Gene 291 (2000) 115±125 www.elsevier.com/locate/gene Neutrality tests of conservative-radical amino acid changes in nuclear- and mitochondrially-encoded proteins David M. Rand*, Daniel M. Weinreich, Brent O. Cezairliyan Department of Ecology and Evolutionary Biology, Box G-W, 69 Brown Street, Brown University, Providence, RI 02912, USA Received 8 June 2000; received in revised form 26 September 2000; accepted 5 October 2000 Received by G. Bernardi Abstract The neutralist-selectionist debate should not be viewed as a dichotomy but as a continuum. While the strictly neutral model suggests a neutralist-selectionist dichotomy, the nearly neutral model is a continuous model spanning strict neutrality through weak selection (Ns , 1) to deterministic selection (Ns . 3). We illustrate these points with polymorphism and divergence data from a sample of 73 genes (31 mitochondrial, 36 nuclear genes from Drosophila, and six Arabidopsis data sets). In an earlier study we used the McDonald±Kreitman (MK) test to show that amino acid replacement polymorphism in animal mitochondrial genes and Arabidopsis genes show a consistent trend toward negative selection, whereas nuclear genes from Drosophila span a range from negative selection, through neutrality, to positive selection. Here we analyze a subset of these genes (13 Drosophila nuclear, ten mitochondrial, and six Arabidopsis nuclear) for polymorphism and divergence of conservative and radical amino acid replacements (a protein-based conservative-radical MK, or pMK, test). The distinct patterns of selection between the different genomes is not apparent with the pMK test. Different de®nitions of conservative and radical (based on amino acid polarity, volume or charge) give inconsistent results across genes. We suggest that segregating ®tness difference between silent and replacement mutations are more visible to selection than are segregating ®tness differences between conservative and radical amino acid mutations. New data on the variation among genes with different opportunities for positive and negative selection are as important to the continuum view of the neutralist-selectionist debate as is the distribution of selection coef®cients within individual genes. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Neutrality test; Mildly deleterious single nucleotide polymorphism; Neutral theory; Natural selection; Molecular evolution; Drosophila; mtDNA; McDonald±Kreitman test 1. Introduction neutral OR selected seems to have greatly overshadowed two crucial words in Kimura's statement: `great majority'. The controversial statement that ª¼the great majority of Just how much is a `great majority'? In an election, 75% of evolutionary changes at the molecular level¼are caused not the votes would be considered a landslide, but Kimura by Darwinian selection but by random drift of selectively would probably not agree that as many as 25% of substitu- neutral or nearly neutral mutantsº (Kimura, 1983, pg. xi) has tions are not neutral. Indeed, he goes on to say that ª¼only been the focal point of the long-running neutralist-selection- a minute fraction of changes at the DNA level are adaptive ist debate. While some evolutionists have taken this view as in nature¼º (Kimura, 1983, pg. xi). Here we argue that the a threat to the foundation of the Modern Synthesis, Kimura neutralist-selectionist debate is over because it is not a quali- clearly quali®es his statement in the next sentence by clar- tative, dichotomous problem. Rather, if the debate is to ifying that he does not deny the role of natural selection in continue it should focus on quantifying the relative sizes adaptive evolution. The issue of whether mutations are of the `great majority' of neutral substitutions and the `minute fraction' of adaptive DNA changes. In recent years the neutral theory has been subjected to a Abbreviations: N.I., Neutrality Index; pN.I., protein Neutrality Index; variety of tests using the growing database of DNA cN.I., Codon Neutrality Index; MK test, McDonald±Kreitman test; pMK sequences that have become available (Dispersion index, test, protein McDonald±Kreitman test; cMK, codon McDonald±Kreitman test; mtDNA, mitochondrial DNA HKA test, Tajima and Fu and Li tests, McDonald±Kreitman * Corresponding author. Tel.: 11-401-863-2890; fax: 11-401-863-2166. tests; see Kreitman and Akashi, 1995). For example, the E-mail addresses: [email protected] (D.M. Rand); dispersion index, or the ratio of the variance to the mean [email protected] (D.M. Weinreich). 0378-1119/00/$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S0378-1119(00)00483-2 116 D.M. Rand et al. / Gene 291 (2000) 115±125 number of substitutions between species is generally greater toward excess amino acid polymorphism (N:I: . 1 for 25/ than the neutral expectation of 1.0 (Gillespie, 1989; 1995; 31 data sets) while nuclear genes show a roughly normal Ohta, 1996). Among a sample of nuclear genes in Droso- distribution centered around neutrality (mean N:I: 1:2; phila, about half of them showed departures from the strict 15/36 data sets with N:I: . 1). We suggested that the low neutral models (Moriyama and Powell, 1996). For mito- recombination environment of mtDNA hinders the ®xation chondrial genes, about half of published data sets also depart of advantageous mutations as they arise on haplotypes from neutral expectations, generally in the direction of carrying accumulated deleterious mutations. In support of negative selection (Nachman, 1998; Rand and Kann, this argument is the observation that ®ve out of six genes 1998). Even silent sites have been shown to deviate from from Arabidopsis thaliana also show N:I: . 1; this plant is neutral evolution (Akashi, 1995; 1996). On balance then, a known to be highly sel®ng, resulting in low effective recom- `sizeable proportion' of the tests from recent data actually bination (but see Kuittinen and Aguade (2000)). reject strict neutral assumptions. Does this mean that the In principle, any two or more functionally distinct classes neutral theory is wrong, or that we just need more insightful of DNA changes can be subjected to the McDonald±Kreit- tests of departures from strict neutrality? (e.g. Kreitman, man test format. These lead logically to a variety of possible 1996; Ohta, 1996). Tests that focus on polymorphic ratio of ratios, or neutrality index (N.I.) values that are sequences sampled from natural populations (Tajima, effective for measuring selection (Sawyer and Hartl, 1992; 1989; Fu and Li, 1993) can `reject' neutrality if the sample Akashi, 1995; Nachman, 1998; Weinreich and Rand, 2000). is, in fact, not a truly random one. But as argued above, the Here we extend these studies by performing a conservative- issue of neutrality vs. non-neutrality will become secondary radical McDonald Kreitman tests (CRMK tests) on a subset to studies that allow one to characterize the distribution of of the genes analyzed in Weinreich and Rand (2000). Only selection coef®cients by placing an individual data set amino acid sequence data are considered, and amino acid somewhere on the continuum from strong purifying selec- changes are classi®ed as conservative or radical depending tion through neutrality to strong positive selection. on charge, volume or polarity (Zhang, 2000). Our intention Studies of polymorphism and divergence in DNA is to examine the relationship between the distribution of sequences allow one to translate empirical data into selec- selection coef®cients within versus between individual tion coef®cients. Following the predictions of Kimura genes. By contrasting the distributions of neutrality index (1983, pg., 44±45) and Sawyer and Hartl (1992), Akashi values based on protein sequences (pN.I. values) with (1995) pointed out that the ratio of polymorphism to diver- neutrality index values based on silent and replacement gence (rpd) scales monotonically with effective selection changes in codons (cN.I. values), we are asking the ques- coef®cient, Ns. High values of rpd are indicative of negative tion: Does polymorphism and divergence in protein selection and low values indicate positive selection. One can sequences reveal the same patterns of genome-speci®c extend this to the McDonald±Kreitman (MK) test and non-neutral evolution as for gene sequences at the DNA express this 2 £ 2 table as a ratio of ratios: rpd-replacement/rpd- level? From these analyses we seek to assess the relative silent (referred to as the Neutrality Index; Rand and Kann, strengths and directions of selection acting on nucleotide 1996). The intention of a neutrality index is to provide a changes with varying levels of functional constraint. A simi- measure of the direction and magnitude of a gene's depar- lar approach has proven effective in analyses of speci®c ture from neutral expectation (Rand and Kann, 1996). N.I. genes. In a study of MHC variation, Hughes et al. (1990) scales monotonically with selection: N:I: , 1 indicates an showed that non-synonymous changes exceeded synon- excess of amino acid ®xations, or positive selection; N:I: . ymous changes in the binding cleft of the molecule, suggest- 1 indicates an excess of amino acid polymorphisms, or ing overdominant selection. They went on to show that negative selection (Rand and Kann, 1996; Nachman, amino acid changes altering side-chain charge occurred 1998; Weinreich and Rand, 2000). One assumption that more frequently than by chance alone, further suggesting follows from Kimura's (1983) analyses is that one class of that that selection was acting to promote a diversity of nucleotide sites is strictly neutral (e.g. silent sites; but see charge pro®les among alleles at MHC (Hughes et al., Akashi, 1995; 1996). Empirically, it becomes relatively 1990). These types of comparisons seek to distinguish straightforward to use MK tests and N.I. values to place between the phenotypic effects of mutations that alter genes on the spectrum from negative to positive selection. codons in a messenger RNA, from mutations that alter Because the MK test focuses on the ratios of counts of amino acids in a protein.