Preliminary analysis of the mitochondrial genome evolutionary pattern in primates

2012·0.34 MB·

by  Zhao, Liang

ZoologicalResearch 33(E3−4):E47−E56 doi:10.3724/SP.J.1141.2012.E03-04E47
Preliminary analysis of the mitochondrial genome
evolutionary pattern in primates
LiangZHAO1,2,XingtaoZHANG1,XingkuiTAO1,WeiweiWANG1,MingLI2,*
1.FacultyofBiology,SuzhouUniversity,SuzhouAnhui 234000,China;
2.InstituteofZoology,theChineseAcademyofSciences,Beijing 100101,China
Abstract:Sincethebirthofmolecularevolutionaryanalysis,primateshavebeenacentralfocusofstudyandmitochondrialDNAis
well suited to these endeavors because of its unique features. Surprisingly, to date no comprehensive evaluation of the nucleotide
substitutionpatternshasbeenconductedonthemitochondrialgenomeofprimates.Here,weanalyzedtheevolutionarypatternsand
evaluatedselectionandrecombinationinthemitochondrialgenomesof44PrimatesspeciesdownloadedfromGenBank.Theresults
revealedthatastrongrateheterogeneityoccurredamongsitesandgenesinallcomparisons.Likewise,anobviousdeclineinprimate
nucleotidediversitywasnotedinthesubunitrRNAsandtRNAsascomparedtotheprotein-codinggenes.Within13protein-coding
genes, the pattern of nonsynonymous divergence was similar to that of overall nucleotide divergence, while synonymous changes
differedonlyforindividualgenes,indicatingthattherateheterogeneitymayresultfromtherateofchangeatnonsynonymoussites.
Codonusageanalysisrevealedthattherewasintermediatecodonusagebiasinprimateprotein-codinggenes,andsupportedtheidea
thatGCmutationpressuremightdeterminecodonusageandthatpositiveselectionisnotthedrivingforceforthecodonusagebias.
Neutralitytestsusingsite-specificpositiveselectionfromaBayesianframeworkindicatednositeswereunderpositiveselectionfor
any gene, consistent with near neutrality. Recombination tests based on the pairwise homoplasy test statistic supported complete
linkage even for much olderdivergentprimate species.Thus, with theexceptionof rate heterogeneityamong mitochondrial genes,
evaluating the validity assumed complete linkage and selective neutrality in primates prior to phylogenetic or phylogeographic
analysisseemsunnecessary.
Keywords:Mitochondrialgenome;Evolutionarypattern;Codonusagebias;Completelinkage;Evolutionneutrality;Primates
The mitochondrial genome of vertebrates exhibits molecule is adaptive. Nonetheless, of the numerous
several peculiar features including maternal inheritance, single mtDNA gene studies and several complete
the presence of single-copy orthologous genes, a lack of mitochondrial genome comparisons published over the
recombination, evolutionary neutrality, and a high past two decades, very few have been able to reject
mutationrate—characteristicsthatmakeitwellsuitedfor neutrality in favor of positive Darwinian selection
evolutionary studies (Pesole et al, 1999; Saccone et al, (García-Martínez et al, 1998; Rand et al, 1994). Indeed,
2000).Among all the characteristics and assumptions of previous studies on vertebrates indicated that different
the mitochondrial genome, the two most important levels of variability are attributable to1functional
aspects for explaining the evolutionary process are the constraints and/or slightly deleterious polymorphisms, a
presumed freedom from positive Darwinian (adaptive)
natural selection and lack of recombination.
Mitochondrial proteins are central to the cellular
Received:19January2012;Accepted:27June2012
oxidative phosphorylation pathway and are functionally
Foundationitems:ThisprojectwassupportedbytheNational Basic
conservedacrossmetazoanphyla(Grayetal, 1999).The
Research Program of China (973 Program: 2007CB411600), the
importance of these mtDNA protein products supports
NaturalScienceFoundationofChina(30630016;30570292)
the hypothesis that some variation in the mtDNA *Correspondingauthor,E-mail:[email protected]
SciencePress Volume33 IssuesE3−4
E48 ZHAO,etal.
pattern consistent with near neutrality (Nachman et al, 1 MaterialsandMethods
1996;Templeton,1996).
1.1 Sequences
Recombination breaks down the correlation in
54Mitogenomicsequencesfrom44primate species
genealogical history between different regions of a
were downloaded from GenBank (Table 1). All 54
genome, which may be the incorrect inference of
sequences were aligned, edited, and compared using the
evolutionary history (Schierup & Hein, 2000). The issue
Sequencher 4.5 (Gene Codes Corporation, Ann Arbor,
of whether recombination occurs in the human
MI). We excluded all gaps and ambiguous alignment
mitochondrial genome remains controversial as the
sites, resulting in 14,764 bp of sequence from the 13
necessary enzymes for recombination are in fact present
protein-coding, 2 rRNA, and 21 of the 22 tRNA genes.
in the mitochondria, and a few paternal mitochondria do
Protein-coding sequences from the L-strand-encoded
penetrate the egg during fertilization (Thyagarajan et al,
genes (ND6 and 8 tRNA genes) were converted into
1996),providingevidencethatsuggestsrecombinationis
complementary strand sequences. The tRNA-Glu gene
possible, at least in humans. Recent broad surveys of
and the Control Region were not included because the
animal mitochondrial genomes have also concluded that
alignments ofthosesequences aredifficultdueto a large
recombination is widespread (Piganeau et al, 2004;
numberofhighlyvariablesites,insertions,anddeletions.
Tsaousis et al, 2005). Another interesting issue about
1.2 Phylogeneticanalysesandsequencediversity
evolutionary patterns of the mitochondrial genome is the
The genealogical relationships between the
codon usage bias in its protein-coding genes. Many
mitochondrial genomes were analyzed by parsimony
studies have analyzed the differences in codon usage
usingPAUP*(Swofford,2002).Bootstrapping(Felsenstein,
betweendifferentorganismsandbetweenproteinswithin
1985) was used to test monophyly. For this study, 1,000
the same organism (Bulmer, 1991; Kanaya et al, 2001;
pseudosamples were generated to estimate the bootstrap
Sharp et al, 1995) and one of their main conclusions is
proportions.
that there are strong correlations between codon usage
Nucleotide diversity (π) was calculated for all 13
andgenomicGCcontent.Moreover,humancodonusage
protein-coding, 2 rRNA, and 21 concatenated tRNA
may be determined solely by GC content and its
genes. Measures of sequence diversity were also
isochores composition (Kanaya et al, 2001). Despite
calculated by the sliding window method using the
suggestive evidence and hypothesis, the relationships
DnaSP 4.10 (Rozas et al, 2003). A window (500 bp in
between codon usage and the underlying evolutionary
length)wasmovedalongthesequencesinstepsof50bp.
constraintsarestillnotfullyunderstood(Pratetal,2009).
Patterns of nucleotide diversity for all positions as well
Since the birth of molecular evolutionary analysis,
as patterns of synonymous and nonsynonymous
theevolutionaryrelationshipsofourownorder,primates,
nucleotidevariationwerecalculatedineachwindow,and
havebeenofcentralinterest.Strangely,nocomprehensive
the value was assigned to the nucleotide at the midpoint
and accurate evaluation for the nucleotide substitution of the window. The gamma parameter alpha (α), which
pattern(s) of the various mtDNA genes (i.e., tRNA and represents the extent of rate heterogeneity among sites,
rRNAgenes, synonymous andnonsynonymous positions was estimated from the individual data sets with TREE-
of protein-coding genes) have been conducted to date. PUZZLE5.2(Schmidtetal,2002)usingthe8-categories
Most primate studies demonstrated mtDNAevolutionary option.
neutrality and no recombination, and generally reported 1.3Codonusagebias
mtDNA still evolves about 5- to 10-fold more rapidly Several parameters related to codon usage bias,
than single-copy nuclear DNA (nDNA) (Brown et al, such as the codon bias index (Morton, 1993), the
1982)atanoverallnucleotidesubstitutionrateof1%per effective number of codons (Wright, 1990), and G +C
millionyears(Brownetal,1979;Wilsonetal,1985). content at second and third positions as well as overall
As large libraries of whole-mtDNA genome wereestimatedforeach primatemitochondrialprotein-
sequences from primates accumulate, we are able to coding genes using DnaSP 4.10 (Rozas et al, 2003).
conduct more sophisticated genome-wide analyses to Furthermore, to determine whether the compositional
obtain solid information on the pattern of whole-mtDNA changes of the nucleotide content in the primate
genomeevolutioninprimates whilealsodeterminingthe mitochondrial protein-coding genes are caused by
relative evolutionary rate of each mitochondrial directional mutation pressure or a result of positive
component. Here, we report on the rates and patterns of selection, we performed a correlation analysis. If the
mtDNA sequence variation and the codon usage in nucleotide bias affects both the synonymous and
primates and describe the results of tests conducted to nonsynonymous sites in protein-coding genes, positive
detectrecombinationandselection.Theseresultswillnot selection could not solely explain this phenomenon,
only be of interest in their own right but will also have because positive selection should not affect silent
implications for the use of mtDNA in evolutionary nucleotide positions. The correlation analysis of GC
studiesofprimates. content at the second codon position (nonsynonymous
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Preliminaryanalysisofthemitochondrialgenomeevolutionarypatterninprimates E49
Table1 PrimatetaxaandGenBankaccessionnumbersformitochondriagenomegenes.
GenBank GenBank
Family Taxon Family Taxon
AccessionNo. AccessionNo.
Galagidae Galagosenegalensis AB371092 Presbytismelalophos DQ355299
Otolemurcrassicaudatus AB371093 Pygathrixnemaeus DQ355302
Lorisidae Nycticebuscoucang AJ309867 Pygathrixroxellana DQ355300
Loristardigradus AB371094 Nasalislarvatus DQ355298
Perodicticuspotto AB371095 Chlorocebuspygerythrus1 EF597501
Daubentoniidae Daubentoniamadagascariensis1 AM905039 Chlorocebuspygerythrus2 EF597500
Daubentoniamadagascariensis2 AB371085 Cercopithecusaethiopssabaeus DQ069713
Indriidae Propithecusverreauxi AB286049 Papiohamadryas Y18001
Lemuridae Vareciavariegata AB371089 Theropithecusgelada FJ785426
Lemurcatta AJ421451 Macacasylvanus AJ309865
Eulemurmacaco AB371088 Macacathibetana EU294187
Eulemurmongoz AM905040 Macacamulatta AY612638
Eulemurfulvusmayottensis AB371087 Macacafascicularis FJ906803
Eulemurfulvusfulvus AB371086 Hylobatidae Hylobateslar X99256
Tarsiidae Tarsiussyrichta AB371090 Pongopygmaeus D38115
Tarsiusbancanus AF348159 Pongoabelii X97707
Cebidae Saguinusoedipus FJ785424 Gorillagorilla1 D38114
Cebusalbifrons AJ309866 Gorillagorilla2 X93347
Aotidae Aotuslemurinus FJ785421 Pantroglodytes1 EU095335
Atelidae Atelesbelzebuth FJ785422 Pantroglodytes2 D38113
Pitheciidae Callicebusdonacophilus FJ785423 Pantroglodytes3 X93335
Cebidae Saimirisciureus1 AB371091 Panpaniscus D38116
Saimirisciureus2 FJ785425 Homosapiens1 AM948965
Cercopithecidae Procolobusbadius DQ355301 Homosapiens2 X93334
Colobusguereza AY863427 Homosapiens3 D38112
Semnopithecusentellus DQ355297 Homosapiens4 AM711903
Trachypithecusobscurus AY863425 Homosapiens5 AF346992
mutations related, GC ) and GC content at third position also employed the pairwise homoplasy test (Φ ), a
2 W
(synonymous mutations related, GC ) with CG content relatively new and robust statistical estimator of
s3
of all protein codon genes (GCc) were implemented recombination (Bruen et al, 2006) that is a coalescent-
usingR2.6.2(Ihaka&Gentleman,1996). based estimator of the genealogical correlation or
1.4 Testsofneutrality compatibility amongsites,which is negativelycorrelated
To investigate protein evolution, we first calculated to recombination. The estimator was calculated, and its
d /d ratios among the set of 54 primate data and tested significance was estimated via a permutation test using
N S
their significance using Z-tests (Nei & Kumar, 2000) for Splitstree4.10(Huson&Bryant,2006).
each of the 13 protein-coding genes as implemented by
MEGA4.0 (Tamura et al, 2007). We also tested for site-
2 Results
specific positive selection with Bayesian posterior
probabilities under the Ny98 fitness regime (0<ω <1, 2.1 Phylogeneticanalysesandsequencediversity
1
ω =1, and ω >1) (Nielsen & Yang, 1998) as described The parsimony tree for primates, based on the
2 3
and implemented by MrBayes 3.1.2 (Huelsenbeck & mitochondrial genome, is shown in Figure 1. Each
Ronquist,2001;Ronquist&Huelsenbeck,2003). internalnodewassupportedbymorethan80%bootstrap
1.5 Testsofrecombination (data not shown). The mtDNApolymorphism data and
We calculated the Kelly (1997) measure of linkage estimates of nucleotide divergence are summarized in
disequilibrium (ZnS); ZnS being the average of R2(Hill Table 2, and the distributions of sequence diversity
& Robertson, 1968) over all pairwise comparisons. We acrossthemitochondrialgenomearepresentedinFigure
KunmingInstituteofZoology(CAS),China ZoologicalSociety Volume33 IssuesE3−4
E50 ZHAO,etal.
Figure1 Parsimonytreeof54primatesbasedonmitochondrialgenome(H~H wereselectednodesforrecombinationtests)
1 6
2.Anobviousdeclineinprimatenucleotidediversitywas acrosstheprimatemitochondrialgenome.
notedin the subunit rRNAs and tRNAs as compared to 2.2 Codonusage
theprotein-codinggenes.Amongthe13protein-coding Severalparametersrelatedtocodonusagebiaswere
genes,ATP8-ATP6andtheNADHdehydrogenase(ND) estimated to check whether synonymous mutations are
complex (with the exception of ND1) showed higher selectively neutral. The codon bias index (CBI) is a
rates of sequence diversity, while the cytochrome measure of the deviation from the equal use of
oxidase(COX)complexexhibitedlowerdiversitywithin synonymouscodonswithvaluesrangingfrom0(uniform
Primates.Notably,thenonsynonymousdivergenceplots useofsynonymouscodons)to1(maximumcodonbias).
weresimilartothoseobtainedfromthetotalnucleotide CBIvalueswereintermediateinourstudy,rangingfrom
divergence,butthetrendwasnotobservedinthesliding- 0.354to0.504(Table3).Theeffectivenumberofcodons
window plot of synonymous divergence.Although the (ENC), which may range from 20 (only one codon is
COX complex exhibited an obvious decline in total used for each amino acid; i.e., the codon bias is
nucleotidedivergenceandnonsynonymous divergence, maximum)to61(allsynonymouscodonsforeachamino
these genes had higher rates of synonymous diversity. acidareequallyused;i.e.,nocodonbias),werelikewise
Basedontheestimatedgammaparameteralpha(α),the intermediate in the primates mitochondrial protein
rate heterogeneity among sites was not even, but the condoning genes (Table 3). GC content at the second
pattern of heterogeneity among sites was distributed position(GC )rangedfrom38.0%to40.4%,GCcontent
2
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Preliminaryanalysisofthemitochondrialgenomeevolutionarypatterninprimates E51
Table2 Characteristicsforeachgenedatasetinthe54 Table3 Summaryofcodonusageindexof13concatenated
Mitogenomicsequencesfrom44primatespecies protein genes from 44 Primates species
Mitogenomicsequences
Genedataset N πtotal πsyn. πnonsyn. α
Proteingenes Organism ENC CBI GC2 GC3s GCc
ND1 954 0.22594 0.56062 0.10797 0.6400 Galagosenegalensis 44.147 0.406 0.393 0.390 0.412
ND2 1032 0.28488 0.55369 0.19488 0.6829 Otolemurcrassicaudatus 43.363 0.428 0.399 0.449 0.441
COX1 1533 0.19501 0.65372 0.04446 0.6678 Nycticebuscoucang 41.565 0.470 0.397 0.400 0.423
COX2 678 0.24309 0.62540 0.11977 0.6908 Loristardigradus 46.337 0.363 0.394 0.386 0.413
Perodicticuspotto 42.444 0.461 0.390 0.348 0.394
ATP8-ATP6 834 0.28099 0.59298 0.17451 0.8017
Daubentoniamadagascariensis1 44.075 0.399 0.383 0.363 0.402
COX3 783 0.21378 0.60615 0.08494 0.7019
Propithecusverreauxi 43.910 0.406 0.399 0.417 0.420
ND3 342 0.26373 0.58340 0.15246 0.9531
Vareciavariegata 44.723 0.389 0.389 0.327 0.388
ND4L 294 0.25770 0.61033 0.13563 0.6112
Lemurcatta 45.347 0.383 0.394 0.373 0.407
ND4 1359 0.25191 0.56225 0.14085 0.6478 Eulemurmacaco 44.925 0.383 0.393 0.381 0.410
ND5 1788 0.25525 0.56755 0.14766 0.7309 Eulemurmongoz 45.215 0.380 0.394 0.382 0.410
ND6 486 0.25715 0.50608 0.17487 0.6829 Eulemurfulvusmayottensis 45.045 0.396 0.391 0.331 0.388
Cytb 1134 0.22837 0.57817 0.11319 0.7729 Eulemurfulvusfulvus 43.987 0.406 0.390 0.346 0.392
13concatenated Tarsiussyrichta 45.321 0.395 0.392 0.348 0.399
11217 0.24287 0.58072 0.13166 0.7410
proteingenes
Tarsiusbancanus 45.541 0.364 0.388 0.338 0.393
RNAgenes
Saguinusoedipus 42.664 0.409 0.383 0.339 0.389
12srRNA 864 0.15497 0.6910
Cebusalbifrons 42.905 0.398 0.385 0.307 0.379
16srRNA 1424 0.16676 0.6700 Aotuslemurinus 44.815 0.367 0.385 0.334 0.390
21concatenatedtRNA
Atelesbelzebuth 44.227 0.385 0.385 0.325 0.388
genes(excluding 1259 0.13533 0.5689
lysinetRNA) Callicebusdonacophilus 44.608 0.367 0.387 0.331 0.391
Allgenes 14764 0.22121 0.7229 Saimirisciureus1 44.552 0.368 0.387 0.331 0.390
N:Numberofsites;πtotal:nucleotidediversityforallsites;πsyn.:nucleotide Procolobusbadius 45.041 0.420 0.401 0.458 0.441
diversityforsynonymoussites;πnonsyn.:nucleotidediversityfornonsynonymous Colobusguereza 44.081 0.427 0.397 0.447 0.434
sites;α:rateheterogeneityindex/Gammaparameter.
Semnopithecusentellus 44.262 0.415 0.403 0.428 0.429
Trachypithecusobscurus 46.006 0.394 0.382 0.328 0.388
at third codon position (GC ) ranged from 30.7% to
s3 Presbytismelalophos 46.065 0.357 0.389 0.379 0.407
50.4%, and overall CG content of the protein codon
Pygathrixnemaeus 46.553 0.373 0.387 0.321 0.384
genes (GCc) ranged from 37.4% to 46.5%. Correlation
Pygathrixroxellana 46.949 0.354 0.387 0.333 0.387
analysisindicatesthatthenucleotidebiasaffectsboththe
Nasalislarvatus 45.514 0.394 0.384 0.311 0.380
synonymous and nonsynonymous sites in protein-coding
Chlorocebuspygerythrus1 44.783 0.415 0.400 0.434 0.431
genes. Likewise, there is a highly significant correlation
between the GC3s and GCc (R2 =0.9932, P﹤0.001; Cercopithecusaethiopssabaeus 44.474 0.426 0.399 0.439 0.436
Figure3 A). The correlation between GC and GCc is Papiohamadryas 44.504 0.425 0.396 0.440 0.434
2
alsohighlysignificant(R2=0.7772,P﹤0.001;Figure3B). Theropithecusgelada 44.556 0.426 0.400 0.442 0.436
2.3 NeutralityTests Macacasylvanus 44.741 0.434 0.400 0.438 0.434
Asaconservativemeasureofselection,Z-testswere Macacathibetana 45.325 0.375 0.387 0.338 0.389
conducted for each protein-coding gene in all species. In
Macacamulatta 45.586 0.377 0.380 0.307 0.374
allcases,d <d washighlysignificant(P=0),consistent
N S Macacafascicularis 46.576 0.374 0.387 0.324 0.385
withstrongpurifyingselectionofmitochondrialgenesin
Hylobateslar 43.281 0.457 0.397 0.498 0.461
these comparisons. The d /d ratios differed among
N S Pongopygmaeus 43.111 0.436 0.397 0.455 0.442
genes(Figure4)andthesedifferencesmaypotentiallybe
Pongoabelii 43.123 0.435 0.398 0.454 0.443
attributable to positive selection or variation in levels of
functionalconstraintamonggenes.TheATP8,ND2,and Gorillagorilla1 43.177 0.435 0.398 0.454 0.442
ND6 genes were characterized by a dN/dS ratio well Pantroglodytes1 44.396 0.427 0.394 0.456 0.443
above the average, while the COX1 gene ratios were the Panpaniscus 41.489 0.504 0.404 0.504 0.465
lowestamongthespecies.Site-specificpositiveselection
Homosapiens1 42.622 0.457 0.397 0.474 0.451
inferred from Bayesian posterior probabilities indicated
ENC:Effectivenumberofcodons(Wright,1990);CBI:Codonbiasindex
that no sites were under positive selection for any gene
(Morton,1993);GC2:GCcontentatthesecondposition;GCs3:GCcontent
ofanyofthespecies.
atthirdcodonposition;GCc:overallCGcontent.
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E52 ZHAO,etal.
Figure3 CorrelationofGCcontentatthethirdcodonposition
(GC ),andGCcontentatthesecondposition(GC)
s3 2
withoverallCGcontent(GCc)
(A) Correlation of GCs3and GCc of coding sequences; (B)
CorrelationofGC2andGCcofcodingsequences.
test, but the Φ test statistic provided no evidence of
W
Figure2 Sequencediversityofthemitochondrialgenomeby recombination for each individual data set. Based on our
nucleotideposition data sets, the assumption of complete linkage of the
Diversitymeasurewascalculatedforaslidingwindowof500 mtDNA genome appears to be justified only between
bpwithastepsizeof50bp.(A)Patternsoftotal nucleotide morerecentlydivergentspecies.
diversity ( π total) for all positions; (B) patterns of
nonsynonymousnucleotidediversity(πnonsyn.)forconcatenated 3 Discussion
protein genes; (C) patterns of synonymous nucleotide diversity
(πsyn.)forconcatenatedproteingenes. Primate mitochondrial genomes exhibit striking
patterns of both polymorphism and substitution rate
2.4 TestsofRecombination heterogeneity among sites and genes, as evidenced by
The measures of linkage disequilibrium are sliding-window estimates of sequence diversity,
summarized in Table 3. The average R2 (Hill & nonsynonymous and synonymous divergence, and the
Robertson1968)acrossallpairwisecomparisons(ZnS) estimatedgammaparameteralpha(α)(Table2,Figure2).
foreachindividualdatasetandthepooleddataprovided Less primate nucleotide diversity was observed in the
evidence of recombination between relatively distant subunit rRNAs and tRNAs than that in the protein-
divergent species but not for more recent divergent coding genes.Among the 13 coding genes, rates were
species.The pairwise homoplasy test statistic (Φ )for higher in the ND genes and theATP8-ATP6 gene and
W
thepooleddataalsoprovidedsimilarresultstotheZnS- lower in the COX complex genes, consistent with the
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Preliminaryanalysisofthemitochondrialgenomeevolutionarypatterninprimates E53
Figure4 Ratiosofnonsynonymous/synonymousratepergeneinthemitochondrialgenomesof54primates.
Dottedlineindicatestheaverageratio.
Table4 Recombinationtestforprimatemitochondrialgenomes.
Internalsubclades(node)
Recombin-
Gene
ationtest H1 H2 H3 H4 H5 H6
ZnS 0.0405** 0.0458** 0.0588** 0.0770** 0.2884 0.7266
ND1
ΦW 1.2488 1.0927 0.8219 0.6476 0.1496 0.0659
ZnS 0.0489** 0.0566** 0.0699** 0.0810* 0.2874 0.7251
ND2
ΦW 1.4217 1.2213 0.9230 0.7143 0.1779 0.0579
ZaS 0.0424** 0.0487** 0.0611** 0.0784** 0.2823 0.7544
COX1
ΦW 1.6117 1.4236 1.1128 0.8265 0.1662 0.0345
ZnS 0.0795** 0.0874** 0.1147 0.0947* 0.2966 0.7880
COX2
ΦW 1.3825 1.1986 0.9053 0.8271 0.1300 0.0000
ZnS 0.0479** 0.0558** 0.0696** 0.0845** 0.3012 0.6755
ATP8-ATP6
ΦW 1.4795 1.2841 0.9917 0.8155 0.1424 0.0772
ZnS 0.0407** 0.0553** 0.0575** 0.0730** 0.2273 0.7828
COX3
ΦW 1.4295 1.2397 0.9946 0.7820 0.2722 0.0029
ZnS 0.0375** 0.0493** 0.0610** 0.0790* 0.2893 0.7436
ND3
ΦW 1.5716 1.3769 0.9752 0.7419 0.2004 0.0751
ZnS 0.0406** 0.0489** 0.0628** 0.0676** 0.2877 0.7621
ND4L
ΦW 1.3633 1.2171 0.9726 0.7554 0.1937 0.0811
ZnS 0.0445** 0.0513** 0.0579** 0.0787* 0.2811 0.7561
ND4
ΦW 1.4206 1.2099 0.9199 0.7240 0.1476 0.0101
ZnS 0.0401** 0.0458** 0.0608** 0.0770** 0.2812 0.7088
ND5
ΦW 1.3903 1.2037 0.9311 0.7230* 0.1596 0.0472
ZnS 0.0644* 0.0726* 0.0815* 0.0750** 0.2754 0.7926
ND6
ΦW 1.264 1.1078 0.9061 0.7237 0.1910 0.0163
ZnS 0.0381** 0.0430** 0.0545** 0.0683* 0.2680 0.7586
CYTB
ΦW 1.3633 1.2238 0.9792 0.7067 0.2383 0.0398*
ZnS 0.0442** 0.0504** 0.0640** 0.0770** 0.2835 0.7394
All-Pro.
ΦW 1.2291** 1.0311** 0.9620** 0.7483 0.1691 0.0411
*indicatesp<0.05;**indicatesp<0.01
results from various mammals (Kumazawa et al, 2004). mitochondrialgenomeevolutionattheintraspecificlevel
Mostnotably, thenonsynonymous divergenceplots were in humans (Elson et al, 2004) and both the inter- and
similartothoseobtainedfromthepooleddatanucleotide intraspecific levels in Drosophila lard (Ballard, 2000),
divergence, while the synonymous changes were only where the synonymous distribution was more
slightly different between individual genes of mtDNA homogeneous and the nonsynonymous distribution was
(Figure 2). Accordingly, rate heterogeneity may result more heterogeneous. Additionally, the results obtained
fromtherateofchangeatnonsynonymoussites. here differed from the inter- and intraspecific studies
The observed pattern of heterogeneity across the basedonwholegenomeinGadusmorhuaanditsrelative
mitochondrial genome was similar to the pattern of species (Marshall et al, 2009). Interestingly, all
KunmingInstituteofZoology(CAS),China ZoologicalSociety Volume33 IssuesE3−4
E54 ZHAO,etal.
mitochondrial genome comparisons mentioned above Figure 3A) and the correlation between GC and GCc is
2
showed significant rate heterogeneity and site specific also highly significant (R2 =0.7772, Figure 3B). Because
hyper-mutability. Possible explanations for rate positive selection should not affect silent nucleotide
heterogeneity in the mitochondrial genome include positions, our results support the idea that adaptive
selection pressure, variation in the mutation rate of each evolution is not the driving force for the codon usage
gene (involving base composition bias and properties of bias, indicating that GC mutation pressure may
the L-strand during mtDNA replication) (Marshall et al, determine codon usage in mitochondrial genomes of
2009), and the consequence of neutral mutational effects differentprimatespecies.
(Galtier et al, 2006). The detected evolutionary patterns Using a conservative measure of selection, Z-tests
ofthemitochondrialgenomewillinfluencethechoiceof indicated that d <d was highly significant (P = 0) in all
N S
mtDNA regions used for phylogeographic studies, since cases; this result is consistent with purifying selection of
the more variable regions of the genome harbor excess mitochondrial genes. A similar result was first found
homoplasy relative to the more slowly evolving regions duringanalysisofthemitochondrialgenomesofhumans,
(Galtier et al, 2006). Marshall et al (2009) proposed that chimpanzees, and gorillas (Hasegawa et al, 1998) and
the more variable regions of the mitochondrial genome also reported in additional studies on fruit flies, humans
harbor excess homoplasy relative to the more slowly and Atlantic cod (Ballard, 2000; Elson et al, 2004;
evolving regions, so choosing to examine more slowly Marshall et al, 2009). However, site-specific positive
evolvingregionsforevolutionaryanalysisamongspecies, selection from a Bayesian perspective indicates that no
andevenwithinspecies,withanincreaseinthesequence sites are under positive selection for any gene of any of
lengthmaybewiser.Basedontheprimatemitochondrial the species, indicating that most mutations are nearly
genome comparison in this study, we suggest that the neutral or slightly deleterious, a pattern consistent with
subunit rRNAs genes, tRNAs genes, and the COX the nearly neutral theory of molecular evolution
complex genes may be promising sites for future proposed by Ohta (1973). Our results therefore indicate
investigationsofprimatephylogeography. that the evolutionary pattern of primate mitochondrial
CBIs estimated in this studyrevealed thattherewas genomes and each single mitochondrial gene did not
intermediate codon usage bias in primate protein-coding severelydeviatefromevolutionaryneutrality.
genes (Table.3). Several evolutionary processes have A presumed absence of recombination is another
been postulated as the major factors that determine key feature of mtDNA that makes it a useful
codonusage.Foranumberofdifferentorganisms,itwas phylogeographic molecule (Marshall et al, 2009). The
suggested that codon usage was best explained by uniparental inheritance of mtDNA indicates that
selection for tRNA abundance, gene expression levels, recombination should be rare or absent, but a broad
and translational optimization (Duret, 2000). In other survey of 267 published animal mtDNA data sets by
cases, the codon usage was explained by mutation rate, Piganeau et al(2004) provided indirect evidence of
mutation preference (Powell & Moriyama, 1997), recombination in more than 14% of cases. Based on the
environmental conditions (Goodarzi et al, 2008), pairwise comparisons (ZnS) and the pairwise homoplasy
generationtime(Subramanian,2008),andrecombination test statistic (Φ ) for the 13 concatenated protein genes,
W
rates (Meunier & Duret, 2004). Although the the assumption of complete linkage of the mtDNA
relationships between codon usage and the underlying genome appears to be justified only between more
evolutionary constraints are still not fully understood, recently divergent species. However, no evidence of
twomajorparadigms ofcodon usagehavebeenfoundin recombination among the individual genes was detected
most species (Prat et al, 2009). Natural selection is with the pairwise homoplasy test statistic (Φ ) even
W
expected to yield a correlation with codon bias in more among the more divergent species, indicating that
highly expressed genes. Alternatively, codon bias could recombination is perhaps a result of substitution rate
be generated by strictly neutral processes, such as heterogeneity in the primate mtDNA genomes.
mutational biases, where some mutations occur more Additionally, as the lineage divergence increases, the
than others across the genome by local variations in the divergence of each individual gene may produce
base composition (Duret, 2002). Correlation analysis artificial significant ZnS values based on all pairwise
indicates that the nucleotide bias affects both the comparisons from the pooled data and may lead to false
synonymous and nonsynonymous sites in primate detection of recombination. Thus, it appears unnecessary
protein-coding genes. There is a highly significant to consider the effects of recombination on the evolution
correlation between the GC3s and GCc (R2 =0.9932, ofmtDNAinprimates.
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