The nematode Caenorhabditis elegans is a facultative hermaphrodite that reproduces either by virtue of self-fertilization or cross-breeding with a male (androdioecious reproductive system). The hermaphrodites are somatically female but produce a limited number of sperm during their late larval development before switching to the production of eggs. The sperm is stored in the spermatheca and can be used to fertilize the newly formed eggs. Except for a very few males (around 0.2% in the standard laboratory strain N2) that arise spontaneously as the result of X chromosome non-disjunction, the entire self-progeny is hermaphroditic. Males can mate with hermaphrodites and give rise to 50% males in the cross-progeny. Cross-fertilization is not possible among hermaphrodites [1, 2]. Male derived sperm is also stored in the spermatheca where it competes with the hermaphrodite's own sperm for the fertilization of the oocytes. Male sperm is usually larger and therefore has a competitive advantage over the hermaphrodite's sperm [3, 4].
If hermaphrodites can reproduce by self-fertilization, males are superfluous. In fact, they could even represent a burden, decreasing individual fitness, in analogy with the two-fold cost of males in theories on the evolution of sex [5, 6]. Therefore, the persistence of males represents an important puzzle for our understanding of C. elegans biology. Its explanation is expected to advance a more general insight into the evolution of androdioecy. To date, the function of C. elegans males has been addressed using two main approaches: (i) experimental evolution in the laboratory, and (ii) analysis of (male-dependent) outcrossing rates in wild populations.
The experimental evolution of laboratory populations of the standard strain N2 (as well as mutants derived from this strain) uniformly demonstrated that initial male frequencies of either 50% or 33% rapidly and steeply decline to less than 10% within ten to 15 generations [7–11]. Furthermore, compared with the dioecious species Caenorhabditis remanei, mating behaviour was severely compromised in the N2 strain, i.e. males often fail to find hermaphrodite mates, possibly due to limited production and/or degeneracy of the hermaphrodite's sex pheromone [[7, 12] but see, [13, 14]]. Taken together, these results suggested that males represent evolutionary relics without any particular function, which are only still present, because of a relatively recent switch to hermaphroditism and selfing in the lineage leading to C. elegans . Interestingly, however, other natural isolates show clear differences to N2. The spontaneous production of males is a pre-requisite for male maintenance and it reaches values of more than 3% of the total offspring in some isolates – clearly more than N2 with a value of less than 0.5% [11, 15]. Similarly, males are able to persist in populations of some strains, e.g. the Hawaiian strain CB4856 and the Oregon strain PX174 . This effect seems to be enhanced in these two strains (but not others) if worm populations are subjected to fluctuating environmental conditions like variable exposure to different mutagens . Similarly, populations with deficient DNA repair and thus increased mutations rates also maintain males at higher frequencies . These results suggested that males are beneficial to ensure frequent outcrossing, which is favored under variable environmental conditions and/or high deleterious mutation rates [17–22].
An alternative albeit indirect route to assess the function of males is to infer outcrossing rates in natural populations. Several recent studies analysed new C. elegans isolates from different parts of the world using a variety of molecular markers such as microsatellites, AFLPs, or DNA sequence polymorphisms. They unanimously demonstrate that outcrossing does occur, but that it is usually extremely rare [23–27]. The only exception is an inferred outcrossing rate of 0.2 , whereas all other studies suggest it to range in between 10-5 up to 0.02 [23–25]. Consequently, males leave a genetic footprint in natural populations. In consistency with the conclusions from experimental evolution in the lab, rare outcrossing may be sufficient to eliminate mutational load and/or maintain genetic diversity required for rapid adaptation to fluctuating environments [17, 29, 30].
In the first part of this publication we describe the decline of the proportion of males in eight different natural isolates under standard laboratory conditions. The fact that males are lost at very different rates even if the strains are maintained under the same conditions indicates that the difference is genetically determined and is therefore a putatively selectable trait. In the second part we evaluate the possible reasons for the difference in male persistence between the two common laboratory strains N2 and CB4856. Several behavioral and physiological factors could account for this difference, for example: i) the mating efficiency of the males, ii) the mating efficiency of the hermaphrodites (this includes the attractiveness of the hermaphrodites for males), iii) the competitive advantage of the male derived sperm, iv) the number of sperm transferred, v) the difference between the maximum number of progeny a hermaphrodite can produce with and without mating. In order to address these points, we performed a systematic analysis of intra- and inter-strain crosses between N2 and CB4856- to our knowledge for the very first time in this context. Our results indicate that CB4856 males are capable of mating successfully with more hermaphrodites than N2 males and that N2 hermaphrodites produced a higher number of (all-hermaphrodite) self-progeny even after mating. Both these effects result in a higher proportion of males in the next generation for CB4856 if compared with N2, thus potentially explaining male persistence in the former but not the latter strain.