Proc. Nati. Acad. Sci. USA Vol. 91, pp. 10034-10038, October 1994 Evolution Phylogeny of hard- and soft-tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences WILLIAM C. BLACK IV*t AND JOSEPH PIESMANt *Department of Microbiology, Colorado State University, Fort Collins, CO 80523; and tDivision of Vector-Borne Infectious Diseases, Centers for Disease Control, P.O. Box 2087, Fort Collins, CO 80522 Communicated by George B. Craig, Jr., June 13, 1994 ABSTRACT Ticks are parasitiform mites that are obligate in different instars appeared to be correlated with the host hematophagous ectoparasites of amphibians, reptiles, birds, species parasitized by each instar and concluded that adap- and mammals. A phylogeny for tick familie, subfamie, and tation to hosts played a major role in tick evolution. This genera has been described based on morphological characters, adaptation was assumed to lead to host specificity and life histories, and host actions. To test the exring phy- eventually to parallel evolution (cospeciation) between ticks logeny, we sequenced -460 bp from the 3' end of the mito- and their hosts. Hoogstraal suggested that the ancestral ticks, chondrial 16S rRNA gene (rDNA) in 36 hard- and soft-tick resembling the present-day Argasidae, arose in the late species; a ti mite, Dernanyssus gafinae, was used Paleozoic or early Mesozoic associated with "slow-moving, as an outgroup. Phylogenies derived using distance, ximum- smooth-skinned reptiles" (5). The Prostriata was among the parsimony, or maximum-likelihood methods were congruent. earliest line to differentiate from those ancestral forms. The existing phylogeny was largely supported with four excep- Within the Metastriata, Hoogstraal proposed that the Am- tions. In hard ticks (Ixodidae), members of Haemaphysalinae blyomminae originated on reptiles in the late Permian and were monophyletic with the primitive Amblyomminae and radiated on those hosts during the Triassic and Jurassic. The members of Hya.ommnae g ped within the Rhipkephali- Haemaphysalinae appeared on reptiles later in the Triassic, nae. In soft ticks (Argasidae), the derived phylogeny failed to whereas the Hyalomminae evolved on early mammals late in support a monophyletic relationship among members of Or- the Cretaceous. The Rhipicephalinae did not appear until the nithodorinae and supported placement of Argasnae as basal to Tertiary and, like the Hyalomminae, evolved primarily on the Ixedidae, suggesting that hard ticks may have originated mammals. from an Argas-like ancestor. Because most Arlas species are The fossil record provides few clues to tick evolution. The obligate bird ectoparasites, this result supports earlier sugges- first mite fossils date from the Devonian and closely resemble tions that hard ticks did not evolve until the late Cretaceous. extant taxa. However, all representatives of that fauna be- long to the suborder Acariformes. Fossils of the Parasiti- Ticks are classified in the suborder Ixodida of the order formes, and in particular of ticks, are quite rare and much Parasitiformes, one of the two orders of mites (Acari) (1). more recent. For example, hard ticks appear They are unique among Acari in possessing a large body size only in amber (2-30 mm) and specialized mouthparts. All ticks are he- from the Eocene and Oligocene (9-11). Direct evidence matophagous, obligate ectoparasites of terrestrial verte- regarding the time of origin of ticks is therefore absent, and brates including amphibians, reptiles, birds, and mammals. scenarios which vary markedly from Hoogstraal's have been The group is relatively small, consisting of about 850 species proposed. Oliver (2) suggested an even earlier age for the divided into two major families: the Argasidae ("soft" ticks) evolution of ticks, by assuming an origin close to that of the and the Ixodidae ("hard" ticks) (2, 3). The third family, Parasitiformes. He established the age of the latter group by Nuttalliellidae, contains only a single species, which shares comparison with the known age of its sister group, the characters of both Argasidae and Ixodidae in addition to Acariformes. He suggested a possible origin on amphibians. having many derived features (4). Alternatively, Russian workers have rejected amphibians or The family Ixodidae is divided into the Prostriata and reptiles as ancestral hosts and suggested a much more recent Metastriata. The Prostriata (subfamily Ixodinae) comprise origin of at least the Prostriata. The Russian school proposed about 240 species in a single genus, Ixodes. The Metastriata that the Ixodidae arose in the Cretaceous because nearly all are divided into four subfamilies (5): the Amblyomminae (125 extant Ixodidae occur on mammals or birds and "'primitive" species in two genera), Haemaphysalinae (147 species), Hy- Ixodidae occur on primitive mammals (marsupials and alomminae (22 species), and Rhipicephalinae (119 species in monotremes) (12, 13). eight genera). The family Argasidae contains about 170 Hoogstraal and Aeschlimann's phylogeny for hard- and species divided into two subfamilies, Argasinae (56 species) soft-tick genera has never been tested with a formal cladistic and Ornithodorinae (114 species in three genera). analysis. The purpose of this study was to use sequence The most commonly cited phylogeny among tick families, variation from the 3' end ofthe mitochondrial 16S rRNA gene subfamilies, and genera is that of Hoogstraal and Aeschli- ("16S" rDNA)§ to derive a molecular phylogeny for hard- mann (6) (Fig. 1). Hoogstraal's conception of the long-term and soft-tick families, subfamilies, and genera. Molecular evolution of ticks combined a scenario ofbroad cospeciation characters provide an objective means to test the existing on specific hosts with an assumption that ticks are a group of phylogeny and, in particular, afford a neutral background ancient derivation (5-8). Hoogstraal and earlier workers against which to examine the morphological characters and suggested that various structural modifications ofthe mouth- cospeciation mechanisms used by Hoogstraal. Objective parts and coxae were associated with specialization for phylogenies also permit examination of alternative modes of particular hosts. They noted that changes in these characters speciation not considered by Hoogstraal, including habitat The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank database (accession nos. L34292-L34330). 10034 Downloaded by guest on September 28, 2021 Evolution: Black and Piesman Proc. Natl. Acad. Sci. USA 91 (1994) 10035 40C. Negative controls (no template) were always run simul- birodo taneously and reaction mixtures were discarded when any Amblyol a DNA appeared in the negative control. Ixodkdan Amblyw_ I DNA Sequencing. Amplified DNA was sequenced directly Aponomma in all taxa. The amplified DNA was purified by Magic PCR H.mphylaIs Preps (Promega) according to manufacturer protocols and resuspended in 20 ,ul of 10 mM Tris HCl/1 mM EDTA, pH Hyalomma 8.0. Double-stranded DNA sequence was determined by cycle sequencing (fmol system; Promega). Six primers were used for sequencing. The original PCR Coamlomma primers were used for sequencing from the ends. In addition, two primers overlapping and complementary to the 16S-2 Noeomma and 16S+2 primers were designed: 16S+3 (5'-ATAC- TCTAGGGATAACAGCGT-3') and 16S - 3 (5'-AAAT- Rhipbephhlii TCATAGGGTCTTCTTGTC-3'). When the entire 460-bp Anomalohlmalaya fragment was amplified, six separate sequencing reactions Rhipientor were used to sequence over the entire length on both strands. When the region was amplified in two overlapping halves, Boophilue four primers were used to sequence over each half on both Maergaropue strands so that eight separate sequencing reactions were run NutidlSkda on one taxa. The 16S+1/16S-2 half was sequenced with NuttafiIliba these primers in addition to the 16S+2 and 16S-3 primers, whereas the 16S-1/16S+2 halfwas sequenced with the PCR Onhodorle Otob primers in addition to the 16S-2 and 16S+3 primers. Omitodoros Antrkola Sequence Alignments and Phylogenetic Inferences. Se- quences were read manually into a computer from autora- Nothoapla diographs using SEQAID II 3.6 (16). These were initially Arg" aligned using CLUSTALV (17). Nucleotides that were obvi- ously misaligned were manually shifted. Distance, maxi- ArgaSnae mum-parsimony, and maximum-likelihood methods were FIG. 1. Phylogeny offamilies, subfamilies, and genera of soft and used in phylogeny reconstruction. For distance analysis, a hard ticks proposed by Hoogstraal and Aeschlimann (6) and based on neighbor-joining tree (18) was generated from a Kimura morphology, life history, and host associations. two-parameter distance matrix (19) using MEGA (20) or PHYLIP 3.5C (21) with NEIGHBOR and DNADIST. Maximum- adaptation and vicariant speciation following biogeographi- parsimony analysis was performed with PHYLIP 3.5C using cal separation. DNAPARS. Support for derived phylogenies was examined with PHYLIP 3.5C using bootstrapping over 1000 replications. MATERIALS AND METHODS Maximum-likelihood analysis (22) was performed with PHYLIP 3.5C using DNAML. PCR Amplification. DNA was isolated by freezing and crushing individual ticks (14) in 60 ,ul of homogenization buffer (15). Amplification was initially