International Journal of Aquaculture, 2025, Vol.15, No.3, 116-134 http://www.aquapublisher.com/index.php/ija 119 during evolution. We compared 13 protein-coding genes of marble goby and other fish such as the spotted tail sea bass and found that their sequences differed by an average of about 10% to 15%. Some genes change quickly, such as ATP8 and ND4L, which differ by more than 20%, while more stable genes such as COI and CYTB have smaller differences, about 8% to 10%. These differences have a certain correspondence with the relationship between species and can also provide some quantitative data for molecular systematic studies. For example, we used the COI gene to calculate genetic distances and found that the K2P distance between marble goby and common tang fish was about 0.12, and the distance with spotted tail sea bass was 0.10. In contrast, the distance between different individuals of marble goby itself is generally less than 0.005 (Figure 1) (Syaifudin et al., 2021). This pattern of "big differences between different species, small differences within the same species" is actually the same as the principle of DNA barcoding, that is, it can be used to determine whether two individuals belong to the same species. Figure 1 Genetic distance between species marble goby based onCOI gene (Adopted from Syaifudin et al., 2021) We noticed that the variation in certain specific regions may have phylogenetic significance. For example, the control region, as the most variable segment, has slightly different lengths and repeat sequence copy numbers in different species, which can be used to identify closely related species. The length of the control region of the marble goby is comparable to that of other sharp pond fish, but is slightly longer than that of the common pond fish by about 50 bp, and it is speculated that it may contain an additional set of microsatellite repeats. In the rRNA gene region, the 12S and 16S rRNA sequences of the marble goby differ from those of other species by less than 5%, supporting the consistency of its taxonomic status. In general, the mitochondrial genome of the marble goby is highly consistent with closely related species in terms of overall layout, but the accumulated mutations at the sequence level reflect the evolutionary distance between species, which provides a lot of information for phylogenetic reconstruction and molecular classification. 2.3 Exploring evolutionary divergence from the perspective of mitochondrial variation The variation of the mitochondrial genome can not only be used to identify differences between species, but also reveal the differentiation and evolutionary history of intraspecific lineages. In this study, we explored the intraspecific genetic structure and evolutionary divergence of marble goby samples from different geographical sources by analyzing the mitochondrial sequence variation of marble goby samples. The results showed that marble goby had high consistency within the species, and the mitochondrial gene sequences of most individuals were almost identical, especially the genetic distance of individuals from the same water system was negligible. However, a certain degree of genetic variation was still detected when comparing across basins. For example, the mitochondrial whole genome sequence difference between marble goby samples from the Mekong River basin and marble goby samples from the Chao Phraya River basin in Thailand was about 1.2%, slightly higher than the typical intraspecific variation level. This suggests that there may be long-term isolation and limited gene exchange between different geographical populations. To further quantify intraspecific variation, we focused on analyzing the differences in mitochondrial COI barcode sequences among different groups of marble goby. Overall, the COI sequences of marble goby populations are
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