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Analysis of ancestry heterozygosity suggests that hybrid incompatibilities in threespine stickleback are environment dependent

['Ken A. Thompson', 'Department Of Zoology', 'Biodiversity Research Centre', 'University Of British Columbia', 'Catherine L. Peichel', 'Division Of Evolutionary Ecology', 'Institute Of Ecology', 'Evolution', 'University Of Bern', 'Bern']

Date: 2022-02 

Hybrid incompatibilities occur when interactions between opposite ancestry alleles at different loci reduce the fitness of hybrids. Most work on incompatibilities has focused on those that are “intrinsic,” meaning they affect viability and sterility in the laboratory. Theory predicts that ecological selection can also underlie hybrid incompatibilities, but tests of this hypothesis using sequence data are scarce. In this article, we compiled genetic data for F 2 hybrid crosses between divergent populations of threespine stickleback fish (Gasterosteus aculeatus L.) that were born and raised in either the field (seminatural experimental ponds) or the laboratory (aquaria). Because selection against incompatibilities results in elevated ancestry heterozygosity, we tested the prediction that ancestry heterozygosity will be higher in pond-raised fish compared to those raised in aquaria. We found that ancestry heterozygosity was elevated by approximately 3% in crosses raised in ponds compared to those raised in aquaria. Additional analyses support a phenotypic basis for incompatibility and suggest that environment-specific single-locus heterozygote advantage is not the cause of selection on ancestry heterozygosity. Our study provides evidence that, in stickleback, a coarse—albeit indirect—signal of environment-dependent hybrid incompatibility is reliably detectable and suggests that extrinsic incompatibilities can evolve before intrinsic incompatibilities.

If ecological selection on trait mismatch is operating in the field but not in the lab, selection for increased ancestry heterozygosity should be specific to the field (or at least stronger than in the lab). If mismatch is deleterious, the fitness landscape is assumed to be saddle or ridge like (see [ 11 ]); hybrids with mismatched phenotypes are displaced along the steep sides orthogonal to the axis of parental divergence and have lower fitness than individuals with relatively “matched” trait values (whether parental or somewhat intermediate). Thus, we predicted that we would observe elevated excess ancestry heterozygosity in samples from the field compared to those from the lab.

In this study, we compare patterns of selection on ancestry heterozygosity between F 2 hybrid stickleback raised indoors in aquaria to those from the same cross types raised in the field in experimental ponds (see Table 1 for overview of data sources). We first consider hybridization between sympatric benthic and limnetic populations of threespine stickleback. These populations, which are reproductively isolated species due to strong assortative mating [ 19 ] and experience reduced hybrid fitness due to extrinsic selection pressures [ 20 ], have evolved independently in at least 5 watersheds in British Columbia, Canada [ 21 , 22 ]. Although reproductively isolated in the wild, the species pairs have no known intrinsic barriers that reduce fitness in the lab [ 20 ]. Second, we consider hybridization between allopatric populations of anadromous and solitary freshwater stickleback. As with the benthic–limnetic species pairs, these populations are recently diverged and can readily hybridize. The benthic × limnetic crosses are between specialist populations that are at extreme opposite ends of the range of trophic phenotypes observed among this region’s native stickleback in fresh water. The marine × freshwater cross involves a limnetic-like marine population and a generalist freshwater population (Cranby Lake) that is intermediate between the limnetic and benthic populations [ 23 , 24 ] and is therefore a less divergent cross with respect to trophic characters—however, the populations differ in other traits involved in marine–freshwater divergence [ 25 ].

Both panels depict results from a representative simulation run of adaptive divergence and hybridization between 2 populations. We consider an organism with 2 traits that have both diverged as a result of selection. Colored points are individual hybrids, with darker colors indicating higher heterozygosity. Panel (A) depicts the distribution of 500 F 2 hybrid phenotypes in two-dimensional trait space. Large black points are the 2 parent phenotypes, which are connected by a black line indicating the “axis of divergence.” Panel (B) depicts the relationship between individual excess ancestry heterozygosity and trait “mismatch” of individual hybrids [ 13 ]. Excess ancestry heterozygosity is the observed heterozygosity minus the expected heterozygosity based on ancestry proportion—0 is the expected mean in the absence of selection (approximately observed heterozygosity frequency of 0.5). Mismatch is calculated as the shortest (i.e., perpendicular) distance between a hybrid’s phenotype and the black line connecting parents in (A). Variation parallel to this axis connecting parents in (A) captures variation in the “hybrid index.” The plot shows that trait mismatch is lower in more heterozygous F 2 hybrids. Heterozygosity values are discrete because a small number of loci underlie adaptation in the plotted simulation run. Simulations are outlined in the Methods. The “mismatch”–heterozygosity relationship is stronger, although less intuitive, in organisms with greater dimensionality (i.e., more traits; see S1 Fig for a case with 10 traits following [ 18 ]). The data and code required to recreate this figure may be found at https://doi.org/10.5061/dryad.h18931zn3 .

Recent theoretical advances, however, suggest ways to test for and measure the net effect of hybrid incompatibilities using experimental crosses. Specifically, selection against hybrid incompatibilities in an F 2 hybrid cross causes an increase in ancestry heterozygosity—the number of sites in the genome that carry both parents’ alleles at ancestry informative sites—at the population level [ 16 , 17 ]. This is expected because F 2 hybrids have a hybrid index of approximately 0.5—deriving half of their alleles from one parental species and half from the other. Thus, individuals with high heterozygosity relative to their hybrid index have fewer pairs of homozygous loci with opposite ancestry compared to relatively more homozygous individuals with a similar hybrid index. Assuming that most alleles affect the phenotype additively and have noninfinitesimal effect sizes, having many loci with opposite homozygous ancestry can result in hybrids with maladaptive “mismatched” phenotypes, whereas highly heterozygous individuals are expected to have less mismatched phenotypes ( Fig 1 , S1 Fig ). Whether “mismatch” affects fitness, however, ultimately depends on the ecology of the system and the underlying fitness landscape. Such coarse approaches—coarse because they use summary statistics rather than direct mapping—are a promising means to identify the presence of small effect hybrid incompatibilities at the genetic level using field experiments.

Ecological selection could underpin incompatibilities if particular allele combinations render hybrids unable to function in their ecological environment, such as in avoiding predators or capturing prey. Several recent studies have shown patterns consistent with this effect, wherein hybrids have “mismatched” trait combinations and reduced fitness as a result [ 11 – 13 ]. Such studies have successfully demonstrated that incompatibilities due to trait mismatch exist, but links to the underlying genetics have not been made. Perhaps the most significant barrier to progress in studying the genetics of “ecological” hybrid incompatibilities is the unique difficulty of detecting them. The ability to detect individual incompatibilities depends on the genetic architecture underlying traits—that is, whether quantitative trait loci (QTL) have small or large phenotypic effects [ 14 , 15 ]. For example, Arnegard and colleagues [ 11 ] found that combining divergent jaw traits together in F 2 threespine stickleback fish (Gasterosteus aculeatus L.) hybrids likely reduced their fitness because these traits interacted in a manner that reduced suction feeding performance. The interacting jaw components map to several regions of the genome that individually explain a small fraction (<10%) of the phenotypic variance (and most variance was unexplained), thus rendering it difficult to study their individual epistatic fitness effects.

Hybrid incompatibilities—interactions among divergent genetic loci that reduce the fitness of hybrids—are a key component of reproductive isolation between diverging lineages [ 1 ]. Incompatibilities have been studied most intensively in the context of sterility and mortality, in part because phenotyping in the laboratory is reliable and because such incompatibilities can have simple genetic architectures involving few loci [ 2 , 3 ]. These sorts of interactions have come to be called “intrinsic” hybrid incompatibilities due to the fact that there are conflicts within the hybrid genome that are expected to impact hybrids in most environmental contexts (although note that the strength of selection against some intrinsic incompatibilities can vary across environments [ 4 , 5 ]). Studies have shown that the number of loci involved in intrinsic incompatibilities tends to increase with genetic divergence between the parent species [ 6 – 8 ] and that incompatibilities can be common throughout the genomes of isolated conspecific populations [ 9 , 10 ]. Collectively, evolutionary biologists have made substantial progress toward identifying generalities about the evolutionary genetics of intrinsic hybrid incompatibilities.

Panel (A) shows the main test of group differences for (left) benthic × limnetic crosses and (right) marine × freshwater crosses. Drawings above the panels were done by K. Chu and show the first listed population on top. These values are extracted from our statistical model using visreg (colored points and violins; [ 31 ]) and emmeans (black estimates of means and CIs; [ 32 ]). Panel (B) shows raw data (i.e., not from a statistical model) for each data source ( Table 1 ; LCR × CRN is Little Campbell River × Marine) separately, with colors representing lab (red) versus pond (blue) as in (A). Violin overlays show the full distribution of the data, and small colored points show values for individual fish. Large black points are the group means and 95% CIs. The data and code required to recreate this figure may be found at https://doi.org/10.5061/dryad.h18931zn3 .

In support of our prediction, mean individual excess ancestry heterozygosity—the deviation from Hardy–Weinberg expectations based on the relative frequency of alternative ancestry alleles in the genome—was significantly elevated among pond-raised fish compared to aquarium-raised fish. This was the case in both the benthic × limnetic data ( = 0.021 ± 0.0081 [magnitude of excess ancestry heterozygosity ± SE], z = 2.62, P = 0.009) ( Fig 2 A—left; also see S2 Fig for plots of individual hybrid index and heterozygosity) and the marine × freshwater data ( Fig 2 A—right; = 0.038 ± 0.0065 [SE], z = 5.86, P < 0.0001). Patterns were similar for all studies in the dataset—each study that contributed data from pond experiments found significant excess ancestry heterozygosity, and each study that contributed data from aquaria found that excess ancestry heterozygosity did not differ from 0 ( Fig 2B ). The signal of excess ancestry heterozygosity was variable among chromosomes, although the majority had values exceeding 0 ( S3 Fig ).

Discussion

Ecological selection acting on hybrids is a critical determinant of gene flow between diverging lineages [33]. Yet, detecting how divergent alleles interact to mediate hybrid fitness in ecological contexts has proven difficult due to the effect sizes of interacting loci and the massive experiments required to achieve sufficient power [34]. Here, we tested whether a coarse-grained signal of selection against hybrid incompatibilities—elevated excess ancestry heterozygosity [17]—differed between laboratory and field replicates of genetic crosses between the same populations. We found that excess ancestry heterozygosity was elevated in recombinant stickleback hybrids raised in experimental ponds compared to those from similar crosses raised in aquaria. This result is consistent with the hypothesis that certain ecologically mediated hybrid incompatibilities between recently diverged stickleback populations act more strongly in field settings than in the lab. Our finding implies that individual stickleback with a greater mismatch in parental traits are less likely to survive than those with lesser mismatch. Below, we consider whether other processes could plausibly explain this result, discuss the relevance of our findings for speciation, and highlight opportunities for future research.

The lack of excess ancestry heterozygosity in hybrids raised in aquaria is an expected result given what is known about “intrinsic” hybrid incompatibilities in stickleback. Previous studies of benthic × limnetic hybrids have found no evidence for intrinsic inviability in F 2 crosses using measures of embryo development and hatching success [22,20,35] or lifetime fitness [20]. A recent review summarizing the literature on reproductive isolation in threespine stickleback [36] reports that “intrinsic” barriers are typically weak to nonexistent. Both marine (i.e., anadromous) × freshwater and benthic × limnetic crosses had no evidence for intrinsic inviability, whereas the authors found evidence for hybrid ecological inviability in both systems [36]. Thus, it would have been surprising if we had recovered any signal of selection on excess ancestry heterozygosity in lab-raised hybrids.

Alternative causes of excess ancestry heterozygosity We hypothesize that selection against trait mismatch (i.e., ecological hybrid incompatibility) caused the observed patterns of selection on ancestry heterozygosity among surviving individuals in the ponds, but several other mechanisms could possibly underlie such a pattern. These alternative mechanisms involve processes operating at single loci rather than interactions among loci. Ultimately, the data presented here have limited ability to conclusively distinguish between single-locus processes like heterosis and multilocus processes like incompatibilities, but we discuss the strength of evidence for different possible alternative mechanisms below. Heterosis refers to a case where, for a given locus, the heterozygote has greater fitness than the parental genotypes. This could obviously lead to selection for elevated ancestry heterozygosity. In the benthic × limnetic crosses, environment-specific heterosis is unlikely based on prior knowledge about hybrid fitness in this system. If heterosis were common, then F 1 hybrids should have higher fitness than parents. However, F 1 and reciprocal backcross hybrids have lower growth and/or survival than both parent taxa in field experiments [20,37,38]. These patterns are opposite to what would be expected as a result of field-specific heterosis, suggesting that it is unlikely to be acting in the benthic × limnetic crosses. Less is known about heterosis in marine × freshwater crosses, although Hagen [39] found evidence for strong ecology-based selection against hybrids in the hybrid zone between the Little Campbell River anadromous population and the resident freshwater-adapted stream population. In sum, the available evidence from observing selection on F 1 hybrids in the field versus lab is inconsistent with there being environment-specific heterozygote advantage. The above patterns also suggest that alleles conferring a fitness advantage are not dominant to lower fitness alleles and thus that environment-specific dominance for fitness does not result in pond-specific selection for increased ancestry heterozygosity. It is conceivable that heterosis could act in the lab via body condition and general health, but only affects mortality in the field and thus would not affect mean excess ancestry heterozygosity in aquaria. Previous studies have found that the growth rate of F 1 benthic × limnetic hybrids in the lab matches the additive expectation of parents [20,40], consistent with there being no intrinsic hybrid vigor. In the present study, we find no relationship between body size and excess ancestry heterozygosity in any of the aquarium-raised crosses (S4 Fig; robust within-family regression analyses are not possible in ponds due to the small number of individuals within families; see [11] for discussion of using body size as a component of fitness). Thus, we conclude that there is no evidence supporting a relationship between ancestry heterozygosity and vigor in the laboratory. We can also use predictions that are specific to the genetics of heterosis versus hybrid incompatibilities to differentiate between them. Specifically, heterosis depends on interactions within a locus, while incompatibilities depend on interactions between loci. If the benefit of ancestry heterozygosity was due to interactions within loci alone, we would expect to see no relationship between genome-wide admixture proportion (i.e., hybrid index) and excess ancestry heterozygosity—this is because all heterozygosity is beneficial regardless of the genetic background. By contrast, if excess heterozygosity was attributable to interactions among loci, as in the hybrid incompatibility model, we would expect diminishing benefits of excess ancestry heterozygosity as genome-wide ancestry proportions became more parent like (i.e., as hybrid index deviates from 0.5). As expected under the incompatibility model, excess ancestry heterozygosity declines as the hybrid index of pond-raised individuals deviates from 0.5 (Spearman ρ = −0.059; P = 0.0006), while there is no relationship in the lab (ρ = −0.012; P = 0.77) (S5 Fig). While this observation does not eliminate the possibility that some of the observed excess heterozygosity is driven by environment-specific heterosis, it is consistent with our hypothesis that excess ancestry heterozygosity largely results from selection against extrinsic incompatibilities. Inbreeding depression could also generate our observed pattern if it only affected fitness in ponds [41–43]. If inbreeding depression led to selection on ancestry heterozygosity in our study, we would expect inbred individuals to have higher mean excess ancestry heterozygosity than outbred individuals. To test this specific prediction, we used the data from Arnegard and colleagues [11] where due to the study design, some F 2 hybrids resulted from mating between full siblings (n = 74), and some individuals were produced by mating between unrelated individuals (n = 541). We find that these 2 groups do not differ in mean excess ancestry heterozygosity (F 1,612 = 0.0734, P = 0.787; S6 Fig). Furthermore, the direction of the nonsignificant difference in mean (estimate of outbred − sibling difference = 0.0035 ± 0.0129 [SE]) is the opposite of what is expected under environment-specific inbreeding depression. We note, however, that this analysis does not account for the possibility of any fixed deleterious alleles in the populations [44,45]. Thus, the data suggest that environment-specific inbreeding depression is not causing heterosis in this system. Finally, an additional analysis indirectly provides evidence of phenotype-based hybrid incompatibilities in the data from Arnegard and colleagues [11]. Specifically, Arnegard and colleagues [11] classified F 2 hybrids into 4 groups (“A,” “B,” “L,” and “O”) based on individual niche use. “B,” “O,” and “L” individuals had benthic-like, intermediate, and limnetic-like diets, respectively. “A” individuals, however, had unusual diets, were smaller, and had a greater extent of mismatched trait combinations compared to the other groups. The authors hypothesize that trait mismatch caused these fish to grow more slowly than more “matched” individuals. Our analysis reveals that “A” group individuals have lower excess ancestry heterozygosity than non-“A” individuals (S7 Fig)—as expected if lower excess ancestry heterozygosity correlates with higher trait mismatch. This reanalysis suggests a link between trait mismatch, ancestry heterozygosity, and fitness in stickleback hybrids raised in a natural environment.

Relation to other studies of incompatibilities and ancestry heterozygosity Our results contribute to a growing understanding of the biology of environment-dependent hybrid incompatibilities. In natural hybrid populations of swordtail fishes, Schumer and colleagues [46] estimated that dozens of incompatibilities separate parent species [47]. The authors also suggested that many of these are likely subject to natural or sexual selection [46]. Previous studies on hybrid stickleback [11,48] have estimated fitness landscapes that are consistent with the hypothesis that mismatched trait combinations are selected against, and our analysis of genetic data supports this hypothesis. We also note that selection against mismatched combinations of traits has the same genetic basis as selection against single phenotypes that express maladaptive transgressive values after hybridization [18,49,50]. Thus, studies focused on only a single trait under stabilizing selection might still find selection against incompatibilities if hybrids have trait values that are below or above the optimum value [51]. More broadly, our results are consistent with predictions generated from theoretical models of speciation and adaptation [17]. Thus, although stickleback is an excellent system in which to test these predictions, the mechanisms underlying our results are likely general. Our findings also highlight differences from previous analyses of selection on hybrids in different environments. In yeast, selection for low ancestry heterozygosity is common in hybrids when tested in the lab [52,53]. This difference (which does not occur as a result of aneuploidy) between the yeast studies and our study of stickleback might result from the fact the lab media that yeast were raised in are novel environments, and transgressive traits suited to these environments result from excess ancestry homozygosity (see S1 Fig; also see [54] for how novel environments can result in directional selection on ancestry). Stickleback populations in postglacial lakes are specialists on zooplankton or benthic invertebrates when they coexist with a competing fish species [23,55,56] or are generalist populations that make use of both niches [57]. Freshwater stickleback populations in this region span a range of phenotypes along a limnetic–benthic axis, and no specialists along novel trophic axes are known to occur. Fish in the experimental ponds that we considered herein have diets that are largely representative of what they consume in nature [11], and there is no evidence supporting the hypothesis that ponds contain novel adaptive peaks. Qualitative patterns of selection against hybrids driving excess ancestry heterozygosity might therefore depend on the availability and nature of novel ecological niches.

[END]

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