Global cooling and hot springs may have induced autotetraploidy and autohexaploidy in Schizothorax ancestors, and its implications for polyploid breeding

The establishment of artificial polyploid fish lines is highly significant, but it is difficult to obtain ploidy doubling fish lines. There are abundant natural polyploids in fish, and studying their polyploid origin may provide new implications for polyploid breeding. Polyploidy can be divided into autopolyploidy and allopolyploidy, and they can be distinguished by the cloning of single-copy nuclear genes. In this study, we found that both tetraploid and hexaploid Schizothorax fishes are autopolyploids, as evidenced by cloning 4 single-copy nuclear genes. In addition, we constructed a time-scaled phylogenetic tree based on the oldest Schizothorax fossil (early Oligocene), and found that the tetraploidization (2n → 4n) and hexaploidization (4n → 6n) of Schizothorax occurred at 30.43–39.64 Ma and after 2.50 Ma, respectively. Both polyploidization events occurred during the transition of two geologic periods (Eocene to Oligocene, 33.9 Ma; Pliocene to Pleistocene, 2.58 Ma) and both experienced global cooling. Through ancestral geographical reconstruction, we found that both polyploidizations most likely originated from the southeastern Tibetan Plateau with relatively high elevation and high-density hot springs. After global cooling, the hot spring zone may become a refuge and new spawning ground for ancestral Schizothorax fishes, increasing the probability of fertilized eggs being accidentally heat shocked by the hot spring, leading to the formation of polyploids. In addition, according to the above implications, we proposed two innovative methods for breeding autohexaploids and allotetraploids from autotetraploid fish. First, autotetraploid can create autohexaploid by inhibiting the extrusion of the second polar body from their self-fertilized eggs. Second, crossing autotetraploid (♂) and other diploid fish (♀) can create allotetraploid by inhibiting the extrusion of the second polar body of their fertilized eggs. Although it is difficult to obtain autotetraploid lines, there are still a few successful examples through distant hybridization or physical shock. In addition, there are a few autotetraploids in nature. These autotetraploid lines can provide core parental resources for the creation of autohexaploids and allotetraploids. This interdisciplinary study bridges evolutionary biology and fish genetic breeding, providing new insights into the origin of polyploid species and methods for polyploid breeding.