Hybrid breakdown in male reproduction between recently diverged Drosophila melanogaster populations has a complex and variable genetic architecture

Abstract

Secondary contact between formerly isolated populations may result in hybrid breakdown, in which untested allelic combinations in hybrids are maladaptive and limit genetic exchange. Studying early-stage reproductive isolation may yield key insights into the genetic architectures and evolutionary forces underlying the first steps toward speciation. Here, we leverage the recent worldwide expansion of Drosophila melanogaster to test for hybrid breakdown between populations that diverged within the last 13,000 years. We found clear evidence for hybrid breakdown in male reproduction, but not female reproduction or viability, supporting the prediction that hybrid breakdown affects the heterogametic sex first. The frequency of non-reproducing F2 males varied among different crosses involving the same southern African and European populations, as did the qualitative effect of cross direction, implying a genetically variable basis of hybrid breakdown and a role for uniparentally inherited factors. The levels of breakdown observed in F2 males were not recapitulated in backcrossed individuals, consistent with the existence of incompatibilities with at least three partners. Thus, some of the very first steps toward reproductive isolation could involve incompatibilities with complex and variable genetic architectures. Collectively, our findings emphasize this system's potential for future studies on the genetic and organismal basis of early-stage reproductive isolation.

Keywords: Drosophila melanogaster; genetic incompatibilities; genetic variation; hybrid breakdown; male reproduction; reproductive isolation.

Figure 1.

Failure in male reproductive success is the predominant mode of hybrid breakdown among F2 hybrids between various Drosophila melanogaster populations. Left: F2 combined cross values and significance for average percent inviability (A), and for female (B) and male (C) reproductive failure rates. Line color denotes significance of differences in inviability/reproductive failure between-population’s crosses relative to both within-population cross groups, where pink indicates p < .05, yellow indicates .05< p < .1, and blue indicates p > .1. Within-population cross averages (gray) are listed adjacent to the population label. P-values were generated from bootstrapping (see Materials and methods section) and are listed underneath each between-population percentage. Individual cross averages are supplied in Supplementary Tables S1–S3. (D) Map of D. melanogaster population expansion and estimated divergence times from Sprengelmeyer et al. (2020). Divergence is given in thousands of years (kya) and each value represents the initial expansion estimate between nodes. Expansion out of ancestral ranges in southern-central African occurred roughly 12.8 kya. Populations rapidly colonized west Africa (12.6 kya), and later other regions of central Africa including our Ethiopian sample (9.5 kya). An additional estimated split (2.7 kya) between low- and high-altitude Ethiopian populations, the latter being used in the present study, is not depicted. Trans-Saharan migration occurred roughly 12.4 kya, during which populations experienced a moderately strong population bottleneck. Migration from the Middle East into cooler European regions occurred in the more recent past (1.8 kya). Coloration based on average annual temperature (scale bottom right) highlights the dramatic environmental differences between regions.

Figure 2.

Male reproductive failure rates between French and Zambian Drosophila melanogaster crosses deviate significantly from within-population crosses, and have a variable and sometimes cross-direction-specific pattern. (A) Histogram depicting percentage of male reproductive failure for between-population (blue) and within-population (yellow) cross directions. Values on the x-axis are the percentage of males that failed in all four reproductive assays, each involving one female from each parental population (binned by rounding percentages up to nearest whole number). Within-population cross averages are displayed for France (purple line) and Zambia (orange line), as well as between-population average (light blue line). In total, 36 of the 50 between-population crosses have reproductive failure rates higher than both within-population averages (bins right of both lines), and the rate of reproductive failure between these groups is significantly different (bootstrap p < 1.0 × 10⁻⁶). Individual cross counts are supplied in Supplementary Tables S4 and S5. (B) Male F2 reproductive failure rates among all possible crosses between five France and five Zambia strains, for both cross directions. Each cross is represented by a separate male failure rate for each cross direction (FR maternal top-left, ZI maternal bottom right). Individual cross directions with significant failure rates compared to within-population controls are bolded. Crosses whose direction-combined cross failure rates have significant raw p-values (p < .05) are highlighted pink, and those with .05 < p < .1 are highlighted yellow. These highlighted crosses were tested for differences between cross directions in failure rate, and crosses with significant (p < .05) differences are denoted by a blue solid line.

Figure 3.

Male reproductive failure in backcrosses is reduced relative to F2 males, suggesting that a two-locus BDMI model is not appropriate for incompatibilities involving uniparental factors. Left: Backcross design to test for mitochondrial (top) or Y (bottom) by X/autosome two-locus recessive BDMIs between two populations (blue and orange). Above, an example cross design to test for BDMIs involving the blue mitochondrial haplotype. Below, an example design to test for BDMIs involving the blue Y haplotype. Right: Reproductive failure rates among BC males. Under the hypothesis of a single, fully penetrant two-locus recessive BDMI, these backcrosses increase the probability of uniparental by hemizygous X or recessive autosomal genotypes, and the expected rate of reproductive failure among backcrosses should increase (blue bars). Out of the six crosses in Figure 2B that had cross direction effects, three crosses each produced reproductive failure at higher rates in the France or Zambia maternal direction. This allowed us to test for BDMIs involving four uniparental elements (France or Zambia, mitochondria or Y), as indicated by labels above yellow bars. Backcrosses are written in the format of “(F1 Maternal Parent / F1 Paternal Parent) x F0 partner”, where the F0 partner is male for backcrosses targeting the mitochondria (top) and female for backcrosses targeting the Y chromosome (bottom). Individual cross counts are supplied in Supplementary Table S7. Male reproductive failure was virtually absent in all six focal backcrosses assayed (yellow bars), differing drastically from expected values under two-locus BDMI models (blue bars). These results suggest that pairing a uniparental chromosome with hemizygous/homozygous genotypes from the opposite population is not sufficient to unmask the incompatibilities involved in cross direction asymmetry of F2 hybrid breakdown, indicating that these do not reflect classic two-locus BDMIs.