Advance Publication by J-STAGE Genes & Genetic Systems Received for publication: April 2, 2017 Accepted for publication: February 5, 2018 Published online: April 10, 2018 1 Original paper 2 Signature of positive selection in mitochondrial DNA 3 in Cetartiodactyla 4 5 Satoko Mori1 and Masatoshi Matsunami1,2* 6 7 8 1Laboratory of Ecology and Genetics, Graduate School of Environmental Science, 9 Hokkaido University, N10W5, Kita-ku, Sapporo, Hokkaido 060-0810, Japan 10 2Graduate School of Medicine, University of the Ryukyus, 207, Nishihara-cho, 11 Okinawa 903-0215, Japan 12 13 *Corresponding author. 14 Masatoshi Matsunami 15 207, Nishihara-cho, Okinawa 903-0215, Japan 16 TEL/FAX +81-98-895-1766, Email: [email protected] 17 18 Running head: Positive selection in mtDNA of Cetartiodactyla 19 20 21 1 22 Abstract 23 Acceleration of the amino acid substitution rate is a good indicator of positive 24 selection in adaptive evolutionary changes of functional genes. Genomic information 25 about mammals has become readily available in recent years, as many researchers have 26 attempted to clarify the adaptive evolution of mammals by examining evolutionary rate 27 change based on multiple loci. The order Cetartiodactyla (Artiodactyla and Cetacea) is 28 one of the most diverse orders of mammals. Species in this order are found throughout 29 all continents and seas, except Antarctica, and they exhibit wide variation in 30 morphology and habitat. Here, we focused on the metabolism-related genes of 31 mitochondrial DNA (mtDNA) in species of the order Cetartiodactyla using 191 mtDNA 32 sequences available in databases. Based on comparisons of the dN/dS ratio (ω) in 12 33 protein-coding genes, ATP8 was shown to have a higher ω value (ω = 0.247) throughout 34 Cetartiodactyla than the other 11 genes (ω < 0.05). In a branch-site analysis of ATP8 35 sequences, a markedly higher ω value of 0.801 was observed in the ancestral lineage of 36 the clade of Cetacea, which is indicative of adaptive evolution. Through efforts to detect 37 positively selected amino acids, codon positions 52 and 54 of ATP8 were shown to have 38 experienced positive selective pressure during the course of evolution; multiple 39 substitutions have occurred at these sites throughout the cetacean lineage. At position 52, 40 glutamic acid was replaced with asparagine, and, at position 54, lysine was replaced 41 with non-charged amino acids. These sites are conserved in most Artiodactyla. These 42 results imply that the ancestor of cetaceans underwent accelerated amino acid changes 43 in ATP8 and replacements at codons 52 and 54, which adjusted metabolism to adapt to 44 the marine environment. 45 46 Key words: ATP8, Cetartiodactyla, dN/dS ratio (ω), mtDNA, positive selection 47 2 48 Introduction 49 Identification of genetic changes leading to morphological and physiological 50 adaptations is one of the central goals of evolutionary biology. An accelerated rate of 51 amino acid substitutions in a particular gene and during a particular evolutionary period 52 are evidence that the gene plays an important role for adaptive evolution in a given 53 species (Messier and Stewart, 1997). An accelerated rate of amino acid substitutions in 54 protein-coding genes can be assessed by the dN/dS ratio (ω), which is the ratio of the 55 nonsynonymous substitution rate (dN) and the synonymous substitution rate (dS) (Yang, 56 1998; Yang and Nielsen, 1998). The value is used as an indicator of selective pressure 57 acting on the protein-coding genes; values of < 1, = 1, and > 1 indicate purifying 58 selection, neutral evolution, and positive selection, respectively (Yang, 2007). Recent 59 studies in mammals have attempted to identify genes that contributed to adaptive 60 evolution in particular species by comparing the substitution rates in multiple loci. For 61 example, Chikina et al. (2016) detected accelerated substitution rates in genes involved 62 in sensory, structure, and metabolism in marine mammals, providing clues to better 63 understand their adaptive evolution to a particular environment. 64 One could assume that natural selection does not operate over an entire coding 65 region but rather at specific amino acid sites that are essential for changing the function 66 of a given gene. Therefore, care should be taken to identify specific amino acid 67 substitutions that affect functional changes in the genes (positively selected sites) in the 68 process of elucidating the genes involved in adaptive evolution. Most studies only 69 discuss acceleration of the substitution rate, while some studies have detected positive 70 selection and positively selected sites (Finch et al., 2014; Tian et al., 2016). 71 Mitochondrial genomes, which are circular molecules of 14,000-20,000 bp in 72 animals (Kolesnikov and Gerasimov, 2012), are now available in databases for many 3 73 organisms. Mitochondrial DNA (mtDNA) in mammals encodes 13 proteins that 74 constitute oxidative phosphorylation (OXPHOS) complexes: ND1-4, 4L, 5 and 6 of 75 complex I (NADH dehydrogenase); cytochrome b (Cytb) of complex III (bc1 complex); 76 COX1-3 of complex IV (cytochrome c oxidase); and ATP6 and ATP8 of complex V 77 (ATP synthase) (Wallace, 2007). Most cellular energy is produced through the 78 OXPHOS pathway, which takes place in the protein complexes embedded in the inner 79 mitochondrial membrane (Saraste, 1999). Mutations of mtDNA greatly affect the 80 metabolic activity of organisms, suggesting that mtDNA plays an important role in 81 morphological evolution and environmental adaptation. It is known that purifying 82 selection is one of the dominant forces of evolution on mitochondrial OXPHOS genes 83 (Tomasco and Lessa, 2011). On the other hand, evidence of adaptive evolution acting on 84 mtDNA has already been detected in previous reports. For instance, da Fonseca et al. 85 (2008) carried out comparative sequence analysis on protein-coding genes of 86 mitochondrial genomes across 41 mammal species and detected a number of 87 substitutions that alter the biochemical properties of functional sites of specific proteins. 88 Because mammals adapt to different habitats by changing their metabolic processes, an 89 accelerated substitution rate in the mtDNA might be tightly linked with the adaptive 90 evolution of some metabolic processes. 91 One of the most diverse orders of mammals, Cetartiodactyla, has 332 extant 92 species grouped into 132 genera (IUCN, Hassanin et al., 2012). This order includes 93 artiodactyls (ruminants, pigs, peccaries, hippos, camels, and llamas) and cetaceans 94 (whales, dolphins, and porpoises) which are found throughout all continents and seas, 95 except Antarctica. Within this order, there is large morphological variation and great 96 habitat diversity, as seen with cetaceans. As the ancestor of Cetacea originally lived on 97 land (Gatesy et al., 2013), an adaptation to water with a change in metabolism was 4 98 required to make the transition to aquatic living (Tomanek, 2014). Other examples are 99 found within tribes Lamini (belongs to family Camelidae) and Caprini (belongs to 100 family Bovidae); most species in these tribes live in high-altitude mountains and adapt 101 to low levels of oxygen, cold temperature, and scarce food supply. Such stresses may 102 promote specific directional evolution of mtDNA (Hassanin et al., 2009). Large 103 variation in body size is also widely observed in this order, which includes very small 104 species such as family Tragulidae (weighs less than 3 kg) (Rössner, 2007) and 105 large-bodied animals such as Giraffini (adult giraffes weigh in excess of 1000 kg) 106 (Brown et al., 2007) and whale (some species have a length greater than 10 m) 107 (Ridgway, 1997). In general, difference in body size is strongly related to metabolism 108 (Martin and Palumbit, 1993). Therefore, the order Cetartiodactyla is a good model for 109 inferring the molecular evolution of mtDNA that is associated with environmental 110 adaptation and morphological evolution, which are linked to metabolic processes. A few 111 previous studies reported adaptive evolution of mtDNA with a focus on specific taxa 112 belonging to Cetartiodactyla. Although Hassanin et al. (2009) showed that the ω of 113 ATPase increased during the evolution of Caprini, which live at high altitudes, they did 114 not demonstrate the statistical significance of this increase. In addition, Caballero et al. 115 (2015) identified that codon site 297 in the ND2 gene in the three “river dolphins” 116 (Families Pontopoeidae, Lipotidae, and Inidae) is under positive selection related to 117 adaptation to the freshwater environment. However, significant positive selection in 118 Cetartiodactyla has not been reported and comprehensive studies of this order are 119 lacking. 120 In this study, we used the dN/dS ratio (ω) to identify genes that show an 121 accelerated evolutionary rate and positive selection of mtDNA in Cetartiodactyla. Then, 122 the relationship between molecular evolution of mtDNA and environmental adaptation, 5 123 such as change of body size and habitat, was elucidated. Amino acid substitutions under 124 positive selection were identified to infer functional changes of genes that correlated 125 with adaptive evolution in specific lineages of Cetartiodactyla. 126 Materials and Methods 127 Phylogenetic analyses 128 The complete mitochondrial genomes from 210 species described in Hassanin 129 et al. (2009) covering most of the family Cetartiodactyla were retrieved from the NCBI 130 database (Supplementary Table S1). Additionally, we downloaded two mitochondrial 131 genomes, Orcinus orca and Equus caballus (Xiufeng and Arnason, 1994; Morin et al., 132 2010). From these sequences, we excluded 21 species for which gene annotations were 133 incomplete and portions of protein coding sequences were missing. We used 191 134 well-annotated mitochondrial genomes for further analysis. These whole sequences 135 were aligned using MAFFT with default settings (Katoh and Standley, 2013). A 136 phylogenetic tree was constructed using the maximum likelihood (ML) analysis 137 implemented in RAxML v8.2 (Stamatakis, 2014) with the GTR+CAT model.