We previously found that populations starting experimental evolution with little genetic diversity evolved increased outcrossing rates, together with increased male fitness, during a period of 100 generations
. Here we were interested in finding if outcrossing is adaptive and testing which fitness components could explain the increase in outcrossing rates. Approaching an answer to these two questions would explain why outcrossing can be adaptive in novel environments. Using competitive assays expected to encompass the full life cycle of selection during experimental evolution, we found that population-wide fitness increased during 100 generations, thus indicating that increased outcrossing rates facilitate adaptation. During the same period, hermaphrodite fitness components expressed under selfing may have increased as well but there was little if any evolution of hermaphrodite fitness components expressed under outcrossing. These results suggest that males were the primary drivers in the adaptive evolution of outcrossing.
Correlated evolution between population fitness and male fitness might indicate that sexual selection explains the evolution of outcrossing rates. In particular, sexual selection could have occurred through an increase in either the mating or cross-fertilization success of males
[32, 33], for example by the evolution of larger sperm
, or the ability to immobilize hermaphrodites
. Consistent with the idea of sexual selection would be the existence of a negative genetic correlation between male fitness and hermaphrodite mating success, as the two sexes have different reproductive interests, cf.,
[36, 37]. We measured such negative correlation between male fitness and hermaphrodite mating success, but found it not to be significant. In addition, and inconsistent with a significant role for sexual selection, there was a positive genetic correlation between hermaphrodite self-fitness and male fitness. These positive correlations suggest that male and hermaphrodite traits were mostly determined by the same loci. The expression of these loci would give little opportunity for the operation of sexual selection, but see
Such arguments about the role of sexual selection in the evolution of outcrossing rates are limited to populations that segregate standing genetic diversity or that are subject to the recurrent mutational input of deleterious alleles
[31, 38–40]. Although conditional on the distributions of fitness effects, it is unlikely that many deleterious alleles accumulated in our experimental evolution. This is because under the life-cycles employed, the population sizes were high enough for reproductive mode to be irrelevant for the efficiency of selection, as we have previously shown in populations starting experimental evolution with standing genetic diversity
Interpreting the sign of genetic correlations between fitness components as we did presupposes that mutation rates and their distribution of effects on the several fitness components are equivalent. Unfortunately, there is little empirical data to confirm the validity of this assumption. In C. elegans, deleterious recessive mutations tend to be pleiotropic, affecting in a similar direction hermaphrodite life history and morphology
[26, 43]. Virtually nothing is known about rates of mutation to male traits, though much of the C. elegans genome is exclusively expressed in males
. Indirect evidence comes from comparing mutation rates among male-hermaphrodite and male–female Caenorhabditis species
. In this particular study, Baer and colleagues found that lifetime reproduction of males and hermaphrodites/females had a 4-fold higher deleterious mutation rate in male–female species than in male-hermaphrodite species; see also
[46, 47]. If these comparative results hold at the intra-population level, it is tempting to speculate that in our evolution experiment there was higher mutational variance for male traits than hermaphroditic traits.
Based on the sign of the correlations between hermaphrodite fitness components and population-wide fitness, hermaphrodite self-fitness should have augmented during experimental evolution and hermaphrodite mating success and outcross-fitness should have declined. Yet, there were little evolutionary dynamics in hermaphrodite mating success and fitness. This asymmetry is first justified with the potentially lower number of developmental and behavioural mutational targets in outcrossing traits than selfing traits, as C. elegans hermaphrodites are only slightly modified females with a few sperm cells. Second, and perhaps more significantly, since males only reached appreciable frequencies in mid-experimental evolution there was little chance for outcrossing traits to be expressed in hermaphrodites and thus they could not have greatly contributed to population-wide fitness. It is therefore not surprising that population-wide fitness and hermaphrodite self-fitness were positively correlated.
The competitive population-wide assay must have revealed the evolution of hermaphrodite self-fitness. Since the scoring procedure employed in the assay was only based in the presence/absence of GFP expression, we were unable to score for GFP/wild-type heterozygotes and had to assume random mating between the competitors in order to estimate an haploid fitness coefficient, which we took as a surrogate of population-wide fitness. As a result, increased performance under outcrossing will be underestimated since the direction of outcrossing, between GFP males with experimental hermaphrodites and vice-versa, is unknown. The only situation where increased outcrossing performance will not be underestimated is if there was assortative mating within the experimental populations. In this case, homozygous non-GFP genotypes would be more common than expected with genetic drift; see
 for comparable population-wide competitive assays and results in C. elegans populations of different reproduction systems when subject to experimental evolution in novel environments.
Lack of evolution in hermaphrodite fitness when outcrossing, but maybe also when selfing, could indicate that traits expressed under selfing and outcrossing cancelled each other out in their population-wide fitness effects. Congruent with this idea is the fact that hermaphrodite mating success was negatively correlated with both population-wide fitness and hermaphrodite self-fitness. Negative correlations among fitness components in hermaphrodites could in turn explain the reduced influence of hermaphrodites in the evolution of outcrossing rates: specifically, it would explain why the evolution of self-fitness did not prevent the evolution of increased outcrossing rates.
Fitness tradeoffs between selfing and outcrossing might have resulted from the continued antagonistic coevolution between males and hermaphrodites
[49, 50]. For example, it would have been possible for increased mating ability of males to have countered increased resistance to mating by hermaphrodites. However, there were no negative genetic correlations between male fitness and hermaphrodite mating success, or between male fitness and hermaphrodite outcross-fitness, as would be expected with the antagonistic coevolution scenario. Fitness tradeoffs between selfing and outcrossing might have instead resulted from developmental/ecological resource allocation constraints between male and female functions in hermaphrodites, despite males
[51, 52]. Congruent with this hypothesis, we found a negative genetic correlation between hermaphrodite self-fitness with hermaphrodite mating success.
Given more time, would outcrossing rates increase to levels close to those found in male–female species? Such outcome is possible, but as soon as males become more frequent there is increased opportunity for the outcrossing traits of hermaphrodites to be expressed and to contribute to population-wide fitness. Hermaphroditic tradeoffs between selfing and outcrossing fitness components could then dominate the adaptive evolution of outcrossing rates, instead of male fitness components. In these circumstances, it could be possible for selfing and outcrossing to be maintained at intermediate frequencies