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5) cultures grown from single eggs or single individuals after 10 serial passages through sterile water in microtitre wells. Culture conditions and DNA extraction procedures are described elsewhere (21, 36). DNA was extracted from a single colony of S. socialis and from single field collections of E. ehrenbergi and S. nebaliae (10). Primer sequences and PCR protocols are available from the authors or at Science Online at www.sciencemag.org/feature/data/1050064.shl. Each sequence found was represented in at least five cloned amplicons from at least two amplifications, except hsp82 copy 2 of P. roseola, which was found only in the cosmid library. Occasionally, a single PCR clone differed from a set of otherwise identical clones at one or two sites, which could be attributed to an error rate of about 0.0003 per nucleotide, similar to the error rate reported in other PCR cloning experiments (37, 38). The hsp82 region in 36 phage lambda clones and 25 cosmid clones selected from genomic libraries of P. roseola was also sequenced.
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0342335911
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note
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DNA sequences were aligned (10) and analyzed with the Wisconsin Package 10.0 (Genetics Computer Group) and the PHYLIP v3.57 package of J. Felsenstein. Uncorrected difference at fourfold degenerate sites (D4) was determined from pair-wise comparisons with DIVERGE. Phylogenetic analyses used all codon third positions belonging to conserved fourfold or threefold degenerate codon classes plus third positions in those few cases where a codon in a single bdelloid sequence differed from a fourfold or threefold degenerate class by no more than a single substitution. Distances for neighbor-joining were adjusted for multiple mutations with the Kimura two-parameter model with rates following a gamma distribution with a coefficient of variance (CV) of 1.41 estimated for both genes from the maximum likelihood algorithm in PAUP*. The same tree topologies, with similar bootstrap support, are found with a range of CV.
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44
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0343641107
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note
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We thank E. Lemyre and D. Kunimoto for assistance in early stages of this work; I. Arkhipova, J. Crow, G. Giribet, J. Mark Welch, B. Normark, and F. Stahl for critically reading the manuscript; and the Eukaryotic Genetics Program of the NSF for consistent support D.M.W. was supported by an NSF Graduate Research Grant. This article is dedicated to G. Evelyn Hutchinson. GenBank accession numbers for the reported sequences are AF143849 to AF143858 and AF249985 to AF250004.
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