Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Activity and flexibility of alcohol dehydrogenase in organic solvents

The oxidation of cinnamyl alcohol to cinnamaldehyde by horse liver alcohol dehydrogenase (LADH) was carried out in nearly anhydrous organic solvents and in solvents containing from 0.1 to 10% added water. In nearly anhydrous solvents containing less than 0.02% water, the oxidation rate increased as the water solubility in the solvent decreased, but the reaction did not require active LADH. Moreover, the highest activity in nearly anhydrous heptane was obtained by lyophilizing the enzyme from a solution of pH 2.0, even though LADH exhibits virtually no enzymatic activity in water at this pH. The catalytic activity of LADH was restored and increased dramatically as small amounts of water were added to each solvent. In conjunction with the activity measurements, electron paramagnetic resonance (EPR) spectroscopy and two active-site directed spin labels were used to examine solvent-dependent structural features of LADH. The EPR spectra indicated that LADH became more rigid as the dielectric constant of the solvent decreased. The degree of rigidity also depended on the pH from which the enzyme was lyophilized, indicating that the ionization state of the enzyme can have an important influence on its dynamics in organic solvents. Finally, adding 1% water to organic solvents had no apparent effect on the enzyme's conformation or flexibility near the spin label, even though enzyme activity was an order of magnitude higher when 1% water was present.