International Journal of Marine Science, 2025, Vol.15, No.3, 144-153 http://www.aquapublisher.com/index.php/ijms 147 environmental DNA macrobarcode research because its fragments are short and facilitate high-throughput amplification. The Cytb gene, the mitochondrial cytochrome b gene, is one of the fastest-evolving coding genes in the mitochondrial genome. Cytb sequence variation is abundant and is often used for phylogenetic analysis of population genetic structures and related species (Figure 1) (Jena et al., 2023). Figure 1 Workflow of phylogenetic analysis of Cynoglossus linguabased onCOI andCytb genes (Adopted from Jena et al., 2023). 4.2 Comparison of label applicability and polymorphism Choosing the right molecular marker is critical to obtaining reliable classification and phylogenetic results. Different genes have different recognition abilities and polymorphisms at different classification levels due to their different degree of evolutionary conservatism. When targeting species identification, labels are required to differ significantly between species and small intraspecies. A large number of studies and practices have shown that COI genes meet this requirement in most fish species, and their interspecies regret distances are usually much higher than those intraspecies, making them an ideal species identification marker. Comparison of data from freshwater fish in Hainan Island shows that the interspecies genetic distance threshold of COI is about 0.69%, and the discrimination accuracy can reach 94.96%, which is significantly better than the 12S and 16S genes. The 16S rRNA gene has a slightly slow evolution rate and relatively low interspecies, which may not be sufficiently distinguished in some close species, but as a supplementary marker, it can improve the robustness of the identification. The degree of variation of the Cytb gene is comparable to or higher than that of COI, and is especially suitable for analyzing intraspecies lineages and population structures. However, high variation also means that sequence alignment and model selection need to be more cautious, otherwise deep relationship judgments may be affected by synonymous replacement saturation (Silva-Santos et al., 2018). Therefore, when reconstructing phylogenetic relationships, it is often preferable to use multiple markers in combination: including slower evolution 16S, etc. to stabilize higher-order relationships, and also including COI, Cytb, etc. to capture low-order differentiation. Nuclear genes such as RAG1 are slowly evolving, and are suitable for exploring long-term evolution of family-level genes, but they lack the ability to distinguish close relatives, and because diploid genes may have allelic sequences, the analysis is relatively complicated. 4.3 Scientific basis for selected molecular markers Based on the above comparison, we selected mitochondrial COI and Cytb genes as main molecular markers in this study, and verified some species with the 16S gene. The COI gene has mature databases and extensive application
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