3 million years ago: the formation of a land bridge between North and South America.
180 million years ago, when Pangea broke apart into several landmasses, North and South America separated from each another. North America became part of Laurasia in the northern hemisphere, and South America became part of Gondwana in the southern hemisphere.
Different species evolved in each region, isolated from each other with no genetic interchange. Horses and camels appeared in North America while giant sloths appeared in South America.
Around 15 million years ago, the collision of tectonic plates produced volcanic activity in the region, creating many islands. These islands enlarged until 3 million years ago when they formed a contiguous strip of land that united North and South America in the present Isthmus of Panama.
The bridge made it easy for animals and plants to migrate between the two continents. But there was also speciation in the sea around the Isthmus of Panama since a strip of land separated some species. Currently, there are seven different shrimp species on each side of the Isthmus. For each one of them, their most closely related species is across the Isthmus.