International Journal of Aquaculture, 2025, Vol.15, No.3, 116-134 http://www.aquapublisher.com/index.php/ija 129 lineage. It may also be affected by factors such as skewed male-female ratio, genetic drift, and even hybrid asymmetry, which may lead to deviations in species relationships (Yu and He, 2012). In order to solve these problems, it is necessary to integrate nuclear genome data in future studies to build a more comprehensive and reliable phylogenetic framework. The nuclear genome contains a lot of data, including autosomal gene sequences, gene fragments, and even SNPs of the whole genome. These data come from both parents and will undergo recombination, unlike mitochondria, which only look at the maternal line. It can provide evolutionary information from another perspective. When studying marble goby, if we add nuclear genome data, we can use it to test the accuracy of the mitochondrial tree. If the nuclear gene data is similar to the mitochondrial results, the classification conclusion will be more reliable. But if the results are different, for example, the mitochondria show one branch, but the nuclear genes are divided into two groups, it may mean that we have to reconsider our judgment of the species. Nuclear DNA can also tell us how the populations separated, how long ago they separated, and whether the genes are still flowing. This "common ancestor analysis" is more accurate than mitochondria. Take marble goby as an example. We can use nuclear gene data to see if there is a little gene exchange between upstream and downstream, which helps us explain why mitochondria look so "fuzzy". In addition, nuclear gene data can also detect hybridization. Mitochondria are a line, and they may have been mixed a long time ago even if they look clean. Sometimes the mitochondria of a group look independent, but in fact they may have been replaced after hybridization with other groups. Nuclear gene data, especially multi-locus data, can show whether there are mixed signals and reveal the hybridization history behind them. Integrating nuclear genome data requires methodological improvements and combinations. Currently, target capture, RAD-seq and other methods are more commonly used to obtain information on hundreds to thousands of nuclear gene sites. These methods have been successfully applied to fish phylogeny. For example, by combining mitochondrial and nuclear gene data, the evolutionary relationship of Antarctic fish has been clarified (Li et al., 2024). For marble goby, we can try to develop specific multi-locus probes to capture representative sequences on each chromosome and construct a high-resolution phylogenetic tree. In addition, whole genome resequencing has also become possible as costs decrease. Whole genome data can not only provide richer variation information, but also be used for genome scanning to find adaptive genetic markers (such as differentiation of different groups on certain genes, indicating local adaptation). This information is also very valuable for understanding the mechanism of speciation. It is worth noting that the joint analysis of mitochondrial and nuclear genes requires the use of appropriate models. It is generally believed that joint analysis can effectively identify species and solve the problem of phylogenetic relationships between species, and the results obtained are more reliable. For example, when clarifying species boundaries, the species tree method can be used instead of a simple gene tree, so as to simultaneously consider the consistency and difference of multiple loci and estimate the true divergence relationship between species. In the future, we plan to introduce several nuclear genes (such as classic nuclear genes such as Rag1, Rag2, IRBP, or UCE ultra-conservative element sequences) based on the existing mitochondrial data for joint phylogenetic analysis. If the nuclear genome evidence of marble goby still supports that it is a single species and there is no obvious isolation between the groups, we will be more confident in maintaining the current classification; on the contrary, if the nuclear genome reveals hidden gene flow patterns or deep divergence, it may be necessary to re-evaluate its taxonomic status and consider species splitting. 6.2 Standardization of phylogenetic and taxonomic schemes In the next study, we think it is necessary to unify the phylogenetic analysis and classification methods. This unification mainly includes two aspects: methods and data. In terms of methods, if everyone uses similar processes and parameters, the research results will be easier to compare and integrate. In terms of data, if there is a unified sharing platform and reference database, it will not only save time, but also make the analysis more accurate and efficient.
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