Bioscience Methods 2025, Vol.16, No.3, 137-153 http://bioscipublisher.com/index.php/bm 143 abalone of the Eastern Pacific are each in their own branch. In this phylogenetic tree, the topological structure between species has a close correspondence with their geographical distribution. For example, the tropical species of ear abalone (H. asinina) first merged with another tropical species of sheep abalone (H. ovina), and then formed a branch with the small abalone (H. diversicolor) in the warm temperate zone; this branch then merged with large temperate species such as red abalone (H. rubra) and European abalone (H. tuberculata). This result is generally consistent with the judgment of the distance between species based on traditional morphological classification, and is also consistent with previous research results based on genes such as 16S and COI. It is worth noting that the support rate (such as Bootstrap value) of whole genome phylogenetic analysis is generally high, and many key branches have received > 90% bootstrap value support, which shows that mitochondrial whole genome data is sufficient and reliable for analyzing the deeper systematic relationships of the genus Haliotis. In addition, some previously controversial taxa have also been clarified on the molecular phylogenetic tree. For example, regarding the question of whether the Taiwan nine-hole abalone and the mainland variegated abalone belong to the same species, the whole genome data show that the genetic distance between the two is very close, and they are mixed in the phylogenetic tree, which supports that they actually belong to different geographical populations of the same species. In contrast, another small abalone in the Indo-Pacific region (produced in Indonesia) is significantly different from the Taiwanese nine-hole abalone, and evidence supports its identification as an independent species (possibly H. squamata), a finding also reflected by the length of the long branches on the phylogenetic tree. It can be seen that the phylogenetic analysis based on the whole mitochondrial genome provides a clear branching structure and quantitative support for the evolutionary relationship between abalone species, laying the foundation for solving taxonomic problems and discovering potential new species. In the process of constructing a phylogenetic tree, commonly used methods include maximum likelihood (ML) and Bayesian inference (BI), and multiple sequence alignment and model selection are used to ensure the reliability of the results. Due to the base composition bias and evolutionary rate heterogeneity of mitochondrial genes, it is usually necessary to partition different genes and apply corresponding substitution models during analysis to improve the accuracy of the phylogenetic tree (Liu et al., 2018; Zhao and Wu, 2024). 4.2 Divergence nodes and lineage clustering The phylogenetic tree of the abalone genus reveals several major evolutionary branches and their differentiation nodes. This information can help us reconstruct the evolutionary history and geographical diffusion process of the abalone genus. According to mitochondrial genetic evidence, the genus Haliotis may have originated in the Tethys Ocean in the Middle Paleozoic and then spread to the coasts of the world. The base node of the phylogenetic tree divides the abalone species into two major lineages: one is the Atlantic-Eastern Pacific lineage, including European abalone and American abalone, and the other is the Indo-Pacific lineage, including various abalones in Asia and Oceania (Mamat et al., 2025). This differentiation node occurred approximately in the early Cenozoic, coinciding with the geological events of the closure of the Tethys Ocean and the isolation of the oceans from each other. Within the Indo-Pacific lineage, it can be further divided into several clusters, such as: East Asian cluster (Haliotis discus, Haliotis diversicolord, Japanese abalone, etc.), Oceania cluster (Australian green abalone, abalone red, etc.) and East Pacific cluster (Mexican abalone, etc.). The differentiation node ages of these clusters may correspond to changes in ocean climate or ocean current patterns. For example, the common ancestor of the East Asian cluster may have spread through the Kuroshio system in the Pliocene, while the differentiation of the Australian cluster is related to the South Pacific warm current. Phylogenetic cluster analysis shows that species close to each other on the phylogenetic tree often have similar geographical distributions and ecological habits. This confirms the role of geographic isolation and adaptive radiation in abalone evolution. For example, large temperate abalone (Halion discus, European abalone, etc.) cluster together, while small tropical abalone (Haliotis scrofa, etc.) form another cluster, suggesting that environmental selection pressure shapes the genetic convergence of species with similar ecological niches (Zhang et al., 2025). 4.3 Correlation between mitochondrial variation and geographic distribution The spatial pattern of abalone species and populations shapes the distribution characteristics of their mitochondrial genetic variation. By analyzing the association between mitochondrial variation and geographical factors, the
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