Loci important to acidic adaptation and their allele frequencies across Atlantic stickleback
Our analysis of genetic structure revealed striking genome-wide evidence of selection, including between acidic and basic ecomorphs. To investigate how polymorphisms important to acidic adaptation are maintained as SGV in marine stickleback, we searched for loci consistently involved in acidic adaptation based on the genome-wide comparison of acidic versus basic poolSeq data (Figure 2; differentiation profiles across all chromosomes are presented in Figure S2). This identified 50 independent adaptive SNPs nearly fixed for alternative alleles between the two freshwater ecomorphs (AFD 0.851 - 0.960; genome-wide median differentiation was 0.145) (Figure 3a; all adaptive SNPs are characterized in Table S3, and associated genes listed in .Table S4). These adaptive SNPs recovered many of the genome regions identified as important to acidic-basic differentiation in Haenel et al. (2019), based on partly independent specimen panels and a different analytical approach. Specifically, 15 of the 19 regions of highest acidic-basic differentiation inferred in Haenel et al. 2019 (i.e., the regions containing the ‘top core SNPs’ in that study) also exhibited a marker qualifying as adaptive SNP in the present investigation (Figure 3a, Figure S3). However, given the much higher (whole-genome) marker resolution, the present study also identified numerous novel regions (Figure 3a, Figure S2). Haplotype networks derived from genotypes phased across 5 kb around three exemplary adaptive SNPs indicated that these markers generally represent longer DNA tracts differentiated between the ecomorphs (Figure 3b). Across these exemplary regions, acidic stickleback populations generally shared closely related haplotypes distinct from the haplotypes prevailing in marine (and basic) fish, although sometimes acidic individuals exhibited marine haplotypes (chromosome IX and XI) and vice versa (chromosome XI).
At the adaptive SNPs, marine stickleback generally exhibited lower frequencies for the alleles characteristic of acidic fish (acidic alleles; median frequency across all SNP by marine sample combinations: 0.30) than for the alleles typical of the basic populations (median frequency 0.70) (Figure 4a; Table. S3). Also, the acidic alleles occurred at a lower overall frequency at the adaptive SNPs than at the baseline SNPs not under consistent acidic-basic divergence (median frequency across all baseline SNP by marine sample combinations: 0.46). These findings are in line with observations in Haenel et al. (2019) and indicate that alleles presumably important for the adaptation to ecologically highly derived acidic habitats tend to be unfavorable in ancestral marine stickleback when occurring at high frequency. Interestingly, however, we found no indication that the frequency of the acidic allele at the adaptive SNPs was elevated in marine samples collected around North Uist compared to samples from more distant locations (Figure 4a; compatibility intervals for the median frequency of the acidic alleles for all samples are presented in Figure S4); the frequency of these alleles was highly stable across all our marine samples. This key finding was reproduced when considering exclusively the subset of adaptive SNPs at which the acidic allele proved the minor allele withinall marine samples (n = 21; indicated in Table S3) (Figure 4b, Figure S4; median frequency across all SNP by marine sample combinations: 0.10); that is, the subset of markers at which purifying selection in marine stickleback appears particularly plausible because acidic adaptation involves a particularly strong shift away from the ancestral allele frequency.
The finding of similar frequencies of alleles important to adaptation to acidic waters across Atlantic marine stickleback challenges perpetual antagonism between gene flow and purifying selection (Schluter & Conte 2009; Bassham et al. 2018; Galloway et al. 2020) as a sufficient explanation for the maintenance of adaptive SGV in the ocean. Instead, we propose that acidic alleles can persist neutrally in marine populations when occurring at moderate to low frequencies. Purifying selection certainly plays a role, but primarily by impeding these alleles from rising to high frequency in marine stickleback. Note that the average frequency of the acidic alleles in the ocean was still around 0.3 (Figure 4a, Figure S4); at many adaptive loci, a substantial proportion of marine stickleback are thus expected to be homozygous for the acidic allele, so that purifying selection should still be effective even when these alleles were recessive. We therefore argue that the reason for the persistence of acidic alleles in marine populations is not their recessivity, but their selective neutrality when relatively uncommon. This interpretation supports quantitative genetic models under which polygenic adaptation can be achieved by moderate allele frequency shifts (Latta 1998; Kremer & Le Corre 2012; Le Corre & Kremer 2012).
An important caveat to consider is that although acidic habitats and the associated stickleback ecomorphs (Figure 1a) are exceptionally common on North Uist and rare elsewhere (Campbell 1985; Bourgeois et al. 1994; Klepaker et al. 2013), the potential of marine populations to hybridize with acidic-adapted freshwater stickleback was not explicitly manipulated or controlled among our Atlantic marine samples. Is it plausible that gene flow from acidic-adapted to marine stickleback is more widespread than we assume, sufficiently so to raise acidic alleles to substantial frequencies in marine stickleback all across the Atlantic despite purifying selection? In our view, the marine samples from the North Sea (DE, NL) refute this concern: western mainland Europe is densely populated and its Ichthyofauna is well investigated, but acidic stickleback ecomorphs have to our knowledge not been reported. Gene flow of acidic alleles into marine fish thus appears highly unlikely across this region, and yet the frequencies of acidic alleles are not reduced in these specific marine samples (Figure 4, Figure S4), consistent with the selective neutrality of these alleles when occurring at the frequencies observed in marine fish. Similar reasoning applies to marine stickleback around Iceland, because highly acidic freshwater habitats seem to be absent in Iceland (Magalhaes et al. 2021).