G-四联体（G-quadruplex）是一种特殊的核酸结构，主要形成于富含鸟嘌呤碱基的序列中。与双螺旋结构不同，G-四联体由四个鸟嘌呤碱基通过氢键连接形成的平面单元堆叠而成，形成一个稳定的四股螺旋结构。G-四联体的稳定性通常需要一价金属阳离子的参与。在线粒体这样的特定生理环境中，这些结构尤其倾向于形成。潜在形成G-四联体的序列（putative quadruplex sequence, PQS）指那些在适当条件下可以形成G-四联体的核酸序列。DNA和RNA均可以形成G-四联体，并与双螺旋等其他核酸结构之间实现动态平衡，从而在诸多生物学过程中扮演重要角色。

线粒体G-四联体在细胞的能量中心——线粒体中形成，对基因表达的调控和DNA复制过程产生重要影响。这些结构在调控线粒体基因转录的启动和终止以及复制引物的形成和复制过程的终止中起到关键作用。此外，线粒体G-四联体通过调节mRNA的剪切、翻译和稳定性，进一步调控线粒体基因表达及其与核基因表达的相互作用，这对于细胞内部信号网络的整合及其对环境变化的适应性响应极为关键。基于这些功能，本研究推测线粒体G-四联体可能通过提高生物调控网络的复杂性，而在生物复杂性的形成和进化中扮演了关键角色。

为了深入探索线粒体G-四联体与生物复杂性之间的潜在联系，本研究构建了涵盖多种动物和真菌的大规模线粒体基因组数据集，测量了线粒体PQS密度、PQS密度与GC含量比值及PQS序列长度占比，并使用多种方法量化生物复杂性，包括大类群尺度上的远缘物种进化顺序和基于生物结构的复杂性，以及在脊椎动物中通过相对脑容量衡量。

研究结果表明，动物和真菌中线粒体的PQS密度、在排除GC含量影响后的比值及PQS序列长度占比与其生物复杂性显著相关。特别是在脊椎动物中，即使排除了GC含量增加的影响，PQS密度依然显著提高，暗示了G-四联体在生物复杂性形成中可能扮演关键角色。而且真菌、无脊椎动物和脊椎动物间PQS特征存在显著差异。线虫和刺胞动物的线粒体PQS情况与其他无脊椎动物有所不同，可能与它们的特殊生境和线粒体基因组特性相关。

在考虑系统发育关系后，羊膜动物及其子类群的相对脑容量与线粒体PQS特征之间未显示出显著的相关性。可能是因为在近缘物种间生物复杂性的量化不足以显现明显差异。灵长目和鸟类的PQS特征显著富集，但其相对脑容量的增加更多地与祖先状态相关。灵长目、翼手目和齿鲸小目，与其近缘类群相比，线粒体G-四联体的富集与相对脑容量的增加呈现同步性。此外，尽管鸟类的线粒体PQS存在明显地富集现象，研究结果显示这与其飞行能力之间并无显著关联，暗示线粒体G-四联体的富集可能不直接源于对增强线粒体供能能力的需求。

这些发现不仅提供了线粒体G-四联体在生物复杂性中可能作用的新见解，还强调了在未来研究中需要整合生物信息学、遗传学及分子生物学等多方面数据，以全面理解生物复杂性。这些综合性的观点为未来探索线粒体PQS在其他生物功能中的作用提供了参考，并为深入研究生物复杂性的分子基础开辟了新的方向。

G-quadruplexes are unique nucleic acid structures predominantly formed in guanine-rich sequences. Unlike the double helix structure, G-quadruplexes consist of guanine tetrads that are stabilized through Hoogsteen hydrogen bonds, stacking into a stable four-stranded helical formation. The stability of these G-quadruplexes generally requires the presence of monovalent metal ions. Mitochondria, specific physiological environments, are particularly conducive to the formation of these structures. Putative quadruplex sequences (PQS) refer to nucleic acid sequences that can transition into G-quadruplex structures under suitable conditions. Both DNA and RNA can form G-quadruplexes, which maintain a dynamic equilibrium with other nucleic acid structures such as double helices, playing a crucial role in various biological processes.

Mitochondrial G-quadruplexes form within the powerhouse of the cell — the mitochondria, significantly influencing gene expression regulation and DNA replication processes. These structures are instrumental in regulating the initiation and termination of mitochondrial gene transcription, as well as the formation of replication primers and the termination of replication processes. Additionally, mitochondrial G-quadruplexes regulate mRNA splicing, translation, and stability, further modulating the expression of mitochondrial genes and their interaction with nuclear gene expression. This regulation is vital for the integration of internal signaling networks and the cell's adaptive response to environmental changes. Based on these functions, this study hypothesizes that mitochondrial G-quadruplexes may enhance the complexity of biological regulatory networks, playing a key role in the development and evolution of biological complexity.

To explore the potential link between mitochondrial G-quadruplexes and biological complexity, this study constructed a large-scale mitochondrial genome dataset covering various animals and fungi. It measured mitochondrial PQS density, the ratio of PQS density to GC content, and the proportion of PQS sequence length, utilizing multiple methods to quantify biological complexity. This included phylogenetic diversification at broad taxonomic scales and structural complexity, as well as relative brain size in vertebrates.

Results indicate that in animals and fungi, mitochondrial PQS density, the adjusted ratio excluding GC content influences, and the proportion of PQS sequence length are significantly correlated with biological complexity. Notably, even after excluding the effects of increased GC content, the PQS density in vertebrates remained significantly elevated, suggesting a potential key role for G-quadruplexes in the emergence of biological complexity. Furthermore, significant differences in PQS characteristics were observed between fungi, invertebrates, and vertebrates. The mitochondrial PQS profiles of nematodes and cnidarians differed from other invertebrates, potentially due to their unique ecological niches and mitochondrial genome traits.

Phylogenetically adjusted analyses showed no significant correlation between the relative brain size and mitochondrial PQS features in amniotes, possibly due to the inability of quantitative measures of biological complexity to reveal distinct differences among closely related species. The PQS features of primates and birds were significantly enriched, although the increase in their relative brain sizes was more closely associated with ancestral states. In primates, Chiroptera, and Odontoceti, the enrichment of mitochondrial G-quadruplexes coincided with increases in relative brain size compared to their closely related groups. Additionally, despite the apparent enrichment of mitochondrial PQS in birds, there was no significant correlation with their flight capabilities, suggesting that the accumulation of mitochondrial G-quadruplexes might not directly stem from a need for enhanced mitochondrial energy capacity.

These findings not only provide new insights into the potential role of mitochondrial G-quadruplexes in biological complexity but also highlight the need for future research to integrate data from bioinformatics, genetics, and molecular biology to fully understand the genetic regulatory mechanisms of biological complexity. This comprehensive perspective paves the way for further exploration of mitochondrial PQS in other biological functions and opens new avenues for in-depth studies of the molecular basis of biological complexity.