The colonization of land by tetrapod ancestors is one of the major questions in the evolution of vertebrates. Despite intense molecular phylogenetic research on this problem during the last 15 years, there is, until now, no statistically supported answer to the question of whether coelacanths or lungfish are the closest living relatives of tetrapods. We determined DNA sequences of the nuclear-encoded recombination activating genes (Rag1 and Rag2) from all three major lungfish groups, the Australian Neoceratodis forsteri, the South American Lepidosiren paradoxa and the African lungfish Protopterus dolloi, and the Indonesian coelacanth Latimeria menadoensis. Phylogenetic analyses of both the single gene and the concatenated data sets of RAG1 and RAG2 found that the lungfishes are the closest living relatives of the land vertebrates. These results are supported by high bootstrap values, Bayesian posterior probabilities, and likelihood ratio tests.S ince the discovery in 1938 of the ''living fossil,'' the coelacanth Latimeria chalumnae, a representative of a group of lobe-finned fish thought to have gone extinct Ϸ80 million years ago (1, 2), there has been remarkable interest in this legendary fish by the public and scientists alike. However, the evolutionary relationships of the coelacanths to the other two living groups of lobe-finned fish (Sarcopterygii), the lungfish (Dipnoi), and the land vertebrates (Tetrapoda) remain debated until today. Since its discovery, many comparative morphologists and paleontologists considered the coelacanth to be the closest living relative of the land vertebrates (1-4), although the lungfish were historically thought, and continued to be thought by some researchers, to hold that claim. More recently, however, several analyses began to challenge the sistergroup relationship between the coelacanth and the tetrapods, first on morphological and paleontological grounds (5-7) and later based on molecular phylogenetic analyses (8,9). Palaeontological studies are limited to studying morphological features, and strong phylogenetic inferences are often hindered because of missing data in incomplete fossils. The most recent paleontological evidence demonstrated that the lungfish represent an ancient lineage and that several of the features defining this group remained highly conserved throughout the entire evolutionary history of land vertebrates (10). The majority of palaeontological studies published during the last decade suggest that lungfish (Dipnoi) are the closest living relatives of the tetrapods or, alternatively, that coelacanths and lungfish form a monophyletic group that is equally closely related to the land vertebrates (11,12).A wealth of molecular phylogenetic studies addressed the tetrapod origin question, first based on mitochondrial DNA data by using partial gene sequences, single genes, or a few genes, and more recently, based on complete mitochondrial genomes (9,(13)(14)(15)(16)(17)(18). Most of these mitochondria-based molecular phylogenetic studies favored the lungfish a...