Polymorphic and Fixed Differences in the Mel, Sim, and Yak Lineages

Polymorphic and fixed differences in the mel, sim, and yak lineages

In D. melanogaster and D. simulans, five alleles were available for eight of the 22 genes (the sets of genes overlap but are not identical). Accession numbers for these data are given in supplemental Table 1. Two outlier genes, Osbp and AP-50, that were excluded in the single allele study (see below) were also excluded from the polymorphism analysis. Two alleles at each of 18 genes were examined (Amy-p was excluded from the analysis because orthology was difficult to determine between the duplicated loci). Homology searches to the assembled D. yakuba genome did not reveal close paralogs for any of the 22 genes [except Adh which matches the recently evolved jgw gene (LONG and LANGLEY 1993)]. Because ancestral state reconstruction under BASEML requires a single gene tree (no recombination), we assigned polymorphic codons to bifurcated lineages. Analyses of polymorphism data were conducted separately for D. melanogaster, D. simulans, and D. yakuba on trees with seven extant nodes. These trees assume bifurcation of a lineage following its split with its sibling species. For D. yakuba, the two alleles for each gene were assumed to have diverged following the split between the D. yakuba and D. teissieri lineages. For analysis of greater than two alleles, polymorphic codons were “collapsed” onto the two sequences resulting from the bifurcation. Codons that were identical among alleles were assigned to sequences at both nodes. For aligned positions where two different codons were segregating among the five alleles, the two codons were assigned randomly to the two sequences. Codons polymorphic for three or more states were excluded from the analysis. Information from the site frequency spectrum of mutations is not employed in this method of ancestral state reconstruction. Reconstructed changes assigned to a lineage prior to the bifurcation were considered fixed in the sample, and those that were assigned to either of the within-species lineages were considered polymorphic. Note that the term “fixed” difference will be used to refer to changes inferred to be fixed in the sample of alleles. Such mutations may be at high frequency, but not fixed, in the population. The performance of this method of reconstructing ancestral states within species was not examined. The mel lineage showed the strongest decline of MCU in the single allele analysis and similar pu:up ratios on pre- and post-MRCA parts of the lineage are consistent with a lack of translational selection. If silent changes are pooled and compared to protein changes, there is a marginally significant difference in ratios of polymorphism to divergence, rpd, which can be interpreted as either an excess of synonymous polymorphism or of fixed amino acid differences. Support for neutral silent evolution suggests that the pattern reflects adaptive protein evolution. The main contributors to the excess of replacement fixations are genes for which evidence for adaptive protein evolution has been noted previously: Amy-p (ARAKI et al. 2001) and Zw (EANES et al. 1993). When only fixed differences in mel are considered, all six genes that show a difference in WS:SW ratios for replacement changes show dWS,SW < 0. In the sim lineage, greater pu:up ratios for polymorphic than for fixed changes are consistent with previous findings (AKASHI 1995; 1999) and support the maintenance of near-equilibrium codon bias under weak selection. The contrast between silent and protein evolution is similar to that observed in mel; rpd is higher for pooled silent changes than for replacement mutations (the difference is only marginally statistically significant). However, in this lineage, selection at silent sites must be considered in the interpretation of the MCDONALD-KREITMAN test (1991). Slightly deleterious unpreferred changes contribute disproportionately to polymorphism resulting in elevated rpd for silent sites relative to neutral expectations (AKASHI 1995, 1999). This can lead - 2 - to false positives for adaptive protein evolution, but the effect appears to be small for genes with intermediate MCU values (AKASHI 1999). Thus, adaptive protein evolution may also have been prevalent in the sim lineage. Note, however, that almost all the inferred replacement fixations occurred at Zw; this acceleration of protein evolution on the sim lineage was noted by EANES et al. (1996). In the yak lineage, findings are limited by analysis of only two alleles per gene. pu:up ratios are higher for polymorphic than for fixed mutations, but the difference is not statistically significant. rpd is similar for pooled silent sites and amino acid changes, but no firm conclusions can be drawn from these small sample sizes. Interestingly, among replacement fixations, all seven genes that show differences in the numbers of inferred WS and SW changes show an excess of WS fixations; dWS,SW may deviate in opposite directions for silent and replacement changes in this lineage.

Supplemental Table 11. Polymorphic and fixed changes in the D. melanogaster subgroup

# silent replacement lin codons tot up pu dup,pu tot WS SW dWS,SW mel 3267 poly 106.3 13.7 80.7 -0.71 13.0 2.1 7.9 -0.59 fix 98.6 9.4 61.4 -0.74 25.1 1.3 13.8 -0.83

rpd 1.1 1.5 1.3 0.5 sim 3260 poly 162.0 34.9 89.0 -0.44 21.3 7.2 6.7 0.04 fix 53.5 19.4 21.5 -0.05 15.8 3.1 7.2 -0.40

rpd 3.0 1.8 4.1 1.3 yak 9048 poly 173.2 35.1 102.6 -0.49 22.9 6.8 6.1 0.05 fix 270.0 73.6 140.2 -0.31 30.5 14.6 7.6 0.31

rpd 0.6 0.5 0.7 0.8

Inferred silent (preferred and unpreferred) and replacement changes were partitioned into poly (polymorphic) and fix (fixed) differences (see text for method). Numbers of codons and changes are summed across five alleles of eight genes for mel and sim and two alleles of 18 genes in yak. rpd, the ratio of the numbers of polymorphic and fixed differences, is shown for classes where #poly + #fixed > 25.