Genetic variation for adaptive traits is associated with polymorphic inversions in Littorina saxatilis

Chromosomal inversion polymorphisms, segments of chromosomes that are flipped in orientation and occur in reversed order in some individuals, have long been recognized to play an important role in local adaptation. They can reduce recombination in heterozygous individuals and thus help to maintain sets of locally adapted alleles. In a wide range of organisms, populations adapted to different habitats differ in frequency of inversion arrangements. However, getting a full understanding of the importance of inversions for adaptation requires confirmation of their influence on traits under divergent selection. Here, we studied a marine snail, Littorina saxatilis, that has evolved ecotypes adapted to wave exposure or crab predation. These two types occur in close proximity on different parts of the shore. Gene flow between them exists in contact zones. However, they exhibit strong phenotypic divergence in several traits under habitat-specific selection, including size, shape and behaviour. We used crosses between these ecotypes to identify genomic regions that explain variation in these traits by using QTL analysis and variance partitioning across linkage groups. We could show that previously detected inversion regions contribute to adaptive divergence. Some inversions influenced multiple traits suggesting that they contain sets of locally adaptive alleles. Our study also identified regions without known inversions that are important for phenotypic divergence. Thus, we provide a more complete overview of the importance of inversions in relation to the remaining genome.

Methods
Parental snails were collected on the Swedish West Coast at Ängklåvebukten (58.8697°, 11.1197°), where both ecotypes occur in close proximity. Two virgin Crab-females were crossed with two Wave-males resulting in two F1-families. F1-families were crossed to produce the F2 generation which was genotyped and phenotyped.

DNA was extracted from a small piece of foot and targeted re-sequencing was performed using a total of 25,000 (120 bp) enrichment probes.

Phenotypes measured included weight, shell length, shell thickness (mean of three measurements per snail), relative thickness (thickness/shell length), size independent parameters describing shell shape and the aperture, shell colour, and boldness behaviour (Bold.Score = log of time until crawling out after disturbance, higher Bold.Score means an individual is less bold) that were previously found to differ between ecotypes.

染色体倒位多态性（chromosomal inversion polymorphisms）是指染色体片段发生方向反转、在部分个体中呈现反向排列的结构变异，长期以来被认为在本地适应中发挥关键作用。这类变异可降低杂合个体中的重组率，从而有助于维持本地适应等位基因的组合。在众多生物类群中，适应不同生境的种群其倒位构型的频率存在差异。然而，要全面阐明倒位对适应的重要性，需验证其对歧化选择下性状的影响。本研究以滨螺（Littorina saxatilis）为研究对象，该物种已演化出适应波浪冲击或蟹类捕食的生态型。这两种生态型在潮间带的不同区域近距离共存，在接触带中二者存在基因交流，但二者在生境特异性选择下的多个性状上呈现显著的表型分化，包括体型、形态与行为。本研究通过对这些生态型进行杂交，结合数量性状基因座（QTL, quantitative trait locus）分析和连锁群间的方差分解，鉴定了与这些性状变异相关的基因组区域。研究证实，此前已发现的倒位区域参与了适应性分化。部分倒位可影响多个性状，表明其携带本地适应等位基因的组合。本研究同时鉴定出未被报道存在倒位的、对表型分化具有重要作用的基因组区域。因此，本研究更为全面地阐明了倒位相对于其余基因组的适应意义。

## 方法
亲本滨螺采集于瑞典西海岸的Ängklåvebukten（58.8697°，11.1197°），该区域两种生态型近距离共存。将2只未交配过的蟹型生态型雌螺与2只浪型生态型雄螺杂交，获得2个F1家系。将F1家系进行杂交以获得F2代群体，随后对其进行基因分型与表型分型。
从滨螺的足组织小块中提取DNA，使用总计25000条（120 bp）富集探针进行靶向重测序。
测定的表型包括体重、壳长、壳厚（每只螺三次测量的平均值）、相对壳厚（壳厚/壳长）、描述壳形与壳口的体型独立参数、壳色，以及大胆度行为（Bold.Score为扰动后螺爬出的时间的对数，Bold.Score值越高则个体大胆度越低），这些性状此前已被证实存在生态型间差异。