Phenotypic plasticity is produced and maintained by processes regulating the transcriptome. While differential gene expression is among the most important of these processes, relatively little is known about other sources of transcriptional variation. Previous work suggests that alternative splicing plays an extensive and functionally unique role in transcriptional plasticity, though plastically spliced genes may be more constrained than the remainder of expressed genes. In this study, we explore the relationship between expression and splicing plasticity, along with the genetic diversity in those genes, in an ecologically consequential polyphenism: facultative diapause. Using 96 samples spread over two tissues and 10 timepoints, we compare the extent of differential splicing and expression between diapausing and direct developing pupae of the butterfly Pieris napi. Splicing differs strongly between diapausing and direct developing trajectories but alters a smaller and functionally unique set of genes compared to differential expression. We further test the hypothesis that among these expressed loci, plastically spliced genes are likely to experience the strongest purifying selection to maintain seasonally plastic phenotypes. Genes with unique transcriptional changes through diapause consistently had the lowest nucleotide diversity, and this effect was consistently stronger among genes that were differentially spliced compared to those with just differential expression through diapause. Further, the strength of negative selection was higher in the population expressing diapause every generation. Our results suggest that maintenance of the molecular mechanisms involved in diapause progression, including post-transcriptional modifications, are highly conserved and likely to experience genetic constraints, especially in northern populations of P. napi.

表型可塑性是通过调节转录组的过程产生和维持的。虽然差异基因表达是这些过程中最重要的过程之一，但人们对转录变异的其他来源知之甚少。先前的研究表明，选择性剪接在转录可塑性中发挥着广泛且功能独特的作用，尽管可塑性剪接基因可能比其余表达基因受到更多限制。在这项研究中，我们探索了表达和剪接可塑性之间的关系，以及这些基因的遗传多样性，在生态上具有重要意义的多态性：兼性滞育。使用分布在两个组织和 10 个时间点的 96 个样本，我们比较了滞育蝴蝶和直接发育的蛹之间的差异剪接和表达程度。剪接在滞育和直接发育轨迹之间存在很大差异，但与差异表达相比，改变了一组更小且功能独特的基因。我们进一步检验了这样的假设：在这些表达位点中，可塑性剪接的基因可能会经历最强的纯化选择以维持季节性可塑性表型。在滞育期间具有独特转录变化的基因始终具有最低的核苷酸多样性，并且与在滞育期间仅具有差异表达的基因相比，在差异剪接的基因中这种效应始终更强。此外，每一代表达滞育的群体中负选择的强度都较高。我们的结果表明，涉及滞育进展的分子机制的维持，包括转录后修饰，是高度保守的，并且可能受到遗传限制，特别是在北方的纳皮种群中。