International Journal of Aquaculture, 2025, Vol.15, No.3, 99-115 http://www.aquapublisher.com/index.php/ija 103 3.2 Identification of highly variable regions and functional interpretation Based on the analysis of variation frequency, several highly variable regions can be identified in the mitochondrial genome of Snakehead. In addition to the control region (D-loop), some protein-coding gene fragments also show high variability and are considered "intergenic hypervariable regions". For example, studies have shown that the 5' end of the ND2 gene, the 3' end region of the large subunit rRNA (16S rRNA), and a sequence of tRNAPhe adjacent to the control region are fragments with high variability in the mitochondrial genome of the black fish genus (Wang et al., 2023; 2024). The high variability of the ND2 fragment may be related to its relatively weak functional restriction; the base changes in the terminal region of 16S rRNA have little effect on the secondary structure of rRNA, so more mutations are accumulated. The control region is not uniform. Among its three subregions, the terminal conserved region and the middle tandem repeat region have the most variation. These hypervariable regions are often rich in phylogenetic information and are often used as molecular markers to analyze species and population relationships. The functional interpretation of the hypervariable region needs to be combined with the role of the gene in which the sequence is located. For example, highly variable protein genes such as ND2 and ND5 encode subunits of mitochondrial respiratory chain complex I. Moderate amino acid substitutions may affect the performance of the enzyme complex, thereby producing phenotypic effects (Garg and Dohre, 2024). However, in most cases, the effects of these variations on individual survival adaptability may be subtle or neutral. Therefore, in phylogenetic analysis, the main contribution of highly variable regions is to improve the ability to distinguish, and it does not necessarily correspond directly to trait changes. On the contrary, if some conservative regions vary, they often indicate important functional effects. For example, the COX1 gene has almost no variation among species of the genus Snakehead. Once amino acid substitutions are detected, it may mean major functional consequences. Therefore, COX1 is often used for DNA barcoding to identify species but is not suitable for inferring intraspecific relationships. For non-coding highly variable regions, such as control regions, although they do not directly encode proteins, their variations may affect replication initiation and transcriptional regulation. Variations in the control region of Snakehead include length polymorphism and base substitutions. Some length variations may change the secondary structure of DNA and thus affect the efficiency of the replication origin. However, since this region is not absolutely necessary for individual survival, its variation is mostly tolerated by natural selection and is therefore used as a neutral marker in evolutionary analysis. Similarly, in the tRNA gene region, some highly variable sites fall in the unpaired region of the arm, which does not affect the folding and function of tRNA, and therefore accumulate differences between species and can be used as phylogenetic signals. If the mutation falls in the anticodon or stem pairing site, it may have significant functional consequences and is rarely preserved in evolution. 3.3 Potential phenotypic effects of non-synonymous mutations Base substitutions in mitochondrial protein-coding genes can be divided into synonymous mutations and non-synonymous mutations. The former does not change the amino acid sequence, while the latter leads to amino acid substitutions, which may affect protein function and phenotype. In the mitochondrial genome of the genus Snakehead, most of the variation sites are synonymous mutations, but there are still a considerable number of non-synonymous mutations that deserve attention. For example, Ou et al. (2021) compared the mitochondrial genes of multiple geographical populations of Channa argus and found that there were several fixed amino acid substitutions in the ND5 gene, with significant frequency differences between northern and southern populations, which may be related to the adaptation of populations to temperature differences. This speculation is consistent with the strong survival ability of Channa argus in cold environments: the northern population of Channa argus may have accumulated mitochondrial mutations that are beneficial to low-temperature metabolism through evolution, thereby improving its cold tolerance. Similarly, Channa maculata and Channa argus differ in distribution area and ecological habits. Channa maculata has poor cold tolerance but may have strong disease resistance. In the comparison of the mitochondrial genomes of the two, some non-synonymous mutations that may
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