Bacteria belonging to the genus Wolbachia are associated with a variety of reproductive anomalies in arthropods. Allele-specific polymerase chain reaction (= Standard PCR) routinely has been used to amplify Wolbachia DNA from arthropods. While testing the two-spotted spider mite Tetranychus urticae and other arthropods known to be infected with Wolbachia, Standard PCR frequently produced false negatives, perhaps because the DNA from the arthropod host interfered with amplification by Taq DNA polymerase. Long PCR, which uses two enzymes (Taq and Pwo), consistently amplified Wolbachia DNA and a sensitivity analysis indicated that Long PCR was approximately six orders of magnitude more sensitive than Standard PCR in amplifying plasmid DNA spiked into insect genomic DNA. A survey indicated that 76% of sixty-two arthropod species and two subspecies in thirteen orders tested positive for the Wolbachia wsp sequence by Long PCR, which is considerably higher than the rate of 16.9% obtained previously for the ftsZ sequence using Standard PCR (Werren, J.H., Windsor, D. and Gao, L. (1995a) Proc R Soc Lond B 262: 197-204). A subsample of Long PCR products from fourteen arthropod species and two subspecies were sequenced, both directly and after cloning. Two A- and eleven B-Wolbachia strains were detected and their wsp sequences displayed a maximum of 23.7% sequence divergence at this locus. Two new groups (named Fus and Ten) were identified in addition to nineteen reported earlier (Zhou, W., Rousset, F. and O'Neill, S.L. (1998) Proc R Soc Lond B 265: 1-7; van Meer, M.M.M., Witteveldt, J. and Stouthamer, R. (1999) Insect Mol Biol 8: 399-408), because they displayed more than 2.5% sequence divergence from other Wolbachia wsp sequences. PCR products from seventeen of twenty-nine (59%) arthropod species analysed could not be sequenced directly due to apparent infection by multiple Wolbachia strains. The wsp sequences cloned from two such species (Plutella xylostella and Trichoplusia ni) indicated both A- and B-Wolbachia were present in a single individual. Hence, superinfection also may be more widespread than the 1.2% incidence previously estimated.

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Spiders, scorpions, mites and ticks (chelicerates) form one of the most diverse groups of arthropods on land, but their origin and times of diversification are not yet established. We estimated, for the first time, the molecular divergence times for these chelicerates using complete mitochondrial sequences from 25 taxa. All mitochondrial genes were evaluated individually or after concatenation. Sequences belonging to three missing genes (ND3, 6, and tRNA-Asp) from three taxa, as well as the faster-evolving ribosomal RNAs (12S and 16S), tRNAs, and the third base of each codon from 11 protein-coding genes (PCGs) (COI-III, CYTB, ATP8, 6, ND1-2, 4L, and 4-5), were identified and removed. The remaining concatenated sequences from 11 PCGs produced a completely resolved phylogenetic tree and confirmed that all chelicerates are monophyletic. Removing the third base from each codon was essential to resolve the phylogeny, which allowed deep divergence times to be calculated using three nodes calibrated with upper and lower priors. Our estimates indicate that the orders and classes of spiders, scorpions, mites, and ticks diversified in the late Paleozoic, much earlier than previously reported from fossil date estimates. The divergence time estimated for ticks suggests that their first land hosts could have been amphibians rather than reptiles. Using molecular data, we separated the spider-scorpion clades and estimated their divergence times at 397 +/- 23 million years ago. Algae, fungi, plants, and animals, including insects, were well established on land when these chelicerates diversified. Future analyses, involving mitochondrial sequences from additional chelicerate taxa and the inclusion of nuclear genes (or entire genomes) will provide a more complete picture of the evolution of the Chelicerata, the second most abundant group of animals on earth.

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