Crossing over between homologous chromosomes during meiosis repairs programmed DNA double-strand breaks, ensures proper segregation at meiosis I [], shapes the genomic distribution of nucleotide variability in populations, and enhances the efficacy of natural selection among genetically linked sites []. Between closely related Drosophila species, large differences exist in the rate and chromosomal distribution of crossing over. Little, however, is known about the molecular genetic changes or population genetic forces that mediate evolved differences in recombination between species []. Here, we show that a meiosis gene with a history of rapid evolution acts as a trans-acting modifier of species differences in crossing over. In transgenic flies, the dicistronic gene, mei-217/mei-218, recapitulates a large part of the species differences in the rate and chromosomal distribution of crossing over. These phenotypic differences appear to result from changes in protein sequence not gene expression. Our population genetics analyses show that the protein-coding sequence of mei-218, but not mei-217, has a history of recurrent positive natural selection. By modulating the intensity of centromeric and telomeric suppression of crossing over, evolution at mei-217/-218 has incidentally shaped gross differences in the chromosomal distribution of nucleotide variability between species. We speculate that recurrent bouts of adaptive evolution at mei-217/-218 might reflect a history of coevolution with selfish genetic elements.

Results and Discussion

1 Egel R.

Lankenau D.-H. Recombination and Meiosis: Crossing-Over and Disjunction. 5 Fisher R.A. The Genetical Theory of Natural Selection. 6 Goldberg M.L.

Sheen J.Y.

Gehring W.J.

Green M.M. Unequal crossing-over associated with asymmetrical synapsis between nomadic elements in the Drosophila melanogaster genome. 7 Petrov D.A.

Fiston-Lavier A.S.

Lipatov M.

Lenkov K.

González J. Population genomics of transposable elements in Drosophila melanogaster. 8 Miller D.E.

Smith C.B.

Kazemi N.Y.

Cockrell A.J.

Arvanitakas A.V.

Blumenstiel J.P.

Jaspersen S.L.

Hawley R.S. Whole-genome analysis of individual meiotic events in Drosophila melanogaster reveals that noncrossover gene conversions are insensitive to interference and the centromere effect. 9 Koehler K.E.

Boulton C.L.

Collins H.E.

French R.L.

Herman K.C.

Lacefield S.M.

Madden L.D.

Schuetz C.D.

Hawley R.S. Spontaneous X chromosome MI and MII nondisjunction events in Drosophila melanogaster oocytes have different recombinational histories. Despite its functional and evolutionary benefits [], crossing over entails risks. First, selfish repetitive DNA sequences (e.g., transposons) distributed throughout the genome present the risk of non-homologous ectopic exchange [], which can give rise to deleterious de novo duplications and deletions in ≥2% of meioses in D. melanogaster []. Second, crossovers in centromere- and telomere-proximal regions can increase the risk of improper chromosomal segregation, resulting in breakage and nondisjunction []. The optimal rate and distribution of crossing over may therefore evolve to balance the benefits of recombination against the costs of ectopic exchange and missegregation.

10 True J.R.

Mercer J.M.

Laurie C.C. Differences in crossover frequency and distribution among three sibling species of Drosophila. 10 True J.R.

Mercer J.M.

Laurie C.C. Differences in crossover frequency and distribution among three sibling species of Drosophila. 11 Lindsley D.L.

Sandler L. The genetic analysis of meiosis in female Drosophila melanogaster. 10 True J.R.

Mercer J.M.

Laurie C.C. Differences in crossover frequency and distribution among three sibling species of Drosophila. 3 Dapper A.L.

Payseur B.A. Connecting theory and data to understand recombination rate evolution. Between Drosophila melanogaster and its closely related species, D. mauritiana, appreciable differences in the rate and chromosomal distribution of crossing over have evolved despite comparable genome sizes and karyotypes []. In D. mauritiana, the total genetic map lengths of the three major chromosomes, X, 2, and 3, are 1.7-, 1.5-, and 2.1-fold longer, respectively, than those in D. melanogaster []. Some of these differences in genetic map length are attributable to differences in the chromosomal distribution of recombination: crossing over is suppressed at considerable distances from telomere- and especially centromere-proximal regions in D. melanogaster [], whereas the range of these effects is narrower in D. mauritiana []. How and why genetic maps evolve is almost entirely unknown [].

11 Lindsley D.L.

Sandler L. The genetic analysis of meiosis in female Drosophila melanogaster. 12 Baker B.S.

Carpenter A.T.C. Genetic analysis of sex chromosomal meiotic mutants in Drosophilia melanogaster. 13 Mehrotra S.

Hawley R.S.

McKim K.S. Synapsis, double-strand breaks, and domains of crossover control in Drosophila females. 13 Mehrotra S.

Hawley R.S.

McKim K.S. Synapsis, double-strand breaks, and domains of crossover control in Drosophila females. 14 McKim K.S.

Dahmus J.B.

Hawley R.S. Cloning of the Drosophila melanogaster meiotic recombination gene mei-218: a genetic and molecular analysis of interval 15E. 15 Bhagat R.

Manheim E.A.

Sherizen D.E.

McKim K.S. Studies on crossover-specific mutants and the distribution of crossing over in Drosophila females. N (0.094) and d N /d S (0.632; N and d N /d S are in the 99.97% and 97% percentiles, respectively; 16 Kohl K.P.

Jones C.D.

Sekelsky J. Evolution of an MCM complex in flies that promotes meiotic crossovers by blocking BLM helicase. 12 Baker B.S.

Carpenter A.T.C. Genetic analysis of sex chromosomal meiotic mutants in Drosophilia melanogaster. 14 McKim K.S.

Dahmus J.B.

Hawley R.S. Cloning of the Drosophila melanogaster meiotic recombination gene mei-218: a genetic and molecular analysis of interval 15E. 15 Bhagat R.

Manheim E.A.

Sherizen D.E.

McKim K.S. Studies on crossover-specific mutants and the distribution of crossing over in Drosophila females. 17 Carpenter A.T. Recombination nodules and synaptonemal complex in recombination-defective females of Drosophila melanogaster. 15 Bhagat R.

Manheim E.A.

Sherizen D.E.

McKim K.S. Studies on crossover-specific mutants and the distribution of crossing over in Drosophila females. 18 Sekelsky J.J.

McKim K.S.

Chin G.M.

Hawley R.S. The Drosophila meiotic recombination gene mei-9 encodes a homologue of the yeast excision repair protein Rad1. 19 Cromie G.A.

Hyppa R.W.

Taylor A.F.

Zakharyevich K.

Hunter N.

Smith G.R. Single Holliday junctions are intermediates of meiotic recombination. We sought to determine the genetic basis and evolutionary causes of these species differences in crossover rate and distribution. To identify candidate genes, we surveyed the molecular evolution of genes previously identified in classical screens for mutations that disrupt meiosis in D. melanogaster []. These mutations disrupt genes that function in synaptonemal complex formation, double-strand break (DSB) formation, DSB repair, establishment of crossover intermediates, and resolution of crossover intermediates []. We generated sequence alignments for a set of 35 meiosis genes and performed an evolutionary screen for unusually high protein-coding sequence divergence between D. mauritiana and D. melanogaster. Among the 35 genes, mei-218 is an outlier with the highest d(0.094) and d/d(0.632; Table S1 ), placing it among the most diverged protein-coding sequences in the genome (dand d/dare in the 99.97% and 97% percentiles, respectively; Figure S1 ). Previous analyses have established that the MEI-218 protein has a mini-chromosome maintenance (MCM) domain and interacts with several other meiosis-specific MCM proteins to form a so-called mei-MCM complex []. In mei-218 mutant females, synaptonemal complex formation, DSB formation, and recombination via gene conversion all proceed normally, whereas the rate of crossing over is reduced by ≥90%, the number of spherical recombination nodules is reduced (with those remaining often having abnormal morphology), and the rate of chromosomal nondisjunction is elevated accordingly []. During repair of DSBs, mei-218 appears to function after strand invasion but prior to crossover resolution []. The MEI-218 protein is thus necessary for the establishment and/or stabilization of heteroduplex crossover intermediates []. Its inferred function and rapid sequence evolution together suggest that mei-218 is a reasonable candidate contributor to the evolved species difference in crossing over between D. mauritiana and D. melanogaster.

20 Liu H.

Jang J.K.

Graham J.

Nycz K.

McKim K.S. Two genes required for meiotic recombination in Drosophila are expressed from a dicistronic message. 1 loss-of-function mutant genetic backgrounds (1; P[mei-217/-218mel], which serves as a positive control, and mei-2181; P[mei-217/-218mau] (1; net ho dp b pr cn/+ + + + + +; P[mei-217/-218mel]/+ females (n = 13 crosses; 2,103 progeny) and, separately, from replicate mei-2181; net ho dp b pr cn /+ + + + + +; P[mei-217/-218mau]/+ females (n = 13 crosses; 2,369 progeny; 10 True J.R.

Mercer J.M.

Laurie C.C. Differences in crossover frequency and distribution among three sibling species of Drosophila. Figure 1 The mei-217/mei-218 Gene Region and Genotypes Assayed for Crossing Over Show full caption 20 Liu H.

Jang J.K.

Graham J.

Nycz K.

McKim K.S. Two genes required for meiotic recombination in Drosophila are expressed from a dicistronic message. (A) Within the ∼8.5-kb region cloned (gray), the mei-217/-218 gene (light blue) on chromosome X (15E5) gives rise to a single dicistronic transcript (green) that encodes both the MEI-217 and MEI-218 proteins from different exons and different translation initiation sites (purple) []. 1 allele contains a nonsense mutation [ 14 McKim K.S.

Dahmus J.B.

Hawley R.S. Cloning of the Drosophila melanogaster meiotic recombination gene mei-218: a genetic and molecular analysis of interval 15E. (B) Three genotypes were used to test whether the D. mauritiana and D. melanogaster alleles of mei-217/-218 mediate species differences in rates of crossing over: females with no transgene (a negative control); females with a transgene of a D. melanogaster mei-217/-218 allele (a positive control); and females with a transgene of a D. mauritiana mei-217/-218 allele. The two transgenes were inserted into the same position on chromosome 3L (75A10). The endogenous mei-218allele contains a nonsense mutation []. Crossover frequencies were scored among six visible markers spanning the left arm of chromosome 2 and the centromere: net (net), decapentaplegic (ho), dumpy (dp), black (b), purple (pr), and cinnabar (cn). For additional details on genotype construction, see STAR Methods and Figure S2 To test the functional consequences of interspecific sequence divergence at mei-218, we assayed the effects of wild-type D. melanogaster and D. mauritiana alleles using a transgenic approach ( Figures 1 A and 1B ). A dicistronic gene encodes both the MEI-217 and MEI-218 proteins from a single transcriptional unit with open reading frames that overlap by seven codons, in different reading frames, and with separate translation initiation sites ( Figure 1 A) []. We therefore cloned homologous mei-217/mei-218 (hereafter, mei-217/-218) gene regions, including the mei-217 and mei-218 coding sequences and all 5′- and 3′-flanking noncoding sequence from D. melanogaster and D. mauritiana into separate attB-P[acman] vectors and used site-specific integration to place each transgene construct into a common attP insertion site on chromosome arm 3L (75A10) in D. melanogaster ( Figures 1 A and 1B; see STAR Methods for details). We crossed the transgenes into separate but largely identical homozygous mei-218loss-of-function mutant genetic backgrounds ( Figure S2 ), yielding two D. melanogaster stocks: mei-218; P[mei-217/-218], which serves as a positive control, and mei-218; P[mei-217/-218] ( Figure 1 B). We then estimated crossover frequencies among six visible markers that span chromosome arm 2L and the centromere, scoring progeny from replicate mei-218; net ho dp b pr cn/+ + + + + +; P[mei-217/-218]/+ females (n = 13 crosses; 2,103 progeny) and, separately, from replicate mei-218; net ho dp b pr cn /+ + + + + +; P[mei-217/-218]/+ females (n = 13 crosses; 2,369 progeny; Figure 1 B; see STAR Methods for details). In wild-type D. mauritiana, the total genetic distance between net and cn is ∼1.4-fold longer than in D. melanogaster [].

mau rescues the mei-218 mutant phenotype and produces D. melanogaster-like rates of crossing over; (2) the divergent mei-217/-218mau allele might be non-functional in D. melanogaster—a kind of molecular incompatibility between species—and fail to rescue the mei-218 mutant phenotype; or (3) mei-217/-218mau might rescue the mei-218 mutant phenotype and produce elevated, D. mauritiana-like rates of crossing over. As expected, crossing over is reduced in mutant mei-218 females ( 12 Baker B.S.

Carpenter A.T.C. Genetic analysis of sex chromosomal meiotic mutants in Drosophilia melanogaster. 14 McKim K.S.

Dahmus J.B.

Hawley R.S. Cloning of the Drosophila melanogaster meiotic recombination gene mei-218: a genetic and molecular analysis of interval 15E. mel, which fully rescues the mutant mei-218 phenotype (mel transgene is sufficient to rescue wild-type genetic map distances in otherwise mei-218 mutant females [ 14 McKim K.S.

Dahmus J.B.

Hawley R.S. Cloning of the Drosophila melanogaster meiotic recombination gene mei-218: a genetic and molecular analysis of interval 15E. mau transgene also rescues the mei-2181 mutant phenotype but significantly increases the rate of crossing over relative to the positive control. The total genetic map length is increased 1.23-fold in mei-217/-218mau females relative to mei-217/-218mel females ( Table 1 The mei-217/-218 Allele of D. mauritiana Alters the Rate and Patterning Crossing Over Relative to that of D. melanogaster Genetic Interval mei-2181 mei-218mel mei-218mau mel versus mau Fold Change p Value (Power) net-ho 0 (0) 2.01 (1.45) 3.70 (1.38) 1.84 0.005 (0.72) ho-dp 0 (0) 5.28 (1.95) 6.02 (1.94) 1.14 0.349 (0.15) dp-b 0.77 (1.07) 22.20 (4.13) 24.27 (5.04) 1.09 0.250 (0.20) b-pr 0.41 (0.57) 10.65 (2.62) 14.53 (2.93) 1.36 0.001 (0.83) pr-cn 0.84 (1.15) 4.13 (1.32) 5.49 (1.94) 1.33 0.034 (0.41) Total map length 2.02 (1.72) 44.27 (6.00) 54.01 (5.38) 1.23 0.0002 (0.90) Total progeny 330 2,103 2,369 – E 0 0.958 0.205 0.130 0.64 E 1 0.042 0.685 0.686 1.00 E 2 0 0.107 0.160 1.51 E 3 0 0.004 0.024 6.21 1 mutant and two transgenic genotypes (see 0 , E 1 , E 2 , and E 3 are the estimated frequencies of tetrads with zero, one, two, and three inferred crossovers, respectively (see For each genotype, the means and SDs (in parentheses) of crossover frequencies for five genetic intervals measured in mei-218mutant and two transgenic genotypes (see STAR Methods and Figure S2 ). p values are derived from unpaired t tests, and we estimated the power (in parentheses) associated with each test. E, E, E, and Eare the estimated frequencies of tetrads with zero, one, two, and three inferred crossovers, respectively (see STAR Methods for details). There are three possible outcomes: (1) despite considerable interspecific sequence divergence, the two alleles might be functionally equivalent, such that mei-217/-218rescues the mei-218 mutant phenotype and produces D. melanogaster-like rates of crossing over; (2) the divergent mei-217/-218allele might be non-functional in D. melanogaster—a kind of molecular incompatibility between species—and fail to rescue the mei-218 mutant phenotype; or (3) mei-217/-218might rescue the mei-218 mutant phenotype and produce elevated, D. mauritiana-like rates of crossing over. As expected, crossing over is reduced in mutant mei-218 females ( Table 1 ) [], yielding a genetic map length that is reduced by 95% relative to that of the positive control transgene mei-217/-218, which fully rescues the mutant mei-218 phenotype ( Table 1 ). This finding confirms that a single copy of the mei-217/-218transgene is sufficient to rescue wild-type genetic map distances in otherwise mei-218 mutant females []. The mei-217/-218transgene also rescues the mei-218mutant phenotype but significantly increases the rate of crossing over relative to the positive control. The total genetic map length is increased 1.23-fold in mei-217/-218females relative to mei-217/-218females ( Table 1 ; 95% confidence intervals = 1.13–1.31; p = 0.0002), accounting for ∼82% of the wild-type species difference in the total net to cn map distance.

mau-mediated increase in genetic map length is not uniform across genetic marker intervals. Those intervals with significantly increased crossover rates occur in telomere- and centromere-proximal regions (1.84-fold for net-ho and 1.36-fold for b-pr) or span the centromere (1.33-fold for pr-cn; 10 True J.R.

Mercer J.M.

Laurie C.C. Differences in crossover frequency and distribution among three sibling species of Drosophila. mau than mei-217/-218mel females, neither differs significantly (p ≥ 0.2501; mel and mei-217/-218mau transgenic females ( Notably, the mei-217/-218-mediated increase in genetic map length is not uniform across genetic marker intervals. Those intervals with significantly increased crossover rates occur in telomere- and centromere-proximal regions (1.84-fold for net-ho and 1.36-fold for b-pr) or span the centromere (1.33-fold for pr-cn; Table 1 ). No difference is expected in crossover rates in the medial regions of 2L [] and, although the two medial intervals scored have higher rates of crossing over in mei-217/-218than mei-217/-218females, neither differs significantly (p ≥ 0.2501; Table 1 ). (We note, however, that our statistical power is relatively weak for these two non-significant intervals, ≤0.20; Table 1 ). We next tested whether species differences in mei-217/-218 gene expression might mediate these differences in crossing over using qRT-PCR. Assaying expression in ovaries from 3-to 5-day-old females, we find no difference in gene expression between wild-type D. melanogaster and D. mauritiana females or mei-217/-218and mei-217/-218transgenic females ( Figure S3 ). These findings suggest that the observed differences in the rate and distribution of crossing over are attributable to evolution of the mei-217/-218 protein-coding sequence, not to its gene expression level.

13 Mehrotra S.

Hawley R.S.

McKim K.S. Synapsis, double-strand breaks, and domains of crossover control in Drosophila females. 21 Hawley R.S.

McKim K.S.

Arbel T. Meiotic segregation in Drosophila melanogaster females: molecules, mechanisms, and myths. 22 Wang S.

Zickler D.

Kleckner N.

Zhang L. Meiotic crossover patterns: obligatory crossover, interference and homeostasis in a single process. mel and mei-217/-218mau transgenes (χ2 test; df = 5; p ≤ e−200; mel and mei-217/-218mau females (χ2 test; df = 3; p ≤ e−80; mau females versus 0.91 in mei-217/-218mel females. We tested whether the mei-217/-218mau-mediated increase in the average number of crossovers per tetrad is achieved by decreasing the incidence of tetrads with no crossovers (E 0 ), increasing the incidence of those with single crossovers (E 1 ) or multiple crossovers (E ≥2 ) or a combination [ 23 Weinstein A. The theory of multiple-strand crossing over. 1 tetrads is the same for mei-217/-218mau and mei-217/-218mel females ( 0 tetrads in mei-217/-218mau females is only 0.64-fold that in mei-218mel females, whereas the incidences of E 2 and E 3 tetrads are 1.5- and 6.2-fold higher, respectively (mau than for mei-217/-218mel females (0.508 versus 0.793; Mann-Whitney p = 0.0085). These results show that the mei-217/-218mau transgene simultaneously strengthens crossover assurance and weakens crossover interference. The number of crossovers formed among homologous chromosomes of a tetrad is highly regulated []. Crossover assurance mechanisms promote the formation of one crossover per tetrad, and crossover interference mechanisms inhibit the formation of multiple crossovers in close proximity on a chromosome arm []. Consistent with regulation, we find that the distributions of the number of crossovers per tetrad are under-dispersed relative to Poisson expectations for both mei-217/-218and mei-217/-218transgenes (χtest; df = 5; p ≤ e Table 1 ). The number of crossovers per tetrad also differs between mei-217/-218and mei-217/-218females (χtest; df = 3; p ≤ e Table 1 ). An average of 1.08 crossovers per tetrad occurs in mei-217/-218females versus 0.91 in mei-217/-218females. We tested whether the mei-217/-218-mediated increase in the average number of crossovers per tetrad is achieved by decreasing the incidence of tetrads with no crossovers (E), increasing the incidence of those with single crossovers (E) or multiple crossovers (E) or a combination []. We find that the incidence of Etetrads is the same for mei-217/-218and mei-217/-218females ( Table 1 ). However, the incidence of Etetrads in mei-217/-218females is only 0.64-fold that in mei-218females, whereas the incidences of Eand Etetrads are 1.5- and 6.2-fold higher, respectively ( Table 1 ). The resulting increase in the occurrence of multiple crossovers accounts for ∼59% of the observed increase in genetic map length. Estimating crossover interference for the two largest adjacent intervals (dp-b-pr) shows that interference is ∼36% weaker for mei-217/-218than for mei-217/-218females (0.508 versus 0.793; Mann-Whitney p = 0.0085). These results show that the mei-217/-218transgene simultaneously strengthens crossover assurance and weakens crossover interference.

14 McKim K.S.

Dahmus J.B.

Hawley R.S. Cloning of the Drosophila melanogaster meiotic recombination gene mei-218: a genetic and molecular analysis of interval 15E. 15 Bhagat R.

Manheim E.A.

Sherizen D.E.

McKim K.S. Studies on crossover-specific mutants and the distribution of crossing over in Drosophila females. 16 Kohl K.P.

Jones C.D.

Sekelsky J. Evolution of an MCM complex in flies that promotes meiotic crossovers by blocking BLM helicase. 24 Tye B.K. MCM proteins in DNA replication. mau females, then more heteroduplexes might achieve second-end capture and be resolved as crossover events versus dissolve and result in non-crossover gene conversion events. Given the shared genetic backgrounds of our transgenic flies, we infer that mei-217/-218mau increases the probability that a DSB will be repaired as a crossover (versus a non-crossover gene conversion) than mei-217/-218mel. As a result, crossover assurance is strengthened (fewer E 0 tetrads), whereas the intensities of crossover interference and centromere (telomere) suppression are diminished [ 22 Wang S.

Zickler D.

Kleckner N.

Zhang L. Meiotic crossover patterns: obligatory crossover, interference and homeostasis in a single process. As our transgenic flies are genetically identical (or nearly so), the observed differences in crossover rate and distribution are not readily attributable to differences in genetic background or to any aspect of meiosis not affected by mei-217/-218. How mei-217/-218 regulates the number and distribution of crossovers is not known []. One possibility is that, just as the canonical MCM complex functions as a holoenzyme to facilitate DNA synthesis into replication forks [], the mei-MCM complex might facilitate DNA synthesis into the forks of heteroduplex DNA structures as required for the formation and stabilization of crossover intermediates. If heteroduplex structures are stabilized more effectively in mei-217/-218females, then more heteroduplexes might achieve second-end capture and be resolved as crossover events versus dissolve and result in non-crossover gene conversion events. Given the shared genetic backgrounds of our transgenic flies, we infer that mei-217/-218increases the probability that a DSB will be repaired as a crossover (versus a non-crossover gene conversion) than mei-217/-218. As a result, crossover assurance is strengthened (fewer Etetrads), whereas the intensities of crossover interference and centromere (telomere) suppression are diminished [].

28 McDonald J.H.

Kreitman M. Adaptive protein evolution at the Adh locus in Drosophila. 29 Wilson D.J.

Hernandez R.D.

Andolfatto P.

Przeworski M. A population genetics-phylogenetics approach to inferring natural selection in coding sequences. Table 2 Population Genetic Evidence for Positive Selection at mei-218, not mei-217 D. mauritiana (mau) D. melanogaster (mel) n 8 20 Gene region (kb) 8.2 8.2 θ W 0.0064 0.0044 π 0.0058 0.0059 Tajima’s D −0.530 −1.044 mau mei-217 mau mei-218 mel mei-217 mel mei-218 Coding sequence length (bp) 840 3,561 840 3,561 Nonsynon. polymorphisms 2 30 5 17 Synonymous polymorphisms 10 15 7 18 Nonsynon. substitutions 3 67 8 53 Synonymous substitutions 7 39 7 22 Lineage-specific MK test, p FET 0.624 0.715 0.704 0.033 p 0.50 (γ > 0) 0 130 1 99 p 0.75 (γ > 0) 0 101 0 80 p 0.95 (γ > 0) 0 43 0 14 W and π) [ 25 Watterson G.A. On the number of segregating sites in genetical models without recombination. 26 Nei M.

Li W.H. Mathematical model for studying genetic variation in terms of restriction endonucleases. 27 Tajima F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. 28 McDonald J.H.

Kreitman M. Adaptive protein evolution at the Adh locus in Drosophila. Summary statistics for the mei-217/mei-218 gene region and the two coding sequences for D. mauritiana and D. melanogaster samples. The combined gene region was used to obtain summaries of the level of polymorphism (θand π) [] and the site frequency spectra (Tajima’s D) []. Lineage-specific McDonald-Kreitman [] tests were performed with the coding sequences of D. yakuba mei-217 and mei-218 as outgroup sequences to polarize substitutions along the D. melanogaster and D. mauritiana lineages. Consistent with the absence of a gene expression difference (see main text; Figure S3 ), McDonald-Kreitman tests contrasting polymorphisms and fixed differences from noncoding sequences (5′ UTR, 3′ UTR, and introns) with those at synonymous positions revealed no evidence for recurrent positive selection. Positions with evidence of multiple substitutions were excluded, as the inferred ancestral state is ambiguous by simple parsimony. We used gammaMap to estimate the number, posterior probability, and location of positively selected substitutions in mei-217 and mei-218 (see Figure 2 A). For additional details, see STAR Methods Table S1 , and Figure S1 Figure 2 The Distribution of Positively Selected Codons in mei-217 and mei-218 in D. melanogaster and D. mauritiana Show full caption 30 Manheim E.A.

Jang J.K.

Dominic D.

McKim K.S. Cytoplasmic localization and evolutionary conservation of MEI-218, a protein required for meiotic crossing-over in Drosophila. (A) The N-terminal basic and central acidic regions of MEI-218 are encoded by codons 1–500 and 500–800, respectively, and the MCM domain is encoded by codons 1,019–1,124 []. Nearly all of the positively selected codons fall within the first 800 codons of mei-218, and none occur in the MCM domain. In mei-217, a single codon in D. melanogaster has a 0.52 probability of positive selection, whereas no codons in D. mauritiana mei-217 have a ≥0.50 probability of positive selection. Codon substitutions are indicated as red (nonsynonymous) and blue (synonymous) circles. (B) The standardized density of SNPs per site in 50-kb windows across chromosome 2 plotted for D. melanogaster (blue) and D. mauritiana (red) with loess-smoothed curves. For each chromosome arm (2L and 2R) and species, SNP densities were standardized by the respective maximum value. Gray triangles show the positions of the six visible markers used to score crossover frequencies. See STAR Methods for more details. Why mei-218 has evolved so rapidly between these closely related species is unclear. Rapid sequence evolution can result from relaxed functional constraints or from divergent positive natural selection. To investigate the population genetic forces responsible for the rapid evolution of mei-218, we studied nucleotide polymorphism and divergence in resequence data obtained from 20 D. melanogaster samples from Rwanda and 8 D. mauritiana samples from Mauritius. There is no evidence for recent hard selective sweeps in the mei-217/-218 gene regions, as levels of polymorphism and the site frequency spectra are typical for these species ( Table 2 ). However, two analyses provide evidence for a history of recurrent positive natural selection. First, using lineage-specific McDonald-Kreitman tests [], we find that D. melanogaster mei-218, but not mei-217, has an excess of nonsynonymous substitutions ( Table 2 ). Second, to localize the signals of positive selection, we implemented gammaMap [], a powerful phylogenetics-population genetics method that combines information from lineage-specific substitutions and the site frequency spectrum from each species to infer the posterior probability of positive selection at individual codons. The gammaMap results show that the probability of positive selection is >0.5 for 99 and 130 codons of mei-218 in D. melanogaster and D. mauritiana lineages, respectively ( Figure 2 A; Table 2 ). These signals of positive selection are restricted to mei-218 almost exclusively, as only one codon in mei-217 shows evidence of positive selection ( Figure 2 A; Table 2 ). Within mei-218, positively selected codons are concentrated in regions encoding the N-terminal basic region and the middle acidic region but appear absent from the C-terminal MCM-domain region ( Figure 2 A). The small MCM-domain itself has no detected positively selected substitutions in either lineage.

1 males are fertile [ 12 Baker B.S.

Carpenter A.T.C. Genetic analysis of sex chromosomal meiotic mutants in Drosophilia melanogaster. 31 Brandvain Y.

Coop G. Scrambling eggs: meiotic drive and the evolution of female recombination rates. 32 Dowsett A.P.

Young M.W. Differing levels of dispersed repetitive DNA among closely related species of Drosophila. 33 Kent T.V.

Uzunović J.

Wright S.I. Coevolution between transposable elements and recombination. 33 Kent T.V.

Uzunović J.

Wright S.I. Coevolution between transposable elements and recombination. 34 Montgomery E.

Charlesworth B.

Langley C.H. A test for the role of natural selection in the stabilization of transposable element copy number in a population of Drosophila melanogaster. 35 Charlesworth B.

Sniegowski P.

Stephan W. The evolutionary dynamics of repetitive DNA in eukaryotes. 36 Charlesworth B.

Barton N.H. Recombination load associated with selection for increased recombination. To explain recurrent bouts of adaptive evolution at mei-218, which has accumulated 218 fixed nonsynonymous differences between species, would seem to require a model of adaptation to a moving fitness optimum. One possibility is that adaptive evolution at mei-218 results from selection on a function other than recombination in females. The mei-217/-218 gene is expressed at high levels in testes, although its function in males, which are achiasmate, is unknown (mei-218males are fertile []). Another possibility is that mei-217/-218-mediated change in recombination rates may have evolved in response to a history of recurrent meiotic drive in the female germline, either increasing or decreasing the rate of crossing over, depending on the timing of drive (MI or MII) and the genetic linkage between mei-217/-218 and drive alleles []. Finally, mei-217/-218-mediated change in recombination rates could reflect adaptation to species differences in transposon abundance. There are two competing models here. First, as the transposon content of the D. melanogaster genome is several-fold higher than that of D. mauritiana [], reduced rates of crossing over in D. melanogaster may have evolved to mitigate a higher risk of ectopic exchange between non-homologous transposon insertions []. Under this model, the rate and distribution of recombination might evolve frequently to balance the benefits of crossing over versus the risk of ectopic exchange arising from historically fluctuating, species-specific transposon loads []. Second, and alternatively, once transposon copy numbers reach equilibrium, selection may favor the evolution of increased crossover rates, facilitating the elimination of transposons via ectopic exchange [].