DataSheet_1_Stronger selective constraints on the mitochondrial genome in flying fishes.docx

Flying fishes, which use their wing-like pectoral fins and hypocercal caudal fin to glide through the air to avoid underwater predators, have independently evolved flight behavior, making them ideal for the study of adaptive evolution. To investigate the adaptation of flight behavior in flying fishes and the origin of Beloniformes fishes, this study obtained the complete mitochondrial genomes of Cheilopogon nigricans and Oxyporhamphus micropterus and constructed the DNA sequences extracted from these newly sequenced mitochondrial genomes with the DNA sequences of 32 previously published mitochondrial genomes into a dataset for reconstructing the phylogenetic relationships of Beloniformes fishes. The phylogeny that emerged strongly supported the possibility that flying fishes developed from halfbeaks and the progressive transition of flying fishes from two-wing to four-wing gliding. The divergence time analysis showed that the split between the suborder Belonidei and the family Adrianichthyidae occurred roughly 77.08 Mya, which fell within the period of evolution of the Indian plate in the late Cretaceous. Selection analyses revealed that flying fishes have a lower dN/dS ratio than the other members of Beloniformes, indicating that flying fishes experienced stronger purifying selection to eliminate deleterious mutations to maintain efficient energy metabolism to adapt to flight behavior. Moreover, this work found the positively selected signal in the ND4 gene, suggesting that different mitogenomic genes might have undergone different selective patterns during adaptive evolution.

飞鱼凭借翼状胸鳍与下歪形尾鳍（hypocercal caudal fin）在空中滑行以躲避水下捕食者，其飞行行为为独立演化所得，因此成为适应性进化研究的理想对象。为探究飞鱼飞行行为的适应性演化机制以及颌针鱼目（Beloniformes）鱼类的起源，本研究获取了黑鳍飞鱼（Cheilopogon nigricans）与小鳍尖嘴鱵（Oxyporhamphus micropterus）的完整线粒体基因组（mitochondrial genomes），并将本次新测序得到的线粒体基因组DNA序列，与此前已发表的32条线粒体基因组DNA序列进行整合，构建得到用于重建颌针鱼目鱼类系统发育关系的数据集。所得系统发育结果强力支持两项结论：飞鱼由鱵类演化而来，以及飞鱼的滑翔方式经历了从双翼到四翼的渐进式过渡。分化时间分析显示，颌针鱼亚目（Belonidei）与异鳉科（Adrianichthyidae）的分化时间约为7708万年前（Mya），该时间节点处于白垩纪晚期印度板块的演化时期内。选择压力分析表明，飞鱼的dN/dS比值（dN/dS ratio）较颌针鱼目其他类群更低，提示飞鱼经历了更强的纯化选择以清除有害突变，从而维持高效的能量代谢以适应飞行行为。此外，本研究在ND4基因（ND4 gene）中检测到正选择信号，表明在适应性演化过程中，线粒体基因组的不同基因可能经历了不同的选择模式。