Scientists can infer that snakes once had legs through a variety of methods, despite not having direct DNA samples from snakes 150 million years ago.
One approach is to study the fossil record. Fossils of ancient snake relatives, such as primitive snakes like Najash rionegrina and Pachyrhachis problematicus, have been discovered with well-preserved limb bones. These fossils exhibit clear evidence of reduced but functional limbs, providing a link between snakes and their legged ancestors.
Comparative anatomy is another powerful tool. By examining the anatomy of modern snakes, scientists can identify vestigial structures, such as pelvic spurs and remnants of hind limb bones, which are remnants of their legged past. These structures serve no functional purpose in snakes but are homologous to the limbs of other reptiles.
Additionally, developmental biology studies contribute to our understanding. Embryological studies of snakes have shown that during early stages of development, snake embryos display limb buds similar to other reptiles.
However, these limb buds regress and do not fully develop. By comparing this process with other reptiles' limb development, scientists can deduce that snakes have a genetic program for limb development that has been modified over time.
Combining evidence from fossils, comparative anatomy, and developmental biology, scientists can confidently conclude that snakes once possessed legs and subsequently underwent evolutionary changes resulting in the loss of their limbs around 150 million years ago.
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