Reviewer #4 (Public Review):
In "A Toxin-Antidote Selfish Element Increases Fitness of its Host", Long et al. attempt to address an outstanding question in the evolution of toxin-antidote (TA) systems in primarily selfing species: How do TA systems escape drift and spread in a primarily selfing species? The authors use simulations to show that at outcrossing rates similar to that observed in C. elegans a TA element, like the peel-1/zeel-1 element, has a high probability of being lost to genetic drift. However, the authors show that the peel-1 gene provides a fitness advantage to strains harboring it, providing evidence for a dual role for this gene and insights into how this element might have escaped being lost to genetic drift.
Strengths:
The experiments in this paper are well-framed. The authors use simulations to show that the observed frequency of the peel-1/zeel-1 TA element in the C. elegans population is highly unlikely given the inferred outcrossing rates of species.
The authors clearly show that the 140-370kb CB4856 introgression into N2 lowers relative fitness, number of eggs laid, and animal size, relative to N2.
The authors generated null alleles of peel-1 and zeel-1 and showed that a truncated version of PEEL-1 confers a detrimental fitness effect when compared to N2. Furthermore, the authors show that the fitness effect associated with peel-1 is independent of the antidote (zeel-1) component of this TA element.
Weaknesses:
1) The reference N2 strain has been cultivated in the lab for decades and many different versions of this strain exist. The different versions of N2, which might have slightly different genomes, are likely to have different fitness in laboratory conditions. It is unclear whether the N2 strain used to construct QX1198 is the same N2 strain used to construct CX12311, PTM229, and PTM377 (and others derived from these). The potential difference in the N2 strain used for the construction of these strains might contribute to the large discrepancy between the relative fitness shown in Figure 2A (~0.25) and Figure 3E-F (~0.07). Alternatively, the other CB4856-specific variants present in the 140-370 kb introgression in the QX1198 strain might cause this large discrepancy.<br />
Regardless of the potential discrepancy among N2 strains used as the genetic background, the claim that the presence of peel-1 confers higher relative fitness is supported by Figure 3E because PTM377/409 were presumably derived from the same N2 strain.
2) For Figures 2B and 3C, the authors report the number of eggs laid per animal. C. elegans strains can lay embryos that do not hatch and therefore fail to develop into reproductive adults. Does the difference between N2 and N2(peel-1(0)) remain when considering the number of reproductively mature progeny? Presumably, eggs laid translate to reproductive adults because a relative fitness increase is observed when peel-1 is present.
3) The authors did not perform whole-genome sequencing of the peel-1 and zeel-1 CRISPR edited strains or mention any backcrossing done to eliminate potential off-target editing events. Therefore it is difficult to conclude whether off-target effects might influence the quantified traits presented in Figure 3. This concern is somewhat alleviated by the reciprocal competition assay presented in Figure 3E (4th boxplot), but a potential off-target editing event that lowers fitness could have segregated with the silent dpy-10 and peel-1 edits.<br />
The same concern is present with the zeel-1-independence experiment, however, this experiment does not have reciprocal competition experiments.
4) In Figure 3C-D, the authors show that a homozygous truncated version of PEEL-1 confers a reduction in eggs laid per animal (proxy for brood size) and animal length (proxy for developmental speed). However, the authors do not show whether a heterozygous truncated PEEL-1 strain (N2 peel-1/peel-1(kah126)) confers the same reduction in eggs laid or animal size. Would the allele frequency dynamics derived from the simulations be affected by a fitness advantage only being conferred by the presence of two copies of peel-1?
5) The authors show a fitness advantage associated with peel-1 in laboratory conditions. It is obviously extremely difficult to extend these observations to the wild, however, the authors do not take their observations that peel-1 confers a fitness advantage in the lab and apply their empirical observations to the simulation framework. If the laboratory fitness advantage of peel-1 did extend to the wild, one might expect the element would fix in the population in the simulation framework.
6) It seems possible that a truncated version of the PEEL-1 protein might have unknown deleterious fitness consequences that are independent of any beneficial effect the full-length protein might have. The same is true for the truncated ZEEL-1 protein, though potentially less concerning because there are only 5 amino acids.
