TWO cypripedioid orchid genera, Paphiopedilum and Phragmipedium, are listed in Appendix I of CITES and are restricted from international trade. Because of their morphological similarity to other genera, however, they may be disguised as belonging to one of the other cypripedioids listed along with other orchids in Appendix II of CITES. Sequence analysis was performed on the internal transcribed spacer region (ITS) of ribosomal DNA of cypripedioid orchids to develop a molecular marker system capable of discriminating among rare species in trade. Molecular analyses concentrated on rare cypripedioid orchids from the genera Paphiopedilum and Phragmipedium, which are known to be poached from the wild and smuggled across international borders disguised as common species. A total of 48 taxa representing two genera (Paphiopedilum, N = 43; Phragmipedium, N = 5) have been sequenced and compared for distinctiveness. Phylogenetic analyses clearly distinguish between these two genera and among other cypripedioid genera, with 5-10 fixed nucleotide differences reported between genera. Within a genus, sections of closely related taxa are recoverable in phylogenetic analyses, in most cases, with low sequence divergence within sections. ITS sequences available in GenBank have been aligned with data generated for this project, resulting in a comprehensive sequence library of 151 sequences representing all genera of cypripedioid orchids: 70 Paphiopedilum taxa, 16 Phragmipedium taxa, and 14 Cypripedium taxa, as well as representatives from Selenipedium and the monotypic genus Mexipedium (Phragmipedium) xerophyticum. Additionally, several organelle intron regions have been screened for variation among genera and species. Both the chloroplast trnS-M and the mitochondrial NAD1 intron regions, which varied between genera in nucleotide substitutions and indels, hold promise for increasing ability to distinguish between these orchids. The set of DNA markers examined for this project are diagnostic of these genera, appear to be robust, and are suitable for rapid assay to avoid unnecessary complication in the legitimate trade of orchids listed in CITES Appendix II.
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