VARIABLE SUBSTITUTION RATES OF THE 18 DOMAIN SEQUENCES IN ARTEMIA HEMOGLOBIN

The Artemia hemoglobin is a dimer comprising two nine-domain covalent polymers in quaternary association. Each polymer is encoded by a gene representing nine successive globin domains which have different seque nces and are presumed to have been copied originally from a single-dom ain gene. Two different polymers exist as the result of a complete dup lication of the nine-domain gene, allowing the formation of either hom odimers or the heterodimer. The total population size of 18 domains co mprising nine corresponding pairs, coupled with the probability that t hey reflect several hundred million years of evolution in the same lin eage, provides a unique model in which the process of gene multiplicat ion can be analyzed. The outcome has important implications for the re liability of local molecular clocks. The two polymers differ from each other at 11.7% of amino acid sites; however when corresponding indivi dual domains are compared between polymers, amino acid substitution fl uctuates by a factor of 2.7-fold from lowest to highest. This variatio n is not obvious at the DNA level: Domain pair identity values fluctua te by 1.3-fold. Identity values are, however, uncorrected for multiple substitutions, and both silent and nonsilent changes are pooled. Ther efore, to determine the variability in relative substitution rates at the DNA level, we have used the method of Li (1993, J Mol Evol 36:96-9 9) to determine estimates of nonsynonymous (K-A) and synonymous (K-S) substitutions per site for the nine pairs of domains. As expected, the overall level of silent substitutions (K-S of 56.9%) far exceeded non silent substitutions (K-A of 6.7%); however, for corresponding domain pairs, K-A fluctuates by 2.3-fold and K-S by 1.7-fold. The large discr epancies reflected in the expressed protein have accrued within a sing le lineage and the implication is that divergence dates of different g enera based on amino acid sequences, even with well-studied proteins o f reasonable size, can be wrong by a factor well in excess of 2.