International Journal of Aquaculture, 2025, Vol.15, No.3, 116-134 http://www.aquapublisher.com/index.php/ija 122 The phylogenetic position of the marble goby confirms that it belongs to one of the core lineages of the Channaidae family, corresponding to a large ambush carnivorous fish group that mainly lives in freshwater bodies. In contrast, representatives of the Loachidae family (such as the mudskipper Periophthalmus genus) are located in a more peripheral position on our constructed tree, and only merge with the branch of the marble goby at a higher level (the level of the goby suborder). This is consistent with the characteristics of the Loachidae family, which is adapted to amphibious habitats and has specific morphology. For example, the Dermogenys family (such as the Dermogenys), which is widely distributed in Asia and Africa, does not aggregate with the Channa-goby fish at all, because they actually belong to the Salmoniformes, which verifies the rationality of our outgroup selection. The phylogenetic analysis we did this time not only clarified the taxonomic position of the marble goby, but also gave us a better understanding of its evolutionary history. Looking at its current lineage, it is very likely that it first appeared in the inland waters of the subtropical Southeast Asia. Later, due to geological changes and climate fluctuations, it slowly spread to surrounding areas. From the phylogenetic tree, its relationship with other closely related fish provides us with some clues about the order of species differentiation. The spread of a branch like the marble goby may have occurred in the late Mesozoic or early Cenozoic, when the water system in Southeast Asia was also changing. And the differentiation between it and those typical marine gobies is estimated to have occurred even earlier. These speculations still need to be further verified in combination with molecular clock analysis. However, as far as the current phylogenetic tree results are concerned, it has provided us with a lot of important information for studying the evolutionary process of the marble goby. 3.3 Intraspecific divergence and its impact on speciation Although phylogenetic analysis mainly focuses on the relationship between species and genus levels, we also paid attention to intraspecific divergence in the marble goby lineage. As mentioned in the mitochondrial variation analysis above, there is a certain degree of genetic differentiation between marble goby of different geographical populations. This intraspecific divergence may have an important impact on speciation. On the phylogenetic tree, we included marble goby individuals from multiple regions in the analysis separately, and found that they were all clustered in the monophyletic branch of marble goby, without any mixing with any known related species. However, within the branch of marble goby, clusters subdivided by geographic origin can be vaguely seen. For example, samples from the Mekong River basin tend to cluster together, while samples from the Chao Phraya River in Thailand and the southern part of the Malay Peninsula form another subcluster. Although the separation of the two subclusters is not very significant and the node support rate is low, it is consistent with the genetic distance analysis, suggesting that regional populations may be separated along their own evolution (Syaifudin et al., 2021). Intraspecific divergence is a prelude to speciation. When gene flow between populations is blocked by geographical or reproductive barriers, genetic differences will gradually accumulate. When the differences reach a sufficient level, the populations may no longer mate and reproduce, thus forming new species. Marble goby are currently still considered a single species, but if its different populations continue to be isolated, the possibility of new species in the future cannot be ruled out. For example, if the ice age or crustal movement causes the Mekong River system to be isolated from the Malay Peninsula system for a long time, then the respective marble goby populations may accumulate independent adaptive characteristics and genetic differences after hundreds of thousands of years, and eventually move towards species differentiation. This has been seen in many freshwater fishes, such as some widely distributed carps that have evolved into closely related sister species in different watersheds. However, judging from the current situation, the differentiation in the marble goby fish has not yet reached the level of becoming a new species. He et al. (2022) also found that although genetic differences have appeared in populations in different watersheds, they have not yet really divided into new species. The situation of marble goby fish and C. alba is a bit similar. We did see signs of lineage differentiation in it, but we also found that many genetic loci are still shared between different populations. This shows that the gene exchange between them may
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