Testes vary widely in mass relative to body mass across species, but we know very little about which genes underlie and contribute to such variation. This is partly because evidence for which genes are implicated in testis size variation tends to come from investigations involving just one or a few species. Contemporary comparative phylogenetic methods provide an opportunity to test candidate genes for their role in phenotypic change at a macro-evolutionary scale—across species and over millions of years. Previous attempts to detect genotype-phenotype associations across species have been limited in that they can only detect where genes have driven directional selection (e.g. brain size increase). Here, we introduce an approach that uses rates of evolutionary change to overcome this limitation to test whether any of twelve candidate genes have driven testis size evolution across tetrapod vertebrates—regardless of directionality. We do this by seeking a relationship between the rates of genetic and phenotypic evolution. Our results reveal five genes (Alkbh5, Dmrtb1, Pld6, Nlrp3, Sp4) that each have played unique and complex roles in tetrapod testis size diversity. In all five genes, we find strong significant associations between the rate of protein-coding substitutions and the rate of testis size evolution. Such an association has never, to our knowledge, been tested before for any gene or phenotype. We describe a new approach to tackle one of the most fundamental questions in biology: how do individual genes give rise to biological diversity? The ability to detect genotype-phenotype associations that have acted across species has the potential to build a picture of how natural selection has sculpted phenotypic change over millions of years.

中文翻译：

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四足动物睾丸大小进化的基因

相对于不同物种的体重，睾丸的质量差异很大，但我们对哪些基因构成并促成这种变异知之甚少。这部分是因为哪些基因与睾丸大小变异有关的证据往往来自仅涉及一个或几个物种的调查。当代比较系统发育方法提供了一个机会来测试候选基因在宏观进化尺度上的表型变化中的作用——跨越物种和数百万年。以前检测跨物种基因型-表型关联的尝试受到限制，因为它们只能检测基因驱动定向选择的位置（例如大脑大小增加）。这里，我们引入了一种方法，该方法使用进化变化率来克服这一限制，以测试 12 个候选基因中的任何一个是否驱动了四足脊椎动物睾丸大小的进化——无论方向性如何。我们通过寻找遗传和表型进化率之间的关系来做到这一点。我们的结果揭示了五个基因（Alkbh5、Dmrtb1、Pld6、Nlrp3、Sp4），每个基因在四足动物睾丸大小多样性中都发挥着独特而复杂的作用。在所有五个基因中，我们发现蛋白质编码替换率与睾丸大小进化率之间存在强烈的显着关联。据我们所知，这种关联从未针对任何基因或表型进行过测试。我们描述了一种解决生物学中最基本问题之一的新方法：单个基因如何产生生物多样性？检测跨物种作用的基因型-表型关联的能力有可能建立一幅自然选择如何在数百万年中塑造表型变化的图景。