Bioscience Methods 2025, Vol.16, No.3, 137-153 http://bioscipublisher.com/index.php/bm 138 with a total length of about 16 kb, and the gene organization is relatively conservative, which facilitates cross-species gene comparison and phylogenetic analysis. The mitochondrial gene mutation rate is high, and it can accumulate sufficient intra- and inter-specific variation to distinguish closely related species or populations (Li et al., 2021). Especially in shellfish classification, traditional morphological methods sometimes make it difficult to accurately identify species due to morphological plasticity or different developmental stages. The application of mitochondrial DNA sequences (such as COI gene barcodes) provides an efficient and reliable molecular means for identifying cryptic species, new species, and larval stage individuals (Jung et al., 2015; Szyp-Borowska and Sikora, 2019). This study will systematically review the evolutionary characteristics of the abalone mitochondrial genome and its application progress in species identification, summarize the basic structure of the abalone mitochondrial genome, including characteristics such as gene composition, length, gene arrangement, and variation patterns in control regions and non-coding regions, analyze the distribution patterns of variation types (such as SNPs and InDels) in the abalone mitochondrial genome, explore the adaptive evolution that non-synonymous mutations may reflect, and compare the gene conservation and differences between different species. Based on the phylogenetic reconstruction results of the mitochondrial whole genome sequence, the phylogenetic structure and differentiation nodes of the main evolutionary branches within the genus Haliotis are explained, and the relationship between mitochondrial variation and geographical isolation is analyzed. Through specific case studies, the systematic relationships of abalone species populations in the southeastern coast of China are compared, the genomic differences between the introduced Japanese abalone and the local abalone are evaluated, and the phylogenetic relationships and taxonomic disputes between Japanese, Australian and East Asian abalone are discussed. This study hopes to provide a comprehensive and in-depth reference for abalone taxonomy and molecular identification research to promote abalone species protection, genetic breeding and sustainable development of the industry. 2 Structural Features of the Abalone Mitochondrial Genome 2.1 Genome composition, size, and gene order The abalone mitochondrial genome is a closed circle, generally between 16 kb and 17 kb in length. For example, the mitochondrial genome of Haliotis discus is about 17 037 bp in length and contains 37 genes, including 13 protein-coding genes, 22 tRNA genes and 2 rRNA genes. This gene composition is consistent with the mitochondrial genomes of most invertebrates (especially gastropods), reflecting the high conservation of the mitochondrial genome. The gene arrangement of the abalone mitochondrial genome remains unchanged among different species: the relative order and chain polarity of the genes are basically the same. Studies have compared the mitochondrial genome arrangements of multiple abalone species and found that except for a few species (such as European abalone H. tuberculata), the mitochondrial gene arrangements of abalone species are all homologous, and no major rearrangements have occurred (Pu et al., 2020). It has been reported that the positions of tRNASer (AGN) and tRNAPhe in the mitochondrial genome of H. tuberculata tuberculata have been swapped relative to other species, suggesting that the genome structure is extremely stable during the evolution of abalone, and only a few lineages have undergone minor rearrangements. This highly conservative gene arrangement facilitates the comparative alignment of mitochondrial genomes and the identification of homologous sequences in different abalone species. The GC content of the abalone mitochondrial genome is approximately 35%-41%. For example, the base composition of the mitochondrial genome of H. asinina is 35.3% A, 24.3% T, 13.3% G, and 27.1% C, showing the common characteristics of A+T bias (Mamat et al., 2025). The composition and organizational structure of the abalone mitochondrial genome are highly similar among species within the genus, which lays the foundation for subsequent interspecific variation and phylogenetic analysis. 2.2 Variation patterns in control and non-coding regions In the mitochondrial genome, the control region (D-loop) is the longest non-coding region and is usually the region with the most sequence variation. The mitochondrial control region of abalone is rich in AT bases and contains replication origins and transcriptional regulatory elements. Its length and sequence may vary significantly between different species and even between different individuals of the same species. For example, in different individuals of Haliotis diversicolor, the length of the control region can vary by tens of bases, often due to changes in the
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