Levels of DNA polymorphism and divergence yield important insights into evolutionary processes.

A recent study of Eanes et al. (1) exemplifies an approach that promises to go some way toward resolving a longstanding question in population genetics. The question is: To what extent is intraand interspecific variation in DNA and protein sequences due to molecular noise? The molecular noise hypothesis, more commonly referred to as the neutral theory, maintains that most molecular polymorphism within species and most molecular divergence between species are a consequence of neutral mutations, that is, mutations resulting in functionally equivalent variants, that randomly drift in populations and gradually accumulate over evolutionary time. The alternative to the neutral theory is that most variation within populations is maintained by various forms ofbalancing selection and/or that the molecular differences between species reflect adaptive changes shaped by natural selection. Ultimately, the issue is a quantitative question; that is, we would like to know how much variation within species and what proportion of changes between species can be attributed to natural selection as opposed to genetic drift of functionally equivalent variants. At present, however, it is not even possible to reject either of the extreme positions (for reviews, see refs. 2 and 3). It should be made clear, however, that both sides in this neutralist/selectionist controversy accept the importance of natural selection as a conservative force, maintaining certain molecular features, by eliminating strongly deleterious mutations quickly after they arise. The conservation of many protein coding sequences and regulatory sequences is strong evidence in support of this aspect of the process. Over the last 25 years, this controversy has been stewing, with the input of data coming mainly from surveys of enzyme polymorphism and sequencing of proteins. Statistical analysis ofenzyme polymorphism data has been useful with respect to this controversy in some cases (for example, see ref. 4) but on the whole has not been very effective in resolving the issue. The power of these analyses is apparently just not up to the task. The clock-like behavior of the amino acid substitution process, as revealed by comparisons of protein sequences from different species, has been used as support for the neutral model. However, careful examination of the data suggests that the substitution process at many loci is much too variable to be compatible with the usual simple versions of the neutral model (3). More complex neutral models have been proposed to explain this property of the substitution process and these models are being debated and contrasted with alternative models that incorporate selection (5-7). With recent advances inDNA technology, reasonably large surveys of variation at the DNA level are possible. For example, Eanes et al. (1) sequenced 32 copies of the G6pd gene sampled from natural populations of Drosophila melanogaster and 12 copies of the gene from Drosophila simulans. Such surveys of DNA variation provide detailed information that can be used in very powerful tests of the neutral model. In particular, the DNA data provide information about the amount of divergence between different copies of the gene sampled from the same species. This divergence within species, or polymorphism, can be directly compared to the level of divergence between copies obtained from different species. In addition, one can compare the levels of divergence within and between species for different kinds of nucleotide sites. For example, for protein coding loci, one can compare the variation at first, second, and third positions of codons. The neutral model makes some very specific predictions about data of this form, and these predictions lead to powerful tests of the model. McDonald and Kreitman (8) have proposed such a test and applied it to data from the Adh locus. Eanes et al. (1) have now used this test to analyze their sequence data. The test ofMcDonald and Kreitman (8) contrasts the level of amino acid divergence and polymorphism with the level of divergence and polymorphism which is silent. (Nucleotide sequence changes that do not alter the protein sequence are referred to as silent changes.) The test is basically a 2 x 2 test of independence in which the four observations employed are (i) the number of "fixed" amino acid replacements between species, (ii) the number of "faed" silent changes between species, (iii) the number of amino acid polymorphisms within species, and (iv) the number of silent polymorphisms within species. A "fixed" substitution is a mutation that has resulted in all sampled sequences from one species differing from all sampled sequences from another species. (The quotes indicate that "fixed" in this case is only with respect to the particular sample obtained, and another sample might show that what was considered "fixed" in the first sample is a polymorphism in the second sample.) The rows in the 2 x 2 table would be labeled "fixed" and "polymorphic within species," and the columns would be labeled "amino acid" and "silent. " These observed numbers correspond to mutations that have occurred on different parts of the gene genealogy, or gene tree, that represents the history ofthe sampled genes. To see this, it is useful to consider an example. Consider a hypothetical data set consisting of the sequences of four copies of a protein coding locus, three copies being obtained by sampling from species A and one copy obtained from species B. [Unlike this example, the data set ofEanes et al. (1) consists of multiple copies from each species. ] For simplicity, let us assume that no recombination occurs within the locus. In this case, the genealogical history of these sampled sequences can be represented by a simple tree, such as that drawn in Fig. 1. It is important to distinguish between this genealogy, which represents the history of a small sample of copies of the gene, and a tree drawn to represent the phylogenetic history of a set of species. In the gene genealogy, or gene tree, each line represents a single lineage, and any point on a line corresponds to a single copy of the gene that is ancestral to one or more sampled copies. In contrast each line of a phylogenetic tree represents a sequence of many generations of an entire population, and a point on the line corresponds to an entire population. The variation that is observed in this hypothetical sample is necessarily due to mutations that occurred along the lineages of the gene tree. On the gene tree in