Bioscience Methods 2025, Vol.16, No.3, 137-153 http://bioscipublisher.com/index.php/bm 141 showed that most genes were under strong purifying selection, with Ka/Ks far less than 1, but the ND2, ND6 and ATP8 genes showed an increase in Ka/Ks ratios in some evolutionary branches. This suggests that these genes may have positive selection sites, which promote the adaptation of species to specific environments. For example, ATP synthase plays a key role in energy metabolism. Although the small subunit of ATP8 is short in length, it evolves rapidly. Changes in its specific amino acids may affect the stability of the enzyme complex under different temperatures or pH conditions (Deng et al., 2019). For another example, cytochrome oxidase (COX) subunits are extremely conserved among abalone species, but some amino acid substitutions have been detected in a few heat-resistant species. It is speculated that these substitutions improve the catalytic efficiency of the enzyme at high temperatures. It should be pointed out that it is still challenging to identify adaptive mutation sites in mitochondrial genes because genetic drift and pedigree history often mask selection signals. However, with the accumulation of more genome sequences and environmental data of abalone species, researchers have begun to apply methods such as phylogenetic generalized least squares to incorporate evolutionary tree information into the analysis to distinguish which molecular changes are significantly associated with environmental factors. Current evidence supports some associations between non-synonymous variations in the abalone mitochondrial genome and adaptive evolution. For example, temperature gradients may drive subtle adjustments in the base composition and protein sequence of some mitochondrial genes, thereby improving the adaptability of aerobic metabolism to different water temperatures (Zhang et al., 2025). In the future, through larger-scale comparative genomic studies and functional experiments (such as measuring the activity of different mutant enzymes), it is hoped that the adaptive significance of specific mutations will be clarified. 3.3 Conserved and divergent regions among abalone species Abalone species are evolutionarily closely related, and their mitochondrial genomes are generally highly conserved, but there are still clear genetic differences between different species. This feature of "differences in overall similarity" provides an important basis for abalone classification and species identification. In terms of conservation, all abalone species share the same set of mitochondrial genes, the gene order is almost completely consistent (except for the aforementioned individual exceptions), and the nucleotide and amino acid sequences of many key genes are highly similar. For example, the green abalone (H. laevigata) and the red abalone (H. rubra) are both closely related species from Australia. The similarity of their mitochondrial genome sequences is about 92%, and most genes have only a few base differences between the two species. For another example, the mitochondrial sequences of the Chinese population of Haliotis discus and the population native to Japan/Korea are almost identical, with a similarity of more than 98.5%. These high similarities reflect that the genetic information of the common ancestor of the abalone genus has been largely preserved during the differentiation process. However, different species have also accumulated enough variation to distinguish them from each other. Especially in the COI gene fragment commonly used in DNA barcoding, the genetic distance between different abalone species is usually more than 10%, while the intraspecific distance is generally less than 2%. Therefore, the COI sequence can reliably distinguish the species identity of abalone (Figure 1) (Chiappa et al., 2022). Not only COI, but other genes or regions in the mitochondrial genome also provide genetic signals to distinguish species. For example, 16S rRNA fragments or ND4 genes with large differences can be selected as auxiliary markers to improve the accuracy of identification. A notable pattern of differentiation is that according to the phylogeny of mitochondrial sequences, abalones on the west coast of the Pacific Ocean (such as Haliotis discus, Haliotis diversicolord) and abalones in the Atlantic and Europe (such as Haliotis tuberculata) belong to different evolutionary lineages and have a large genetic distance from each other. This is consistent with the transmission pathways and reproductive isolation in geological history. In addition, species in the same geographical area are often more closely related to each other. For example, several species distributed near the coast of China and Japan (Haliotis discus, Haliotis diversicolord, etc.) are clustered into one branch, while species in Australia (Haliotis tuberculata, etc.) are clustered into another branch (Mamat et al., 2025). These differences indicate that different abalone species have accumulated species-specific mitochondrial DNA variations during long-term evolution, thus forming a clear differentiation pattern. The
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