Phylogeny of All Major Groups of Cetaceans Based on DNA Sequences from Three Mitochondrial Genes

Phylogeny of All Major Groups of Cetaceans Based on DNA Sequences from Three Mitochondrial Genes Michel C. Milinkovitch,* Axe1 Meyer) and Jeffrey R. Powell * *Department of Biology, Yale University and +Department of Ecology and Evolution, State University of New York at Stony Brook Traditionally, living cetaceans (order Cetacea) are classified into two highly distinct suborders: the echolocating toothed whales, Odontoceti, and the filter-feeding baleen whales, Mysticeti. A molecular phylogeny based on 1,352 base pairs of two mitochondrial ribosomal gene segments and the mitochondrial cytochrome b gene for all major groups of cetaceans contradicts this long-accepted taxonomic subdivision. One group of toothed whales, the sperm whales, is more closely related to the morphologically highly divergent baleen whales than to other odontocetes. This finding suggests that the suborder Odontoceti constitutes an unnatural grouping and challenges the conventional scenario of a long, independent evolutionary history of odontocetes and mysticetes. The superfamily Delphinoidea (dolphins, porpoises, and white whales) appears to be monophyletic; the Amazon River dolphin, Inia geofiensis, is its sister species. This river dolphin is genetically more divergent from the morphologically similar marine dolphins than the sperm whales are from the morphologically dissimilar baleen whales. The phylogenetic relationships among the three families of Delphinoidea remain uncertain, and we suggest that the two cladogenetic events that generated these three clades occurred within a very short period of time. Among the baleen whales, the bowhead is basal, and the gray whale is the sister species to the rorquals (family Balaenopteridae). The phylogenetic position of beaked whales (Ziphioidea) remains weakly supported by molecular data. Based on molecular clock assumptions, the mitochondrial-DNA data suggest a more recent origin of baleen whales (-25 mya) than has been previously assumed (>40 mya). This revised phylogeny has important implications for the rate and mode of evolution of morphological and physiological innovations in cetaceans. Introduction Whales are among the most specialized of all mam- 1993; Milinkovitch et al. 1993), and the phylogenetic mals and include the largest animals that ever lived. The distinctness of the extinct suborder Archaeoceti is prob- movement of the ancestral cetaceans from the terrestrial lematic (Fordyce 1989; Wyss 1990). The fossil record of to an aquatic environment involved extensive remod- cetaceans is incomplete and has not provided unequiv- eling of the morphological, physiological, and behavioral ocal evidence on whether the archaeocetes gave rise to systems (Barnes and Mitchell 1978; Gingerich et al. one, both, or neither suborder of living whales (see, e.g., 1983; Barnes 1984a). The order Cetacea is generally Barnes and Mitchell 1978; Barnes 1984~; Barnes et al. considered to be a monophyletic group, although a sep- 1985; Fordyce 1992; McLeod et al. 1993). arate origin of the two morphologically highly divergent Phylogenetic studies of extinct and extant cetaceans suborders of living whales, the Odontoceti (toothed are complicated by their highly modified morphology. whales) and the Mysticeti (baleen whales), has been fa- However, a close phylogenetic relationship between ce- vored by others (e.g., Yablokov 1965). The origin of and taceans and ungulates was first suggested more than 100 evolutionary relationships among fossil and extant ce- yr ago (Flower 1883) and was more recently confirmed taceans are disputed (Barnes 19840; Barnes et al. 1985; by paleontological (Van Valen 1966; Szalay 1969; Gin- Heyning and Mead 1990; Fordyce 1992; McLeod et al. gerich et al. 1983, 1990; Thewissen and Hussain 1993) and molecular studies (Goodman et al. 1985; Miyamoto Key words: phylogenetics, sperm whales, baleen whales, cyto- and Goodman 1986; McKenna 1987; Czelusniak et al. chrome b, mitochondrial ribosomal DNA, my = million years, mya 1990; Irwin et al. 199 1; Milinkovitch 1992; Milinkovitch = million years ago. et al. 1993). Several independent approaches support a Address for correspondence and reprints: Michel C. Milinkovitch, sister-group relationship of cetaceans with artiodactyl Department of Genetics, Facultk de M6decine V&!xinaire, Sart Tilman ungulates. Accordingly, artiodactyls are more closely re- B-43, UniversitC,de L2ge, 4000 Li*ge, Belgium. lated to cetaceans than they are to perissodactyl ungulates Mol. Biol. Evol. 11(6):939-948. 1994. 0 1994 by The University of Chicago. All rights reserved. (see, e.g., Czelusniak et al. 1990; Gingerich et al. 1990; 0737-4038/94/I iO6-0012$02.00 Irwin et al. 199 1; Milinkovitch et al. 1993). 939 Y4u Mmsovitcn et al. Extant cetaceans are assigned to the suborder Mys- taxa (Milinkovitch et al. 1993). Here, we present a larger ticeti (baleen whales), which is comprised of 11 species, data set that includes, for the same genes, five additional or to the suborder Odontoceti (toothed whales), with key species of cetaceans and three artiodactyl species as about 67 species. These two groups are generally con- outgroups. These eight species have been chosen to break sidered to have diverged from the extinct suborder Ar- up long branches on the tree and hence to facilitate chaeoceti more than 40-45 mya (see, e.g., Barnes et al. proper polarization of the characters. In addition, we 1985; Fordyce 1992; McLeod et al. 1993). However, the determined the DNA sequences of portions of the cy- paleontological evidence for this date is equivocal since tochrome b gene (424 base pairs including 22 base pairs the assignment of key fossils to a particular whale lineage of the adjacent tRNA-Glu) for the same 21 cetacean is often uncertain (see, e.g., Marples 1956; Keyes 1973; species and three outgroups. Fordyce 1989). One of the earliest “modern” whale fos- Material and Methods sils (ZMT62) is from the early Oligocene (probably 35 We determined DNA sequences from the species my old) and has been tentatively interpreted as a prim- listed in the Appendix. Tissue samples were obtained itive mysticete (Fordyce 1989). However, the specimen from delphinaria or from stranded or by-catch animals. is very incomplete (a piece of mandible and three teeth), DNA was extracted from blood, skin, spleen, or liver and, consequently, we regard it as dubious and of un- tissue from frozen or DMSO-preserved specimens. The certain subordinal status (contra Novacek 1993). Fossils primers used for PCR amplification and direct sequenc- from lineages of the living whales are more recent ing (Kocher et al. 1989) of part of the 12s gene were (Barnes et al. 1985). The oldest known fossils of extant modified L1091 and H1478 (Kocher et al. 1989) pri- families, from the Oligocene-Miocene boundary (about mers. Primers for the 16s gene were 16sar-L and 16sbr-H 23 mya), are sperm whales and right whales from the (Palumbi et al. 199 I), and for cytochrome b were same deposit (Barnes 1984b). modified L 14724 (Paabo 1990) (5’-TGACATGAA- The classification of cetaceans into odontocetes and AAAYCAYCGTTG) and H 15 149 (Kocher et al. 1989) mysticetes is based on morphological differences that (5’-CCCTCAGAATGATATYTGTCCTCA). Details of include the presence of teeth and baleen, respectively. the protocol have been reported previously (Kocher et Some extinct mysticete whales, however, were toothed al. 1989; Palumbi et al. 199 1). The DNA sequences were and did not possess baleen. In addition, some living determined with an automatic sequencer (Applied Bio- odontocete whales lack teeth (e.g., the females in ziphiids systems 373A) following the manufacturer’s protocols. and narwhal), while baleen whale embryos exhibit ves- In all cases, both strands were sequenced. tigial teeth. Clearly, teeth are an ancestral characteristic For each DNA segment, sequences from all taxa for all whales and therefore are not phylogenetically in- were aligned using the multiple-alignment program formative for subordinal classification. The lack of de- MALIGN (Wheeler and Gladstein 1993). Indels (inser- finitive morphological characters and the paucity and tions and deletions) were not detected in cytochrome b, usually poor quality of key cetacean fossils make the and large differences in parameter settings did not sig- assignment of these specimens to the mysticete, odon- nificantly influence the alignment of the ribosomal gene tocete or archaeocete lineages challenging (see, e.g., For- sequences. Data were analyzed by neighbor-joining (NJ) dyce 1989). (Saitou and Nei 1987), maximum parsimony (MP) Character-based phylogenies including most major (Swofford 1993), and maximum-likelihood (ML) (Fel- lineages of living cetaceans are rare (Milinkovitch et al. senstein 198 1) methods, and the robustness of the phy- 1993). Here, we present an extensive molecular-phylo- logenetic hypotheses was tested by bootstrapping (Fel- genetic hypothesis for all major groups of cetaceans, in- senstein 1985). Previous phylogenetic studies including cluding the “boto” or Amazon river. dolphin (Inia geof- more distantly related orders of mammals have dem- frensis), the largest of the platanistoid dolphins. The onstrated that artiodactyls are the sister group to ceta- analyses are based on three mitochondrial gene seg- ceans (Czelusniak et al. 1990; Irwin et al. 199 1; Milin- ments, the efficacy of which has been well documented kovitch et al. 1993); we therefore used three very for molecular-phylogenetic reconstruction in mammals divergent artiodactyl species as outgroups to

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