Methods for identifying species by using short orthologous DNA sequences, known as ''DNA barcodes,'' have been proposed and initiated to facilitate biodiversity studies, identify juveniles, associate sexes, and enhance forensic analyses. The cytochrome c oxidase 1 sequence, which has been found to be widely applicable in animal barcoding, is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals. We therefore propose the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as potentially usable DNA regions for applying barcoding to flowering plants. The internal transcribed spacer is the most commonly sequenced locus used in plant phylogenetic investigations at the species level and shows high levels of interspecific divergence. The trnH-psbA spacer, although short (Ϸ450-bp), is the most variable plastid region in angiosperms and is easily amplified across a broad range of land plants.Comparison of the total plastid genomes of tobacco and deadly nightshade enhanced with trials on widely divergent angiosperm taxa, including closely related species in seven plant families and a group of species sampled from a local flora encompassing 50 plant families (for a total of 99 species, 80 genera, and 53 families), suggest that the sequences in this pair of loci have the potential to discriminate among the largest number of plant species for barcoding purposes.angiosperm ͉ internal transcribed spacer ͉ Plummers Island ͉ species identification ͉ trnH-psbA

Sister species separated by the Isthmus of Panama have been widely used to estimate rates of molecular evolution. These estimates are based on the assumption that geographic isolation occurred nearly simultaneously for most taxa, when connections between the Caribbean and eastern Paci¢c closed approximately three million years ago. Here we show that this assumption is invalid for the only genus for which many taxa and multiple genetic markers have been analysed. Patterns of divergence exhibited by allozymes and the mitochondrial COI gene are highly concordant for 15 pairs of snapping shrimp in the genus Alpheus, indicating that they provide a reasonable basis for estimating time since cessation of gene £ow. The extent of genetic divergence between pairs of sister species varied over fourfold. Sister species from mangrove environments showed the least divergence, as would be expected if these were among the last habitats to be divided. Using this pair yields a rate of sequence divergence of 1.4% per one million years, with implied times of separation for the 15 pairs of 3^18 million years ago. Many past studies may have overestimated rates of molecular evolution because they sampled pairs that were separated well before ¢nal closure of the Isthmus.