International Journal of Marine Science, 2025, Vol.15, No.1, 1-14 http://www.aquapublisher.com/index.php/ijms 5 3.2 Molecular clock analysis and divergence time estimation With the help of phylogenetic trees and combined with molecular clock models, this study further estimated the differentiation time of the main evolutionary lineages of the genus Abalone. Using fossil records and the differentiation time of other gastropods as calibration points, this study infers that the genus Abalone originated in the Late Cretaceous about 100 million years ago, and then experienced multiple species radiations in the Paleogene and Neogene. For example, the divergence between abalones in the northern Pacific (Halion discus, Abalone japonensis, etc.) and abalones in the southern hemisphere (Abalone occidentalis, Abalone occidentalis, etc.) occurred approximately at the end of the Cretaceous about 70 million years ago. Botwright et al. (2019) also obtained a similar differentiation time estimate (about 71 million years ago) by comparing the genomes of Abalone occidentalis and Abalone occidentalis. This suggests that the early differentiation of the abalone genus may be related to plate movement and ocean separation. Later, during the Miocene, regional radiation of abalone occurred along the coasts of different continents. For example, this study estimates that the common ancestor of the species of the American abalone branch (red abalone, black abalone, etc.) lived about 30 million years ago. During the Miocene warm period, changes in ocean circulation may have contributed to the formation of these species (Tshilate et al., 2023). Similarly, the differentiation between African abalone and Australian abalone was roughly 15 million years ago, which coincides with changes in the Indian Ocean-Atlantic water system. It should be pointed out that due to the limited reliable fossil calibration, there is a certain uncertainty in the molecular clock inference, but the overall time frame is consistent with geological events. According to the time tree constructed in this study, the evolutionary trajectory of the abalone genus can be depicted: the genus originated in the Tethys Sea and then split into two major lineages in the north and south with continental drift. The northern hemisphere lineage diversified in the northwest Pacific, and the wrinkled abalone group appeared; the southern hemisphere lineage evolved into warm-resistant species in South Africa and Australia. The rise and fall of sea levels and changes in ocean currents during the glacial period have shaped the recent distribution pattern of species. For example, the Quaternary glacial period caused some abalone populations to be isolated, thus forming closely related species. The evolutionary history of abalone is closely related to changes in the global marine environment. 3.3 Revisions in abalone taxonomy based on genomic evidence Based on the high-resolution phylogenetic tree and differentiation time information obtained in this study, some revision suggestions can be made for the classification of the genus Abalone. Given that the genomes of the wrinkled disc abalone (from Hokkaido, Japan to the northern coast of China) and the Japanese abalone (H. discus) are very different, it is supported to regard the two as the same species or at least very closely related evolutionary units. Traditionally, Japanese abalone is sometimes considered to be a nominative subspecies of the wrinkled disc abalone. The results of this study further confirm that its taxonomic status should be merged, which will simplify the classification system of the genus Abalone. The phylogenetic tree of this study shows that the nine-hole abalone (H. diversicolor) and its variants (such as the variegated abalone H. diversicolor supertexta) form an independent branch and are far away from the wrinkled disc abalone, supporting its treatment as an independent species or subgenus. For a long time, the classification of Haliotis diversicolor and Haliotis discus has been controversial. Based on genomic evidence, Haliotis diversicolor can be classified as a new sub-classification unit such as "Haliotis diversicolor" to reflect its genetic independence. For some newly described species in recent years, such as the yellow and pink abalone (H. corrugata, H. fulgens, etc.) in Mexico, the genomic analysis of this study supports the validity of their respective species. Although these species are morphologically similar, their genomic differences are clear and their species status should be retained (Vega-García et al., 2015; Mares-Mayagoitia et al., 2023). This study found that although the green abalone (H. laevigata) and black-lipped abalone (H. rubra) in Australia overlap geographically, their genomes are significantly different, which proves that they are not the same species, but a pair of different species known to hybridize naturally. This is also reflected in aquaculture practice - the fertile offspring of green abalone and black abalone are used for breeding, but the genomic differences between the parents still maintain the species boundaries (Kube
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