Genomic analysis suggests that mitonuclear coevolution proceeds over rapid timescales in the Amazonian Pipra manakin complex

Mitonuclear coevolution is defined as reciprocal selection between the nuclear and mitochondrial genomes. It is necessary to maintain compatibility between nuclear- and mitochondrially-encoded products that interact during mitochondrial processes, including mitochondrial genome replication, transcription, and translation, and oxidative phosphorylation. Theory predicts that mitonuclear coevolution may play a crucial role in the early phases of speciation by generating strong genetic incompatibilities between recently diverged taxa that have evolved unique mitochondrial-mitonuclear haplotypes. However, the timescale over which mitonuclear coevolution proceeds remains unclear, making it difficult to definitively link this process with early speciation. Here, we test for expected genomic signals of mitonuclear coevolution across the Amazonian Pipra manakin complex, which includes recently and more deeply diverged avian lineages. Using dN/dS ratio analyses, we compared signals of positive selection in mitonuclear gene categories and functionally equivalent nuclear gene categories that do not participate in mitonuclear coevolution for each pair of Pipra lineages separately and for all the lineages simultaneously. For the ribosomal protein and aminoacyl tRNA synthetase (AARS) gene categories, we identified genomic patterns consistent with stronger positive selection in mitonuclear versus nuclear genes which is suggestive of mitonuclear coevolution having occurred across the Pipra complex. Significantly, we determined that expected genomic signals of mitonuclear coevolution could be identified between lineages that diverged as recently as 0.35-0.4 MYA. This time span is in keeping with the initial stages of avian speciation and suggests that mitonuclear coevolution may operate on a timescale that would allow it to play an important role during early speciation.

Methods
The methods employed in this study can be found in the linked 2025 Molecular Ecology publication with additional information specified in the provided scripts and data files. To summarize, DNA was extracted from twenty-five Pipra manakin tissue samples and sequenced using a whole-genome sequencing methodology. The produced genetic data was processed and then mitonuclear and nuclear protein-coding gene sequences were obtained for each individual. Pairwise dN/dS comparisons and maximum likelihood codon evolution models were employed to assess positive selection in mitonuclear and functionally equivalent nuclear gene categories where stronger positive selection associated with mitonuclear gene categories is a putative signal of mitonuclear coevolution having occurred in the system. The scripts necessary to complete these analyses are included in this repository along with all required metatdata files.

核线粒体共进化（mitonuclear coevolution）被定义为核基因组与线粒体基因组之间的互惠选择。维持线粒体过程中相互作用的核编码与线粒体编码产物间的兼容性至关重要，这些过程涵盖线粒体基因组复制、转录、翻译以及氧化磷酸化。理论预测，核线粒体共进化可能在物种形成早期阶段发挥关键作用，通过在演化出独特线粒体-核单倍型的新近分化类群间产生强烈的遗传不相容性来实现。然而，核线粒体共进化的发生时间尺度仍不明确，这使得将该过程与早期物种形成明确关联存在一定难度。
本研究以亚马孙娇鹟属（Pipra）复合体为研究对象，该类群包含新近分化及更深层分化的鸟类支系，借此检验核线粒体共进化的预期基因组信号。我们采用dN/dS比值（dN/dS ratio）分析方法，分别针对每一对伞鸟支系以及所有支系同时，比较了核线粒体基因类别与不参与核线粒体共进化的功能等价核基因类别的正选择信号。针对核糖体蛋白及氨酰tRNA合成酶（aminoacyl tRNA synthetase, AARS）基因类别，我们鉴定出与核线粒体基因相较于核基因受到更强正选择相符的基因组模式，这暗示核线粒体共进化已在娇鹟属复合体中发生。尤为重要的是，我们发现即便在分化时间仅为0.35~0.4百万年的支系间，也能检测到核线粒体共进化的预期基因组信号。该时间跨度与鸟类物种形成的初始阶段相符，这表明核线粒体共进化的作用时间尺度足以使其在早期物种形成过程中发挥重要作用。
方法
本研究采用的实验方法详见已发表的2025年《Molecular Ecology》论文，额外信息可参见随附的脚本与数据文件。简要而言，研究人员从25份娇鹟属（Pipra）组织样本中提取DNA，并通过全基因组测序技术完成测序。对所得遗传数据进行处理后，获取了每个个体的核线粒体与核蛋白编码基因序列。我们采用成对dN/dS比较及最大似然密码子演化模型（maximum likelihood codon evolution models），评估核线粒体基因类别与功能等价核基因类别的正选择情况——其中核线粒体基因类别伴随更强正选择被视为该系统中发生核线粒体共进化的推定信号。本仓库中包含完成上述分析所需的全部脚本及必要的元数据（metadata）文件。