BRIEF COMMUNICATIONS Evolution, 56(1), 2002, pp. 191–198 MOLECULAR CORRELATES OF REPRODUCTIVE ISOLATION BENJAMIN M. FITZPATRICK Population Biology Graduate Group, Section of Evolution and Ecology, University of California, Davis, California 95616 E-mail: benfitz@ucdavis.edu Abstract. Evolution of reproductive isolation as a byproduct of genetic divergence in isolated populations is the dominant (albeit not exclusive) mode of speciation in sexual animals. But little is known about the factors linking speciation to general divergence. Several authors have argued that allopatric speciation should proceed more rapidly if isolated populations also experience divergent selection. Reproductive isolation between allopatric populations is not subject to direct selection; it can accumulate only by random drift or as a fortuitous byproduct of selection on other traits. Here I present a novel analysis of published data, demonstrating that pre- and postmating isolation of Drosophila species are more tightly correlated with allozyme divergence than with silent DNA divergence. Inasmuch as proteins are more subject to the action of natural selection than are silent DNA polymorphisms, this result provides broad support for a model of selection-mediated allopatric speciation. Key words. Allozymes, DNA hybridization, Drosophila, genetic distance, speciation, synonymous substitutions. Received November 21, 2000. Several authors have demonstrated that reproductive isolation is correlated with genetic divergence between species (e.g., Ayala et al. 1974; Parker et al. 1985; Coyne and Orr 1989, 1997; Tilley et al. 1990; Knowlton et al. 1993; Gleason and Ritchie 1998; Sasa et al. 1998). Such a correlation is to be expected as both kinds of divergence should accumulate over time (Muller 1942). Indeed, these studies have generally conceived of molecular genetic divergence as a proxy for divergence time and interpreted their results as demonstrating gradual evolution of reproductive isolation. But we are well aware that although some classes of molecular markers may evolve according to a stochastic clock model, others are more subject to natural selection and may evolve episodically at different rates in different lineages (Gillespie 1991). Ideally, we would like to control for time and see if there is any residual correlation between reproductive isolation and certain kinds of molecular divergence. A residual correlation could arise if both kinds of divergence are accelerated by positive selection and/or retarded by stabilizing selection. That is, episodes of adaptive divergence or population bottlenecks could have concordant influences on nonneutral molecular divergence and reproductive isolation. Conventional wisdom has it that mutations causing changes in amino acid sequences of translated products are far more ‘‘visible’’ to selection than synonymous mutations that produce no change at the protein sequence level (Gillespie 1991; Futuyma 1998). Thus, synonymous divergence is expected to be more clock-like than amino acid divergence. Even the architects of the neutral theory of molecular evolution agreed that amino acid substitutions are more likely to be caused by selection than silent substitutions (Ohta 1972; Kimura 1983). This difference has been supported by several empirical analyses (Gillespie 1991; Golding 1994). DNA sequence analyses provide some of the best evidence that amino acid substitutions are more sensitive to selection than are silent substitutions (McDonald and Kreitman 1991; Aguade et al. 1992; Eanes et al. 1993). On the other hand, silent sequence variation and DNA hybridization divergence (which includes Accepted September 4, 2001. noncoding sequence divergence) appear to conform more closely to predictions of the neutral theory (Gillespie 1991; Ohta 1995; but see Zeng et al. 1998). In this paper, I use DNA divergence from a variety of sources as a proxy for time and divergence in electrophoretically detectable enzyme allele (allozyme) frequencies as a proxy for divergence that could be affected by selection. Once DNA divergence is taken into account, I evaluate the residual correlation between reproductive isolation and allozyme divergence for Drosophila species-pairs. Some authors have cautioned against conflating species differences with speciation (Templeton 1981; Wu and Hollocher 1998). Reproductive isolating mechanisms are a particularly important and interesting class of species differences with a significant and unique relationship to speciation. Differences that cause reproductive isolation cause speciation (species concepts differ only how they allow other kinds of differences to determine species status). Following Wu and Hollocher (1998) and Coyne and Orr (1998), I am satisfied that, by learning about the evolution of reproductive isolation, I am learning something of general importance to processes of speciation. The notion that natural selection can accelerate allopatric speciation was adumbrated during the modern synthesis (Dobzhansky 1937; Mayr 1942; Muller 1942), and has been made more explicit by recent authors (Rice and Hostert 1993; Rosenzweig 1995; Orr and Smith 1998; Schluter 1998). A model of selection-mediated geographic speciation enjoys some empirical support (Rice and Hostert 1993; Funk 1998), but its theoretical basis has rarely been discussed. Mayr (1970) asserted that most ecological adaptation involves several traits, most traits are polygenic, and most genes are pleiotropic. Therefore any response to selection may reverberate through large portions of the genome and affect some loci with pleiotropic effects on reproductive isolation. That is, the footprint of divergent selection may be found in a large number of genes and in the degree of intrinsic reproductive isolation between taxa. By the same token, purifying selection 191 q 2002 The Society for the Study of Evolution. All rights reserved. 192 BRIEF COMMUNICATIONS maintaining function in complex metabolic and developmental pathways ought to work against the accumulation of intrinsic postzygotic isolation. These propositions, rooted in the idea that organisms are composed of coadapted gene complexes (Dobzhansky 1937), lead to the prediction that the rate of evolution of reproductive isolation should be more tightly correlated with functional protein divergence than silent DNA divergence. In addition to pleiotropy, hitchhiking due to physical linkage and linkage disequilibria will contribute to concordance in evolutionary rates (Rice and Hostert 1993). Pleiotropic premating isolation is an obvious consequence of adaptive changes in reproductive phenology and habitat choice (Rice 1984; Rice and Salt 1988, 1990; Feder 1998; Funk 1998. Pleiotropic postzygotic isolation will automatically result if ecologically intermediate hybrids find themselves unable to make a living (Rice and Hostert 1993; Hatfield and Schluter 1999). But Mayr’s argument implies that the influence of pleiotropy/hitchhiking may be more subtle and far reaching. It suggests that any functional genetic change increases the chance of substituting an allele that contributes to reproductive incompatibility. Further, if isolated populations are selected to remain in their mutual ancestral state (as in deleterious mutation theories), they should retain reproductive compatibility longer than if they were allowed to drift apart at random. This scenario is clearly distinguished from parallel selection, which may contribute to genetic and reproductive divergence if isolated populations fix different changes in response to similar selection (Muller 1942; Cohan and Hoffman 1989). Laboratory assays of reproductive isolation in Drosophila have minimized ecological factors that could contribute to environment-dependent reproductive isolation. Yet, I demonstrate that reproductive isolation of Drosophila species in the laboratory is correlated with allozyme divergence even after conditioning on divergence time, as estimated by silent DNA sequence divergence. I argue that the residual variation in allozyme divergence is best explained by natural selection, and the residual correlation of reproductive isolation with allozyme divergence is due to pleiotropy/hitchhiking. METHODS My central analysis is to test for correlation between allozyme divergence and reproductive isolation after factoring out the dependence of both quantities on divergence time, as estimated by silent and non-coding DNA divergence. This is done by regressing both allozyme divergence and a measure of reproductive isolation on silent DNA divergence, and then testing for an association between the resulting residuals of isolation and the residuals of allozyme divergence. All of the data on reproductive isolation and allozyme divergence, DNei (Nei 1987), used in this study were compiled from the literature by Coyne and Orr (1989, 1997). They calculated an index of premating isolation as 1 2 (# heterospecific matings)/(# homospecific matings). Postmating isolation was scored more coarsely. Potential offspring of interspecific matings were broken into four classes—male and female offspring from each of the two reciprocal crosses. The index takes on values of 0, 1/4, 1/2, 3/4, or 1 when 0, 1, 2, 3, or all 4 of the categories of F1 hybrids are completely inviable/ FIG. 1. Hypothetical phylogenetic tree used to illustrate phylogenetic correction method. Independent distances are AB, EF, (CA 1 CB)/2, and {[(EA 1 EB)/2 1 EC]/2 1 [(FA 1 FB)/2 1 FC]/ 2}/2. sterile. Both indices of reproductive isolation top out at one (complete isolation), and therefore they are not linearly related to the actual number of genic incompatibilities. The number of incompatibilities should ‘‘snowball’’ faster than linearly with time (Orr 1995; Menotti-Raymond et al. 1997; Orr and Turelli 2001), but no such expectation is warranted for these simple indices of isolation. Allozyme studies were typically based on 20–40 loci. DNA hybridization divergences were reported as patristic distances by Powell and DeSalle (1995; Powell 1997). All data were checked against the original sources whenever possible. DNA data from the mitochondrial cytochrome oxidase subunit I (COI) and the nuclear alcohol dehydrogenase (Adh) and glycerol-3-phosphate dehydrogenase (Gpdh) were obtained from Genbank. Exons from these loci were aligned in Clustal X and adjusted by eye. Synonymous sequence divergence was estimated using Nei and Gojobori’s (1986) method, as implemented in the computer program MEGA2. Phylogenetically independent contrasts (PIC’s) for each measure were calculated by Coyne and Orr’s (1989) modification of Felsenstein’s (1985) procedure (Fig. 1) using published phylogenies (MacIntyre and Collier 1986; Powell and DeSalle 1995; Powell 1997). Some have advocated Mantel’s matrix permutation test for these kinds of data. But Mantel’s test only accounts for dependence due to redundancy and ignores dependence due to common ancestry (Legendre et al. 1995). That is, Mantel’s test accounts for dependence between the distances AE and AF but not for the dependence between AE and BF (Fig. 1). The PIC procedure accounts for common ancestry by reducing all pairwise comparisons across a node to a single comparison. Redundancy (e.g., taxon A contributes to three of the four comparisons in Fig. 1) BRIEF COMMUNICATIONS 193 FIG. 2. Relationships between allozyme divergence (DNei) and four DNA datasets. Vertical lines illustrate the residuals, which were saved for further analysis. should not be a problem if it is safe to assume that withinclade divergence is independent of between-clade divergence. Further, properly nested averaging makes the method somewhat robust to violation of this assumption (Omland 1997). The rate of evolution of premating isolation is considerably accelerated by reproductive character displacement between sympatric species, but no difference in the pattern of postmating isolation in sympatric versus allopatric taxa is expected or observed (Coyne and Orr 1989). Therefore, when analyzing premating isolation, geographical relationships must be taken into account. For the two largest datasets, Adh and Gpdh, all sympatric comparisons were simply removed prior to analysis (i.e., comparisons between sympatric species pairs were not included when computing PICs—this may not completely account for historical sympatry between clades). This practice resulted in unacceptably small sample sizes for the DNA hybridization and COI datasets, so I did not consider prezygotic isolation in the analysis of these datasets. Preliminary analysis of prezygotic isolation without accounting for geography gave results qualitatively similar to postzygotic isolation (not shown). For each of the phylogenetically corrected datasets (DNA hybridization, Adh, Gpdh, and COI), DNei and reproductive isolation were regressed on synonymous DNA divergence and the residuals from these regressions were saved as linear transformations of the dependent variables (Figs. 2 and 3). In order to assure linear relationships and better fit the nor- mality assumption of regression analysis, logarithmic and arcsine transformations were employed as necessary (see axis labels in Figs. 2–4). This purely phenomenological approach to curve fitting is intended to avoid systematic biases that may be introduced by assuming particular models of evolution. Relationships between residual DNei and residual reproductive isolation were assessed by Spearman rank correlation. Analysis of residuals is an excellent approach when there is a priori knowledge of a confounding third variable (Afifi and Clark 1996). Bivariate regressions of reproductive isolation on both DNei and DNA divergence were used as additional descriptive analyses. I used rate heterogeneity comparisons to test whether silent sequence divergence and DNei differed in performance as molecular clocks. Relative rates tests (Li 1997) on individual datasets failed to reject a molecular clock null hypothesis (data not shown). Nevertheless, one type of genetic distance may be systematically more clocklike. I constructed unrooted phylograms for independent species pairs with outgroups (no species was included in more than one comparison). Let x and y denote the genetic distances between the two in-group species and their most recent common ancestor. A standardized measure of branch length heterogeneity was calculated for both DNA and allozyme divergence: B 5 (x 2 y)/(x 1 y). Statistical properties of this index are discussed in the Appendix. Heterogeneity indices for DNA and allozymes were compared using Wilcoxon signed-rank tests for paired 194 BRIEF COMMUNICATIONS FIG. 3. Relationships between postzygotic isolation and four DNA datasets. Vertical lines illustrate the residuals, which were saved for further analysis. data (Zar 1984), with each independent phylogram yielding one pair of Bs. The DNA hybridization, Adh, and COI datasets yielded 9, 11, and 9 independent phylograms, respectively. If allozymes are generally neutral, population bottlenecks can inflate DNei by increasing homozygosity (Chakraborty and Nei 1977). Bottlenecks might also increase the rate of evolution of reproductive isolation (Mayr 1970; Templeton 1981; but see Barton and Charlesworth 1984, Turelli et al. 2001). If an association between population size and homozygosity h as significantly influenced patterns of allozyme differentiation, then relatively long branches should, on average, be associated with relatively low heterozygosities. I used 26 unrooted phylograms, as above, to test for a correspondence between allozyme-based branch-length variation and heterozygosity variation. That is the quantities (x 2 y) for DNei and H should usually have opposite signs if population bottlenecks have been important in accelerating allozyme divergence. This was evaluated with a sign test for deviation from the null expectation of having the same sign half of the time. I also evaluated the correlation between DNei and average log homozygosity. This follows from the definition of DNei (Nei 1987): DNei 5 2ln Jxy 1 (ln Jx 1 ln Jy)/ 2, where Jxy is the probability of identity of alleles between taxa x and y, and Jx and Jy are the within-taxon probabilities of identity (homozygosities). RESULTS AND DISCUSSION Allozyme divergence was positively correlated with all DNA-based divergences (Fig. 2). Reproductive isolation was positively correlated with DNA hybridization and synonymous Adh divergence but not significantly correlated with synonymous COI or Gpdh divergence (Figs. 3 and 4). Where analyzed, results for premating isolation were concordant with those for postmating isolation (Tables 1 and 2, Figs. 3–5). In all datasets, reproductive isolation was more strongly correlated with allozyme divergence than DNA divergence (Table 1). Knowlton et al. (1993) reported a similar result from species pairs of Alpheus shrimp. After regression on DNA divergence, residual reproductive isolation was significantly positively associated with residual allozyme divergence (Table 2, Figs. 4 and 5). Conversely, after regression on allozyme distance, residual isolation was not significantly associated with residual DNA divergence (Spearman and Pearson correlation, all P-values . 0.05, not shown). Both kinds of genetic distance are statistically adequate molecular clocks (relative rates tests, not shown) and both are correlated, via a mutual dependence on time, with degree of reproductive isolation. But there appears to be an additional source of association between allozyme divergence and reproductive isolation that is not shared by silent DNA divergence. 195 BRIEF COMMUNICATIONS FIG. 4. The top two panels present relationships between premating isolation and synonymous DNA divergence from Adh and Gpdh. Vertical lines illustrate the residuals, which are plotted against residual allozyme divergence in the lower two panels. DNA hybridization incorporates information from all single copy and some repetitive DNA in the genome (Powell 1997). According to Miklos and Rubin (1996), there is about 90Mb of single copy DNA in the Drosophila genome, comprising about 55% of the total genome. Exons comprise about 24.1Mb (Adams et al. 2000). According to data summarized in Li (1997) an average of about 12% of substitutions in codons actually change amino acids (based on comparisons between the D. melanogaster and D. obscura groups). As- suming (conservatively) that substitution rates in introns and intergenic spacers are the same as for synonymous sites, we can calculate an approximate proportion of single copy DNA differences that are nonsynonymous as 0.12 3 (24.1 Mb 4 90 Mb) ø 0.032. Thus, DNA hybridization represents the broadest sample of the genome, and incorporates predominantly noncoding differences. As such, the results from DNA hybridization are probably the most compelling. Results from single sequences TABLE 1. Bivariate regressions of reproductive isolation on DNA- and allozyme-based genetic distances. Coefficients are standardized so that their relative magnitudes directly reflect their relative ‘‘strength’’ in the regression model. There is variation among datasets in the observed association between allozyme divergence (DNei) and reproductive isolation because no two datasets included exactly the same pairs of taxa. Allozyme, DNA hybridization, and Adh distances were ln transformed. Dataset DNA hybrid Adh COI Gpdh Independent variable Post Post Preb Post Post Preb DNAa coefficient 0.0541 0.1175 0.4840* 20.5795 20.4193 20.0192 Allozyme coefficient 0.8502** 0.7604* 0.5085* 1.1545** 0.8107* 0.3740 r2 F P 0.7862 0.7260 0.7953 0.6019 0.3753 0.1286 18.3847 13.2506 21.3757 6.8040 3.9053 1.1072 0.000447 0.001543 0.000162 0.01585 0.04697 0.3560 All DNA divergence, except for DNA hybridization, is based solely on synonymous sequence differences (Nei and Gojobori 1986). Prezygotic isolation was analyzed using only allopatric species pairs. Significance of individual coefficients (t-test): * P , 0.05; ** P , 0.01. a b 196 BRIEF COMMUNICATIONS TABLE 2. Results of Spearman rank correlation analyses corresponding to Figures 2–5. Variation among datasets is largely attributable to the fact that each includes a slightly different subset of the pairs of taxa in Coyne and Orr (1989, 1997). ‘‘Post’’ and ‘‘Pre’’ refer to post- and premating reproductive isolation, respectively. Dataset DNA hybridization Adh Gpdh COI Variables Residual Residual Residual Residual Residual Residual Post vs. Residual DNei Post vs. Residual DNei Pre vs. Residual DNei Post vs. Residual DNei Pre vs. Residual DNei Post vs. Residual DNei might be attributed to the fact that DNei is based on a large sample of loci and, therefore, might provide a better molecular clock than single loci. To verify that allozymes are not more clocklike than DNA, I used rate heterogeneity comparisons (see Methods) to test branch length variation in phylograms constructed from the different molecular datasets. Allozyme-based branch lengths were more heterogeneous than DNA-based branch lengths in the majority of comparisons, although the difference was statistically significant only in the DNA hybridization dataset (Wilcoxon signedrank tests, Table 3). My interpretation of the central analysis of the paper (Table rs P 0.7253 0.7253 0.6879 0.5912 0.2143 0.8322 0.005023 0.005023 0.006540 0.1588 0.6103 0.000785 2, Figs. 4 and 5) relies on the important differences between synonymous DNA and allozyme divergence discussed previously. Silent DNA sequence divergence should behave much more like a stochastic clock than allozyme divergence. Allozyme divergence (as measured by DNei) is likely to be significantly affected by selection and, possibly, bottleneck effects (which perturb the interaction between stabilizing selection and drift). Regression of DNei on synonymous DNA divergence is, coarsely, regression of allozyme divergence on time. The residual variation in allozyme divergence not accounted for in such a regression should be due to selection and/or changes in effective population size (e.g., founder effects). Similarly, re- FIG. 5. Residual relationships between postmating isolation and allozyme divergence for four DNA datasets; lines were fitted by least squares. 197 BRIEF COMMUNICATIONS TABLE 3. Results of Wilcoxon signed-rank tests for paired branchlength heterogeneity indices estimated from independent three-taxon phylograms (see text). BD and BDNA are branch length heterogeneities based on allozymes (DNei) and DNA, respectively. Dataset DNA hybridization Adh Gpdh COI Number Number with with Rank sum BD . BDNA BD , BDNA 41 33 9 30 7 8 2 6 2 3 5 4 Exact probability 0.027 0.175 0.469 0.846 sidual variation in reproductive isolation after regression on DNA divergence should reflect these same factors. My analyses provide no evidence that variance in heterozygosity has had a systematic effect on variance in DNei. Taxa with longer branches had lower H in only 10 of 26 comparisons (sign-test P 5 0.3269) and DNei was not significantly correlated with average log homozygosity (Spearman rank correlation 5 0.0182, P 5 0.960). These results suggest that bottleneck effects on neutral allozyme variation do not provide a satisfactory explanation for variance in DNei. However, there are other ways that variation in effective population size could influence DNei. If most electrophoretically detectable variation evolves according to deleterious mutation theories, small population size could increase the rate of divergence without substantially affecting heterozygosity (Ohta 1972, 1992; Gillespie 1999). By contrast, if isolated populations are continually substituting different advantageous alleles, divergence will be faster in large populations, but heterozygosity will still be insensitive to population size (Gillespie 1999). In neutral, overdominant, and fluctuating selection models, substitution rates are expected to be fairly constant while heterozygosity increases with population size (Gillespie 1999). Significant residual correlation between allozyme divergence and reproductive isolation after factoring out divergence time (as estimated by synonymous DNA) is consistent with the venerable proposition that ‘‘positive selection’’ can accelerate speciation via pleiotropy/hitchhiking. It is equally consistent with deleterious mutation theories, which suggest that ‘‘negative selection’’ would tend to retard divergence. Both models predict ‘‘correlated progression’’ among different parts of the genome over long spans of time (Bell 2000). My results should not be taken as evidence against alternative models that invoke fluctuating selection or idiosyncratic variation in modes of evolution among different loci. They simply suggest that correlated progression—beyond what is expected due to time—is common enough to leave a statistical footprint in comparative allozyme and reproductive isolation data from Drosophila. This is not merely another confirmation of the fact that genetic divergence is associated with speciation. Rather, my analysis shows that one kind of genetic divergence (electrophoretically detectable enzyme differences) is more tightly correlated with reproductive isolation than is another (silent DNA differences). This is all the more noteworthy given that DNA sequences are now the preferred source of data for addressing questions about taxonomic status, species boundaries, and modes of speciation. The result is indicative of a pervasive role of natural selection in influencing the rate of evolution of reproductive isolation. ACKNOWLEDGMENTS I would like to thank the participants in the evolution discussion group at the University of California, Davis for constructive criticism. M. Turelli, H. B. Shaffer, J. H. Gillespie, D. J. Begun, and two anonymous reviewers provided excellent advice and editorial comments. My research is supported by an NSF Grant (DEB 0089716) to M. Turelli and an EPA STAR Graduate Fellowship (U-91572401). LITERATURE CITED Adams, M. D., S. E. Celniker, R. A. Holt, C. A. Evans, J. D. Grocayne et al. 2000. The genome sequence of Drosophila melanogaster. Science 287:2185–2195. Afifi, A. A., and V. Clark. 1996. Computer-aided multivariate analysis. Chapman and Hall, London. Aguade, M., N. Miyashita, and C. H. Langley. 1992. Polymorphism and divergence in the Mst26A male accessory gland gene region in Drosophila. Genetics 132:755–770. Ayala, F. J., M. L. 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Let x and y denote the branch lengths, in units of actual substitutions per site (or codon substitutions per locus for allozymes), from the most recent common ancestor to species 1 and 2, respectively. Define B5 x2y . x1y (A1) The delta method gives approximate expressions for the expectation and variance, assuming Cov(x, y) 5 0: 1 E(B) ù V(x) 2) ]2B ]2B 1 V(y) 2 2]x 2]y 2 5 0, (A2) E(x),E(y) assuming the expectations and variances of x and y are equal. And [ 2 1 ]x 2 V(B) ù V(x) ]B ] 1 ]y 2 ) 1 V(y) ]B 2 5 E(x),E(y) V(x 1 y) . [E(x 1 y)] 2 (A3) If the clock is Poisson, then E(x 1 y) 5 V(x 1 y) 5 2lt, so V(B) ù 1 2l t (A4) where l is the substitution rate and t is the number of generations since the most recent common ancestor. Under a Poisson molecular clock, all measures of genetic distance used in this paper are estimates of 2lt. DNei is in units of codon substitutions per enzyme locus (Nei 1987), while DNA distances are in numbers of nucleotide substitutions per site (Nei and Gojobori 1986; Nei 1987). DNA hybridization distance, measured by DTM (Powell 1997), is linearly related to the proportion of nucleotide differences p 5 c(DTM), where c is between 0.010 and 0.015 (Nei 1987). Thus, DNA hybridization distances estimate 2lt/c. The numerical value of DNei for a given pairwise comparison is always greater than the numerical values of the DNA-based estimates of 2lt in my datasets. Thus, under a Poisson clock null model, B calculated from DNei is expected to have a lower variance than B based on DNA distances. This would bias my rate heterogeneity comparisons toward finding that DNei is a better clock than DNA. Despite this bias, I found modest evidence that DNei has more variable rates than DNA distances (Table 3).