Proc. Natl. Acad. Sci. USA Vol. 96, pp. 2177–2182, March 1999 Evolution Molecular studies suggest that cartilaginous fishes have a terminal position in the piscine tree (vertebrate evolutionyAmniotayChondrichthyesystarry skateymolecular dating) ANN-SOFIE RASMUSSEN AND ULFUR ARNASON* Department of Genetics, Division of Evolutionary Molecular Systematics, University of Lund, So¨lvegatan 29, S-223 62 Lund, Sweden Communicated by Albert de la Chapelle, The Ohio State University, Columbus, OH, December 24, 1998 (received for review October 30, 1998) ABSTRACT The Chondrichthyes (cartilaginous fishes) The conclusions based on the teleostean and chondrich- are commonly accepted as being sister group to the other thyan rooting have been challenged in two recent molecular extant Gnathostomata (jawed vertebrates). To clarify gnatho- studies (8, 9) in which the gnathostome tree was rooted by stome relationships and to aid in resolving and dating the using non-gnathostome taxa. The first study indicated that the major piscine divergences, we have sequenced the complete lungfishes have a basal position in the piscine tree and that the mtDNA of the starry skate and have included it in phyloge- separation between extant bony fishes and amniotes preceded netic analysis along with three squalomorph chondrichthy- the divergence of the extant bony fishes. The second study ans—the common dogfish, the spiny dogfish, and the star refuted the commonly held belief that the Chondrichthyes are spotted dogfish—and a number of bony fishes and amniotes. basal to other gnathostomes. This analysis, with only the spiny The direction of evolution within the gnathostome tree was dogfish, Squalus acanthias, representing squalomorph chon- established by rooting it with the most closely related non- drichthyans, did not, however, resolve the relationship between gnathostome outgroup, the sea lamprey, as well as with some the coelacanth, the chondrichthyans, and the teleosts. To more distantly related taxa. The analyses placed the chon- examine this relationship in greater detail, we have, in the drichthyans in a terminal position in the piscine tree. These present study, broken up the chondrichthyan branch by in- findings, which also suggest that the origin of the amniote cluding mitochondrial genes of three other chondrichthyans, lineage is older than the age of the oldest extant bony fishes the common dogfish, Scyliorhinus canicula, (10), the star (the lungfishes), challenge the evolutionary direction of sev- spotted dogfish, Mustelus manazo, (11), and the starry skate, eral morphological characters that have been used in recon- Raja radiata, (present study). Thus, the chondrichthyans are structing gnathostome relationships. Applying as a calibra- represented by a total of three squalomorphs and one bato- tion point the age of the oldest lungfish fossils, 400 million morph. years, the molecular estimate placed the squalomorphy The divergence between squalomorphs (sharks) and bato- batomorph divergence at '190 million years before present. morphs (skates and rays) is paleontologically dated to the early This dating is consistent with the occurrence of the earliest Jurassic (6, 7, 12). Even though this is the minimum age for the batomorph (skates and rays) fossils in the paleontological squalomorphybatoid divergence, the inclusion of the skate, in record. The split between gnathostome fishes and the amniote addition to strengthening the phylogenetic analysis, makes it lineage was dated at '420 million years before present. possible to test a molecular estimate of the divergence time between the squalomorphs and the skate against the paleon- The relationship between gnathostomous fishes and their tological record of the Batomorphii. terrestrial relatives is of fundamental importance for the understanding of vertebrate evolution. Molecular analyses of MATERIALS AND METHODS this relationship have addressed in particular the question of whether, among extant fishes, the lungfishes or the coelacanth Enriched mtDNA was isolated from frozen liver of the starry are the sister group to terrestrial vertebrates. However, al- skate, Raja radiata, following described procedures (13). The though these analyses have differed with respect to the taxa specimen was collected in Faxafloi, Iceland, by Oskar Gud- included, a teleostean (1–4) or chondrichthyan (5) rooting of mundsson. The mtDNA was digested separately with BlnI and the gnathostome tree has been a common characteristic, and BclI. Digested DNA fragments were separated on an agarose these studies have, in general, supported a sister group rela- gel and were excised, electroeluted, and ligated. With the tionship between lungfishes and amniotes (or tetrapods). exception of parts of the NADH2 and NADH5 genes, which Because the application of rooting automatically gives evolu- were PCR-amplified and direct-sequenced, natural clones tionary direction to a tree, it is essential that rooting is covered the whole molecule. The mtDNA of the starry skate performed by using an outgroup that is unambiguously posi- has been deposited in the GenBank database with accession tioned without the ingroup taxa. The commonly applied number AF106038. Users of the sequence are kindly requested teleostean rooting of the vertebrate tree is incompatible with to refer to the present paper and not only to the accession piscine paleontology (6, 7) whereas the chondrichthyan rooting number of the sequence. is subjective in the sense that it assumes a priori that chon- The phylogenetic analyses included all published piscine drichthyans are the sister group of all other extant gnathos- mtDNAs together with a comprehensive selection of taxa tomes. Therefore, the application of either the teleostean or represented by complete mtDNAs, namely sea lamprey, Petro- chondrichthyan rooting is inconsistent with the criterion that myzon marinus (14); African lungfish, Protopterus dolloi (3); unequivocal outgroups should be used to establish the polarity of phylogenetic trees. Abbreviations: MYBP, million years before present; ML, maximum likelihood; MP, maximum parsimony; NJ, neighbor joining; NADH1-6, The publication costs of this article were defrayed in part by page charge subunits 1–6 of nicotinamide adenine dinucleotide dehydrogenase. Data deposition: The sequence reported in this paper has been payment. This article must therefore be hereby marked ‘‘advertisement’’ in deposited in the GenBank database (accession no. AF106038). accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. e-mail: ulfur. PNAS is available online at www.pnas.org. [email protected]. 2177 Downloaded by guest on October 2, 2021 2178 Evolution: Rasmussen and Arnason Proc. Natl. Acad. Sci. USA 96 (1999) bichir, Polypterus ornatipinnis (2); coelacanth, Latimeria cha- mitochondrial H-strand, excluding the L-strand encoded gene lumnae (4); starry skate, Raja radiata, (present study); spiny NADH6 (the composition of which deviates from that of the dogfish, Squalus acanthias (9); common dogfish, Scyliorhinus H-strand encoded genes). The different genes of each taxon canicula (10); star spotted dogfish, Mustelus manazo (11); were combined into one supergene because, as has been loach, Crossostoma lacustre (15); rainbow trout, Onchorhyn- demonstrated by statistical analysis (30), in this manner, the chus mykiss (16); carp, Cyprinus carpio; Atlantic cod, Gadus stochastic effects of limited sequence data are reduced. The morhua (17); alligator, Alligator mississippiensis (18); ostrich, analyses were performed primarily on a data set that, in Struthio camelus (19); chicken, Gallus gallus (20); wallaroo, addition to the gnathostomes, included the sea lamprey Petro- Macropus robustus (21); and cow, Bos taurus (22). Analyses myzon marinus (14), but rooting of the gnathostome tree also also were carried out by using the same taxa with an addition was tested separately with the hagfish Myxine glutinosa (8), the of different outgroup sequences: the hagfish, Myxine glutinosa lancelet Branchiostoma floridae (23), and three echinoderms— (8), the lancelet, Branchiostoma floridae (23), and three echi- one starfish and two sea urchins—Arbacia lixula (24), Strongy- noderm sequences, Arbacia lixula (24), Strongylocentrotus pur- locentrotus purpuratus (25), and Asterina pectinifera, (26). puratus (25), and Asterina pectinifera (26). After exclusion of gaps and ambiguous sites adjacent to gaps, The phylogenetic analyses [maximum likelihood (ML) (27), the length of the lamprey alignment was 7,959 nt or 2,653 aa. neighbor joining (NJ) (28), and maximum parsimony (MP) The distance values of the amino acid data set are given in (29)] were performed on amino acid as well as nucleotide Table 1. Relative rate test between the gnathostomous fishes alignments of the concatenated sequences of 12 mitochondrial and the sea lamprey and between the gnathostomous fishes protein-coding genes. The nucleotide analyses were based on and the amniotes showed that the evolutionary rate of the the combined data sets of both first (excluding synonymous lungfish is 10–15% higher than that of other gnathostomous leucine transitions) and second codon positions. fishes. This difference in evolutionary rate was compensated for in the subsequent molecular estimates of evolutionary RESULTS AND DISCUSSION divergence times by using the age of lungfish fossils as a calibration point. The distance values in Table 1 are consistent General Features of the Mitochondrial Genome of the with