International Journal of Marine Science, 2025, Vol.15, No.3, 144-153 http://www.aquapublisher.com/index.php/ijms 146 by the north and south, indicating that there is significant spatial heterogeneity in the fish community structure in local waters (Xu et al., 2023). In terms of freshwater fish in tropical islands, Lin et al. (2022) used mitochondrial Cytb, ND2 and COI multiple genes to analyze the genetic structure of Opsariichthys hainanensis, and found that the population of vegetation fish in Hainan Island and the Pearl River system on the mainland was significantly differentiated. Regardless of seawater fish or island freshwater fish, molecular phylogenetics has been successfully applied to analyzing population pattern and genealogical relationships. This provides valuable experience and reference for subsequent research on fish diversity in Hainan offshore. 3 Classification Status of Wild Fish Offshore in Hainan 3.1 Limitations of traditional morphological classification There are many species of fish offshore in Hainan, and traditionally, they are mainly classified and identified based on morphological characteristics. However, in practice, morphological classification faces many difficulties and limitations. On the one hand, different fish may have morphological phenomena such as "also-species" and "also-species" in terms of morphology, especially in tropical waters such as coral reefs. The body colors and markings of fish species are extremely diverse, and it is often difficult to distinguish close species in a timely and accurate manner based on external forms alone (Gravier-Bonnet et al., 2016). On the other hand, the larval and adult morphology of fish may vary greatly, and many fish lack reliable morphological characteristics in the early life history stages, resulting in traditional methods being unable to identify fish eggs and juvenile fish (Grealy et al., 2016). In addition, performing fine fish morphology classification requires long-term accumulation of experience from taxonomic experts, which has a high threshold for non-professional personnel. In an area off the coast of Hainan, where species are rich and relatively weak in research, the above problems are more prominent. In the past, the list of species records based solely on morphological records may have been omitted or misunderstood. 3.2 Review of existing molecular classification research In view of the shortcomings of morphological identification, molecular classification research on fish in Hainan and adjacent waters has been gradually carried out and preliminary results have been achieved in recent years. Some studies have used DNA barcode technology to conduct species identification and classification status verification on fish in the sea area around Hainan Island. Du et al. (2021) used DNA taxonomy methods to evaluate the fish diversity throughout the South China Sea. In combination with COI sequence alignment, they found that some fish traditionally classified as widely distributed species actually had significant intraspecies genetic differentiation, suggesting that multiple undescribed new species may be implicit. There are also some molecular classification explorations in specific groups: for the South China Sea economic fish Grouper family and some species of the order Performin, researchers analyzed their phylogenetic relationships through Cytb and other sequences, and the results showed new insights into the family affiliation of certain species (Zeng et al., 2022). 4 Commonly Used Molecular Markers and Their Application in Fish Systemology 4.1 Introduction to commonly used molecular markers (such as COI, 16S, Cytb) In molecular systems research, different gene markers are suitable for analysis at different classification levels due to their different evolutionary rates and functional characteristics. In fish studies, mitochondrial DNA has become the most widely used source of molecular markers due to its high mutation rate, maternal haploid inheritance and no recombination. The most commonly used markers include: COI gene, mitochondrial cytochrome oxidase I gene, with a total length of about 650 bp, and is internationally recognized as the standard DNA barcode sequence. COI has the advantages of high discrimination at the species level and strong versatility of amplification primers, and is widely used in species identification and classification research. For fish, COI fragments can usually clearly distinguish most of the relative species and are the preferred sequence for establishing species libraries. 16S rRNA gene, mitochondrial small subunit ribosomal RNA gene, sequence conserved regions and variable regions interphase distribution. The 16S sequence evolution rate is slightly slower than COI, but it performs well in the classification above the department level and the analysis of older branches. In addition, 16S is often used in
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