To assess whether protein sequences were actively transcribed, we employed several methods. Numerous promoter-prediction programs were used, but none was sufficiently consistent across species or discriminatory to be useful in the prediction of pseudogenes. The ratio of non-synonymous to synonymous (Ka/Ks) nucleotidesubstitution rates was used as a measure of selective pressure on each individual gene. Rates were calculated using homologous genes for all species in the current analysis, in order to determine ancestral states using the Bergen Center Ka/Ks Calculation Tool (http://services.cbu.uib.no/tools/kaks/) and default values, with the exception that the tree method was set to maximum likelihood [17].
