International Journal of Aquaculture, 2025, Vol.15, No.3, 116-134 http://www.aquapublisher.com/index.php/ija 121 through the fast bootstrap method, and evaluated the node support rate through the non-parametric bootstrap method. The Bayesian analysis uses MrBayes software to run independent MCMC chains until convergence to obtain the posterior probability support value. The consistency of the topological structures obtained by the two methods will also be used as the basis for judging the reliability of the results. In addition, we also constructed an NJ (neighbor-joining) tree based on mitochondrial barcode sequences to compare the consistency of the barcode tree and the whole genome tree in inferring species relationships. For the detection of intraspecific differentiation, we used network analysis software (such as Network) to construct a haplotype network to intuitively display the haplotype differences and evolutionary relationships of different geographical groups. It is worth mentioning that in the species definition analysis, we introduced a combination of the threshold method and the model method in molecular systematics. In terms of the threshold method, referring to the 2% genetic distance species discrimination standard commonly used in fish (Roul et al., 2021), we examined whether the COI genetic distance between each lineage in the phylogenetic tree exceeded this threshold; in terms of the model method, we tried to apply the GMYC (generalized mixed Yule-Coalescent) model to analyze the phylogenetic tree of the COI sequence to objectively divide the evolutionary lineage. Although methods such as GMYC have high sampling requirements, our analysis can be used as an auxiliary reference to verify the conclusions of traditional classification and threshold judgment. Through a multi-pronged analysis strategy, we try to improve the credibility of phylogenetic inference and species definition. 3.2 Phylogenetic position of marble goby and its related groups Through the phylogenetic tree constructed by the above method, we clearly determined the position of marble goby in the evolutionary tree of gobies. The results showed that marble goby (Oxyeleotris marmorata) and several species of sharp tang fish (such as high-fin sharp tang fish O. altipinna, O. heterodon, etc.) of the same genus were clustered into a branch, supporting the genus of sharp tang fish as a monophyletic group. Within the genus, the genetic distance between marble goby and O. urophthalmus (eye-spotted sharp tang fish, distributed in the Mekong River basin) is the closest, and the two form a sister group relationship on the phylogenetic tree. This result is consistent with the geographical distribution, because the eye-spotted sharp tang fish has been historically considered to be a close relative of marble goby, and the two are also morphologically similar. On the other hand, this lineage of the genus Channa is clearly separated from the species of the genus Eleotris on the phylogenetic tree, and the node support rate is extremely high (ML bootstrap value>95%, BI posterior probability=1.00), indicating that there are clear differentiation boundaries between the genera within the family Channaidae. In particular, the genus Channa, where the marble goby belongs, is closer to another freshwater Channa family group, the genus Oxyeleotris, and is farther away from the Oxudercidae (including amphibious gobies such as mudskippers) and the traditional Gobiidae. This also confirms the current taxonomic understanding of the family-level relationship within the suborder Gobiidae: the Eleotridae and the typical Gobiidae are two parallel branches, together constituting most of the diversity under the suborder Gobiidae, while the Oxudercidae and others are the base groups that diverged earlier. Specifically, in the phylogenetic tree we made, all fish of the family Channa are clustered on the same branch, and the support of this branch is also very high. Under this large branch, it can be divided into two small clusters. One cluster is fish living in freshwater, such as the genera Oxyeleotris and Eleotris; the other cluster is mainly fish living by the sea or in brackish water, such as Hypseleotris in Australia. Marble goby belongs to the first cluster, which is one of the core members of the freshwater Channa (Wantania et al., 2025). In this cluster, the relationship between different genera is basically the same as the traditional result of classification based on appearance. For example, we found that several fish of the genus Channa are very closely related, and they are also differentiated from the fish of the genus Channa within the family. We also compared the results of the phylogenetic tree with the appearance characteristics of the fish and found that the two aspects are also quite consistent. Those species that cluster together on the tree often have similar appearances and ecological habits. This also shows that the systematic relationships analyzed from the mitochondrial genome are consistent with the traditional classification methods (Li et al., 2024).
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