Outcrossing and the maintenance of males within C. elegans populations

Abstract

Caenorhabditis elegans is an androdioecious nematode with both hermaphrodites and males. Although males can potentially play an important role in avoiding inbreeding and facilitating adaptation, their existence is evolutionarily problematic because they do not directly generate offspring in the way that hermaphrodites do. This review explores how genetic, population genomic, and experimental evolution approaches are being used to address the role of males and outcrossing within C. elegans. Although theory suggests that inbreeding depression and male mating ability should be the primary determinants of male frequency, this has yet to be convincingly confirmed experimentally. Genomic analysis of natural populations finds that outcrossing occurs at low, but not negligible levels, and that observed patterns of linkage disequilibrium consistent with strong selfing may instead be generated by natural selection against outcrossed progeny. Recent experimental evolution studies suggest that males can be maintained at fairly high levels if populations are initiated with sufficient genetic variation and/or subjected to strong natural selection via a change in the environment. For example, as reported here, populations adapting to novel laboratory rearing and temperature regimes maintain males at frequencies from 5% to 40%. Laboratory and field results still await full reconciliation, which may be facilitated by identifying the loci underlying among-strain differences in mating system dynamics.

Figure 1

Some genetic consequences of outcrossing and selfing. Left: outcrossing maintains heterozygosity, whereas self-fertilization creates an excess of homozygotes. Right: outcrossing can break linkage between alleles at different loci, whereas self-fertilization maintains linkage disequilibria over time. These patterns are generated by the coancestry within and between loci generated by selfing (middle) and are manifest within populations by changing levels of heterozygosity and linkage disequilibrium (bottom). Here, heterozygosity is determined by , where s is the rate of self-fertilization and H₀ is the initial heterozygosity before selfing (scaled here to its maximum value of 0.5; Crow and Kimura 1970). Linkage disequilibria at generation t are given by $D\left(t\right)={(1-\stackrel{`}{r})}^t{D}_0$, where D<sub>0</sub> is the amount of linkage disequilibrium at generation zero (scaled to 1.0 for convenience) and $\stackrel{`}{r}$ is the effective amount of recombination scaled by the rate of selfing within the population: $\stackrel{`}{r}=r\left[1\right(1-s)/2-s)]$ (Nordborg 1997; Barrière and Félix 2005). The actual recombination rate, r, was set to 0.5 for this example.

Figure 2

Sex ratios resulting from self-fertilization and outcrossing in Caenorhabditis elegans. Hermaphrodite eggs (X) can be fertilized by self (X) or male (X or Ø) sperm. Offspring resulting from self-fertilization are nearly all hermaphrodites, with a small fraction of males produced by nondisjunction of the X chromosome (μ). Outcrossed progeny are approximately 50% male and 50% hermaphrodite with a correction for the nondisjunction rate. Individual hermaphrodites can produce both self and male-sired offspring; thus, sex ratio in the progeny depends on how many embryos were sired by each parent. *, Approximate ratios based on N2.

Figure 3

Variation in male frequency among natural isolates of Caenorhabditis elegans. A survey of male frequency in 10 natural isolates of C. elegans reveals variation among strains that spans nearly 2 orders of magnitude (F_9,54 = 43.6, P < 0.0001). Strains were allowed to recover from freezing for 2 generations and then maintained for 10 generations under conditions similar to Manoel et al. (2007) before male frequency was determined. Male frequency was determined for 6 replicate populations per line (each initiated by single, unmated hermaphrodites) by sexing a total of ∼400 worms per replicate. Although differences among replicates were also significant (F_47,54 = 8.2, P < 0.0001), they accounted for only 10.2% of the total variance, whereas strain accounted for 87.1%. Values are least square means ± 1 standard error of the mean.

Figure 4

Evidence of male maintenance in 2 experimental evolution studies. (A) A hybrid population derived by 10 generations of random mating between 2 divergent Caenorhabditis elegans strains, CB4856 and CB4857, at 18.5 °C (the ancestral population) was then reared in 10 replicates at 15 °C or 23 °C for 57 and 69 generations, respectively, before freezing. All 3 lines were thawed simultaneously, allowed to recover at the correct temperature, and scored for male frequency (based on a minimum of 200 worms per replicate). Although male frequency declines from near 30% in the ancestor (likely a by-product of the crosses performed to generate the ancestor), males are maintained at high frequency (∼8%) at both temperatures after more than 57 generations. (B) A genetically heterogeneous ancestral population was derived by systematically crossing 16 strains of C. elegans (AB1, AB3, CB4852, CB4853, CB4855, CB4857, CB4858, N2, PB303, PB306, RC30, JU262, JU345, PX174, PX178, and PX179). Five replicate populations of the ancestral population were evolved independently for 47 generations under conditions similar to Manoel et al. (2007). Male frequency was measured by synchronously thawing stocks of each replicate population from generations 6,12, 24, 36, 47, and the ancestral population. Worms were allowed to recover from freezing for 2 generations and then hatched-off, plated in triplicate, and allowed to develop to adulthood. The frequency of males in each population was determined from a minimum of 700 individuals per line per generation. Generation (F_4,50 = 30.04, P = < 0.0001), line (F_4,50 = 174.33, P < 0.001), and the interaction between generation and line (F_16,50 = 27.22, P < 0.0001) were all significant. The 5 replicate populations are in gray, and average male frequency is in black. Values are least square means ± 1 standard error of the mean.

Figure 5

Summary of male maintenance experiments in Caenorhabditis elegans. Males/outcrossing can be maintained (horizontal arrow) or increase (up arrow) in frequency in experiments initiated with a heterogeneous populations or when isogenic lines are exposed to unique selective laboratory environments. When mutation rates are artificially increased, populations can have more males (>) than nonmutated controls; however, males are still lost from populations over time (diagonal down arrow). Males are otherwise lost (down arrow) from isogenic populations regardless of bacterial food type (Escherichia coli OP50 or HT115), maintenance regime (i.e., hatch-off), and temperature. Factors uniquely observed in concert with maintenance or increased male/outcrossing frequency are highlighted with boxes. Relative to factors listed at left, ✓, applicable; χ, not applicable; *, Starting strain derived by crossing up to 16 unique isogenic lines; ^‡, Included wild-type and him-5 alleles in the N2 background; ?, unspecified.