Evolutionary EST analysis identifies rapidly evolving male reproductive proteins in Drosophila

Abstract

Sequence comparisons of genomes or expressed sequence tags (ESTs) from related organisms provide insight into functional conservation and diversification. We compare the sequences of ESTs from the male accessory gland of Drosophila simulans to their orthologs in its close relative Drosophila melanogaster, and demonstrate rapid divergence of many of these reproductive genes. Nineteen ( approximately 11%) of 176 independent genes identified in the EST screen contain protein-coding regions with an excess of nonsynonymous over synonymous changes, suggesting that their divergence has been accelerated by positive Darwinian selection. Genes that encode putative accessory gland-specific seminal fluid proteins had a significantly elevated level of nonsynonymous substitution relative to nonaccessory gland-specific genes. With the 57 new accessory gland genes reported here, we predict that approximately 90% of the male accessory gland genes have been identified. The evolutionary EST approach applied here to identify putative targets of adaptive evolution is readily applicable to other tissues and organisms.

Figure 1

Differential hybridization of the EST library with ³²P-labeled cDNA from wild-type males (WT) and DTA-E males (both D. melanogaster), the latter having no main cell accessory gland genes expressed. Row 1 shows a sample EST clone that hybridizes strongly with the male wild-type probe but not the DTA-E probe, and is thus a candidate Acp. Rows 2 and 3 show example EST clones with equal (strong or weak, respectively) hybridization to wild type and DTA-E, and thus cannot be identified as potential Acps by this method.

Figure 2

The number of nonsynonymous substitutions per nonsynonymous site (d_N) plotted against the number of synonymous substitutions per synonymous site (d_S) for the D. simulans accessory gland EST library (A), and random nonreproductive proteins (B) compared with D. melanogaster genomic sequence. ● are putative Acps (by differential hybridization or containing signal sequences), whereas ○ cannot be identified as putative Acps by these methods. The line shows the neutral expectation of d_N = d_S. Data for the lower panel are from Moriyama and Powell (44). d_N and d_S were estimated by the maximum likelihood method (50, 51). Similar results are obtained by using the method of Nei and Gojobori (53).