Phylogenetic analysis and divergence time estimation
The phylogenetic position of T. dalaica, within the order Cypriniformes, and its estimated time of divergence were deduced based on single copy ortholog genes from related species. Gene family clustering showed that a total of 23,925 genes in T. dalaicacould be divided into 19,271 gene families, of which 116 were unique toT. dalaica and 4,126 were single copy orthologs across all related species. Based on both the supermatrix constructed using these single copy genes and the maximum likelihood method, a phylogenetic tree of these species was reconstructed with high confidence (Figure 3). It showed that T. dalaica is closer to T. tibetana, withT. siluroides identified as the basal species within theTriplophysa species. Divergence time estimation showed thatT. dalaica and T. tibetana diverged approximately 8.31 million years ago (Mya), with the history of Triplophysa fish likely to be at least 14.6 Mya (Figure 4).