International Journal of Aquaculture, 2025, Vol.15, No.3, 116-134 http://www.aquapublisher.com/index.php/ija 126 Ecological and geographic information is also important when judging species. If two groups that look like different species have obvious differences in their living environment or distribution area, then there is more reason to treat them as separate species. For marble goby, we can look at the ecological differences between different groups, such as their preferred habitats, what they eat, how fast they grow, etc. Maybe we can find some ecological differences related to genetic differences. For example, some groups like to stay in fast-flowing rivers, while others are more often found by slow-flowing lakes. This difference in environment may bring different selection pressures, causing them to take different evolutionary paths. If these differences are really found, they should also be taken into account when making classification judgments. It is worth mentioning that the application of comprehensive taxonomy has been successful in fish research in recent years. For example, Bemis et al. (2023) used morphological identification and DNA barcode comparison for thousands of fish samples in the Philippine Sea to confirm the taxonomic status of thousands of fish species, and some species that have long been in doubt due to the uncertainty of a single method have also been clarified. Similarly, Guimaraes et al. (2020) clarified the cryptic diversity of Characidae fish in the Amazon basin by integrating morphological metrics and molecular systematics, and described two new cryptic species at one time. These studies have proved that the integration of multiple evidences can greatly improve the reliability of taxonomic decisions and avoid misjudgments or omissions due to reliance on a single piece of evidence. 5 Case Study: Phylogeographic Survey of Marble Goby in the Mekong River Region 5.1 Cross-population sample collection and mitochondrial sequencing To verify the previous analysis and to see if geographic isolation has any impact on the population and species division of marble goby, we chose the Mekong River Basin as a case study. The Mekong River Basin spans several countries, including China, Laos, Thailand, Cambodia and Vietnam, and is a place where freshwater fish are particularly abundant. marble goby are widely distributed in this area. They can be seen not only in the upper reaches of the Lancang River in China, but also in the lowland water network of the Mekong Delta in Vietnam. We believe that different tributaries and river sections may separate these fish, and over time, some genetic differences may appear between them. This difference may just provide some new clues for species division (Truong et al., 2025). The researchers compared these sequences with existing marble goby sequences in the GenBank database and confirmed that the measured sequences were consistent with the cloud-spotted sharp-eared tang fish species (Syaifudin et al., 2021). For example, through BLAST comparison, the similarity between the sample COI sequence and the database marble goby sequence was more than 99%, ensuring the accuracy of species identification. During sample collection, we also recorded the habitat information of each sample, such as water environment (river or lake), water quality parameters, coexistence with other fish, etc. These ecological data will help to understand the relationship between population genetic structure and environmental factors in subsequent discussions. 5.2 Phylogenetic inference and species-level discovery Using the molecular data of marble goby in the Mekong River Basin mentioned above, we analyzed the phylogenetic relationship of populations in the region (Figure 3) (Syaifudin et al., 2021). The first phylogenetic tree based on the whole mitochondrial genome was constructed. In this high-resolution regional phylogenetic tree, we observed that the samples of the marble goby formed several clusters according to their geographical origins. For example, the samples of the upper Lancang River clustered into one branch, which was clearly distinguished from the samples of the middle and lower reaches; while the samples of the middle reaches of Laos/Thailand and the samples of the lower reaches of Cambodia/Vietnam were mixed in another branch. This suggests that the upstream population may have been isolated from the downstream population for a long time, and gradually accumulated unique genetic variation. It is worth noting that even the upstream and downstream populations that clustered into different clusters still had a small genetic distance between them (the mitochondrial whole genome K2P distance was about 0.8%), which was only slightly higher than the background value of intraspecific
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