International Journal of Aquaculture, 2025, Vol.15, No.3, 116-134 http://www.aquapublisher.com/index.php/ija 125 samples are added in the future and nuclear gene data are used to support it, the conclusion may change. Therefore, at present we prefer to be conservative and treat the marble goby as a species, but recognize that there is a clear genetic structure within it. In addition to sequence distance and phylogenetic tree, molecular delimitation can also refer to the method of molecular diagnostic sites, that is, looking for nucleotide differences that only appear in one part of the individuals and never appear in another part (Li et al., 2024). We compared the mitochondrial sequences of marble goby from different regions and found that there are still several nucleotide sites that remain consistent in all marble goby samples (i.e., there are no fixed differences), which means that there is no clear molecular trait to divide it into two species. If there are multiple fixed differences (diagnostic sites), it means that the populations may have reached species-level differences. At present, only a few nearly fixed difference sites (such as an insertion and deletion in the control region) have been found between different marble goby populations, but due to the limited sample size, we cannot determine whether these differences are related to species boundaries. In order to improve the reliability of species demarcation, we recommend the use of multi-gene joint analysis and multi-method cross-validation strategies. Specifically, the data of mitochondrial genes and several nuclear genes can be combined to construct a species tree, and Bayesian programs such as BPP can be used to analyze species boundaries. This type of analysis can simultaneously consider factors such as incomplete lineage sorting and is more robust than single genes. Based on molecular evidence, ecological and biogeographic information should also be combined: if the population corresponding to a branch has a unique niche or geographical isolation and does not mate and reproduce with another branch in the wild, it is more reasonable to regard it as a different species. In addition, the comparison of symbiotic organisms or parasite lineages can also assist in determining whether the host is a different species. 4.3 Integrative taxonomy combining morphology and genetics Based on the above analysis, we deeply realize that it is difficult to make accurate classification judgments on complex cases such as marble goby based on a single piece of evidence. To this end, it is necessary to adopt the idea of integrative taxonomy, combining multiple information such as morphology, molecular genetics, and ecology to comprehensively evaluate the taxonomic status of marble goby. Integrative taxonomy emphasizes the consistency of different data sources: species division is more reliable only when evidence such as morphological characteristics, molecular lineages, and ecological distribution support each other. In the case of marble goby, we should first make it clear that multiple morphologically distinguishable evolutionary units have not yet been found. However, morphological similarity does not mean genetic identity. Our molecular analysis suggests the existence of genetic structure, so the next step is to look for morphological differences that may correspond to genetic differentiation. This requires a careful examination of the morphological variation of marble goby in different geographical groups, including measuring more refined morphological indicators (such as scale counts, bone structure, body color pattern patterns, etc.), and using statistical analysis methods to identify potential differences. If certain morphological features are found to strictly correspond to genetic branches, then these features can be used as one of the bases for the division of new species (Bleeker, 1852). For example, if the Mekong population of marble goby has a significantly higher number of dorsal fin spines or a unique arrangement of spots than other populations, then combined with its genetic differences, it can be considered to be described as a new species. In terms of genetics, in addition to mitochondria, we should also introduce nuclear DNA markers for verification. This includes analyzing population structure using microsatellite or SNP data, and using multi-gene collaborative trees to test the reliability of mitochondrial tree results. If nuclear genes also support the division of the same two branches, the evidence for the division of new species will be greatly enhanced (Yu and He, 2012). On the contrary, if nuclear genes show that there are no clear boundaries between the groups of marble goby, it means that mitochondrial differentiation may only be at the population level and is not enough to rise to species.
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