have long been thought to be sister species on the basis of morphology (Feldman-Muhsam and Shechter, 1970; Graham et al., 1972) and molecular markers (Murrell et al., 2000). Rhipicephalus annulatus is found across Southern Europe, Western and Central Asia,
http://dx.doi.org/10.1016/j.ympev.2014.03.017 1055-7903/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: Burger, T.D., et al. Phylogenetic analysis of mitochondrial genome sequences indicates that the cattle tick, Rhipicephalus (Boophilus) microplus, contains a cryptic species. Mol. Phylogenet. Evol. (2014), http://dx.doi.org/10.1016/j.ympev.2014.03.017
64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80
YMPEV 4854
No. of Pages 13, Model 5G
2 April 2014 2 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146
T.D. Burger et al. / Molecular Phylogenetics and Evolution xxx (2014) xxx–xxx
Northern and tropical sub-Saharan Africa (Fig. S1). After a long and costly eradication campaign in the first half of the 20th century, the North American distribution of R. annulatus is now limited to Mexico and the border regions of Texas. Rhipicephalus microplus is found in South, East and Southeast Asia, Central and South America (including Mexico), and Eastern and Southern Africa (Fig. S1; Kolonin, 2009). Recently, cattle ticks from Australia previously known as R. microplus were reinstated as a separate species, Rhipicephalus (Boophilus) australis (Fuller, 1899), and this species was found to be present in Australia, the Philippines, Cambodia, Tahiti, New Caledonia, and the islands of Borneo, Sumatra, Java, and New Guinea (Estrada-Peña et al., 2012). The distributions of two further Rhipicephalus (Boophilus) species are limited to Africa (Fig. S2): Rhipicephalus (Boophilus) decoloratus (Koch, 1844) is found across sub-Saharan Africa, whereas Rhipicephalus (Boophilus) geigyi (Aeschlimann and Morel, 1965) is limited to the tropical region of sub-Saharan Africa (Estrada-Peña et al., 2006). Rhipicephalus (Boophilus) kohlsi (Hoogstraal and Kaiser, 1960) is found in the Middle East and Western Asia (Fig. S2); this species has a preference for sheep and goats rather than cattle. Indeed, we note that most species of the subgenus Boophilus have been reported from sheep, goats, deer, antelope, and horses as well as domestic cattle (Yeruham et al., 1999; Jubb and Campbell, 2002; Kolonin, 2009; de Meeûs et al., 2010; Pound et al., 2010; Madder et al., 2011). The geographic distributions of R. annulatus, R. australis and R. microplus (Fig. S1) have been greatly affected by the trade of live cattle. Though the spread of R. microplus is not well documented, it is thought to have originated in India (Hoogstraal, 1986) and then spread to Madagascar and Southern Africa. There is little evidence for how or when R. microplus spread to the Americas (Labruna et al., 2009), but it was likely on cattle from India or Africa in the 16th and 17th centuries (Barré and Uilenberg, 2010). Rhipicephalus microplus has also recently been reported as an introduced species in Western Africa, where it appears to be replacing the indigenous Rhipicephalus (Boophilus) species, including R. annulatus (Madder et al., 2007, 2011, 2012). Rhipicephalus annulatus (common in Southern Europe) was likely introduced into Mexico by Spanish colonists (Hoogstraal, 1986) and then spread into the United States, some time prior to the first recorded outbreak of cattle fever in 1796 (Angus, 1998). Rhipicephalus australis was apparently introduced into the Northern Territory of Australia on cattle or buffalo from Indonesia in 1829 (Angus, 1996). Cattle from Australia subsequently carried R. australis to New Caledonia in 1942 (De Meeûs et al., 2010). Mating experiments between R. microplus and R. annulatus have established that these two species are reproductively isolated (Graham et al., 1972; Thompson et al., 1981; Davey et al., 1983). A study of the distributions of R. microplus and R. annulatus along the Texas-Mexico border revealed a stable parapatric boundary between the two species, which also suggests the two species are reproductively isolated (Lohmeyer et al., 2011). Rhipicephalus microplus and R. australis have also been shown to be reproductively isolated; the mating of R. microplus from South Africa and R. australis from Australia produced infertile offspring (Spickett and Malan, 1978). More recently, geographically disparate R. microplus strains from Mozambique and Argentina were found to produce fertile crosses, but crosses between either strain and R. australis from Australia were infertile (Labruna et al., 2009). The phylogenetic relationships among Boophilus species are not well studied, and most studies investigating these relationships have relied on partial 12S rRNA and 16S rRNA sequences (Murrell et al., 2000, 2001; Beati and Keirans, 2001; Labruna et al., 2009; Estrada-Peña et al., 2012). However, molecular analysis (Beati and Keirans, 2001) has confirmed the sister species relationship between R. annulatus and R. microplus suggested by morphology (Feldman-Muhsam and Shechter, 1970). The total-evidence phylogeny of Murrell et al. (2001) strongly supported monophyly
of the Boophilus clade, within the genus Rhipicephalus, but could not resolve relationships among Boophilus species, however this study did not include R. microplus s.s.; the ‘R. microplus’ specimen used in the study was from Australia, and thus actually R. australis. Later studies with wide geographic sampling of R. microplus have also supported monophyly of the subgenus Rhipicephalus (Boophilus), and the reinstatement of R. australis (Labruna et al., 2009; Estrada-Peña et al., 2012). Intriguingly, two strains of R. microplus from India and Nepal were shown to be highly divergent from R. microplus from the Americas and Africa (Labruna et al., 2009). However, the phylogenetic placement of R. microplus from India and Nepal was not strongly resolved, though R. microplus from India clustered with R. annulatus in the 16S rRNA analysis (Labruna et al., 2009). Mitochondrial genomes are a rich source of data for investigating phylogenetic relationships (Shao and Barker, 2007), and we Q3 have demonstrated the use of mitochondrial genomes in resolving the phylogentic reltionships among tick lineages (Burger et al., 2012, 2013). To date, the mt genomes of only two rhipicephaline ticks have been sequenced: the entire mt genome of R. sanguineus (Black and Roehrdanz, 1998), and 13 gene boundary fragments of the mt genome of R. australis (Campbell and Barker, 1999). We present complete mitochondrial (mt) genome sequences from Rhipicephalus (Boophilus) australis, Rhipicephalus (Boophilus) geigyi, Dermacentor nitens, and three geographically disparate specimens of Rhipicephalus (Boophilus) microplus from Cambodia, China and Brazil, as well as partial (ca. 70%) mt genome sequences of Rhipicephalus (Boophilus) kohlsi, Rhipicephalus (Boophilus) annulatus, and Rhipicephalus appendiculatus. We use these mt genome sequences, as well as the cox1, 12S rRNA, 16S rRNA, and ITS2 markers, to address the phylogenetic relationships among the species of the subgenus Boophilus. We also compare four common molecular markers, cox1, 12S rRNA, 16S rRNA, and ITS2, to determine which of these markers have the most resolving power to determine relationships among the species of the subgenus Boophilus. We also sequenced partial 18S and 28S nuclear rRNA of R. appendiculatus, R. australis, R. geigyi, R. kohlsi, R. microplus, and D. nitens which we reported in a separate study (Burger et al., 2014); these sequences had very high pairwise identities (99–100%) and were not informative for the species-level analysis in this study.
147
2. Materials and methods
187
2.1. Specimens and DNA extraction
188
Specimens sequenced in the present study are listed in Table 1, along with accession numbers for sequences deposited in GenBank. Specimens of R. microplus and R. annulatus were identified according to Walker et al. (2003). Of the R. microplus SCB collected in China only three specimens were male. All three males had a caudal appendage and long spurs on coxa I, as per R. microplus; R. annulatus males lack a caudal appendage and have short spurs on coxa I (Walker et al., 2003). However, we noted that the male R. microplus from China had distinct (or sharp) adanal plate spurs. Rhipicephalus microplus and R. annulatus have indistinct (or blunt) adanal plates spurs (Walker et al., 2003), as does R. australis (Estrada-Peña et al., 2012), however this feature may be variable among populations (Estrada-Peña et al., 2012). The mouthparts of most of the female specimens of R. microplus from China were either damaged or obscured by host tissue. Thus, we relied on the coxal spurs for identification of females: distinct spurs on coxa I and spurs present on coxae II–III in R. microplus; indistinct spurs on coxa I and spurs absent on coxae II–III, in R. annulatus (Walker et al., 2003). DNA was extracted from tick specimens using the DNeasy Tissue Extraction Kit (QIAGEN). Individual ticks were cut in half
189
Please cite this article in press as: Burger, T.D., et al. Phylogenetic analysis of mitochondrial genome sequences indicates that the cattle tick, Rhipicephalus (Boophilus) microplus, contains a cryptic species. Mol. Phylogenet. Evol. (2014), http://dx.doi.org/10.1016/j.ympev.2014.03.017
148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186
190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208
YMPEV 4854
No. of Pages 13, Model 5G
2 April 2014 3
T.D. Burger et al. / Molecular Phylogenetics and Evolution xxx (2014) xxx–xxx Table 1 Collection data for the tick mitochondrial genomes sequenced in this study. Species
Collection location
Collector
GenBank ID mt genome
GenBank ID ITS2
Rhipicephalus (B.) microplus Rhipicephalus (B.) microplus Rhipicephalus (B.) microplus Rhipicephalus (B.) annulatus Rhipicephalus (B.) australis Rhipicephalus (B.) kohlsi Rhipicephalus (B.) geigyi Rhipicephalus appendiculatus Dermacentor nitens
Cambodia Guiyang, Guizhou, China Pantanal Research Station, MS, Brazil Tulcea County, Romania Bunya, QLD, Australia Salalah, Oman CIRDES colony strain, Burkina Faso Lake Chivero National Park, Zimbabwe Campo Grande, MS, Brazil
John Kopinski Stephen C Barker Stephen C Barker Lidia Chitimia Philip Vitale Stephen C Barker Hassan Adakel William Mazhowu Stephen C Barker
KC503260 KC503259 KC503261 KC503256 KC503255 KC503262 KC503263 KC503257 KC503258
KC503272 KC503274 KC503273 KC503267 KC503268 KC503271 KC503270 KC503269 KC503275
215 216 217
220 221
1
R. (B.) microplus Brazil 14,905 bp R. (B.) microplus Cambodia 14,903 bp R. (B.) microplus China 14,864 bp R. (B.) australis 14,891 bp R. (B.) geigyi 14,948 bp R. (B.) annulatus* 9,756 bp R. (B.) kohlsi* 9,678 bp R. appendiculatus* 9,569 bp Dermacentor nitens 14,839 bp
3
H
ATP8
K D
ATP6
222
P
2
219
Y
COX
218
L1
D6
NA
T
W
X1 CO
214
4L
2
X3
CO
Short (ca. 400–700 bp) regions of the cox1, cytb and 12S rRNA genes were first amplified and sequenced using universal-arthropod primers (Table S1; Simon et al., 1994; Kambhampati and Smith, 1995; Shao et al., 2005). Species-specific primers were then designed for each species from these three regions, and used in conjunction with universal-tick primers in the 12S rRNA and cytb genes, designed from all known tick 12S rRNA and cytb sequences (Table S1). Entire mitochondrial genomes were then amplified in three overlapping fragments, from cytb to cox1, cox1 to 12S rRNA, and 12S rRNA to cytb (Fig. 1). ITS2 regions were amplified using primers in the 5.8S and 28S rRNA genes, designed from a consensus of tick sequences available on GenBank, and sequenced with these PCR primers and species-specific internal sequencing primers (Table S1). Expand Long Range dNTPack kits (Roche) were used to amplify long mitochondrial PCR products (>1000 bp). TaKaRa Ex Taq DNA polymerase kits (Takara Biotechnology) were used to amplify short mitochondrial gene fragments (
