J Mol Evol (1995) 40:260-272 jouRA,oMOLECULAR VOLUTION © Springer-VerlagNew York Inc. 1995 Mammalian Mitochondrial DNA Evolution: A Comparison of the Cytochrome b and Cytochrome c Oxidase II Genes Rodney L. Honeycutt,1 Michael A. Nedbal, 1 Ronald M. Adkins, 1'* Laura L. Janecek 1'2 i Department of Wildlife & Fisheries Sciences, Texas A&M University, 210 Nagle Hall, College Station, TX 77843, USA 2 Savannah River Ecology Laboratory, University of Georgia, Aiken, SC 29801, USA Received: 10 June 1994 / Accepted: 10 October 1994 Abstract. The evolution of two mitochondrial genes, Introduction cytochrome b and cytochrome c oxidase subunit II, was examined in several eutherian mammal orders, with spe- The mitochondrial cytochrome b (COB) and cytochrome cial emphasis on the orders Artiodactyla and Rodentia. c oxidase subunit II (COII) genes have been used in When analyzed using both maximum parsimony, with several recent molecular systematic studies of rodents either equal or unequal character weighting, and neigh- (DeWalt et al. 1993; Ma et al. 1993; Thomas and Martin bor joining, neither gene performed with a high degree of 1993), ungulates (Irwin et al. 1991; Irwin and Wilson consistency in terms of the phylogenetic hypotheses sup- 1992; Miyamoto et al. 1994), marine mammals (Irwin ported. The phylogenetic inconsistencies observed for and Arnason 1994), primates (Ruvolo et al. 1991; Diso- both these genes may be the result of several factors tell et al. 1992; Adkins and Honeycutt 1994), and euthe- including differences in the rate of nucleotide substitu- rian mammal orders (Adkins and Honeycutt 1991, 1993; tion among particular lineages (especially between or- Honeycutt and Adldns 1993). The COB gene, in partic- ders), base composition bias, transition/transversion bias, ular, has been used extensively in the investigation of differences in codon usage, and different constraints and systematic relationships among vertebrates, and the COII levels of homoplasy associated with first, second, and gene studies have focused primarily on primates and third codon positions. We discuss the implications of their presumed relatives. In this paper we examine pat- these findings for the molecular systematics of mam- terns of nucleotide sequence variation in the COB and mals, especially as they relate to recent hypotheses con- COII genes, with an emphasis on determining relation- ceming the polyphyly of the order Rodentia, relation- ships within and among mammalian orders. New nucle- ships among the Artiodactyla, and various interordinal otide sequence data for the COII gene are combined with relationships. existing information from both COB and COII, and the similarities and differences among resultant gene phy- Key words: Cytochrome b -- Cytochrome c oxidase logenies are evaluated. In addition, rates of nucleotide II -- Mammals -- Mitochondrial DNA -- Molecular substitutions, differences in codon usage, and base com- evolution position bias are examined for both genes, using repre- sentative taxa from the mammalian orders Rodentia and Artiodactyla. Materials and Methods * Present address: Museum of Zoology, University of Michigan, 1109 Geddes, Ann Arbor, MI 48109, USA Mitochondrial Genes Examined. A total of 35 COB genes and 37 COIl Correspondence to: R.L. Honeycutt genes were examined for six orders of eutherian mammals as well as 261 one metatherian order (Table 1). In the case of the new COII sequences Table 1. Specimens examined reported in this paper, mitochondrial DNA (mtDNA) was isolated using cesium chloride/propidium iodide gradient centrifugation (Brown Taxa COW COIP 1980), and the entire COII gene was amplified with primers H8320 and L7553 (see Adkins and Honeycutt 1994) by the polymerase chain Infraclass Eutheria reaction (PCR) using the parameters 95°C denaturation (1 rain), 45°C Order Artiodactyla annealing (i rain), and 72°C extension (1.25 rain) for 30 cycles. The Family Antilocapridae amplified COII genes were either sequenced directly using single- Antilocapra americana 10 13 stranded PCR products (Allard et al. 1991a) or the double-stranded Family Bovidae product was ligated into the pBluescript plasmid and then sequenced. In Bos gaurus 18 both cases sequencing followed that of Kraft et aL (1988). Because of Bos grunniens 18 the inherent error rate of Taq polymerase (Saild et al. 1988; Tindall and Bos indieus 18 Kunkel 1988; Keohavang and Thilly 1989), at least two clones were Bos javanicus 18 sequenced for each taxon. In two cases (Geomys, Perognathus) a single Bos taurus 3 3 sequence discrepancy was found. A third clone was sequenced in each Boselaphus tragocamelus 13 case, and the base present in two clones was assumed to be correct. Bubalus depressicornis 18 Capra hircus 10 13 Damaliscus dorcas 18 Data Analysis Gazella spekei 18 Syncerus c. caffer 18 Syneerus c. nanus 18 Phylogenetic analyses were performed by two methods--maximum Tragelaphus imberbis 18 parsimony as implemented by PAUP 3.1 (Swofford 1993) and neigh- Ovis aries 10 bor-joining (Saitou and Nei 1987) as implemented by the MEGA pro- Family Camelidae gram (Kumar et al. 1993). Maximum-parsimony analyses were con- Camelus dromedarius 10 ducted using both equal and unequal character weighting. Equal Family Cervidae weighting consisted of all substitutions regardless of codon position Cervus unicolor 13 being given equal weight. Unequal weighting schemes included the use Dama dama 10 of transversions only as well as a procedure whereby differential Odocoileus heminous 10 weights were assigned to each codon position (e.g., transversions only Odocoileus virginianus 18 at third position, all substitutions at second position, and all substitu- Family Giraffidae tions at first position with changes involving leucine at the first position Giraffa camelopardalis 10 recoded as Y, the generic symbol for a pyrimidine). Neighbor-joining Family Tragulidae analyses were conducted using pairwise distance estimates based on Tragulus napu 10 several models (Jukes and Cantor 1969; Kimura 1980; Tajima and Nei Family Suiidae 1984; Tamura and Nei 1993). In addition, gamma distances were es- Sus scrofa 10 18 timated using MEGA (Kumar et al. 1993) for all the above models Tayassu tajacu 10 except Tajima and Nei (1984). We chose to examine relationships Order Carnivora using these various distances in an effort to circumvent assumptions Phoca vitulina 5 5 specific to any one model (e.g., rate of nucleotide substitution assumed Order Cetacea to be the same for all sites, all nucleotide frequencies equal to 0.25, and Balaenoptera physalus 4 4 no transition bias). Order Lagomorpha Support for individual nodes on a phylogenetic reconstruction was Oryctolagus cuniculus 9 17 evaluated using both the bootstrap option (Felsenstein 1985) and the Order Primates Bremer support index (Bremer 1988), the number of extra steps re- Family Galagidae quired to break up a clade, Tests for rate heterogeneity among divergent Galago crassicaudatus 16 taxa were evaluated using a relative rate test (Mindell and Honeycutt Galago senegalensis 1 1990). Codon usage and base composition values were obtained by the Family Hominidae sequence analysis program MacVector 3.5 (International Biotechnolo- Homo sapiens 2 2 gies, Inc.) and MEGA (Kumar et al. 1993). Order Rodentia Suborder Sciurognathi Family Geomyidae Results and Discussion Cratogeomys c. castanops 7 18 Cratogeomys c. tamaulipensis 7 Cratogeomys fumosus 7 Interordinal Phylogenetic Comparisons Cratogeomys goldmani 7 Cratogeomys gymnurus 7 A total of 18 species of eutherian mammals were used in Cratogeomys merriami 7 the interordinal comparisons, with a marsupial outgroup Geomys bursarius 7 18 (either Didelphis or Monodelphis). These 18 species rep- Pappogeomys bulleri 7 resented six eutherian orders including (1) Primates Family Heteromyidae Perognathus flavus 18 (Homo and Galago); (2) Carnivora (Phoca); (3) Lago- Family Sciuridae morpha (Oryctolagus): (4) Cetacea (Balaenoptera); (5) Marmota flaviventris 15 Rodentia (Geomys, Cratogeomys, Sciurus, Cavia, Mus, Sciurus caroliensis 15 18 Rattus, and either Hystrix or Georychus); and (6) Artio- Spermophilus columbianus 15 dactyla (Bos, Capra, Odocoileus, Antilocapra, Sus, and Spermophilus lateralis 15 Spermophilus richardsoni 15 either Cervus or Dama). Spermophilus tridecemlineatus 15 262 Table 1. Continued (Fig. 1 and Table 2). Both genes also suggested a sister- group relationship between the order Carnivora and the Taxa COB a COIP Artiodactyla/Cetacea clade, which included Sus. These Family Muridae findings are congruent with several independent studies Acomys willsoni 18 including a detailed analysis by Graur and Higgins Apodemus sylvaticus 18 (1994) that examined both nuclear and mitochondrial Malacothrix typica 18 genes and an examination of several complete mitochon- Meriones shawi 18 Microtus pennsylvanicus 14 drial genomes of mammals (Arnason and Johnsson 1992; Mus domesticus 6 6 Honeycutt and Adkins 1993). Although the placement of Peromyscus banderanus 18 Carnivora is somewhat incongruent with morphological Rattus norvegicus 8 8 evidence, the association of the cetaceans as sister to Suborder Hystricognathi ruminant artiodactyls to the exclusion of nouruminants is Family Bathyergidae Georychus capensis 18 not contradicted by morphological evidence (Honeycutt Family Caviidae and Adkins 1993; Granr and Higgins 1994). Cavia apera 18 Third, recent morphological evidence (Novacek 1992; Cavia porcellus 12 Luckett and Hartenberger 1993) and at least one molec- Family Hystricidae ular study of mitochondrial