The long-term objectives of this research are to eliminate the confusion surrounding interpretation of the I. ricinus complex and in general to identify molecular markers that may be used in a broader sense to reduce ambiguities in classifications above the species level, especially with regards to species complexes and subgenera of ticks and other hematophagous arthropods. This research will use mitochondrial and nuclear DNA molecular markers (e.g., 16s, 12s, COI, COII, EF1alpha, and beta-tubulin) to test the genetic boundaries of the Ixodes ricinus species complex, especially those species found in the nearctic (I. scapularis, I. pacificus, I. affinis, I. jellisoni), and palearctic (I. ricinus, I. persulcatus) faunal regions that are known to play a role in the transmission of the Lyme disease agent, Borrelia burgdorferi s.l. In addition to the other 8 species currently considered to be part of the complex, we will also examine other closely related Ixodes species (other members of the subgenus Ixodes) that appear to be vectors. These latter species include I. dentatus, I. minor, I. muris, I. spinipalpus, and may also include other species such as I. cookei and I. holocyclus. Outgroups will consist of other members of the subgenus Ixodes, members of other prostriate (Ixodes) subgenera, and representative species of metastriates. Mitochondrial and nuclear DNA will be initially examined using direct sequencing of genes amplified by the polymerase chain reaction (PCR). Our working hypotheses are that ticks in the I. ricinus complex form a closely related monophyletic group and that vector capacity for Borrelia burgdorferi evolved once in the genus Ixodes, such that vector capacity is governed by genetic factors limited to a single phylogenetic group such as, but not limited to, the I. ricinus complex. Therefore, the Specific Aims for this proposal are 1) to examine phylogenetic relationships among species in the I. ricinus complex, 2) to test for monophyletic relationships among Ixodes species known to transmit Borrelia burgdorferi, and 3) to elucidate markers useful for molecular taxonomic identification Borrelia burgdorferi vectors.