The origin of vertebrates was defined by evolution of a skeleton; however, little is known about the developmental mechanisms responsible for this landmark evolutionary innovation. In jawed vertebrates, cartilage matrix consists predominantly of type II collagen (Col2␣1), whereas that of jawless fishes has long been thought to be noncollagenous. We recently showed that Col2␣1 is present in lamprey cartilage, indicating that type II collagen-based cartilage evolved earlier than previously recognized. Here, we investigate the origin of vertebrate cartilage, and we report that hagfishes, the sister group to lampreys, also have Col2␣1-based cartilage, suggesting its presence in the common ancestor of crown-group vertebrates. We go on to show that lancelets, a sister group to vertebrates, possess an ancestral clade A fibrillar collagen (ColA) gene that is expressed in the notochord. Together, these results suggest that duplication and diversification of ColA genes at the chordate-vertebrate transition may underlie the evolutionary origin of vertebrate skeletal tissues.development ͉ gene duplication ͉ skeleton evolution ͉ chordate ͉ notochord T he phylogenetic relationships of the vertebrates were established largely based on anatomical characters, particularly those of the skeleton. The skeletons of jawed vertebrates (gnathostomes) are composed of cartilage and bone, which contain high levels of type II and type I collagen (Col2␣1 and Col1␣1͞2 protein, respectively). By contrast, the cartilaginous skeletons of lampreys and hagfishes, the only extant jawless fishes (agnathans), have been reported to be noncollagenous and to contain instead the elastin-like proteins lamprin and myxinin (1). This difference in the extracellular matrices of vertebrate skeletons led to the idea that type II collagen became the major structural component of gnathostome cartilage after the divergence of these two lineages (1). However, this view was challenged by our recent report that lampreys have two Col2␣1 orthologs and that both genes are expressed during chondrogenesis. Adult lamprey cartilage was also shown to be rich in Col2␣1 protein (2). Furthermore, we identified a lamprey ortholog of Sox9, a direct transcriptional regulator of Col2␣1 in gnathostomes, and we showed that it is coexpressed with Col2␣1 (2). This discovery revealed that the genetic pathway for vertebrate chondrogenesis predated the divergence of lampreys and gnathostomes, although precisely how early this character arose is unknown. Here, we investigate the evolutionary origin of Col2␣1-based cartilage by expanding our analysis to the most inclusive clade of vertebrates, which includes the hagfishes, and to a sister group to the vertebrates, the lancelets (3, 4). We report that hagfishes also have Col2␣1-based cartilage, suggesting that this type of cartilage was present in common ancestor of all crown vertebrates. Our analysis of lancelets revealed the presence of an ancestral clade A fibrillar collagen (ColA) gene that is expressed in the notochord. Thus, during th...