International Journal of Aquaculture, 2025, Vol.15, No.3, 116-134 http://www.aquapublisher.com/index.php/ija 117 Mitochondrial DNA has been widely used in species identification and phylogenetic analysis due to its maternal inheritance, non-recombination, and high mutation rate (Li et al., 2024). Among them, the DNA barcoding technology constructed by mitochondrial gene sequences has become a standard tool for species molecular identification due to its advantages of rapidity, efficiency, and ease of operation. Fish is an important group for the application of DNA barcoding technology. At present, more than 9.46 million sequences have been included in the global DNA barcode database BOLD, covering a large number of fish species, providing rich references for molecular identification. Studies have shown that the accuracy of fish species identification by DNA barcoding can reach 93% for freshwater fish and 98% for marine fish, which is significantly higher than the traditional method based on morphological identification (Ward, 2012). Therefore, gene sequences such as mitochondrial COI have become effective molecular markers for species identification, playing an important role in fishery resource surveys, food traceability, environmental DNA monitoring, etc. The whole mitochondrial genome can not only be used for species identification, but also for studying species relationships and classification. It is not large in size, about 16 kb, but it contains a lot of information. For example, the 13 protein-coding genes are very commonly used and can help us see the relationship between closely related species (Yu and He, 2012). Now that sequencing technology is becoming more and more advanced, many samples can be tested at a time. Because of this, the mitochondrial genomes of many fish have been sequenced and used to draw phylogenetic trees. For example, a research team analyzed the mitochondrial data of 64 species of schizothorax and finally solved many of the classification problems that have been unclear in this group (Rustam et al., 2022). Generally, people will use mitochondrial data in conjunction with some analysis methods, such as maximum likelihood (ML) or Bayesian (BI). This can make the phylogenetic tree more accurate, and can also roughly estimate the time of species differentiation, helping us understand how these species evolved step by step. However, mitochondrial data is not perfect. Sometimes, different genes evolve at different rates; sometimes there will be interference from paternal inheritance and nuclear genes, and even some closely related species may "borrow" each other's mitochondrial genes, all of which will have an impact. Despite this, if there is no nuclear genome data at hand, or the number of samples is small, people will still give priority to mitochondrial DNA for analysis. This is because it is convenient, and in many cases the information is enough. In order to make the results more reliable, the mitochondrial genome can be disassembled for analysis during research. For example, segment it by gene fragments or regions, and compare which places have changed a lot and which places are stable. In this way, the right fragments can be selected according to needs and used in research at different levels, so that we can see who the species is and how they are related (Song et al., 2016). The mullet is important in evolutionary studies and aquaculture. However, its traditional classification has been somewhat difficult to understand. So this study used the mitochondrial genome to look at the phylogeny and species identity of the mullet. The first thing we did was to sequence the complete mitochondrial genome of the mullet and analyze its characteristics. Then, we compared this data with the genomes of some closely related species to see where the mullet belongs in the goby family and where it ranks. In addition, we compared mullets from different regions to see how much mitochondrial differences there were. This can help us determine whether these fish have shown significant intraspecific differentiation, which is helpful for understanding whether they may be differentiating into new species. Morphologically, mullets are sometimes difficult to distinguish. So we wanted to use genetic data to help see if we could more accurately classify its species range and make its taxonomic position clearer. We also selected the mullet in the Mekong River Basin as a case study to analyze the geographic distribution and phylogeny of the population there. Finally, we also discussed the significance of these results for resource conservation and aquaculture management. Studying the genetic background and adaptability of these alien populations can also help us better understand the evolutionary changes that occur when species migrate and invade. Whether it is basic evolutionary research or practical aquaculture, the study of marble goby is very important.
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