Comparative Genomics of Drosophila mtDNA: Novel Features of Conservation and Change Across Functional Domains and Lineages

  • Kristi L. MontoothEmail author
  • Dawn N. Abt
  • Jeffrey W. Hofmann
  • David M. Rand


To gain insight on mitochondrial DNA (mtDNA) evolution, we assembled and analyzed the mitochondrial genomes of Drosophila erecta, D. ananassae, D. persimilis, D. willistoni, D. mojavensis, D. virilis and D. grimshawi together with the sequenced mtDNAs of the melanogaster subgroup. Genomic comparisons across the well-defined Drosophila phylogeny impart power for detecting conserved mtDNA regions that maintain metabolic function and regions that evolve uniquely on lineages. Evolutionary rate varies across intergenic regions of the mtDNA. Rapidly evolving intergenic regions harbor the majority of mitochondrial indel divergence. In contrast, patterns of nearly perfect conservation within intergenic regions reveal a refined set of nucleotides underlying the binding of transcription termination factors. Sequencing of 5′ cDNA ends indicates that cytochrome C oxidase I (CoI) has a novel (T/C)CG start codon and that perfectly conserved regions upstream of two NADH dehydrogenase (ND) genes are transcribed and likely extend these protein sequences. Substitutions at synonymous sites in the Drosophila mitochondrial proteomes reflect a mutation process that is biased toward A and T nucleotides and differs between mtDNA strands. Differences in codon usage bias across genes reveal that weak selection at silent sites may offset the mutation bias. The mutation-selection balance at synonymous sites has also diverged between the Drosophila and Sophophora lineages. Rates of evolution are highly heterogeneous across the mitochondrial proteome, with ND accumulating many more amino acid substitutions than CO. These oxidative phosphorylation complex-specific rates of evolution vary across lineages and may reflect physiological and ecological change across the Drosophila phylogeny.


Comparative genomics Codon bias Drosophila mtDNA Oxidative phosphorylation tRNA evolution 



The authors gratefully acknowledge constructive discussion with the fly genomics community at large, as well as specific input from Brian Bettencourt, Rob Haney, Rob Kulathinal and Colin Meiklejohn and helpful comments from anonymous reviewers. The shotgun sequence data reported in this manuscript were generated by the Drosophila 12 Genomes Consortium. This work was supported by National Institutes of Health grants GM067862 to DMR and GM076812 to KLM and a Summer Research Fellowship to DMR in the Bay Paul Center for Comparative Molecular Biology and Evolution at the Marine Biological Laboratories.

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  1. Akashi H (1997) Codon bias evolution in Drosophila. Population genetics of mutation-selection drift. Gene 205:269–278PubMedCrossRefGoogle Scholar
  2. Akashi H (2001) Gene expression and molecular evolution. Curr Opin Genet Dev 11:660–666PubMedCrossRefGoogle Scholar
  3. Altschul SF, Gish W, Miller W, Meyers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410PubMedGoogle Scholar
  4. Bachtrog D, Thornton K, Clark A, Andolfatto P (2006) Extensive introgression of mitochondrial DNA relative to nuclear genes in the Drosophila yakuba species group. Evolution 60:292–302PubMedGoogle Scholar
  5. Ballard JW (2000) Comparative genomics of mitochondrial DNA in members of the Drosophila melanogaster subgroup. J Mol Evol 51:48–63PubMedGoogle Scholar
  6. Ballard JW, Kreitman M (1994) Unraveling selection in the mitochondrial genome of Drosophila. Genetics 138:757–772PubMedGoogle Scholar
  7. Ballard JW, Melvins RG, Katewa SD, Maas K (2007) Mitochondrial DNA variation is associated with measurable differences in life-history traits and mitochondrial metabolism in Drosophila simulans. Evolution 61:1735–1747PubMedCrossRefGoogle Scholar
  8. Bazin E, Glemin S, Galtier N (2006) Population size does not influence mitochondrial genetic diversity in animals. Science 312:570–572PubMedCrossRefGoogle Scholar
  9. Beard CB, Hamm DM, Collins FH (1993) The mitochondrial genome of the mosquito Anopheles gambiae: DNA sequence, genome organization, and comparisons with mitochondrial sequences of other insects. Insect Mol Biol 2:103–124PubMedCrossRefGoogle Scholar
  10. Bergstrom CT, Pritchard J (1998) Germline bottlenecks and the evolutionary maintenance of mitochondrial genomes. Genetics 149:2135–2146PubMedGoogle Scholar
  11. Berthier F, Renaud M, Alziari S, Durand R (1986) RNA mapping on Drosophila mitochondrial DNA: precursors and template strands. Nucleic Acids Res 14:4519–4533PubMedCrossRefGoogle Scholar
  12. Birky CW, Walsh JB (1988) Effects of linkage on rates of molecular evolution. Proc Natl Acad Sci USA 85:6414–6418PubMedCrossRefGoogle Scholar
  13. Blier PU, Breton S, Desrosiers V, Lemieux H (2006) Functional conservatism in mitochondrial evolution: insight from hybridization of arctic and brook charrs. J Exp Zoolog B Mol Dev Evol 306:425–432PubMedCrossRefGoogle Scholar
  14. Boore JL (1999) Animal mitochondrial genomes. Nucleic Acids Res 27:1767–1780PubMedCrossRefGoogle Scholar
  15. Boore JL, Brown WM (1994) Complete DNA sequence of the mitochondrial genome of the black chiton, Katharina tunicata. Genetics 138:423–443PubMedGoogle Scholar
  16. Breton S, Burger G, Stewart DT, Blier PU (2006) Comparative analysis of gender-associated complete mitochondrial genomes in marine mussels (Mytilus spp.). Genetics 172:1107–1119PubMedCrossRefGoogle Scholar
  17. Brown WM, Prager EM, Wang A, Wilson AC (1982) Mitochondrial DNA sequences of primates: tempo and mode of evolution. J Mol Evol 18:225–239PubMedCrossRefGoogle Scholar
  18. Bulmer M (1991) The selection-mutation-drift theory of synonymous codon usage. Genetics 129:897–907PubMedGoogle Scholar
  19. Charlesworth B, Morgan MT, Charlesworth D (1993) The effect of deleterious mutations on neutral molecular variation. Genetics 134:1289–1303PubMedGoogle Scholar
  20. Clary DO, Wolstenholme DR (1985) The mitochondrial DNA molecule of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code. J Mol Evol 22:252–271PubMedCrossRefGoogle Scholar
  21. Crooks G, Hon G, Chandonia J, Brenner S (2004) WebLogo: a sequence logo generator. Genome Res 14:1188–1190PubMedCrossRefGoogle Scholar
  22. de Bruijn MH (1983) Drosophila melanogaster mitochondrial DNA, a novel organization and genetic code. Nature 304:234–241PubMedCrossRefGoogle Scholar
  23. Doiron S, Bernatchez L, Blier PU (2002) A comparative mitogenomic analysis of the potential adaptive value of Arctic charr mtDNA introgression in brook charr populations (Salvelinus fontinalis Mitchill). Mol Biol Evol 19:1902–1909PubMedGoogle Scholar
  24. Drosophila 12 Genomes Consortium (2007) Evolution of genes and genomes on the Drosophila phylogeny. Nature 450:203–218CrossRefGoogle Scholar
  25. Duret L (2000) tRNA gene number and codon usage in the C. elegans genome are co-adapted for optimal translation of highly expressed genes. Trends Genet 16:287–289PubMedCrossRefGoogle Scholar
  26. Edmands S, Burton RS (1999) Cytochrome C oxidase activity in interpopulation hybrids of a marine copepod: a test for nuclear-nuclear or nuclear-cytoplasmic coadaptation. Evolution 53:1972–1978CrossRefGoogle Scholar
  27. Ellison CK, Burton RS (2008a) Interpopulation hybrid breakdown maps to the mitochondrial genome. Evolution 62:631–638PubMedCrossRefGoogle Scholar
  28. Ellison CK, Burton RS (2008b) Genotype-dependent variation of mitochondrial transcriptional profiles in interpopulation hybrids. Proc Natl Acad Sci USA 105:15831–15836PubMedCrossRefGoogle Scholar
  29. Felsenstein J (1974) The evolutionary advantage of recombination. Genetics 78:737–756PubMedGoogle Scholar
  30. Friedrich M, Muqim N (2003) Sequence and phylogenetic analysis of the complete mitochondrial genome of the flour beetle Tribolium castanaeum. Mol Phylogenet Evol 26:502–512PubMedCrossRefGoogle Scholar
  31. Gabriel W, Lynch M, Burger R (1993) Muller’s ratchet and mutational meltdowns. Evolution 47:1744–1757CrossRefGoogle Scholar
  32. Garesse R (1988) Drosophila melanogaster mitochondrial DNA: gene organization and evolutionary considerations. Genetics 118:649–663PubMedGoogle Scholar
  33. Gillespie JH (2000) Genetic drift in an infinite population. The pseudohitchhiking model. Genetics 155:909–919PubMedGoogle Scholar
  34. Goldman N, Yang Z (1994) A codon-based model of nucleotide substitution for protein-coding DNA sequences. Mol Biol Evol 11:725–736PubMedGoogle Scholar
  35. Haag-Liautard C, Coffey N, Houle D, Lynch M, Charlesworth B, Keightley PD (2008) Direct estimation of the mitochondrial DNA mutation rate in Drosophila melanogaster. PLoS Biol 6:e204PubMedCrossRefGoogle Scholar
  36. Herbeck JT, Novembre J (2003) Codon usage patterns in cytochrome oxidase I across multiple insect orders. J Mol Evol 56:691–701PubMedCrossRefGoogle Scholar
  37. Hill WG, Robertson A (1966) The effect of linkage on limits to artificial selection. Genet Res 8:269–294PubMedCrossRefGoogle Scholar
  38. Hoffmann AA, Turelli M (1997) Cytoplasmic incompatibility in insects. In: O’Neill S, Hoffmann A, Werren J (eds) Influential passengers: inherited microorganisms and arthropod reproduction. Oxford University Press, New York, pp 42–80Google Scholar
  39. Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755PubMedCrossRefGoogle Scholar
  40. Hutter CM, Rand DM (1995) Competition between mitochondrial haplotypes in distinct nuclear genetic environments: Drosophila pseudoobscura vs. D. persimilis. Genetics 140:537–548PubMedGoogle Scholar
  41. Ikemura T (1985) Codon usage and tRNA content in unicellular and multicellular organisms. Mol Biol Evol 2:13–34PubMedGoogle Scholar
  42. Innan H, Stephan W (2001) Selection intensity against deleterious mutations in RNA secondary structures and rate of compensatory nucleotide substitutions. Genetics 159:389–399PubMedGoogle Scholar
  43. James AC, Ballard JW (2003) Mitochondrial genotype affects fitness in Drosophila simulans. Genetics 164:187–194PubMedGoogle Scholar
  44. Kern AD, Kondrashov FA (2004) Mechanisms and convergence of compensatory evolution in mammalian mitochondrial tRNAs. Nat Genet 36:1207–1212PubMedCrossRefGoogle Scholar
  45. Ko WY, David RM, Akashi H (2003) Molecular phylogeny of the Drosophila melanogaster species subgroup. J Mol Evol 57:562–573PubMedCrossRefGoogle Scholar
  46. Kopp A, True JR (2002) Phylogeny of the Oriental Drosophila melanogaster species group: a multilocus reconstruction. Syst Biol 51:786–805PubMedCrossRefGoogle Scholar
  47. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 5:150–163PubMedCrossRefGoogle Scholar
  48. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132PubMedCrossRefGoogle Scholar
  49. Lewis DL, Farr CL, Farquhar AL, Kaguni LS (1994) Sequence, organization, and evolution of the A+T region of Drosophila melanogaster mitochondrial DNA. Mol Biol Evol 11:523–538PubMedGoogle Scholar
  50. Lewis RL, Beckenbach AT, Mooers AØ (2005) The phylogeny of the subgroups within the melanogaster species group: likelihood tests on COI and COII sequences and a Bayesian estimate of phylogeny. Mol Phylogenet Evol 37:15–24PubMedCrossRefGoogle Scholar
  51. Li WH (1987) Models of nearly neutral mutations with particular implications for nonrandom usage of synonymous codons. J Mol Evol 24:337–345PubMedCrossRefGoogle Scholar
  52. Li WH (1993) Unbiased estimation of the rates of synonymous and nonsynonymous substitution. J Mol Evol 36:96–99PubMedCrossRefGoogle Scholar
  53. Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25:955–964PubMedCrossRefGoogle Scholar
  54. Lynch M (1996) Mutation accumulation in transfer RNAs: molecular evidence for Muller’s ratchet in mitochondrial genomes. Mol Biol Evol 13:209–220PubMedGoogle Scholar
  55. Lynch M, Koskella B, Schaack S (2006) Mutation pressure and the evolution of organelle genomic architecture. Science 311:1727–1730PubMedCrossRefGoogle Scholar
  56. Machado CA, Hey J (2003) The causes of phylogenetic conflict in a classic Drosophila species group. Proc Biol Sci 270:1193–1202PubMedCrossRefGoogle Scholar
  57. Maddison DR, Maddison WP (2000) MacClade 4: analysis of phylogeny and character evolution Version 4.0. Sinauer Associates, Sunderland, MAGoogle Scholar
  58. Martin AP (1995) Metabolic rate and directional nucleotide substitution in animal mitochondrial DNA. Mol Biol Evol 12:1124–1131PubMedGoogle Scholar
  59. Meiklejohn CD, Montooth KL, Rand DM (2007) Positive and negative selection on the mitochondrial genome. Trends Genet 23:259–263PubMedCrossRefGoogle Scholar
  60. Moriyama EN, Powell JR (1997) Synonymous substitution rates in Drosophila: mitochondrial versus nuclear genes. J Mol Evol 45:378–391PubMedCrossRefGoogle Scholar
  61. Muller H (1964) The relation of recombination to mutational advance. Mutat Res 1:2–9Google Scholar
  62. Musto H, Romero H, Zavala A, Jabbari K, Bernardi G (1999) Synonymous codon choices in the extremely GC-poor genome of Plasmodium falciparum: compositional constraints and translational selection. J Mol Evol 49:27–35PubMedCrossRefGoogle Scholar
  63. Nachman MW (1998) Deleterious mutations in animal mitochondrial DNA. Genetica 102–103:61–69PubMedCrossRefGoogle Scholar
  64. Neiman M, Taylor DR (2009) The causes of mutation accumulation in mitochondrial genomes. Proc Biol Sci 276:1201–1209PubMedCrossRefGoogle Scholar
  65. Nigro L, Solignac M, Sharp PM (1991) Mitochondrial DNA sequence divergence in the Melanogaster and oriental species subgroups of Drosophila. J Mol Evol 33:156–162PubMedCrossRefGoogle Scholar
  66. Ojala D, Montoya J, Attardi G (1981) tRNA punctuation model of RNA processing in human mitochondria. Nature 290:470–474PubMedCrossRefGoogle Scholar
  67. Pamilo P, Bianchi NO (1993) Evolution of the Zfx and Zfy genes: rates and interdependence between the genes. Mol Biol Evol 10:271–281PubMedGoogle Scholar
  68. Pamilo P, Nei M (1988) Relationships between gene trees and species trees. Mol Biol Evol 5:568–583PubMedGoogle Scholar
  69. Parsch J, Meiklejohn CD, Hauschteck-Jungen E, Hunziker P, Hartl DL (2001) Molecular evolution of the ocnus and janus genes in the Drosophila melanogaster species subgroup. Mol Biol Evol 18:801–811PubMedGoogle Scholar
  70. Pesole G, Gissi C, De Chirico A, Saccone C (1999) Nucleotide substitution rate of mammalian mitochondrial genomes. J Mol Evol 48:427–434PubMedCrossRefGoogle Scholar
  71. Pollard DA, Iyer VN, Moses AM, Eisen MB (2006) Widespread discordance of gene trees with species tree in Drosophila: evidence for incomplete lineage sorting. PLoS Genet 2:e173PubMedCrossRefGoogle Scholar
  72. Popadin K, Polishchuk LV, Mamirova L, Knorre D, Gunbin K (2007) Accumulation of slightly deleterious mutations in mitochondrial protein-coding genes of large versus small mammals. Proc Natl Acad Sci USA 104:13390–13395PubMedCrossRefGoogle Scholar
  73. Powell JR (1983) Interspecific cytoplasmic gene flow in the absence of nuclear gene flow: evidence from Drosophila. Proc Natl Acad Sci USA 80:492–495PubMedCrossRefGoogle Scholar
  74. Rand DM, Kann LM (1996) Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans. Mol Biol Evol 13:735–748Google Scholar
  75. Rand DM, Kann LM (1998) Mutation and selection at silent and replacement sites in the evolution of animal mitochondrial DNA. Genetica 102–103:393–407PubMedCrossRefGoogle Scholar
  76. Rand DM, Clark AG, Kann LM (2001) Sexually antagonistic cytonuclear fitness interactions in Drosophila melanogaster. Genetics 159:173–187PubMedGoogle Scholar
  77. Reed LK, Nyboer M, Markow TA (2007) Evolutionary relationships of Drosophila mojavensis geographic host races and their sister species Drosophila arizonae. Mol Ecol 16:1007–1022PubMedCrossRefGoogle Scholar
  78. Remsen J, O’Grady PO (2002) Phylogeny of Drosophilinae (Diptera: Drosophilidae), with comments on combined analysis and character support. Mol Phylogenet Evol 24:249–264PubMedCrossRefGoogle Scholar
  79. Reyes A, Gissi C, Pesole G, Saccone C (1998) Asymmetrical directional mutation pressure in the mitochondrial genome of mammals. Mol Biol Evol 15:957–966PubMedGoogle Scholar
  80. Richards S, Liu Y, Bettencourt BR, Hradecky P, Letovsky S, Nielsen R, Thornton K, Hubisz MJ, Chen R, Meisel RP et al (2005) Comparative genome sequencing of Drosophila pseudoobscura: chromosomal, gene, and cis-element evolution. Genome Res 15:1–18PubMedCrossRefGoogle Scholar
  81. Richly E, Leister D (2004) NUMTs in sequenced eukaryotic genomes. Mol Biol Evol 21:1081–1084PubMedCrossRefGoogle Scholar
  82. Roberti M, Polosa PL, Bruni F, Musicco C, Gadaleta MN, Cantatore P (2003) DmTTF, a novel mitochondrial transcription termination factor that recognises two sequences of Drosophila melanogaster mitochondrial DNA. Nucleic Acids Res 31:1597–1604PubMedCrossRefGoogle Scholar
  83. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574PubMedCrossRefGoogle Scholar
  84. Rozas J, Rozas R (1999) DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15:174–175PubMedCrossRefGoogle Scholar
  85. Saccone C, De Giorgi C, Gissi C, Pesole G, Reyes A (1999) Evolutionary genomics in Metazoa: the mitochondrial DNA as a model system. Gene 238:195–209PubMedCrossRefGoogle Scholar
  86. Saccone C, Gissi C, Lanave C, Larizza A, Pesole G, Reyes A (2000) Evolution of the mitochondrial genetic system: an overview. Gene 261:153–159PubMedCrossRefGoogle Scholar
  87. Sackton TB, Haney RA, Rand DM (2003) Cytonuclear coadaptation in Drosophila: disruption of cytochrome c oxidase activity in backcross genotypes. Evolution 57:2315–2325PubMedGoogle Scholar
  88. Schneider T, Stephens R (1990) Sequence logos: A new way to display consensus sequences. Nucleic Acids Res 18:6097–6100PubMedCrossRefGoogle Scholar
  89. Shoemaker DD, Dyer KA, Ahrens M, McAbee K, Jaenike J (2004) Decreased diversity but increased substitution rate in host mtDNA as a consequence of Wolbachia endosymbiont infection. Genetics 168:2049–2058PubMedCrossRefGoogle Scholar
  90. Tajima F (1993) Simple methods for testing molecular clock hypothesis. Genetics 135:599–607PubMedGoogle Scholar
  91. Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10:512–526PubMedGoogle Scholar
  92. Tavare S (1986) Some probabilistic and statistical problems on the analysis of DNA sequences. Lect Math Life Sci 17:57–86Google Scholar
  93. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882PubMedCrossRefGoogle Scholar
  94. Valverde JR, Marco R, Garesse R (1994) A conserved heptamer motif for ribosomal RNA transcription termination in animal mitochondria. Proc Natl Acad Sci USA 91:5368–5371PubMedCrossRefGoogle Scholar
  95. Wong A, Jensen JD, Pool JE, Aquadro CF (2007) Phylogenetic incongruence in the Drosophila melanogaster species group. Mol Phylogenet Evol 43:1138–1150PubMedCrossRefGoogle Scholar
  96. Xia X (2005) Mutation and selection on the anticodon of tRNA genes in vertebrate mitochondrial genomes. Gene 345:13–20PubMedCrossRefGoogle Scholar
  97. Yamauchi MM, Miya MU, Nishida M (2004) Use of a PCR-based approach for sequencing whole mitochondrial genomes of insects: two examples (cockroach and dragonfly) based on the method developed for decapod crustaceans. Insect Mol Biol 13:435–442PubMedCrossRefGoogle Scholar
  98. Yang Z (1997) PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci 13:555–556PubMedGoogle Scholar
  99. Yang Z, Nielsen R (1998) Synonymous and non-synonymous rate variation in nuclear genes of mammals. J Mol Evol 46:409–418PubMedCrossRefGoogle Scholar
  100. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Kristi L. Montooth
    • 1
    Email author
  • Dawn N. Abt
    • 2
  • Jeffrey W. Hofmann
    • 2
  • David M. Rand
    • 2
  1. 1.Department of BiologyIndiana UniversityBloomingtonUSA
  2. 2.Department of Ecology & Evolutionary BiologyBrown UniversityProvidenceUSA

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