Limitations and future directions for ADH research
Although the patterns of gene-expression decoupling we observed inN. lecontei are consistent with the ADH, additional data are needed to: (1) verify that decoupled gene-expression phenotypes are independent at the genetic level, (2) demonstrate that decoupled gene-expression traits contribute to stage-specific adaptations, and (3) establish that metamorphosis is an adaptation for trait decoupling. Here, we discuss strategies for evaluating each of these additional ADH predictions.
First, if stage-specific levels of expression for a particular gene are genetically independent, alleles that contribute to expression variation in one life stage should not have pleiotropic effects on expression in another life stage (and vice versa). One way to evaluate this prediction is to perform quantitative trait locus (QTL) mapping on gene-expression traits at different life stages. Genetic independence would be supported if QTL for gene-expression traits measured at different stages are non-overlapping (e.g., (Freda et al., 2017; Saenko et al., 2012)). One example of this approach is a 2017 study that investigated genetic decoupling of thermal hardiness between larval and adult D. melanogaster (Freda et al., 2017). Whereas D. melanogasterlarvae live in thermally stable rotting fruits and are only present in the warm months, flying adults experience a more variable thermal environment and are exposed to low temperatures during the overwintering generation. Consistent with these strong opposing selection pressures, thermal hardiness is completely decoupled across metamorphosis in this species. Moreover, decoupling of the thermal-hardiness phenotype is mirrored at a genetic level: loci that contribute to cold hardiness in larvae do not appear to have pleiotropic effects on adults, and vice versa (Freda et al., 2017). Interpreted in light of fruit-fly ecology, decoupled thermal hardiness phenotypes and alleles provide strong support for the ADH.
Second, the prediction that decoupled gene-expression traits contribute to stage-specific adaptation could be evaluated using multiple complementary approaches. For example, if decoupled genes contribute disproportionately to adaptation, genes exhibiting the most stage-biased expression patterns should also reveal a history of positive selection (e.g., evidence of recent selective sweeps or elevated rates of non-synonymous substitutions relative to the rest of the genome) (Vitti, Grossman, & Sabeti, 2013). Although this prediction has been confirmed by several studies for sex-biased genes (Assis et al., 2012; Drosophila 12 Genomes et al., 2007; Mank, Nam, Brunstrom, & Ellegren, 2010; Proschel et al., 2006; L. Yang, Zhang, & He, 2016), it has rarely been tested in the context of stage-biased expression across metamorphic boundaries (but see (Perry et al., 2014)). An alternative approach would be to use experimental genomics to connect genetic variants directly to fitness at different life stages (e.g., (Egan et al., 2015; Gloss, Groen, & Whiteman, 2016; Gompert et al., 2019; Ingvarsson, Hu, Lei, & de Meaux, 2017)). Following exposure to a selection regime that favors different traits at different ontogenetic stages, the ADH predicts that genes with the most decoupled expression will exhibit the most pronounced allele frequency shifts.
Third, to more directly test the hypothesis that metamorphosis itself is an adaptation for optimizing trait decoupling, comparative data can be used two evaluate two additional predictions: (1) metamorphosis is favored under ecological conditions that result in pervasive antagonistic pleiotropy across the life cycle and (2) metamorphosis facilitates trait decoupling. To disentangle the ecological and genetic correlates of metamorphosis from shared phylogenetic history, comparative tests of the ADH should focus on lineages that contain multiple independent origins of particular metamorphic phenotypes. For example, within holometabolous insects, hypermetamorphosis has evolved multiple times (Belles, 2011). Likewise, gains and losses of complex life cycles have been demonstrated in numerous taxa and are particularly well documented in insects and amphibians (Badets & Verneau, 2009; Bonett & Blair, 2017; Emmanuelle, Gwenaelle, & Armelle, 2010; Moran, 1994; Poulin & Cribb, 2002; Wiens, Kuczynski, Duellman, & Reeder, 2007).