Phylogenetic analysis of the mitochondrial genomes and nuclear rRNA genes of ticks reveals a deep phylogenetic structure within the genus Haemaphysalis and further elucidates the polyphyly of the genus Amblyomma with respect to Amblyomma sphenodonti and Amblyomma elaphense

Ticks and Tick-borne Diseases 4 (2013) 265–274

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Original article

Phylogenetic analysis of the mitochondrial genomes and nuclear rRNA genes of ticks reveals a deep phylogenetic structure within the genus Haemaphysalis and further elucidates the polyphyly of the genus Amblyomma with respect to Amblyomma sphenodonti and Amblyomma elaphense Thomas D. Burger a , Renfu Shao b , Stephen C. Barker a,∗ a b

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia GeneCology Research Group, School of Science, Education and Engineering, University of the Sunshine Coast, Maroochydore DC, QLD 4558, Australia

a r t i c l e

i n f o

Article history: Received 28 November 2012 Received in revised form 15 February 2013 Accepted 15 February 2013 Available online 18 April 2013 Keywords: Ixodida Phylogeny Mitochondrial genomes 18S rRNA 28S rRNA Haemaphysalis Amblyomma

a b s t r a c t We sequenced the entire mitochondrial genomes of 3 species of metastriate ticks: Haemaphysalis formosensis, H. parva, and Amblyomma cajennense. We also sequenced two thirds (ca. 9500 bp) of the mitochondrial genomes of H. humerosa and H. hystricis. We used these 5 mitochondrial genome sequences together with the 13 tick mitochondrial genomes we sequenced previously and the 2 tick mitochondrial genomes sequenced by Black and Roehrdanz (1998), as well as the nuclear rRNA genes from 84 ticks and mites, in phylogenetic analyses. Our analyses reveal deep phylogenetic structure within the genus Haemaphysalis, with at least 2 species, H. parva and H. inermis that are highly divergent from the rest of the genus Haemaphysalis. We identify a region of the 18S rRNA gene which correlates with this division of the genus Haemaphysalis as well as a novel insertion in the mitochondrial genome of H. parva. We reject the hypotheses of Hoogstraal and Aeschlimann (1982) and Barker and Murrell (2004) on the relationships among metastriate genera. Instead, our analysis provides further evidence for the division of the Metastriata into 2 major lineages: (i) Amblyomma s.s. plus Rhipicephalinae (i.e. Rhipicephalus, Hyalomma, Rhipicentor, and Dermacentor); and (ii) Haemaphysalis plus Bothriocroton plus Amblyomma sphenodonti. We also provide further evidence for the polyphyly of the genus Amblyomma with respect to A. sphenodonti and A. elaphense. The most likely position of A. elaphense is sister to the rest of the Metastriata; the most likely position of A. sphenodonti is sister to the genus Bothriocroton. These 2 species do not belong in the genus Amblyomma, and we propose that new genera are required for A. sphenodonti and A. elaphense. © 2013 Elsevier GmbH. All rights reserved.

Introduction Ticks (Chelicerata: Anactinotrichida: Ixodida) are blood-feeding ectoparasites of terrestrial vertebrates. Hard ticks (Ixodidae) comprise 702 of the 896 valid tick species (Guglielmone et al., 2010) and are organised into 2 groups: Prostriata, containing only the genus Ixodes, and Metastriata, containing the 13 other currently recognised hard tick genera (Hoogstraal and Aeschlimann, 1982; Guglielmone et al., 2010). The metastriate genus Haemaphysalis is the second most specious tick genus, with 166 currently valid species. The genus is distributed globally, though the greatest diversity is found in southeastern Asia (Kolonin, 2009).

∗ Corresponding author. E-mail address: [email protected] (S.C. Barker). 1877-959X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2013.02.002

One of the defining morphological features of the genus Haemaphysalis is the presence of a prominent “blade-like dorsal retrograde process” (Nuttall and Warburton, 1915) on trochanter I (i.e. a large backwards-facing hump or spur on the first segment of the first leg). Also characteristic of the genus are short, wide palps, with the palp femur projecting laterally over the rectangular basis capitulum. The genus Haemaphysalis was studied by Hoogstraal and Kim (1985), who proposed a trend in the evolution of morphology within and among the subgenera of Haemaphysalis from atypical and ‘primitive’ Amblyomma-like forms to typical Haemaphysalis-like forms. Hoogstraal and Kim (1985) ‘graded’ the subgenera of Haemaphysalis into 3 categories: (i) Structurally Primitive, (ii) Structurally Intermediate, and (iii) Structurally Advanced. Hoogstraal and Kim (1985) also placed much emphasis on tick–host specificity and apparent tick–host coevolution; more recent work suggests that the degree to which ticks are host-specific is overestimated and that ecological specificity is more important in tick evolution (Klompen et al., 1996).

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Hoogstraal and Kim’s (1985) hypothesis on the relationships among species of Haemaphysalis have not yet been fully tested by phylogenetic analysis. Klompen et al. (1997) addressed some of the relationships among Haemaphysalis subgenera in a phylogenetic analysis of the Metastriata, based on morphology. Klompen et al. (1997) supported monophyly of Haemaphysalis, but could not resolve the phylogenetic position of the Structurally Primitive subgenera with respect to the rest of the genus Haemaphysalis. Sampling of the genus Haemaphysalis in molecular analysis has been limited; most sequences of Haemaphysalis spp. in GenBank are from Structurally Advanced species. One Structurally Primitive species, H. inermis did not cluster with the rest of the genus Haemaphysalis in analyses of mitochondrial 12S and 16S rRNA (Norris et al., 1999) and nuclear 18S rRNA (Dobson and Barker, 1999; Miller et al., 2007). However, a total evidence analysis of morphology, 18S, partial 16S, and partial 28S rRNA data supported monophyly of Haemaphysalis with respect to H. inermis, though only 2 other Haemaphysalis species (Structurally Primitive H. punctata and Structurally Advanced H. leporispalustris) were included in the analysis (Klompen et al., 2000). Recent analysis of both 18S and partial 28S nuclear rRNA sequences including 6 species of Haemaphysalis had some support for the position of H. inermis as sister to the rest of the Haemaphysalis (Burger et al., 2012). This study also included another Structurally Primitive species, H. punctata, which did not cluster with H. inermis, but was within the main Haemaphysalis clade. A recent phylogenetic analysis of complete mt genomes and nuclear rRNA genes was informative for the genus Amblyomma, suggesting that the genus Amblyomma was paraphyletic with respect to A. sphenodonti and A. elaphense (Burger et al., 2012). However, this analysis could not conclusively resolve the phylogenetic position of A. sphenodonti or A. elaphense, though there was moderate support for A. sphenodonti as sister to the genus Bothriocroton. The most likely position of A. elaphense was either as sister to the rest of the Metastriata or sister to the genus Haemaphysalis (Burger et al., 2012). In addition, though there was no strong evidence for a sister group relationship between A. elaphense and A. sphenodonti, this possibility could not be excluded. Burger et al. (2012) also proposed that the Metastriata comprised 2 clades: (i) Amblyomma s.s. (excluding A. sphenodonti and A. elaphense) plus Rhipicephalinae (Rhipicephalus, Hyalomma, and Dermacentor); and (ii) Haemaphysalis plus Bothriocroton and A. sphenodonti. This division of the Metastriata into 2 clades contrasts with the proposal of Hoogstraal and Aeschlimann (1982) and the working hypothesis of Barker and Murrell (2004), though a previous analysis of nuclear 18S rRNA had moderate support for Amblyomma s.s. plus Rhipicephalinae (Dobson and Barker, 1999), and a total evidence analysis of morphology and rRNA had moderate support for both clades (Klompen et al., 2000). For convenience, in this paper, we call these 2 clades Haematobothrion, for Haemaphysalis plus Bothriocroton plus A. sphenodonti; and Amblyocephalus for Amblyomma s.s. plus Rhipicephalinae. These names are a combination of the names of the 2 major genera of each clade. We do not intend for these clade names to become formal taxonomic ranks, rather we use them to assist in the discussion of the phylogenetic relationships among metastriate lineages. Burger et al. (2012) proposed that analysis of additional mitochondrial genome sequences from the genus Haemaphysalis would help to resolve the phylogenetic positions of A. elaphense and A. sphenodonti. Thus, we sequenced the entire mitochondrial genomes of Haemaphysalis formosensis (Structurally Advanced; SA), Haemaphysalis parva (SA) and Amblyomma cajennense, as well as two thirds of the mitochondrial genome of Haemaphysalis humerosa (SA) and Haemaphysalis hystricis (SA). We also sequenced nuclear 18S rRNA and partial 28S rRNA genes from these 5 species and from Haemaphysalis flava (SA), Haemaphysalis leporispalustris (SA), and

Haemaphysalis sulcata (Structurally Intermediate). We use these sequences (i) to test the monophyly of the genus Haemaphysalis and the relationships between Haemaphysalis subgenera; and (ii) to improve the phylogeny of the metastriate ticks (and particularly the phylogenetic positions of A. sphenodonti and A. elaphense) by improving taxon sampling for this group. Materials and methods Specimens and DNA extraction Specimens sequenced in this study are listed in Table 1, along with accession numbers for sequences deposited in GenBank. DNA was extracted from tick specimens using the DNeasy Tissue Extraction Kit (QIAGEN). A single tick of each species was cut in half longitudinally, using a scalpel under a dissecting microscope; half was used for DNA extraction, and the other half kept as a voucher specimen. Tissue for extraction was snap-frozen in liquid nitrogen and ground with micropestle prior to DNA extraction. Voucher specimens for each mt genome we sequenced were deposited in the Queensland Museum, South Brisbane BC, Queensland 4010, under registration numbers QMS93625-QMS93629. PCR amplification and sequencing 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., 2005b). Species-specific primers were then designed for each species from these 3 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 3 overlapping fragments, from cytb to cox1, cox1 to 12S rRNA, and from 12S rRNA to cytb (Fig. 1). Partial mitochondrial genomes were sequenced from only the first 2 fragments. 18S rRNA and partial 28S rRNA genes were amplified and sequenced as per Burger et al.

Fig. 1. The relative length and arrangement of genes in the mitochondrial genomes of Haemaphysalis formosensis, H. parva, and Amblyomma cajennense and the partial mt genomes of H. humerosa and H. hystricis. Genes illustrated on the outside of the main circle are encoded on the forward or majority (J) strand; genes on the inside of the circle are encoded on the reverse or minority (N) strand. The inner 3 circles represent the size and relative position of the PCR fragments amplified in these 5 species. Only fragments 1 and 2, ca. 9500 bp of the mitochondrial genome, were sequenced in H. humerosa and H. hystricis. Diagram constructed using GenomeVx (Conant and Wolfe, 2008). *H. humerosa and H. hystricis are partial mitochondrial genomes.

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Table 1 Specimens used in this study, along with collection data and GenBank accession numbers for nucleotide sequences deposited in GenBank. Species

Collection locality

Collector

Mt genome GenBank ID

18S and 28S GenBank ID

Haemaphysalis formosensis Haemaphysalis parva Haemaphysalis humerosa Haemaphysalis hystricis Amblyomma cajennense Haemaphysalis flava Haemaphysalis leporispalustris Haemaphysalis sulcata

¯ Kabutoyama, Nishinomiya, Hyogo, Japan Western Romania Pinjarra Hills, Brisbane, QLD, Australia ¯ Kabutoyama, Nishinomiya, Hyogo, Japan Balneário do Sol, Bonito, MS, Brazil ¯ Shiose, Nishinomiya, Hyogo, Japan Lab strain, CDC, Atlanta, GA, USA Western Romania

Matsuo Kobayashi & Kyoko Sawabe Lidia Chitimia Jim Rothwell Matsuo Kobayashi & Kyoko Sawabe Paulo Mira Batista Matsuo Kobayashi & Kyoko Sawabe Centres for Disease Control Lidia Chitimia

JX573135 JX573136 JX573138 JX573137 JX573118 – – –

JX573121 JX573129 JX573125 JX573133 JX573123 JX573131 JX573122 JX573130 JX573119 JX573127 JX573120 JX573128 JX573124 JX573132 JX573126 JX573134

(2012), using previously reported primers (Hillis and Dixon, 1991; Black et al., 1997; Whiting et al., 1997; Dobson and Barker, 1999). Expand Long Range dNTPack kits (Roche) were used to amplify all long mitochondrial PCR products (>1 kb). TaKaRa Ex Taq DNA polymerase kits (Takara Biotechnology) were used to amplify short mitochondrial gene fragments (<1 kb) as well as nuclear rRNA genes. PCR conditions were optimised for each reaction, with the annealing temperature adjusted to suit the primers used, and extension time set to 1 min per kb of expected product size. General PCR conditions for Ex Taq were: 94 ◦ C for 60 s, followed by 40 cycles of 98 ◦ C for 10 s, 60 ◦ C for 30 s, 72 ◦ C for 2 min, and a final extension of 72 ◦ C for 5 min. General PCR conditions for Expand dNTPack were: 92 ◦ C for 120 s, 10 cycles of 92 ◦ C for 10 s, 55 ◦ C for 15 s, 60 ◦ C for 8 min, followed by 25 cycles of 92 ◦ C for 10 s, 55 ◦ C for 15 s, 60 ◦ C for 8 min (increasing by 20 s per cycle), and a final extension of 68 ◦ C for 7 min. PCR products were examined on 1% agarose gel stained with ethidium bromide. DNA Molecular Weight Marker VII (Roche Diagnostics) and Low DNA Mass Ladder (Invitrogen) were used, respectively, to estimate the length and concentration of PCR products. Wizard SV Gel and PCR Clean-up System (Promega) was used to purify PCR products for use in sequencing reactions. Sequencing reactions used the ABI Prism BigDye v3.1 Terminator kit (Applied Biosystems) and an Applied Biosystems 3730xl DNA Analyzer at the Australian Genome Research Facility. Sequencing of purified mitochondrial genome PCR products used an Illumina HiSeq run at BGI-Hong Kong. Contig assembly and mitochondrial genome annotation Geneious Pro 5.6 (Drummond et al., 2010) was used to assemble Sanger and Illumina reads. Protein coding genes were identified by BLAST searches of open reading frames; tRNA genes were identified with tRNAscan-SE (Lowe and Eddy, 1997) and ARWEN (Laslett and Canback, 2008). Non-coding regions and rRNA genes were identified by BLAST search and alignment with other tick mitochondrial genomes. Sequence alignment The mitochondrial genome sequences of 15 tick species were retrieved from GenBank (Table 2). Amino acid sequences of mitochondrial protein coding genes were aligned using Muscle (Edgar, 2004) as implemented in MEGA5 (Tamura et al., 2011), and the alignments were exported from MEGA5 as amino acids and as nucleotides. Individual gene alignments were concatenated into combined amino acid and nucleotide alignments of all mitochondrial protein-coding genes for phylogenetic analysis. Gblocks (Castresana, 2000) was used to trim the amino acid and nucleotide alignments of any ambiguously aligned sites. The trimmed mtAA alignment was 3275 amino acids long, and the mtDNA alignment was 10,530 nucleotides long (Supplementary Data 1 and 2). Mitochondrial 12S and 16S rRNA genes were aligned using MAFFT v 6.814b (Katoh et al., 2002), with the Q-INSI setting which uses RNA secondary structure to aid alignment. Gblocks

(Castresana, 2000) was used to trim the alignments of any ambiguously aligned regions. The trimmed alignments were then concatenated into a single alignment 1557 nucleotides long for phylogenetic analysis (Supplementary Data 2). 18S rRNA sequences from 84 species of ticks and mites were used in our separate analysis of 18S and partial 28S nuclear rRNA genes. 18S rRNA sequences of 66 ticks and 10 Holothyrida and Opilioacarida (Acari: Parasitiformes) were downloaded from GenBank (Table S2) and aligned with the 18S rRNA sequences of 8 tick species sequenced in this study (the 5 mt genome species as well as H. flava, H. sulcata, H. leporispalustris). Partial 28S rRNA sequences for 43 of these species were also aligned: 29 ticks and 6 Holothyrida and Opilioacarida downloaded from GenBank, plus partial 28S rRNA sequences of the 8 ticks from this study. 18S and partial 28S rRNA sequences were aligned using MAFFT as above and were concatenated into a single alignment 4400 nucleotides long for phylogenetic analysis (Supplementary Data 3).

Phylogenetic analysis For our analysis of mt DNA, mt rRNA, and nuclear rRNA sequences, we used GARLI (Zwickl, 2006). Our mtDNA alignment was partitioned by codon position, and 6 separate analyses were run: (1) mt amino acids (mtAA); (2) mt DNA, including all codon positions (mtDNA123); (3) all mtDNA codons and mt rRNA (mtDNA123mtRNA); (4) all mtDNA codons, mt rRNA, and nuclear rRNA (mtDNA123mtRNAnucRNA); (5) all mtDNA codons and nuclear rRNA (mtDNA123nucRNA); and (6) first and second mtDNA codons and nuclear rRNA (mtDNA12nucRNA). jModeltest (Posada, 2008) was used to select optimal substitution models for each partition: GTR+G:4 for mt rRNA, GTR+I+G:4 for first and second codon positions, and nuclear rRNA and HKY+I+G:4 for third codon positions. For the mitochondrial amino acid alignment, ProtTest3 (Darriba et al., 2011) was used to inform the choice of protein evolution model; however, as the optimal model determined by ProtTest (mtArt) is not implemented in GARLI, we used the next best model, mtREV+I+G:4. For each dataset, GARLI was instructed to estimate the free parameters of each model, to treat all partitions as unlinked, and to perform 100 bootstrap replicates, which were summarised with SumTrees, part of the DendroPy package (Sukumaran and Holder, 2010). Our analyses used the GARLI web service (Bazinet and Cummings, 2011), which uses a special programming library and associated tools (Bazinet et al., 2007) and grid computing (Cummings and Huskamp, 2005) through The Lattice Project (Bazinet and Cummings, 2008), which includes clusters and desktops in one encompassing system (Myers et al., 2008). Following the general computational model of a previous phylogenetic study (Cummings et al., 2003), which used an earlier grid computing system (Myers and Cummings, 2003). Files were distributed among hundreds of computers where the analyses were conducted asynchronously in parallel.

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Table 2 Mitochondrial genome sequences retrieved from GenBank. Higher taxon

Species

GenBank ID

Reference

Metastriata

Bothriocroton concolor Bothriocroton undatum Amblyomma sphenodonti Amblyomma elaphense Amblyomma fimbriatum Amblyomma triguttatum Haemaphysalis flava Rhipicephalus sanguineus Ixodes hexagonus Ixodes persulcatus Ixodes uriae Ixodes holocyclus Ornithodoros moubata Ornithodoros porcinus Carios capensis

NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC

Burger et al. (2012) Burger et al. (2012) Burger et al. (2012) Burger et al. (2012) Burger et al. (2012) Shao et al. (2005a) Black and Roehrdanz (1998) Shao et al. (2004) Black and Roehrdanz (1998) Shao et al. (2005b) Shao et al. (2005b) Shao et al. (2005b) Shao et al. (2004) Shao et al. (2005a) Shao et al. (2004)

Prostriata

Argasidae

017756 017757 017745 017758 017759 005963 005292 002074 002010 004370 006078 005293 004357 005820 005291

Fig. 2. The repeat regions in the mt genome of Haemaphysalis parva. Numbers on the top line represent the position in the mt genome relative to tRNA-M, the common start point of chelicerate mt genomes on GenBank. The ca. 147-bp insertion is between the tRNA-E and nad1 genes and has 99 bp of sequence that is identical to the 3 end of 16S rRNA. There is also 24 bp of sequence that is identical to both the 3 end of nad1 and the 3 end of 16S rRNA; a similar stretch of identical or near-identical sequence is present in other metastriate tick mt genomes (Fig. S1).

CONSEL v0.20 (Shimodaira and Hasegawa, 2001) was used to assess the confidence in phylogenetic tree selection by applying the approximately unbiased (AU) test. The AU test is a multiscale bootstrap technique that aims to overcome the biases of other confidence tests, and allows for hypothesis testing of overall tree topology and of specific clades of taxa. We tested the topology of the bootstrap consensus tree as well as 13 different topological constraints in separate GARLI runs allowing for optimisation of model rates and branch lengths, and instructed GARLI to output the site likelihood scores for use in CONSEL.

Phylogenetic analysis of mt genome sequences The Maximum Likelihood (ML) trees inferred from the mtAA, mtDNA123, and mtDNA123mtRNA datasets all have identical topology, differing only in the level of bootstrap support (the mt data tree; Fig. 3). These 3 datasets all have strong bootstrap support (100%) for monophyly of the Metastriata, and of Amblyomma s.s., Bothriocroton, and Haemaphysalis s.s. (excluding H. parva). The level of bootstrap support for the controversial, intergeneric

Results Mitochondrial genome organisation The mitochondrial genomes of H. formosensis, H. parva, and A. cajennense, and the partial mt genomes of H. humerosa and H. hystricis have the same mt gene arrangement as the other known metastriate ticks (Black and Roehrdanz, 1998; Campbell and Barker, 1998; Shao et al., 2004, 2005a; Burger et al., 2012) (Fig. 1 and Table S3). The entire mt genomes range in length from 14,676 bp (H. formosensis) to 14,846 bp (H. parva), and the partial mt genome sequences of H. humerosa and H. hystricis are 9531 bp and 9576 bp, respectively. These 5 mt genomes are considerably GC-poor, as is the case for other metastriate ticks (Black and Roehrdanz, 1998; Shao et al., 2004; Burger et al., 2012) (Table 3). The mt genome of H. parva has an insertion of ca. 144 bp relative to other Haemaphysalis spp. at the junction of the ARNSE block of tRNA genes and nad1 (Fig. 2). The first 24 bp of the insertion in H. parva are identical to both the 3 end of nad1, and the 3 end of 16S rRNA (Fig. S1). A 99-bp region at the 5 end of the insertion (including the 24-bp motif) is also identical to the 3 end of 16S rRNA (Fig. 2). The remaining 48 bp of the insertion are not highly similar to any other region of the mt genome of H. parva. All other metastriate tick mt genomes in GenBank also have 24–27 bp of identical sequence between the ends of nad1 and 16S rRNA (Fig. S1). Thus the 3 copies of the 24-bp motif in H. parva are due to the larger copy of the 3 end of 16S rRNA in the insertion.

Fig. 3. Maximum Likelihood tree inferred from mitochondrial genome datasets. Node support is indicated by the bootstrap percentages at each node for each dataset from left to right: mtAA/mtDNA123/mtDNA123mtRNA. 100% bootstrap support is represented by an asterisk *; 100% support in all 3 datasets is represented by a triple asterisk ***. Mitochondrial genomes sequenced in this study are in bold type. Within the Metastriata, currently recognised genera are colour-coded, and the branches leading to A. elaphense and A. sphenodonti are coloured red to indicate that these 2 species do not cluster with the genus Amblyomma. Controversial nodes tested in the AU test of tree topology are labelled by letter as in Table 5.

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Fig. 4. Maximum Likelihood tree inferred from an alignment of 18S and partial 28S nuclear rRNA genes. Node support is indicated by the bootstrap percentage before the node; bootstrap percentages below 50% are not shown. Species sequenced for 18S and 28S in this study are in bold type. Within the Metastriata, currently recognised genera are colour-coded as indicated by the higher-taxa labels to the right. The branches leading to A. elaphense and A. sphenodonti are coloured red to indicate that these 2 species do not cluster with the genus Amblyomma.

nodes within the Metastriata (A, C, E, G, J; Fig. 3; Table 5) differs among the 3 datasets. Only the mtAA dataset has strong bootstrap support (87%) for A. sphenodonti as sister to H. parva, for A. elaphense as sister to the rest of the Metastriata (91%), and for the monophyly of Amblyocephalus (Rhipicephalus plus Amblyomma s.s.; 91%). However, the mtAA dataset does not have strong

support for the monophyly of Haematobothrion (Haemaphysalis plus Bothriocroton, including A. sphenodonti and H. parva; 38%). The mtDNA123mtRNA dataset, however, does have moderate support for Haematobothrion (74%), though neither mtDNA123 nor mtDNA123mtRNA datasets have strong support for any other controversial node (33–66% and 51–67%, respectively).

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Table 3 Nucleotide composition of the mitochondrial genomes of Haemaphysalis s.l. and Amblyomma s.s. Species

T%

C%

A%

G%

C+G

Haemaphysalis formosensis Haemaphysalis parva Haemaphysalis humerosa a Haemaphysalis hystricis a Haemaphysalis flava b Amblyomma cajennense Amblyomma fimbriatum b Amblyomma triguttatum b

39.7 39.7 41.4 40.6 39.1 39.1 39.6 40.0

12.4 12.1 12.7 13.5 12.9 14.0 12.9 12.2

38.6 39.1 36.3 35.9 37.7 36.8 38.1 38.4

9.3 9.0 9.6 10.0 10.3 10.1 9.4 9.4

21.7 21.2 22.3 23.5 23.3 24.0 22.3 21.6

a Nucleotide composition frequencies from H. humerosa and H. histricis are for partial mt genomes. b Mt genome sequences retrieved from Genbank, displayed for comparison with the species sequenced in this study.

Phylogenetic analysis of nuclear rRNA genes The ML tree inferred from the 76 species of tick and mite 18S and partial 28S nuclear rRNA genes (the nuclear rRNA tree; Fig. 4) does not resolve the controversial, intergeneric nodes within the Metastriata. There is some support in the nuclear rRNA tree for a monophyly of Amblyocephalus, i.e. Rhipicephalinae (including Rhipicephalus, Hyalomma, and Dermacentor) plus Amblyomma s.s. (69%). In contrast to the mt data tree, the nuclear rRNA tree has moderate support for A. sphenodonti as sister to Bothriocroton (70%) and for the monophyly of the genus Haemaphysalis (77%). Within the genus Haemaphysalis, H. parva and H. inermis form a sister-clade to the rest of the genus (100% for both clades). We examined the nuclear rRNA alignment closely and found that the 18S rRNA gene in H. parva and H. inermis lacks a Haemaphysalis-specific marker in the V2 region sensu Gillespie et al. (2005) (Fig. 5). Phylogenetic analysis of combined mitochondrial genome and nuclear rRNA datasets The ML trees inferred from the combined mtDNA and nuclear rRNA datasets (mtDNA123mtRNAnucRNA, mtDNA123nucRNA, and mtDNA12nucRNA) (combined data tree; Fig. 6) also had identical topology, differing from the mt data tree only in the position of A. sphenodonti. The mtDNA123nucRNA data supports monophyly of the genus Haemaphysalis (node B, 82%) with H. parva as sister to the 4 other Haemaphysalis spp. and has moderate support for A. sphenodonti as sister to the genus Bothriocroton (node D, 69%). All of the other controversial, intergeneric nodes within the Metastriata (A, C, E; Fig. 6) are moderately supported (68–84 bp). Including mt rRNA in the analysis (mtDNA123mtRNAnucRNA) decreases support for Amblyomma s.s. plus Rhipicephalus (Amblyocephalus; 84–76%), and monophyly of Haemaphysalis (82–73%). Excluding third codon positions and mt rRNA from the analysis (mtDNA12nucRNA), increased support for Rhipicephalus plus Amblyomma s.s. (84–95%) and the position of A. elaphense (68–88%), though support for the relationships within Haematobothrion (Nodes B, C, and D) is decreased. Approximately unbiased (AU) test of tree topology We performed the AU test of tree topology on the mtDNA123nucRNA and mtAA datasets with 14 topological constraints (Table 4 and S4). We chose these 2 datasets as they had the highest bootstrap support in our ML analysis (Figs. 3 and 6); the results of the AU test performed on the other 4 datasets did not differ significantly from the results of the mtDNA123nucRNA dataset. Topologies 1, 3, 6, 7, and 8 were not rejected by either dataset, topologies 2 and 4 were rejected by mtAA, but not by mtDNA123nucRNA or any other mtDNA dataset, and constraints 5, 9, 10, 11, 12, 13, and 14 were rejected by both datasets. Topologies

Fig. 5. Alignment of 18S rRNA from positions 180–202, displaying a Haemaphysalisspecific marker sequence in the variable region V2 of 18S rRNA. This sequence TTGTGTMTT from positions 187–196 in the alignment is present in all Haemaphysalis 18S rRNA, except H. inermis and H. parva. These 2 species have the ancestralMetastriate sequence in this region, from comparison with the other tick sequences: GAAGCCC. Numbers in brackets after taxon names indicate the number of species of that taxon with identical sequence. The complete alignment of 18S rRNA is available in Supplementary Data 3.

2 and 4 are alternate positions of A. elaphense: sister to Haematobothrion; and sister to A. sphenodonti plus Bothriocroton. The other alternative positions of A. elaphense are the unconstrained position, sister to the rest of the Metastriata (topology 1), and sister to Bothriocroton without A. sphenodonti (topology 7). All other phylogenetic positions of A. elaphense tested were rejected by the AU test, including position with Haemaphysalis s.l. (topologies 5 and 10), Amblyomma s.s. (topologies 11, 14), and forming a monophyletic lineage with A. sphenodonti (topology 9). The different phylogenetic positions of A. sphenodonti in the mt data and combined data trees were not rejected by the AU test. The mtAA tree places A. sphenodonti as sister to H. parva (topology 6), and the mtDNA123nucRNA tree places A. sphenodonti as sister to Bothriocroton (topology 1); neither position can be rejected by either dataset. Two other alternative positions of A. sphenodonti cannot be rejected: sister to monophyletic Haemaphysalis (topology 3); and A. sphenodonti plus H. parva sister to Bothriocroton (topology 8). However, we can reject the placement of A. sphenodonti with Amblyomma s.s. (topologies 13, 14), forming a monophyletic lineage with A. elaphense (topology 9), or forming a lineage with A. elaphense and Haemaphysalis s.l. (topology 10).

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Fig. 6. Maximum Likelihood tree inferred from the combined mtDNA and nuclear rRNA datasets. Node support is indicated by the bootstrap percentages at each node for each data set from left to right: mtDNA123mtRNAnucRNA/mtDNA123nucRNA/mtDNA12nucRNA. 100% bootstrap support is represented by an asterisk *; 100% support in all 3 datasets is represented by a triple asterisk ***. Mitochondrial genomes sequenced in this study are in bold type. Within the Metastriata, currently recognised genera are colour-coded, and the branches leading to A. elaphense and A. sphenodonti are coloured red to indicate that they do not cluster with the genus Amblyomma. Controversial nodes tested in the AU test of tree topology are labelled by letter as in Table 5.

The results of the AU test of the clades (Table 5) that differ among the 14 topological constraints are consistent with the AU test of the overall topologies. However, Clade F (A. elaphense sister to Haematobothrion) is not rejected in the mtAA data (p = 0.056), though the topological constraint containing this clade is rejected in the mtAA data (topology 2; Table 4). Intriguingly, the monophyly of Amblyocephalus (clade A), uniting Amblyomma s.s. and Rhipicephalus sanguineus is strongly supported by the AU test in both datasets

(p ≥ 0.95). The complementary clade of Haematobothrion (clade C) containing Haemaphysalis, Bothriocroton, and A. sphenodonti cannot be rejected, though alternative positions of A. elaphense within Haematobothrion cannot be rejected either (clades H and K). Similarly, while the monophyly of Haemaphysalis is not rejected (clade B), the alternative of a paraphyletic Haemaphysalis, with A. sphenodonti sister to H. parva also cannot be rejected (clades J and L). We can reject monophyly of Amblyomma s.l. (clades O, T, and V),

Table 4 Results of the approximately unbiased (AU) test of confidence in tree selection on 14 topological constraints. Topology constraint (tree)a 1 2 3 4 5 6 7 8 9 10 11 12

13 14 a b c

Unconstrained (combined data tree, Fig. 5) A. elaphense + Haematobothrion A. sphenodonti + Haemaphysalis s.l.b A. elaphense + A. sphenodonti + Bothriocroton A. elaphense + Haemaphysalis s.l. H. parva + A. sphenodonti (unconstrained mt data tree, Fig. 3) A. elaphense + Bothriocroton (H. parva + A. sphenodonti) + Bothriocroton A. sphenodonti + A. elaphense (primitive Aponomma monophyly) A. sphenodonti + A. elaphense (paraphyletic) + Haemaphysalis s.l. A. elaphense + Amblyomma s.s. Working hypothesis of Barker and Murrell (2004), ignoring placement of A. sphenodonti and A. elaphense A. sphenodonti + Amblyomma s.s. A. sphenodonti + A. elaphense + Amblyomma s.s. (monophyly of Amblyomma s.l.)

LnL (mtDNA)

p-Valuec (mtDNA)

LnL (mtAA)

p-Valuec (mtAA)

136,243 136,251 136,254 136,256 136,262 136,262

0.907 0.308 0.334 0.274 0.023 0.250

56,795 56,817 56,789 56,826 56,827 56,779

0.182 0.016 0.358 0.005 0.010 0.612

136,265 136,265 136,272

0.166 0.174 0.017

56,808 56,777 56,827

0.087 0.647 0.006

136,272

0.023

56,817

0.004

136,279 136,298

0.010 0.001

56,847 56,861

<0.001 <0.001

136,327 136,334

<0.001 <0.001

56,862 56,882

<0.001 <0.001

Full topological constraints are available in Newick format (Supplementary Data 4). Haemaphysalis s.l. includes H. Parva. p-Values rejected by the AU test (p < 0.05) are in bold type.

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Table 5 Results of the approximately unbiased (AU) test of confidence in tree selection on the 22 clades differing among the 14 tested topologies. p-Value a (mtDNA)

Topology constraint (clade) A B C D E F G H I J K L M N O P Q R S T U V a b

Amblyomma s.s. + R. sanguineus (monophyly of Amblyocephalus) Monophyly of Haemaphysalis s.l.b A. sphenodonti + Bothriocroton + Haemaphysalis s.l. (monophyly of Haematobothrion) A. sphenodonti + Bothriocroton A. elaphense + Metastriata A. elaphense + Haematobothrion A. sphenodonti + Haemaphysalis s.l. A. elaphense + A. sphenodonti + Bothriocroton A. elaphense + Haemaphysalis s.l. A. sphenodonti + H. parva A. elaphense + Bothriocroton H. parva + A. sphenodonti + Bothriocroton A. sphenodonti + A. elaphense A. sphenodonti + A. elaphense + Haemaphysalis s.l. A. elaphense + Amblyomma s.s. R. sanguineus sister to the rest of the Metastriata R. sanguineus + Haemaphysalis s.l. + Amblyomma s.s. R. sanguineus + Haemaphysalis s.l. R. sanguineus + Haemaphysalis s.l. + Amblyomma s.s. + A. sphenodonti Amblyomma s.s. + A. sphenodonti Bothriocroton + Haemaphysalis s.l. (ex. A. sphenodonti) A. sphenodonti + A. elaphense + Amblyomma s.s. (monophyly of Amblyomma s.l.)

0.994 0.816 0.823 0.845 0.831 0.189 0.215 0.257 0.023 0.184 0.166 0.174 0.017 0.023 0.009 0.009 0.001 0.001 <0.001 <0.001 <0.001 <0.001

p-Valuea (mtAA) 0.999 0.175 0.940 0.158 0.947 0.056 0.476 0.004 0.008 0.825 0.088 0.640 0.006 0.002 0.004 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

p-Values indicating support in the AU test (p > 0.95) are underlined and p-values indicating rejection in the AU test (p < 0.05) are in italics. Haemaphysalis s.l. includes H. Parva.

monophyly of A. sphenodonti + A. elaphense (clade M), placement of A. elaphense as sister to Haemaphysalis s.s. and s.l. (clades I and N), and all clades unique to the ‘working hypothesis’ phylogeny of Barker and Murrell (2004) (clades Q, R and S). Discussion Our analysis of the mtDNA of H. parva places this species as sister to the rest of the genus Haemaphysalis. In our analysis of 18S and 28S rRNA, H. parva forms a clade with H. inermis, sister to rest of the genus Haemaphysalis. Thus, H. parva and H. inermis form a clade which is highly divergent from the rest of the genus Haemaphysalis, and the genus is likely monophyletic with respect to these 2 species. H. parva lacks a Haemaphysalis-specific marker in the 18S rRNA gene, as does H. inermis; both species have the Metastriateancestral sequence at this position (Fig. 5). This indicates that H. parva and H. inermis split off from the rest of the genus Haemaphysalis before this Haemaphysalis-specific marker evolved, but after Haemaphysalis-specific morphology evolved. H. inermis is Structurally Primitive sensu Hoogstraal and Kim (1985), whereas H. parva is Structurally Advanced. Most of the other Haemaphysalis species in our nuclear rRNA analysis are also Structurally Advanced, except for H. punctata, which is Structurally Primitive, and H. sulcata, which is Structurally Intermediate. Both H. punctata and H. sulcata have the Haemaphysalis-specific 18S rRNA marker and are in the main Haemaphysalis clade in our nuclear rRNA tree (Fig. 4). Thus, the Structurally Primitive and Structurally Advanced categories of Hoogstraal and Kim (1985) are likely not monophyletic groups, as each of our 2 clades of Haemaphysalis have both Structurally Primitive and Structurally Advanced species. Phylogenetic analysis of the morphology of metastriate ticks has also suggested that the Structurally Primitive Haemaphysalis are paraphyletic with respect to the more derived subgenera of Haemaphysalis (Klompen et al., 1997). The mitochondrial genome of H. parva differs from that of the other Haemaphysalis species with an insertion of ca. 147 bp at the junction of tRNA-E and nad1 (Fig. 2). Intriguingly, a variable tandem repeat (2–3 copies) has also been reported in this region, the junction of tRNA-E and nad1, in Rhipicephalus (Boophilus) microplus (Campbell and Barker, 1999; Campbell et al., 2001). However, the insertion in H. parva is not a tandem repeat and appears to be a

partial copy of the 16S rRNA. 99 bp of the insertion in H. parva are 100% identical to the 3 end of the 16S rRNA (Fig. 2), suggesting that either the insertion was ancient and that these 2 regions are evolving in concert, as observed in the duplicate control regions of metastriate ticks (Shao et al., 2005b), or that this insertion was recent and has not yet been purged from the mt genome of H. parva. If H. inermis is closely related to H. parva, as suggested by the nuclear rRNA tree (Fig. 4), sequencing the mt genome of H. inermis may be crucial in discerning between these 2 hypotheses: if a similar insertion is found in H. inermis, this would suggest the insertion was ancient, whereas if no insertion is found in H. inermis, this would suggest the insertion was more recent, and unique to H. parva. If this ca. 147-bp insertion is shared between the H. parva and H. inermis, it also could provide a useful marker for this clade of Haemaphysalis, complementary to the Haemaphysalis-specific sequence we identified in the 18S rRNA gene of the main Haemaphysalis clade. The discovery of the partial copy of 16S rRNA in the insertion of H. parva led us to the further discovery of a 24–27-bp motif of identical sequence between the 3 ends of nad1 and 16S rRNA in all metastriate ticks (Fig. 2 and Fig. S1). This 24–27-bp motif may have originated from the nad1 gene, as the region is similar to the end of nad1 in prostriate and soft ticks. The motif also appears to be evolving in concert between the 2 regions, as the single-nucleotide polymorphisms (SNPs) observed in several species are present in both sequences (Fig. S1). The position of A. sphenodonti in the mt data tree is sister to H. parva (Fig. 3). This is not supported in phylogenies inferred from the nuclear rRNA data (Fig. 4) or from the combined mtDNA and nuclear rRNA data (Fig. 6); in both trees the position of A. sphenodonti is sister to Bothriocroton. However, the AU test of the combined data cannot reject the possibility of A. sphenodonti plus H. parva. The high bootstrap support of this node in the mtAA dataset (87%) may be due to amino acid homoplasy, as the mtDNA-only dataset has a lower level of bootstrap support for this node (57%). The clustering of A. sphenodonti and H. parva may also be due to long-branch attraction. If so, addition of more mitochondrial genome sequences closely related to H. parva may be able to resolve the phylogenetic position of both species. The sister group relationship between H. parva and H. inermis in the nuclear rRNA tree indicates that the mt genome of H. inermis could be crucial in resolving the phylogenetic position of H. parva.

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Support for monophyly of Amblyocephalus (Amblyomma s.s. plus Rhipicephalus) was strong in the mtAA and combined datasets and was supported significantly in the AU test. However, R. sanguineus is the only representative of the genus Rhipicephalus in the mt genome data, and the other major rhipicephaline genera, Dermacentor and Hyalomma, are not represented. Thus, further mt genome data from these genera are required to test the sister relationship between Amblyomma s.s. and the Rhipicephalinae. However, combined molecular and morphological analyses also support the monophyly of Rhipicephalinae sensu Murrell et al. (2001) (Mangold et al., 1998; Klompen et al., 2000; Beati and Keirans, 2001). In addition, although our analysis of nuclear rRNA has only weak support for monophyly of the 4 genera of Rhipicephalinae sequenced to date (Rhipicephalus, Rhipicentor, Dermacentor, and Hyalomma), there is moderate support for the monophyly of Amblyocephalus. The phylogenetic positions of the remaining Rhipicephaline genera (Margaropus, Anomalohimalaya, and Cosmiomma) have yet to be tested by molecular data. Margaropus appears to be closely related to the subgenus Rhipicephalus (Boophilus), though the phylogenetic positions of Anomalohimalaya and Cosmiomma with respect to the rest of the Rhipicephalinae are more controversial (Murrell et al., 2001). We predict that mtDNA data from representatives of all the rhipicephaline genera will resolve the phylogenetic relationships among them and support monophyly of the Rhipicephalinae. The complementary clade to Amblyocephalus, Haematobothrion (Haemaphysalis plus Bothriocroton plus A. sphenodonti), was moderately supported in the combined mtDNA and nuclear rRNA dataset, but was not strongly supported by the AU test; the lower level of support for this clade may be due to uncertainty in the phylogenetic position of A. elaphense. In addition, the AU test rejects the relationships between metastriate genera in the working hypothesis phylogeny proposed by Barker and Murrell (2004). Thus, our analysis supports the relationships between metastriate genera (Rhipicephalinae plus Amblyomma and Haemaphysalis plus Bothriocroton) proposed by Klompen et al. (2000), though Klompen et al. did not address the phylogenetic positions of A. sphenodonti and A. elaphense. The apparent polyphyly of the genus Amblyomma with respect to A. sphenodonti and A. elaphense has previously been established (Miller et al., 2007; Burger et al., 2012). Our analysis adds to the work on resolving the phylogenetic position of these 2 species with the inclusion of the type species of the genus Amblyomma, A. cajennense (Fabricius, 1787). We can also now reject the monophyly of A. sphenodonti plus A. elaphense and thus the possibility that these 2 species could be housed in a single genus. We can also reject the possibility that A. elaphense is sister to the genus Haemaphysalis. The most likely position of A. elaphense is sister to the rest of the Metastriata, though A. elaphense may also belong to Haematobothrion. The most likely phylogenetic position of A. sphenodonti is sister to the genus Bothriocroton, though the alternate position as sister to the genus Haemaphysalis cannot be excluded with current data. Regardless of their phylogenetic position within the Metastriata, neither A. elaphense nor A. sphenodonti belong to the genus Amblyomma. The morphology of A. sphenodonti and A. elaphense provides no clues as to their relationships to other tick genera; both species have been described as morphologically ‘primitive’ (Kaufman, 1972; Klompen et al., 2002), though we prefer the term plesiomorphic. A. sphenodonti and A. elaphense have remained in the genus Amblyomma as there are no obvious morphological synapomorphies for Amblyomma s.s. which would exclude these 2 species (Klompen et al., 2002). Neither A. sphenodonti nor A. elaphense have the synapomorphy that defines the genus Bothriocroton: three pairs of large wax glands on segment VIII in larvae (Klompen et al., 2002; Burger et al., 2012). Though morphological analysis of the genus Haemaphysalis is complicated by the presence of the plesiomorphic

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‘Structurally Primitive’ species, A. sphenodonti and A. elaphense lack any of the characters unique to the derived Haemaphysalis species: the “blade-like dorsal retrograde process” on trochanter I; palps projecting laterally over the basis capitulum; or the presence of large wax glands mid-dorsal on segment XI in larvae (Klompen et al., 1997). It seems that the close relatives of A. sphenodonti and A. elaphense are extinct, thus it is possible that further taxon sampling of mt genomes may not improve our understanding of the phylogenetic position of these 2 species. Further sampling of the genus Haemaphysalis, particularly H. inermis, may be informative for A. sphenodonti, but A. elaphense is not closely related to any other extant lineage of Metastriata. Moreover, to leave A. sphenodonti and A. elaphense in the genus Amblyomma makes this genus taxonomically unstable. Thus, A. sphenodonti and A. elaphense must either be transferred to existing genera or new genera must be described for them. With current data, A. sphenodonti may be sister to either Bothriocroton or Haemaphysalis, but A. elaphense is not closely related to any extant genus. Thus, the most taxonomically stable solution to the polyphyly of Amblyomma is to describe 2 new genera for these 2 species. Acknowledgements We thank Peter O’Donoghue (The University of Queensland, Australia) for comments on an earlier draft of this manuscript, and we thank Jim Rothwell (The University of Queensland, Australia), Matsuo Kobayashi, Kyoko Sawabe (National Institute of Infectious Diseases, Japan), Lidia Chitimia (Institute for Diagnosis and Animal Health, Romania), and Paulo Mira Batista (EMBRAPA, Brazil) for providing the specimens sequenced in this study. We also thank the two anonymous reviewers whose comments helped improve this manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ttbdis.2013.02.002. References Barker, S.C., Murrell, A., 2004. Systematics and evolution of ticks with a list of valid genus and species names. Parasitology 129, S15–S36. Bazinet, A.L., Cummings, M.P., 2008. The Lattice Project: a grid research and production environment combining multiple grid computing models. In: Weber, M.H.W. (Ed.), Distributed & Grid Computing – Science Made Transparent for Everyone. Principles, Applications and Supporting Communities. Rechenkraft.net, Marburg, pp. 2–13. Bazinet, A.L., Cummings, M.P., 2011. Computing the tree of life – leveraging the power of desktop and service grids. In: Proceedings of the Fifth Workshop on Desktop Grids and Volunteer Computing Systems (PCGrid 2011). Bazinet, A.L., Myers, D.S., Fuetsch, J., Cummings, M.P., 2007. Grid services base library: a high-level, procedural application program interface for writing Globus-based Grid services. Future Gener. Comput. Syst. 23, 517–522. Black, W., Roehrdanz, R., 1998. Mitochondrial gene order is not conserved in arthropods: prostriate and metastriate tick mitochondrial genomes. Mol. Biol. Evol. 15, 1772–1785. Black, W.C., Klompen, J.S.H., Keirans, J.E., 1997. Phylogenetic relationships among tick subfamilies (Ixodida: Ixodidae: Argasidae) based on the 18S nuclear rDNA gene. Mol. Phylogenet. Evol. 7, 129–144. Beati, L., Keirans, J.E., 2001. Analysis of the systematic relationships among ticks of the genera Rhipicephalus and Boophilus (Acari: Ixodidae) based on mitochondrial 12S ribosomal DNA gene sequences and morphological characters. J. Parasitol. 87, 32–48. Burger, T.D., Shao, R., Beati, L., Miller, H., Barker, S.C., 2012. Phylogenetic analysis of ticks (Acari: Ixodida) using mitochondrial genomes and nuclear rRNA genes indicates that the genus Amblyomma is polyphyletic. Mol. Phylogenet. Evol. 64, 45–55. Campbell, N.J.H., Barker, S.C., 1998. An unprecedented major rearrangement in an arthropod mitochondrial genome. Mol. Biol. Evol. 15, 1786–1787. Campbell, N.J.H., Barker, S.C., 1999. The novel mitochondrial gene arrangement of the cattle tick. Boophilus microplus: fivefold tandem repetition of a coding region. Mol. Biol. Evol. 16, 732–740.

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