Positive Selection and Functional Divergence at Meiosis Genes That Mediate Crossing Over Across the Drosophila Phylogeny

Abstract

Meiotic crossing over ensures proper segregation of homologous chromosomes and generates genotypic diversity. Despite these functions, little is known about the genetic factors and population genetic forces involved in the evolution of recombination rate differences among species. The dicistronic meiosis gene, mei-217/mei-218, mediates most of the species differences in crossover rate and patterning during female meiosis between the closely related fruitfly species, Drosophila melanogaster and D. mauritiana The MEI-218 protein is one of several meiosis-specific mini-chromosome maintenance (mei-MCM) proteins that form a multi-protein complex essential to crossover formation, whereas the BLM helicase acts as an anti-crossover protein. Here we study the molecular evolution of five genes- mei-218, the other three known members of the mei-MCM complex, and Blm- over the phylogenies of three Drosophila species groups- melanogaster, obscura, and virilis We then use transgenic assays in D. melanogaster to test if molecular evolution at mei-218 has functional consequences for crossing over using alleles from the distantly related species D. pseudoobscura and D. virilis Our molecular evolutionary analyses reveal recurrent positive selection at two mei-MCM genes. Our transgenic assays show that sequence divergence among mei-218 alleles from D. melanogaster, D. pseudoobscura, and D. virilis has functional consequences for crossing over. In a D. melanogaster genetic background, the D. pseudoobscura mei-218 allele nearly rescues wildtype crossover rates but alters crossover patterning, whereas the D. virilis mei-218 allele conversely rescues wildtype crossover patterning but not crossover rates. These experiments demonstrate functional divergence at mei-218 and suggest that crossover rate and patterning are separable functions.

Keywords: Drosophila; crossing over; evolution; positive selection; recombination.

Figure 1

Meiotic recombination in Drosophila. During meiotic recombination programmed DSBs are formed, the 5′ ends are recessed and the resulting 3′ single-stranded tails invade the homologous chromosome. After synthesis off the template, the BLM Helicase can unwind the structure which is then resolved via synthesis-dependent strand annealing (SDSA) into a non-crossover gene conversion. If the invading strand synthesizes far enough, second-end capture can occur, creating a crossover intermediate. Most crossover intermediates are processed via the class I pathway in which they are stabilized by the mei-MCM complex (green rings) and resolved into interfering crossovers. A smaller fraction of crossover intermediates enters the class II pathway in which they are resolved as either non-interfering crossovers or non-crossovers with equal probability. The BLM Helicase inhibits crossover intermediate processing though the class II pathway and therefore promotes the class I pathway.

Figure 2

Molecular evolution across the Drosophila phylogeny. (A) Phylogenetic relationship of the 23 species analyzed within the melanogaster, obscura, and virilis species group. (B) PAML analyses for the three species groups were performed separately because the phylogenetic distances among them is so large that d_s is saturated. We report the log-likelihood estimates from a model 7 – model 8 comparison (*P < 0.05, **P < 0.01, ***P < 0.001). (C) A schematic of the structural domains in the five proteins analyzed. In the MEI-MCMs the AAA ATPase MCM domains are shaded in green and the MCM N-terminal domains are shaded in blue. In the BLM Helicase the RecQ DNA Helicase domain in shaded in purple. In MEI-218 and REC, the pins represent codons with evidence for positive selection in the melanogaster group (yellow circle) and the obscura group (red squares).

Figure 3

The mei-217/-218 allele of D. pseudoobscura alters the rate and patterning of crossing over in D. melanogaster. (A) D. melanogaster females containing a transgene of a D. pseudoobscura mei-217/-218 allele inserted on chromosome 3L (75A10) were assayed for crossing over. The endogenous mei-218¹ allele contains a nonsense mutation. Crossover frequencies were estimated among the 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). mei-217/-218^mel data re-produced from Brand et al. (2018). (B) For each genotype, the means and standard deviations [in brackets] of crossover frequency for the five genetic intervals measured in the two transgenic genotypes. The p-values are for unpaired t-tests (*P < 0.05, **P < 0.01, ***P < 0.001). (C) The proportion of total crossovers distributed across the five intervals in the net-cn region in mei-217/-218^mel (blue) and mei-217/-218^pse (green) females. The total number of crossovers scored for mei-217/-218^mel and mei-217/-218^pse females is 956 and 786, respectively (see Table S2).

Figure 4

The mei-217/-218 allele of D. virilis alters the rate and patterning of crossing over in D. melanogaster. (A) D. melanogaster females containing a transgene of a D. virilis mei-217/-218 allele inserted on chromosome 3L (75A10) were assayed for crossing over. The endogenous mei-218¹ allele contains a nonsense mutation. Crossover frequencies were estimated among the 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). mei-218¹ and mei-217/-218^mel data re-produced from Brand et al. (2018). (B) For each genotype, the means and standard deviations [in brackets] of crossover frequency for the five genetic intervals measured in the mei-218 mutant and two transgenic genotypes. p-values are derived for unpaired t-tests. *P < 0.05, **P < 0.01, ***P < 0.001. (C) The proportion of total crossovers distributed across the five intervals in the net-cn region in mei-217/-218^mel (blue), mei-217/-218^vir (orange), and mei-218¹ (gray) females. The total number of crossovers scored for the mei-217/-218^mel, mei-217/-218^vir, and mei-218¹ females is 956, 93, and 7, respectively (see Table S2).