Bioscience Methods 2025, Vol.16, No.3, 137-153 http://bioscipublisher.com/index.php/bm 142 mitochondrial genome of the abalone genus has a highly conserved skeleton, but the differences at the base level are sufficient to delineate species boundaries. High conservation ensures that we can use universal primers to amplify the corresponding genes of different species for direct sequence comparison; and sufficient differences ensure that sequence analysis can identify species. This balance between conservation and difference makes the mitochondrial genome an ideal molecular tool for abalone species identification and phylogenetic analysis. In practical applications, it is only necessary to determine the sequence of one or a few highly variable region genes in the abalone mitochondrial genome, and then compare it with the sequence library of known species to determine the species identity and analyze the kinship. Figure 1 Abalone shell sample. (A) Haliotis tuberculata tuberculata BAU 1391. (B) Haliotis tuberculata tuberculata with lamellae (formerly Haliotis lamellosa) BAU 676.3. (C) Haliotis mykonosensis BAU 657.3. (D) Haliotis tuberculata coccinea BAU 717.3. (E) Haliotis stomatiaeformis, juvenile BAU 699. All scale bars are 1 cm (Adapted from Chiappa et al., 2022) 4 Phylogenetic Reconstruction and Lineage Differentiation 4.1 Construction of mitochondrial genome-based phylogenetic trees With the development of molecular biology technology, the mitochondrial whole genome sequences of more and more abalone species have been determined, providing rich data for phylogenetic research. The phylogenetic tree constructed based on the mitochondrial whole genome (usually taking the concatenated sequence of 13 protein-coding genes) has significantly higher resolution and credibility than the tree constructed with a single gene. Recent phylogenetic analysis covers the main abalone species in the world, and the results support that the genus Haliotis is an evolutionary branch of a monophyletic origin, and it can be divided into several branches. For example, Mamat et al. (2025) reconstructed a phylogenetic tree using the whole genome sequences of 8 abalone species, showing that the Northwest Pacific species such as Haliotis discus, Haliotis diversicolord, and Japanese black abalone (H. discus discus) are clustered into one branch, while in contrast, the green abalone and red abalone of Australia are clustered into another branch, and the ear abalone of the Central Pacific and the Mexican
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