The context and cause of adaptive radiations has been widely described and explored but why rapid evolutionary diversification does not occur in related evolutionary lineages has yet to be understood. One possible answer to this is simply that evolutionary diversification is provoked by environmental diversity, and that some lineages do not encounter the necessary environmental diversity. Three-spined stickleback on the Scottish island of North Uist show enormous diversification, which seems to be associated with the diversity of aquatic habitats. Stickleback on the neighbouring island of South Uist have not been reported to show the same level of evolutionary diversity, despite levels of environmental variation that we might expect to be similar to North Uist. In this study, we compared patterns of morphological and environmental diversity on North and South Uist. Ancestral anadromous stickleback from both islands exhibited similar morphology including size and bony ‘armour’. Resident stickleback showed significant variation in armour traits in relation to pH of water. However, North Uist stickleback exhibited greater diversity of morphological traits than South Uist and this was associated with greater diversity in pH of the waters of lochs on North Uist. Highly acidic and highly alkaline freshwater habitats are missing, or uncommon, on South Uist. Thus, pH appears to act as a causal factor driving the evolutionary diversification of stickleback in local adaptation in North and South Uist. This is consistent with diversification being more associated with ecological constraint than ecological opportunity.

Adaptation to derived habitats often occurs from standing genetic variation (SGV). The maintenance within ancestral populations of genetic variants favorable in derived habitats is commonly ascribed to long-term antagonism between purifying selection and gene flow resulting from hybridization across habitats. A largely unexplored alternative idea based on quantitative genetic models of polygenic adaptation is that variants favored in derived habitats are neutral in ancestral populations when their frequency is relatively low. To explore the latter, we first identify genetic variants important to the adaptation of threespine stickleback fish to a rare derived habitat – nutrient-depleted acidic lakes – based on whole-genome sequence data. Sequencing marine stickleback from six locations across the Atlantic ocean then allows us to infer that the frequency of these derived variants in the ancestral habitat is unrelated to the likely opportunity for gene flow of these variants from acidic-adapted populations. This result is consistent with the selective neutrality of derived variants within the ancestor. Our study thus supports an underappreciated explanation for the maintenance of SGV, and calls for a better understanding of the fitness consequences of adaptive genetic variation across habitats and genomic backgrounds.

1. Quantitative PCR (qPCR) has been commonly used to measure gene expression in a number of research contexts, but the measured RNA concentrations do not always represent the concentrations of active proteins which they encode. This can be due to transcriptional regulation or post-translational modifications, or localisation of immune environments, as can occur during infection. However, in studies using free-living non-model species, such as in ecoimmunological research, qPCR may be the only available option to measure a parameter of interest, and so understanding the quantitative link between gene expression and associated effector protein levels is vital. 2. Here we use qPCR to measure concentrations of RNA from mesenteric lymph node (MLN) and spleen tissue, and multiplex ELISA of blood serum to measure circulating cytokine concentrations in a wild population of a model species, Mus musculus domesticus. 3. Few significant correlations were found between gene expression levels and circulating cytokines of the same immune genes or proteins, or related functional groups. Where significant correlations were observed, these were most frequently within the measured tissue (i.e. the expression levels of genes measured from spleen tissue were more likely to correlate with each other rather than with genes measured from MLN tissue, or with cytokine concentrations measured from blood). 4. Potential reasons for discrepancies between measures, including differences in decay rates and transcriptional regulation networks are discussed. We highlight the relative usefulness of different measures under different research questions, and consider what might be inferred from immune assays.