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The tetrapod double cone is a pair of tightly associated cones called the "principal" and the "accessory" member. It is found in amphibians, reptiles, and birds, as well as monotreme and marsupial mammals but is absent in fish and eutherian mammals. To explore the potential evolutionary origins of the double cone, we analyzed single-cell and -nucleus transcriptomic atlases of photoreceptors from six vertebrate species: zebrafish, chicken, lizard, opossum, ground squirrel, and human. Computational analyses separated the principal and accessory members in chicken and lizard, identifying molecular signatures distinguishing either member from single cones and rods in the same species. Comparative transcriptomic analyses suggest that both the principal and accessory originated from ancestral red cones. Furthermore, the gene expression variation among cone subtypes mirrors their spectral order (red → green → blue → UV), suggesting a constraint in their order of emergence during evolution. Finally, we find that rods are equally dissimilar to all cone types, suggesting that they emerged before the spectral diversification of cones., Competing Interests: Competing Interests The authors declare no competing interests.

Vertebrates rely on rod photoreceptors for vision in low-light conditions. Mammals have a specialized downstream circuit for rod signaling called the primary rod pathway, which comprises specific cell types and wiring patterns that are thought to be unique to this lineage. Thus, it has been long assumed that the primary rod pathway evolved in mammals. Here, we challenge this view by demonstrating that the mammalian primary rod pathway is conserved in zebrafish, which diverged from extant mammals ~400 million years ago. Using single-cell RNA-sequencing, we identified two bipolar cell (BC) types in zebrafish that are related to mammalian rod BCs (RBCs) of the primary rod pathway. By combining electrophysiology, histology, and ultrastructural reconstruction of the zebrafish RBCs, we found that, like mammalian RBCs, both zebrafish RBC types connect with all rods in their dendritic territory, and provide output largely onto amacrine cells. The wiring pattern of the amacrine cells post-synaptic to one RBC type is strikingly similar to that of mammalian RBCs, suggesting that the cell types and circuit design of the primary rod pathway have emerged before the divergence of teleost fish and amniotes. The second RBC type, which forms separate pathways, is either lost in mammals or emerged in fish., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.