Proc. Natl. Acad. Sci. USA Vol. 87, pp. 6703-6707, September 1990 Evolution Molecular phylogeny of Rodentia, Lagomorpha, Primates, Artiodactyla, and Carnivora and molecular clocks (mammalian phylogeny/DNA sequence trees/branching dates) WEN-HSIUNG LI*t, MANOLO GOUY*t, PAUL M. SHARP§, COLM O'HUIGIN*, AND YAU-WEN YANG$ *Center for Demographic and Population Genetics, University of Texas, P.O. Box 20334, Houston, TX 77225; tLaboratoire de Biomdtrie, Universitd Lyon I, 69622 Villeurbanne Cedex, France; §Department of Genetics, Trinity College, Dublin 2, Ireland; and IDepartment of Cell Biology, Baylor College of Medicine, Houston, TX 77030 Communicated by Wyatt W. Anderson, May 14, 1990 (receivedfor review November 15, 1989) ABSTRACT Phylogenetic analysis ofDNA sequences from Lagomorpha was an outgroup to Primates and Rodentia in primates, rodents, lagomorphs, artiodactyls, carnivores, and Shoshani's (13) immunodiffusion comparisons. Finding the birds strongly suggests that the order Rodentia is an outgroup true evolutionary position of Lagomorpha has important to the other four mammalian orders and that Artiodactyla and implications not only for paleontology but also for molecular Carnivora belong to a superordinal clade. Further, there is evolution. Some authors (11, 14) have advocated the gener- strong evidence against the Glires concept, which unites Lago- ation-time effect because they found much higher rates of morpha and Rodentia. The radiation among Lagomorpha, nucleotide substitution in rodents than in humans. Under this Primates, and Artiodactyla-Carnivora is very bush-like, but hypothesis, substitution rates should also be high in rabbits there is some evidence that Lagomorpha has branched offfirst. because, like rodents, they have a short generation time. To Thus, the branching sequence for these five orders of mammals test this hypothesis, we need to determine the evolutionary seems to be Rodentia, Lagomorpha, Primates, Artiodactyla, position of Lagomorpha. and Carnivora. The branching date for Rodentia could be as early as 100 million years ago. The rate of nucleotide substi- DATA AND METHODS tution in the rodent lineage is shown to be at least 1.5 times The genes used (and their abbreviations) are apolipoprotein higher than those in the other four mammalian lineages. (Apo) Al, ApoB (3' part), ApoE, atrial natriuretic factor(ANF), A3/A1 /3-crystallin (BA3CRYST), Na',K+-ATPase a and (3 Despite the efforts of numerous comparative anatomists, subunits (ATPNKA and ATPNKB), brain and muscle creatine paleontologists, and molecular evolutionists (1-8), the kinases (CK-B and CK-M), glutathione peroxidase (GSHPX), branching order of the major eutherian lineages remains growth hormone (GH), hemoglobin a and ,3 (HBA and HBB), highly controversial. In fact, the prevailing view of eutherian interleukin la and and low evolution has always been a bush-like radiation (1, 2, 9). To insulin, 1,3 (ILlA IL1B), density untangle this phylogenetic knot, we have sequenced a num- lipoprotein receptor [LDLR (3' part)], luteinizing hormone ber of apolipoprotein (Apo) genes from several mammalian subunit P (LHB), lysozyme c (LYSOc), muscle nicotinic ace- orders and have compiled DNA sequences ofthese and other tylcholine receptor a subunit (MACHRA), c-myc oncogene genes from data banks and the literature. Our current focus (MYC), nerve growth factor (3 subunit [NGFB (3' part)], is on Primates, Rodentia, Lagomorpha, Artiodactyla, and parathyroid hormone (PTH), phospholipase A2 (PPLA2), pro- Carnivora because there are many more DNA sequence data lactin (PROLAC), protein kinase C subtypes a, (1II, and y from these five orders than from others. [PKINCA (internal part), PKINCB2, and PKINCG (internal Although Artiodactyla and Carnivora are commonly part); named according to ref. 15], proopiomelanocortin thought to have branched offprior to the primate-rodent split (POMC), protein phosphatase 2A catalytic subunit isotypes a (2, 5, 10), some authors (3, 4) maintain that Rodentia is an and ,( [PP2AA and PP2AB (3' part)], and transferrin (Transfer). outgroup to Artiodactyla, Carnivora, and Primates. The All sequence data are from GenBank Release 63 and the EMBL resolution of this issue has important consequences for data library as available by electronic mail, except for the molecular evolutionary studies. For example, using artiodac- following sequences: cow and dog ApoAl and ApoE (ref. 16; tyls and carnivores as outgroups to rodents and primates, Wu unpublished data), cow and mouse BA3CRYST (17), rat and Li (11) estimated that the rate of synonymous nucleotide PP2AA (18), and rabbit ApoE (19). substitution is -2 times higher in the rodent lineage than in We use only the coding regions of protein-coding genes the human lineage. This view was criticized by Easteal (12), because alignment for noncoding regions is difficult. Se- who argued that the rodents might be an outgroup to the other quence alignment was made at the amino acid level by a three orders. Should this be the case, the rate difference multiple alignment algorithm (20). Nucleotide alignment was between the rodent and human lineages would be reduced, then obtained according to the protein alignment. Regions although it is unlikely to be completely nullified. where alignment was uncertain were discarded. The evolutionary position of Lagomorpha has always been We use the method of Li et al. (21) to estimate the numbers controversial. Many paleontologists (4, 5) believe that Lago- of substitutions per synonymous site (Ks) and per nonsyn- morpha is closely related to Rodentia, whereas others (2, 10) onymous site (KA) between two genes. This method assumes think that it separated from Rodentia at the time of eutherian that both substitution rates are uniform over the coding radiation. Great uncertainties are also seen in molecular regions ofthe gene. This assumption may hold approximately studies (6, 7, 13). For example, Lagomorpha and Primates for the synonymous rate but is unlikely to hold well for the were put in one clade in the maximum parsimony analysis of nonsynonymous rate. Currently we are ignorant of how KA protein sequence data by Goodman et al. (6), whereas Abbreviations: Apo, apolipoprotein; MP method, maximum parsi- The publication costs of this article were defrayed in part by page charge mony method; ST method, method of Sattath and Tversky; Myr, payment. This article must therefore be hereby marked "advertisement" million years. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 6703 Downloaded by guest on September 27, 2021 6704 Evolution: Li et al. Proc. Natl. Acad. Sci. USA 87 (1990) varies among sites. Jin and Nei (22) have suggested the use also use the method of Sattath and Tversky (26) (the ST of the r distribution method), which is efficient (27) and is simple to use when only four taxa are involved. Let dij be the evolutionary distance f(A) = [pa/r(a)]e-AAa-1, [1] between taxa i and j. Then taxa 1 and 2 are neighbors (i.e., in one cluster), and so are taxa 3 and 4, if d12 + d34 is smaller where A is the substitution rate, f3 is a trivial scale parameter, than both d13 + d24 and d14 + d23. and a > 0 determines the shape and width ofthe distribution. The density f(A) is unbounded near the origin if a < 1, RESULTS decreases exponentially if a = 1, and becomes bell-shaped if a > 1. Following Jin and Nei (22) and using the two- Evidence Supporting Rodentia as an Outgroup to Primates, parameter method (23), we can show that the mean number Lagomorpha, Artiodactyla, and Carnivora. An important of substitutions per site is given by question concerning the evolutionary relationships among Rodentia, Primates, Lagomorpha, Artiodactyla, and Car- nivora is which order has branched off first? To address this Ka= [2(1-2P-Q 1/a + (1-2Q)-1/a-3], [2] question, we use a bird species (chicken) as an outgroup. To 4 maximize the use of sequence data, we consider only three of the five mammalian orders at one time. As no reliable estimate where P and Q are the proportions of transitional and of Ks can be obtained between a chicken and a mammalian transversional differences between the two sequences. If we gene, we use only KA in the following analysis. Also, in the MP use the one-parameter model, then analysis, we use only nonsynonymous changes. First, we infer the branching order of Rodentia, Primates, 3a and Artiodactyla, by using chicken as an outgroup. We use K - [(1 4p/3)-1/ - 1], [3] 4 14 genes with a total of4347 codons (see footnote to Table 1). In the ST method we consider three conditions for KA: the where p is the proportion of nucleotide differences between nonsynonymous rate is uniform among the coding regions of the two sequences. For simplicity, we have used Eq. 3. a gene, and the rate follows a r distribution with a = 1 or a In phylogenetic analysis we consider four taxa at one time = 0.5. Under each condition, we compute first the KA value because this can greatly increase the number of genes for for each gene and then the weighted average over genes using analysis and therefore can increase the power of a statistical the number ofcodons in a gene as the weight. The results are test. We use the maximum parsimony (MP) method (24); we shown in Table 1, group I. Fig. 1 a-c shows that under all infer either the minimum number of nonsynonymous substi- three conditions, Rodentia is an outgroup to Primates and tutions or the minimum number of all substitutions. In the Artiodactyla and that the internal branch is fairly long. former case we infer the minimum number of nonsynony- In the MP method, there are 265 informative nonsynony- mous changes between two codons among all possible evo- mous sites, of which 136 sites support Rodentia as an lutionary paths. The computer program is a minor modifica- outgroup, whereas only 76 and 53 sites support, respectively, tion ofthe PROTPARS program ofthe PHYLIP package (25).