International Journal of Aquaculture, 2025, Vol.15, No.3, 116-134 http://www.aquapublisher.com/index.php/ija 124 In addition to the weak differences between species, morphological classification also depends on the experience and expertise of the identifier. However, there is currently a shortage of senior taxonomists in the field of fish classification. Due to the lack of experts familiar with local fish classification in many regions, there is insufficient attention to non-economic common species such as marble goby, resulting in identification gaps or errors in local chronicles and survey reports. Even experienced taxonomists need to use a large number of complete adult fish specimens for comparison and identification to deal with such "complex populations". However, in actual environments, the collected samples are often incomplete or incomplete (such as part of the body of the catch, processed products, etc.), which increases the difficulty of pure morphological identification (Panprommin and Manosri, 2022). Therefore, the identification of marble goby by morphological means alone may result in misjudgment or omission. A typical example is that in the early reports of the introduction of the marble goby into my country (late 20th century), due to insufficient understanding of its native population, some places also called similar local Channa fish "marble goby", causing confusion in species records. It was not until later through dissection and DNA analysis that it was confirmed that those fish were not true cloud-spotted sharp-necked tang fish, but other closely related species (Wang et al., 2024). This shows that relying solely on morphology may confuse different species, which is not conducive to scientific management and research (Chen et al., 2002). In addition, the limitations of morphological classification are also reflected in the inability to reveal cryptic diversity between species. If two groups are almost the same in morphology, but have actually been isolated for a long time and have genetic differentiation, morphological methods may never be able to distinguish them, resulting in the neglect of "cryptic species". This is not impossible in a widely distributed species like marble goby. In fact, studies have shown that DNA barcoding technology can discover hidden lineages in many morphologically similar fish. Therefore, it is necessary to be alert to the possibility that there may be undescribed new species of marble goby, which traditional taxonomy has not yet detected. 4.2 Molecular standard delimitation of species With the development of molecular systematics, it has become a trend to define species using DNA sequences. For groups such as marble goby that are difficult to classify by morphology, molecular standards provide an objective and quantitative basis for demarcation. In the classification of fish species, a commonly used principle is the "barcode gap": that is, the genetic distance within a species is significantly smaller than the genetic distance between species. When the sequence differences between two populations exceed the general intraspecific variation range and form their own monophyletic branches, they can be regarded as different species. Specifically for COI barcodes, the empirical threshold is usually set at about 2% residual difference (Roul et al., 2021). In this study, our analysis showed that the COI distance between all marble goby samples was much lower than 2%, while the distance between marble goby and any closely related species (such as other Channa fish) was higher than 8%. This clear gap supports that marble goby belongs to a single species. Therefore, from the perspective of COI sequence, there is no strong evidence to split it into a new species. However, relying on a single gene threshold alone is not foolproof. Many studies have pointed out that different thresholds apply to different groups, and cryptic species are often close to closely related species in genetic distance (Zhang et al., 2019). Therefore, we combine phylogenetic tree morphology and other methods for discrimination. If the marble goby population forms two or more obvious branches, each with high support and no shared haplotypes, then despite the similar morphology, we should also consider that they may represent independent evolutionary units. For example, in our COI phylogenetic tree, although the marble goby samples are generally mixed together, they can be barely divided into the "Mekong River Cluster" and the "Malay Peninsula Cluster". Although the difference between the two is not large, there is no haplotype sharing. According to the GMYC model analysis, the lineages corresponding to the two clusters have similar differentiation times but have not exceeded the species divergence threshold. The model does not judge them as different species (that is, marble goby is still treated as a single species) (Fujisawa and Barraclough, 2013; Talavera et al., 2013). However, if more
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