Genome-wide survey of nuclear protein-coding markers for beetle phylogenetics and their application in resolving both deep and shallow-level divergences

Beetles (Coleoptera) are the most diverse and species-rich insect group, representing an impressive explosive radiation in the evolutionary history of insects, and their evolutionary relationships are often difficult to resolve. The amount of traditional markers' (e.g. mitochondrial genes and nuclear rDNAs) for beetle phylogenetics is small, and these markers often lack sufficient signals in resolving relationships for such a rapidly radiating lineage. Here, based on the available genome data of beetles and other related insect species, we performed a genome-wide survey to search nuclear protein-coding (NPC) genes suitable for research on beetle phylogenetics. As a result, we identified 1470 candidate loci, which provided a valuable data resource to the beetle evolutionary research community for NPC marker development. We randomly chose 180 candidate loci from the database to design primers and successfully developed 95 NPC markers which can be PCR amplified from standard genomic DNA extracts. These new nuclear markers are universally applicable across Coleoptera, with an average amplification success rate of 90%. To test the phylogenetic utility, we used them to investigate the backbone phylogeny of Coleoptera (18 families sampled) and the family Coccinellidae (39 species sampled). Both phylogenies are well resolved (average bootstrap support >95%), showing that our markers can be used to address phylogenetic questions of various evolutionary depth (from species level to family level). In general, the newly developed nuclear markers are much easier to use and more phylogenetically informative than the traditional markers', and show great potential to expedite resolution of many parts in the Beetle Tree of Life.

甲虫（鞘翅目）是最多样化和物种最丰富的昆虫群，代表了昆虫进化史上令人印象深刻的爆炸性辐射，并且它们的进化关系通常很难解决。甲虫系统发育学的传统标志物（例如线粒体基因和核RDNA）的量很小，这些标记通常缺乏足够的信号来解决这种快速辐射的谱系。在这里，基于甲虫和其他相关昆虫物种的可用基因组数据，我们进行了全基因组调查，以搜索适用于甲虫系统发育学研究的核蛋白质编码（NPC）基因。结果，我们确定了1470个候选基因座，该基因座为NPC标记开发提供了甲壳虫进化研究界的宝贵数据资源。我们从数据库中随机选择了180个候选基因座来设计引物，并成功地开发了95个NPC标记，可以从标准基因组DNA提取物中扩增PCR。这些新的核标志物普遍适用于鞘翅目，平均扩增成功率为90％。为了测试系统发育效用，我们用它们研究了鞘翅目（取样18个家族）和Coccinellidae（39种采样）的主链系统发育。两种系统发育均得到很好的解决（平均自举支持> 95％），表明我们的标记可用于解决各种进化深度（从物种水平到家庭水平）的系统发育问题。通常，新开发的核标记比传统标记更容易使用，并且在系统发育方面更具信息性，并具有加快生命甲虫树中许多部分的分辨率的巨大潜力。