Hydrogen isotopic analysis of organic materials has been widely applied in studies of paleoclimate, animal migration, forensics, food and flavor authentication, and the origin and diagenesis of organic matter. Hydrogen bound to carbon (C-H) generally retains isotopic information about the water present during organic matter synthesis and associated biosynthetic fractionations, but hydrogen bound to other elements (O, S, or N) can readily exchange with atmospheric water vapor and reflects recent exposure to water or vapor. These two pools must be separated to obtain meaningful information from isotope ratios of organic materials. Previously published analytical methods either replace exchangeable H chemically or control its isotopic composition, usually by equilibration with water or waters of known isotopic composition. In addition, the fraction of H that is exchangeable can vary among samples and is itself of scientific interest. Here we report an improved and automated double-equilibration approach.Samples are loaded in a 50-position autosampler carousel in an air-tight aluminum equilibration chamber. Water vapor of known isotopic composition is pumped through the chamber at 115 degrees C for at least 6 h. After flushing with dry N(2) and being cooled, the carousel is rapidly transferred from the equilibration chamber to a He-purged autosampler attached to a pyrolysis elemental analyzer connected to an isotope ratio mass spectrometer. By equilibrating two aliquots of each sample with two isotopically distinct waters, it is possible to calculate both (1) the D/H ratio of non-exchangeable H, and (2) the fraction of H that is exchangeable. Relative to previous double-equilibration techniques, this approach offers significant reductions in sample size and labor by allowing simultaneous equilibration of several tens of samples.

Plant molecular systematic studies of closely related taxa have relied heavily on sequence data from nuclear ITS and cpDNA. Positive attributes of using ITS sequence data include the rapid rate of evolution compared to most plastid loci and availability of universal primers for amplification and sequencing. On the other hand, ITS sequence data may not adequately track organismal phylogeny if concerted evolution and high rDNA array copy number do not permit identification of orthologous copies. Shaw et al. (American Journal of Botany 92: 142-166) recently identified nine plastid regions that appear to provide more potentially informative characters than many other plastid loci. In the present study, sequences of these loci and ITS were obtained for six taxonomic groups in which phylogenetic relationships have been difficult to establish using other data. The relative utility of these regions was compared by assessing the number of parsimony informative characters, character congruence, resolution of inferred trees, clade support, and accuracy. No single locus emerged as the best in all lineages for any of these measures of utility. Results further indicated that in preliminary studies, sampling strategy should include at least four exemplar taxa. The importance of sampling data from independent distributions is also discussed.

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