biases in codon usage and transition/transversion rates are substantially more reliable than those that do not. Limitations Although the Ka/Ks ratio is a good indicator of selective pressure at the sequence level, evolutionary change can often take place in the regulatory region of a gene which affects the level, timing or location of gene expression. Ka/Ks analysis will not detect such change. It will only calculate selective pressure within protein coding regions. In addition, selection that does not cause differences at an amino acid level—for instance, balancing selection—cannot be detected by these techniques. Another issue is that heterogeneity within a gene can make a result hard to interpret. For example, if Ka/Ks = 1, it could be due to relaxed selection, or to a chimera of positive and purifying selection at the locus. A solution to this limitation would be to apply Ka/Ks analysis across many species at individual codons. The Ka/Ks method requires a rather strong signal in order to detect selection. In order to detect selection between lineages, then the selection, averaged over all sites in the sequence, must produce a Ka/Ks greater than one—quite a feat if regions of the gene are strongly conserved. In order to detect selection at specific sites, then the Ka/Ks ratio must be greater than one when averaged over all included lineages at that site—implying that the site must be under selective pressure in all sampled lineages. This limitation can be moderated by allowing the Ka/Ks rate to take multiple values across sites and across lineages; the inclusion of more lineages also increases the power of a sites-based approach. Further, the method lacks the capability to distinguish between positive and negative nonsynonymous substitutions. Some amino acids are chemically similar to one another, whereas other substitutions may place an amino acid with wildly different properties to its precursor. In most situations, a smaller chemical change is more likely to allow the protein to continue to function, and a large chemical change is likely to disrupt the chemical structure and cause the protein to malfunction. However, incorporating this into a model is not straightforward as the relationship between a nucleotide substitution and the effects of the modified chemical properties is very difficult to determine. An additional concern is that the effects of time must be incorporated into an analysis, if the lineages being compared are closely related; this is because it can take a