International Journal of Aquaculture, 2025, Vol.15, No.3, 116-134 http://www.aquapublisher.com/index.php/ija 120 highly conserved globally, and the sequence differences of most individuals are less than 0.5%, showing significant single species characteristics. For example, we compared the COI sequences of marble goby in the Mekong Delta of Vietnam and marble goby introduced into China for farming, and found only 0~2 base differences (genetic distance <0.3%), indicating that there is no obvious genetic differentiation between the introduced population and the original population. This is consistent with the results reported in the literature that marble goby cultured populations and wild populations are highly similar. However, marble goby in different regions still show some phylogenetic structure in a wider geographical span. For example, samples from Sumatra in western Indonesia and Papua in eastern Indonesia belong to two branches on the COI phylogenetic tree, and the K2P genetic distance between them is close to 2%. Although this is still near the general species definition threshold, it is enough to show that a certain degree of genetic deviation has occurred in the regional population. From the differences in mitochondria, we can see some clues about the evolution of marble goby. Let's talk about the situation within the population first. The mitochondrial differences between them are very small, which may indicate that these fish have only recently expanded, or there is frequent genetic exchange between different groups. This situation is quite consistent with the living habits of marble goby - they are widely distributed, highly mobile, and it is not difficult to migrate. But from another perspective, there are still some differences between fish in different regions. This difference may have been separated by geographical factors or environmental differences in the past. For example, the Malay Peninsula may have played the role of a "wall" between the Mekong River and the East Indies. Over time, the fish groups in different places gradually became somewhat different. However, these differences are not large now, and are far from becoming a new species, but perhaps this is the first step in species differentiation. In fact, similar situations have been seen in some other widely distributed fish species. The genetic differences between groups are quite obvious, but they are not yet at the point where they can be called new species. For example, He et al. (2022) studied the black carp in different regions of northern and southern China and found that fish in different waters have great genetic differences, but new species have not yet been formed. Therefore, species differentiation is not something that happens overnight. It is usually a gradual accumulation process, and only with a long enough time and obvious isolation conditions can it develop into a real "new species". 3 Phylogenetic Analysis and Evolutionary History 3.1 Phylogenetic study of mitochondrial phylogeny of marble goby In terms of molecular data acquisition, for marble goby, we used high-throughput sequencing to obtain the complete mitochondrial genome sequence and extracted the nucleotide sequences of 13 protein-coding genes for phylogenetic analysis. In addition, to enhance the comprehensiveness of the analysis, we downloaded mitochondrial genome data of several related species from the GenBank database, including representative species of Channaidae and some species of other families of the Goby suborder. Distant species in the Perciformes (such as marine perciformes) were selected as outgroups in the analysis to ensure that the root of the phylogenetic tree was properly oriented. In terms of sequence alignment, we used programs such as MAFFT to perform multiple sequence alignments of mitochondrial gene sequences of multiple species and manually corrected obvious mismatch regions. Given the different lengths and evolutionary rates of mitochondrial protein-coding genes, we used two strategies to construct phylogenetic trees: one was to sequentially connect the 13 protein-coding genes into a long concatenated sequence (total length of about 11 400 bp) for overall analysis; the other was to analyze some representative genes (such as the evolutionarily conservative COI gene and the rapidly evolving ND5 gene) separately to compare the similarities and differences between the single gene tree and the whole genome tree. The sequence evolution model selects the best substitution model through the model detection tool. Generally, the GTR+Γ model is used to adapt to the variation pattern of mitochondrial sequences. Phylogenetic inference uses two methods: Maximum Likelihood (ML) and Bayesian Inference (BI) (Rustam et al., 2022). In the ML analysis, we used software such as RAxML to search for the best tree under the assumed model
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