EXPERIMENTAL-ANALYSIS OF VARIANCE FOR DNA HYBRIDIZATION .2. PRECISION

Using DNAs from the Virginia opossum (Didelphis virginiana), we estima ted the variance components for two classes of replicate hybrids: diff erent drivers matched to the same tracer and different homoduplexes ma de from tracers matched to identical drivers. A nested analysis of var iance (ANOVA) was used to partition total variance among four levels: Individuals, extracts, preparations, and different aliquots from the s ame preparation. The variance contributed by these levels depended on the kind of hybrid replicate (driver or tracer) and on the index of th ermal stability (T(mode), T(m), T50H, or Normalized Percentage Hybridi zation). For replicate drivers, significant variance contributions wer e made by (1) individuals to T(m), (2) extracts to T(mode) and NPH but not T(m), and (3) different preparations to NPH. The composite T50H m easure calculated from both T(m) and NPH revealed effects from both co nstituent indices. For replicate tracers, preparation error was the si ngle most consistent effect across all indices, followed by extract ef fects for those indices that incorporated a measure of percent hybridi zation (T50H, NPH). Total variance of the four indices was qualitative ly similar for both drivers and tracers: T(mode) ranked lowest, follow ed in order by T(m), T50H, and NPH, with the variance of NPH being as much as 100 times greater than for T(mode). These results provide guid elines for the design of experiments to generate DNA hybridization-bas ed phylogenies and to assess their robustness with bootstrapping. Repl icate drivers for a distance matrix based on T(m) should use different individuals, whereas one based on T(mode) could minimally use differe nt extracts from the same individual. Thus, T(mode) may be the index o f choice for DNA hybridization experiments when material, time, and mo ney are limited.