The role of selection in the evolution of human mitochondrial genomes

Toomas Kivisild*,1,4,5, Peidong Shen†,4, Dennis Wall‡3, Bao Do†, Raphael Sung†, Karen

Davis†, Giuseppe Passarino§, Peter A. Underhill*, Curt Scharfe†, Antonio Torroni**,

Rosaria Scozzari††,David Modiano‡‡, Alfredo Coppa§§, Peter de Knijff***, Marcus

Feldman‡, Luca L. Cavalli-Sforza*, Peter J. Oefner†,2,4

*Department of Genetics, Stanford University School of Medicine, Stanford, California

94305, USA.

†Stanford Genome Technology Center, Palo Alto, California 94304, USA.

‡Department of Biological Sciences, Stanford University, Stanford, California 94305,

USA.

§Dipartimento di Biologia Cellulare, Università della Calabria, Rende, Italy.

**Dipartimento di Genetica e Microbiologia, Università di Pavia, Pavia, Italy.

††Dipartimento di Genetica e Biologia Molecolare, Università "La Sapienza", Rome,

Italy.

‡‡Dipartimento di Scienze di Sanità Pubblica, Sezione di Parassitologia, Università "La

Sapienza", Rome, Italy.

§§Dipartimento di Biologia Animale e dell'Uomo, Università "La Sapienza", Rome, Italy.

***Department of Human Genetics, Leiden University Medical Center, Leiden, The

Netherlands.

Present addresses:

Genetics: Published Articles Ahead of Print, published on September 19, 2005 as 10.1534/genetics.105.043901

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1Department of Evolutionary Biology, Tartu University and Estonian Biocenter, 51010

Tartu, Estonia.

2 Institute of Functional Genomics, University of Regensburg, Josef-Engert-Str. 9,

93053 Regensburg, Germany.

3 Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.

4Both authors contributed equally to the manuscript.

Complete coding region sequence information for 277 individual samples have been

submitted to GenBank under accession numbers DQ112686-DQ112962.

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Running title: Evolution of human mitochondrial genomes

Key words: mitochondrial DNA, human population genetics, natural selection, molecular

clock, evolution

5 Corresponding authors: TK, Estonian Biocenter, Tartu, Estonia, 51010, e-mail

tkivisil@ebc.ee, and P.J.O., Institute of Functional Genomics, University of Regensburg,

Josef-Engert-Str. 9, 93053 Regensburg, Germany, e-mail: oefner@genome.stanford.edu.

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ABSTRACT

High mutation rate in mammalian mitochondrial DNA generates a highly

divergent pool of alleles even within species that have dispersed and expanded in size

recently. Phylogenetic analysis of 277 human mitochondrial genomes revealed a

significant (p<0.01) excess of rRNA and non-synonymous base substitutions among

hotspots of recurrent mutation. Most hotspots involved transitions from guanine to

adenine that, together with thymine to cytosine transitions, illustrate the asymmetric bias

in codon usage at synonymous sites on the heavy-strand DNA. The mitochondrion-

encoded tRNAThr varied significantly more than any other tRNA gene. Threonine and

valine codons were involved in 259 of the 414 amino acid replacements observed. The

ratio of non-synonymous changes from and to threonine and valine differed significantly

(P=0.003) between populations with neutral (22/58) and those with significantly negative

Tajima’s D values (70/76), independent of their geographic location. In contrast to a

recent suggestion that the excess of non-silent mutations is characteristic to Arctic

populations implying their role in cold adaptation, we demonstrate that the surplus of

non-synonymous mutations is a general feature of the young branches of the phylogenetic

tree, affecting also those that are found only in Africa. We introduce a new calibration

method of the mutation rate of synonymous transitions to estimate the coalescent times of

mtDNA haplogroups.

INTRODUCTION

Mitochondrial DNA (mtDNA) encodes for 13 proteins, 2 ribosomal genes, and 22

tRNAs that are essential in the energy production of the human cell. Variation in its

5

sequence has provided significant insights into the maternal history of anatomically

modern humans (DENARO et al. 1981; GILES et al. 1980), complementing the paternal

legacy of the Y-chromosome (UNDERHILL et al. 2000). Studies based on restriction

fragment length polymorphism (RFLP) of the coding and direct sequencing of the non-

coding control region have formed the basis of a hierarchical classification of distinct

geographic and ethnic affinities (CHEN et al. 1995; FORSTER et al. 2001; MACAULAY et

al. 1999; TORRONI et al. 1996; TORRONI et al. 1993; WATSON et al. 1997). Studies

addressing sequence variation in the mtDNA coding region have suggested that natural

selection has significantly shaped the course of human mtDNA evolution (CANN et al.

1984; ELSON et al. 2004; INGMAN and GYLLENSTEN 2001; MISHMAR et al. 2003;

MOILANEN et al. 2003; MOILANEN and MAJAMAA 2003; NACHMAN et al. 1996; RUIZ-

PESINI et al. 2004). These studies have disagreed, however, upon whether the distribution

of specific human mtDNA clades or haplogroups is due to an adaptation to different

climates or if their distribution is a function of random genetic drift assisted by purifying

selection that eliminates non-synonymous changes. In an attempt to clarify this

disagreement and to study the mode of natural selection on mtDNA variation in human

populations, we provide here a phylogenetic analysis of a global sample of mtDNAs and

investigate the position, chemical nature and geographic distribution of recurrent and

frequent mutations in the coding region.

MATERIAL AND METHODS

DNA samples: The ascertainment set comprised 277 individuals from the five

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continents, including 129 African (10 Biaka Pygmy, 15 Mbuti Pygmy, 2 Lisongo, 6 San,

2 Mandenka, 4 Ethiopian Jew, 9 Sudanese, 1 Eritrean, 1 Ghanan, 3 Herero, 1 Ovambo, 1

Pedi, 1 Sotho, 2 Tswana, 2 Zulu, 10 Fulbe, 10 Mossi, 10 Rimaibe, 1 Berta, 1 Tuareg, 37

Dominican), 43 Asian (1 Arab, 1 Kazak, 1 Druze, 4 Bedouin, 1 Sepharadim, 1 Yemenite

Jew, 2 Pathan, 5 Sindhi, 2 Burushaski, 1 Baluchi, 1 Brahui, 2 Makran, 2 Hazara, 1 Tamil,

2 Cambodian, 1 Hmong, 1 Atayal, 1 Ami, 4 Han Chinese, 5 Japanese, 4 Korean), 76

European (5 Northern European, 12 Italian, 1 Greek, 2 Finn, 2 Ashkenazi, 1 Georgian, 17

Hungarian, 3 Icelander, 3 Czech, 1 Sardinian, 5 Basque, 1 Iberian, 23 Dutch), 13

Oceanian (4 New Guinean, 3 Melanesian, 6 Australian Aborigines), and 16 Native

American (1 Auca, 1 Guarani, 5 Brazilian Indian, 3 Colombian Indian, 2 Mayan, 1

Piman, 1 Muskogee, 1 Navaho, 1 Quechua) genomic DNA samples (Supplemental Table

1). A subset of 103 sequences of these has been reported elsewhere (SHEN et al. 2005).

DNA was extracted using QIAamp DNA Blood Kit (QIAGEN Inc., Valencia, CA).

Immortalized cell lines have been established for all individuals with the exception of the

17 Hungarians, the 23 Dutch, the 37 Dominicans, the 10 Mossi, the 10 Rimaibe, and the

10 Fulbe.

PCR and DNA sequencing: The 41 primer pairs used for bidirectional

sequencing of mtDNA nucleotides 435 to 16023, the PCR conditions and determined

complete coding region sequence information for 277 individual samples are available at

http://insertion.stanford.edu/primers_mitogenome.html. Amplicons were purified with

Qiagen (Valencia, CA) QIAquick spin columns and sequenced with the Applied

Biosystems (Foster City, CA) Dye Terminator Cycle Sequencing Kit and a model 3700

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DNA sequencer.

Phylogenetic and statistical analyses: An unrooted tree from a median-joining

network (BANDELT et al. 1999) was drawn and labeled following existing mtDNA

haplogroup nomenclature (KIVISILD et al. 2004; KIVISILD et al. 2002; KONG et al. 2003;

KONG et al. 2004; MACAULAY et al. 1999; SALAS et al. 2002; SHEN et al. 2005; TORRONI

et al. 1996; TORRONI et al. 2001; YAO et al. 2002). The tree was rooted using nuclear

inserts of mtDNA retrieved from human gnomic sequence and the consensus sequence of

the three chimpanzee mitochondrial genomes. The accession numbers, mtDNA positional

range and identity measures of the genomic contigs containing the inserts that were used

for rooting are as follows: NT_006713.14 (bps 341-2697; ID 94%); NT_009237.17 (bps

521-2976; ID 94%); NT_006316.15 (bps 2899-3050; ID 94%); NT_077913.3 (bps 3914-

9756; ID 98%); and NT_034772.5 (bps 10269-15487; ID 94%). The GenBank accession

numbers of the two Pan troglodytes and one Pan paniscus sequences that were used are

D38113, X93335, and D38116, respectively. Haplogroup divergence estimates ρ and

their error ranges were calculated as averages of the distances from the tips to the most

recent common ancestor of the haplogroup (FORSTER et al. 1996; SAILLARD et al. 2000).

Two separate measures of non-synonymous (N) to synonymous (S) substitution ratios

were used: first, the MN/MS ratio estimates the number of mutational changes inferred

from the phylogenetic tree (Figure 1), whereas the dN/(dS+constant) refers as in

(MISHMAR et al. 2003) to the ratio of average pair-wise distances of N and S changes in

the given sample. Statistical significance was determined from binomial or χ2

probabilities. Disease implicated substitutions were excluded from these analyses. For

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interspecies comparisons, mammalian mtDNA sequences were retrieved from the

Mitochondriome web site

(http://bighost.area.ba.cnr.it/mitochondriome/Mt_chordata.htm).

Tests for positive selection

Seven primate taxa, namely Homo sapiens, Pan troglodytes, Gorilla gorilla, Papio

hamadryas, Hylobates lar, Pongo pygmaeus, and Macaca sylvanus were chosen from

Genbank (gi|17981852, gi|5835121, gi|5835149, gi|5835638, gi|5835820, gi|5835163,

gi|14010693) and aligned using clustalW (THOMPSON et al. 1994), to test for the historic

occurrence of positive, directional selection on the 13 coding regions of the primate

mitochondrion using the program codeml of the PAML package (YANG 2002). In these

tests, maximum likelihood ratios of non-synonymous to synonymous mutations (ω,

omega) exceeding 1 are consistent with the hypothesis of positive selection, while values

close to 1 indicate selective neutrality, and values converging on 0 suggest strong

purifying selection. We conducted both lineage and site specific tests. For the lineage-

specific tests, we used a model in which all lineages have the same ω (hereafter referred

to as M0) and compared that with a model in which ω is estimated for each lineage

(hereafter referred to as M1). To test for the action of selection among amino acid sites

within a specific lineage, we compared a model that allows for heterogeneity in ω among

sites, but not among lineages, with a model that allows for variation in ω along a

predefined lineage (as in (YANG and NIELSEN 2002)). We assumed the following

unrooted phylogeny (troglodytes, ((((macaca, papio), hylobates), pongo), gorilla),

troglodytes), human). However, results of our analyses were robust to minor fluctuations

9

in the tree.

RESULTS

The deepest splits of the phylogeny constructed from 277 mtDNA complete

coding region sequences (Figure 1) were sustained by African mtDNAs which belonged

to previously defined haplogroups L0-L5 (KIVISILD et al. 2004; MISHMAR et al. 2003;

SALAS et al. 2002; SHEN et al. 2005; TORRONI et al. 2001). A number of new sub-clades

were identified among these that are illustrated in Figure 1. Haplogroup sharing between

distinct geographic regions was generally low. All European sequences could be assigned

to clades N1, X, W, HV, TJ, and U (FINNILÄ et al. 2001; HERRNSTADT et al. 2002;

MACAULAY et al. 1999; TORRONI et al. 1996). Asian, Amerindian, Oceanian, and

Australian Aborigine sequences belonged to region specific haplogroups nested within

macro-clades M and N (FRIEDLAENDER et al. 2005; KIVISILD et al. 2002; KONG et al.

2003; KONG et al. 2004; YAO et al. 2002). All Australian Aborigine M sequences (two

from this study and one from Ingman et al. (INGMAN et al. 2000) share six mutations that

define a new haplogroup M42. The majority of Australian N and R sequences (INGMAN

and GYLLENSTEN 2003) belong to clades S and P defined by transitions at np 8404 and

15607, respectively (FORSTER et al. 2001; FRIEDLAENDER et al. 2005).

The most parsimonious root of the mtDNA tree using nuclear inserts of mtDNA

and the chimpanzee consensus sequence as outgroups, appeared between haplogroup L0

and the rest of the phylogeny (Figure 1). Extensive interspecies homoplasy and

mutational saturation was highlighted by the fact that for more than one third (417/1292)

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of the variable sites, regardless of their phylogenetic position on the tree, the derived

allele among humans corresponded to the chimpanzee allele. In agreement with non-

coding region information (AQUADRO and GREENBERG 1983), a high ratio (21.5 on

average, 34.8 in synonymous positions) of transitions to transversions was observed in

the coding region (577-16023).

Interspecies calibration of the molecular clock over the complete mtDNA

sequence (INGMAN et al. 2000; MISHMAR et al. 2003) is problematic because of

saturation of transitions at silent positions and the effect of selection on the fixation rate

of amino acid replacement mutations (HO et al. 2005). Assuming 6 million years for the

human-chimp species split (GOODMAN et al. 1998) and 6.5 million years for the MRCA

of their mtDNA lineages (MISHMAR et al. 2003) we estimated the average transversion

rate at synonymous and rRNA positions as 2.1x10-9 and 4.1x10-10 per year per position,

respectively. Using the observed relative rates of different substitution types in humans

(Table 1) the average transition rate at 4,212 synonymous positions is 3.5 x10-8 (S.D. 0.1

x10-8) per year per position. Over all genes in mtDNA this would be equivalent to

accumulation of one synonymous transition per 6,764 (S.D. 140) years on average. The

coalescent date of the human mitochondrial DNA tree using this rate is 160,000 (S.D.

22,000) years. This coalescent date is broadly consistent with the dates of the Homo

sapiens fossils recognized so far from Ethiopia (CLARK et al. 2003; MCDOUGALL et al.

2005; WHITE et al. 2003). The most recent common ancestor of all the Eurasian,

American, Australian, Papua New Guinean and African lineages in clade L3 dates to

65,000 ± 8,000 years while the average coalescent time of the three basic non-African

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founding haplogroups M, N, and R is 45,000 years. These estimates, bracketing the time

period for the recent out-of-Africa migration (STRINGER and ANDREWS 1988), are

younger than those based on calibrations involving all coding region sites (INGMAN et al.

2000; MISHMAR et al. 2003) but are still in agreement with the earliest archaeological

signs of anatomically modern humans outside Africa (MELLARS 2004). The differences

with the date estimates of previous studies are most likely due to the overrepresentation

of possibly slightly deleterious non-synonymous mutations in the younger branches of the

tree (ELSON et al. 2004) that introduces a bias to the coalescent approach if all the sites of

the coding region are used.

Of the 1,788 mutations depicted in the tree, 1,758 occurred at 1,292 variable sites

in the coding region, between nps 577-16023. Consistent with previous reports (ELSON et

al. 2004; MISHMAR et al. 2003; MOILANEN and MAJAMAA 2003), there was a significant

excess of synonymous mutations in all genes coded by mtDNA, especially among those

positions that defined the deeper branches of the tree (Tables 2 and 3). In contrast to

Ruiz-Pesini et al. (RUIZ-PESINI et al. 2004), we did not observe any significant regional

(climatic) differences in the rate of non-synonymous changes for mtDNA haplogroups.

This discrepancy likely results from the fact that Ruiz-Pesini et al. compared region-

specific haplogroups of different diversity levels - e.g. the "old" paragroup L in Africans

versus "young" Arctic haplogroups (Table 4). Populations of Asian, European, and West

African origin showed significantly negative Tajima’s D values (Table 3), consistent with

either selection, population growth, and/or population subdivision (RAY et al. 2003). That

population substructure accounts at least for part of the deviation from neutrality is

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obvious from the observation that it decreases upon partitioning of West Africans into a

sample from Burkina Faso and one from the Dominican Republican.

Significant (p<0.05) mutational bias towards specific (NNG to NNA and NNU to

NNC) codon usage was observed in 27 out of 32 pairs of codons that differed by a

transition in the third codon position (Table 5). This relative preference of G to A and T

to C mutations (per existing nucleotide pool in the light strand) extends over the non-

silent positions and is characteristic of the non-coding D-loop region (AQUADRO and

GREENBERG 1983; MALYARCHUK and ROGOZIN 2004). The general strand bias, known,

however, to be reversed in some Metazoan genera, can be related to asymmetric

mutational constraints involving deaminations of A and C nucleotides during the

replication and/or transcription processes (HASSANIN et al. 2005). Importantly, the ND6

gene, encoded by the heavy strand showed the opposite mutational bias suggesting that

the differences of codon usage in human mtDNA might be primarily a function of strand

asymmetry rather than differences in the tRNA pools as generally expected (TANAKA and

OZAWA 1994). Notably, 16/18 nucleotide positions (Table 6) that had undergone five or

more recurrent changes involved the transition of guanine to adenine in the light strand.

Of the 1,292 variable sites, 288 (22.2%) had mutated recurrently. Unexpectedly,

the hotspots that had mutated ≥5 times were predominantly within mitochondrial rRNA

(p<5x10-15) and showed a significantly higher ratio of non-synonymous to silent

mutations (90:32 hits, respectively) than polymorphic sites with lower recurrence

(608:1004) (Table 6). Finally, these hotspots of mutational activity included positions

13

where the human derived allele predominates in the mammalian consensus sequences

(e.g. nps 709, 3010, 10398, and 13928), implying the effect of site-specific positive

selection. Among the six non-synonymous substitutions that have recurred ≥4 times, five

involved threonine (p<6.1x10-7). Overall, threonine and valine codons were involved in

259 of the 414 amino acid replacements observed on the tree.

Lineage-specific tests failed to detect significant positive selection along any

unique lineage in the 7 taxon phylogeny of primates. A model fixing a single ratio of ω to

all lineages (M0) could not be rejected in favor of a model of different ω's on specified

lineages (M1). The ω estimated across all lineages in the phylogeny was 0.35. A test of

the previous model against a model enforcing neutral selection, where ω is expected to be

equal to 1, showed that these data do not deviate significantly from neutrality (M0 was

rejected in favor of model where ω=1; p ≈ 0, df =1). Further tests for lineage specific

variation in ω, including a model that assigned a different omega to the human lineage

from the remaining primates, fit the data worse than M1. However, site specific model

testing revealed significant positive selection across regions of the primate

mitochondrion. A model enforcing a single ω ratio on all codon sites was rejected in

favor of a model allowing for 3 ratios across sites with 3 site classes (p ≈ 0, df = 5). The

3-ratio model identified 16 codon sites to be under significant (posterior probabilities >

0.95; dN/dS = 2.02) positive selection (Table 7). Among these, four codon sites appeared

to be among the non-synonymous sites with recurrent mutation (particularly #114 in ND3

gene, np 10398 with 7 recurrences) in human-human comparisons (Table 6).

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A majority of the mitochondrial disease related mutations have been detected in

the tRNA genes, and they mainly affect the secondary structure of the molecule

((MCFARLAND et al. 2004) and references therein). Our global survey of natural variation

in the tRNA genes showed that there was a seven-fold excess of tRNAThr mutations

(N=28) over other tRNA genes (p<10-19). This finding would suggest, at first glance, that

this gene might have become nonfunctional in mitochondrion and that its encoded tRNA

needs to be imported from the nucleus. Evidence suggesting nuclear tRNA import into

mitochondria in marsupials has been obtained previously (DÖRNER et al. 2001).

However, plotting the observed mutations in the tRNAThr gene against the mammalian

consensus sequences (HELM et al. 2000) showed that none of the mutations we have

observed in 277 humans fell within the 100% conserved regions of the tRNA (Figure 2).

Most pathological mutations affecting tRNAs cluster to highly conserved regions

(MCFARLAND et al. 2004), as illustrated in the case of tRNALeu in Figure 2. Four private

mutations changed the nucleotide that is more than 90% conserved in mammalian

tRNAThr, while most of the frequent and recurrent mutations in the data set affected the

minor fraction of the sites that are not highly conserved in mammalian species. This

argues against the proposition that human mitochondrial tRNAThr has lost function. A

large fraction (12/28) of the mutations affecting tRNAThr occurred at three positions, two

of which have a different allele in consensus human as compared to the 31 mammalian

species analyzed by Helm et al. (HELM et al. 2000). Similarly, a mutational hotspot at

position 5821 in tRNACys showed a majority allele in human different from that found in

consensus mammalian (Figure 2). Surprisingly, in the latter tRNA we observed two

parallel mutations at position 5814 that have been previously reported pathogenic. Yet,

15

because this position is not highly conserved in other mammalian species, its pathological

role has to be questioned. No other tRNA site that has been confirmed as associated with

mitochondrial disease was found to be variable in our data set. The only mutational

hotspot (12172) affecting the human allele that matches the allele conserved in >90% of

mammalian tRNA-s was found in tRNAHis.

A comparison of the amino acid substitutions in the mtDNA encoded proteins in

humans, primates, carnivores, and artiodactyls, revealed that substitutions between

threonine and alanine are significantly over-represented in humans while changes

between methionine and leucine are most common in other mammalian species (Table 8).

The direction of threonine and valine substitution with other amino acids was

significantly different between populations with neutral and significantly negative

Tajima’s D values, respectively (Table 3), and between haplogroups: in H1 sequences

sampled broadly from Europe and Near East, 7 of 11 non-synonymous mutations resulted

in the replacement of threonine and valine with alanine and isoleucine, while only three

mutations resulted in a change towards threonine or valine (Figure 1). In contrast to this

pattern, in haplogroup V sequences from Finland (FINNILÄ et al. 2001), where

populations continued to rely largely on hunting and fishing for subsistence even after the

first contacts with farmers, 6 of 7 replacement polymorphisms resulted in a change to

threonine and valine, and none in the replacement of the latter two amino acids (p<0.01).

Similarly, L3 sequences of West African origin showed significantly lower (p<0.001)

ratio of gains to losses of threonine and valine residues (13/14) than haplogroup L0

sequences from East and South Africa (22/2; Figure 1). East Asian sequences showed an

16

increase of valine codons (9 mutations to and 2 from valine codons), but also a significant

(p<0.01) decrease of threonine, with 11 mutations from and 5 to threonine codons, while

Native Americans had 1 mutation from and 7 to threonine, respectively. Over all

haplogroups and genes, the direction of amino acid change was significantly (p<0.02)

biased toward replacement of isoleucine and methionine to valine, even when considering

the transitional preferences observed in mitochondrial D-loop (TAMURA and NEI 1993).

The strand-specific mutational biases are unlikely to explain this pattern because of the

observed excess of mutations involving valine codons (8/16 in our data set and 13/16 in

the list of ND6 polymorphic sites in MITOMAP) in the ND6 gene that is encoded by the

opposite strand.

DISCUSSION

In phylogenetic analysis of human mitochondrial DNA coding region sequences,

two different spheres of character evolution can be distinguished (HO et al. 2005; PENNY

2005). Firstly, within our species, at the population level, relatively low level of parallel

mutations – as compared to the mtDNA control region based phylogenies – enables the

reconstruction of the unrooted tree from individual sequences without significant

ambiguity. This tree is determined by a substantial fraction of amino acid replacement

mutations whose proportion to synonymous substitutions increases from the average of

0.37 in “older” clades to 0.62 in the “younger” ones. In the second sphere, high level of

homoplasy with chimpanzee, affecting at least one third of the variable sites in humans,

complicates detailed phylogenetic analyses at the interspecies level. Approximately 930

synonymous mutations that can be observed between a human and a chimpanzee mtDNA

17

represent only the visible component of variation between the species while the effective

ratio of non-synonymous to silent mutations is expectedly significantly less than the

observed value of 0.2 due to the hidden load of synonymous mutations. These differences

between the two spheres imply that even among the substitutions that define the deepest

branches of the human mtDNA tree there is a significant excess of non-synonymous

mutations that have not yet been eliminated by purifying selection – assuming, of course,

that they are generally deleterious, after all.

More than half of the amino acid replacements observed in the human mtDNA

tree involved threonine and valine codons. Adaptive correlation with the elevated

mutability in the mitochondrion-encoded tRNAThr could be, in principle, considered as

one explanation for the excess of mutations involving threonine codons. However, none

of the highly conserved sites in the tRNAThr gene was found to be different in humans

from the consensus mammalian and, instead, the excessive variability in this gene could

be ascribed largely to the presence of three hotspot positions. Furthermore, no such

general molecular phenomenon or the characteristic G to A and T to C mutational bias on

the light strand of mtDNA would explain the pattern of differences of amino acid

replacement directions that were observed between human populations.

One factor that could, theoretically at least, explain the different amino acid

replacement patterns observed between populations and between humans and other

mammals is diet. Threonine and valine, essential amino acids that must be taken in the

diet, are abundant in meats, fish, peanuts, lentils, and cottage cheese, but deficient in most

18

grains. Alternatively, or in combination with dietary restriction, other constraints of

selection on slightly deleterious positions during the phases of population expansion and

contraction may be involved. Because of the specific compositional bias in mtDNA

induced by characteristic mutational preferences different from those observed in the

nuclear genome, additional inter- and intraspecies comparisons of mtDNA encoded

amino acid replacement patterns should be examined to gain deeper insights into the non-

synonymous character evolution in metazoan mitochondria, particularly including taxa

with shifted strand symmetry (HASSANIN et al. 2005).

Tests of neutrality based on the comparisons of the ratio of non-synonymous and

synonymous mutations across all sites can detect only major effects of purifying (KN/KS

approaches 0) or directional selection (KN/KS is significantly >1), which affect

simultaneously a large number of codon positions. Consistent with previous studies

(CANN et al. 1984; ELSON et al. 2004; INGMAN and GYLLENSTEN 2001; MISHMAR et al.

2003; MOILANEN et al. 2003; MOILANEN and MAJAMAA 2003; NACHMAN et al. 1996;

RUIZ-PESINI et al. 2004) human mtDNA encoded proteins did not provide evidence of

directional selection. However, several hotspots of mutational activity included non-silent

substitutions susceptible to site-specific positive selection. Comparing the mtDNA

protein encoding genes from several primates (Macaca, Papio, Hylobates, Pongo,

Gorilla, and Pan) with human, we discovered significant positive selection in several

regions, generally non-matching, however, with the codons displaying high KN/KS ratio

in human-human comparisons. This difference might be explained by the dynamic

polarity of the amino acid replacements at the intra- and interspecies levels whereby the

19

constraint of selection is determined in each lineage by the ancestral state of each codon

position.

In conclusion, we have provided new evidence for non-random processes

affecting the evolution of the human mtDNA-encoded proteins. The potential role of

selection in affecting fixation probabilities at different non-silent positions undermines

the appropriateness of using the average mitochondrial clock over all sites in dating

events in human population history. Despite the evidence of departures from neutrality

and high levels of homoplasy at the interspecies level, the phylogenetic approach for

analyzing mtDNA sequence data at the intraspecies level remains viable because the

reconstruction of the basic branches is robust and the excess of non-synonymous

substitutions affects mainly the terminal branches of the tree.

20

ACKNOWLEDGEMENTS

We thank Richard Villems for useful comments. This work was supported by NIH grants

GM28428, GM63883, and GM55273, EC DG Research grant ICA1CT20070006,

Progetto CNR- MIUR Genomica Funzionale-Legge 449/97, and Telethon-Italy E.0890.

We thank Rita Horvath for providing DNA samples.

21

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25

Figure legends

Figure 1. Maximum parsimony tree of 277 human mtDNA coding region sequences.

Locations of branch-defining mutations are listed relative to the revised reference

sequence (ANDREWS et al. 1999). Transversions are specified in capital letters, del

indicates deletion, + insertion. Amino acid replacements are specified in parentheses with

threonine and valine affecting changes highlighted in red and blue, respectively. ~t

indicates change in tRNA, ~r in rRNA gene. Haplogroup labels follow existing

classification (ACHILLI et al. 2004; BANDELT et al. 2001; CHEN et al. 1995; FORSTER et

al. 2001; FRIEDLAENDER et al. 2005; KIVISILD et al. 2004; KIVISILD et al. 2002; KONG et

al. 2003; MACAULAY et al. 1999; PALANICHAMY et al. 2004; SHEN et al. 2004; TANAKA

et al. 2004; TORRONI et al. 1996; TORRONI et al. 2001; TORRONI et al. 1993; WATSON et

al. 1997). Substitutions at nps 709, 3693, and 4715 in haplogroups B4, L2, and Z,

respectively, are reconstructed less parsimoniously, yet in accordance with the

phylogenetic relations suggested by additional data (FINNILÄ et al. 2001; KIVISILD et al.

2002; KONG et al. 2003; TORRONI et al. 2001). Coalescent estimates of haplogroups,

shown in thousands of years in italics beside clade labels, are based on the average

number of synonymous transitions to the root of the clade. The tree was rooted using as

outgroup the nuclear inserts of mtDNA and the majority consensus of complete

sequences of two Pan troglodytes and one Pan paniscus (for details see Material and

Methods). The numbers in triangles indicate the additional mutational steps required in

comparison to the optimally rooted tree using only the chimpanzee outgroup with the red

font indicating the root that was rejected (p<0.01) under the assumption of equal

26

evolutionary rate over branches. The positions at which the derived character states in

humans and the chimpanzee consensus match are shown above the triangles. Disease

implicated substitutions confirmed by two independent studies (www.mitomap.org) are

indicated by #. Ad, Af, Am, As, Eu and Oc correspond to African descent, African,

Amerindian, Asian, European and Oceanian, respectively. The continent of origin is

distinguished for each individual sample by box color and sub-continental affiliation with

letters below the boxes where N, E, W, S, C, SW refer to north, east, west, south, central,

and southwest parts of the continents, respectively; BP and MP refer to Biaka and Mbuti

Pygmies, respectively, A – Australian, P – Papuan, M – Melanesian, H – Hungarian

LHON patients, G - Georgian. The two African mtDNAs (L0a2 and L1b1) found in West

Asia (Pakistan) are likely due to recent admixture as indicated by their low genetic

distance to related African samples presented in the tree and by the fact that they were

detected in a Makrani and a Sindhi. According to Quintana-Murci et al. (QUINTANA-

MURCI et al. 2004) the Makrani harbor an extremely high frequency (39%) of African

haplogroups L3d, L3b, L2a, and L1a, most likely as a result of the forced migration of

slave women from Africa that began in the 7th century and increased considerably during

the Omani Empire. European gene flow, on the other hand, may account for the presence

of European K and X sequences in one Aboriginal Australian and one Native American

(Muskogee), respectively.

Figure 2. Natural and pathological variation in human mitochondrial species of tRNAs.

Mutations observed in the current study are shown in yellow, those with a status

of confirmed pathological variation (www.mitomap.org) in red triangles.

27

Nucleotide positions that are 100% and 90% conserved in mammalian species

(HELM et al. 2000), are highlighted in green and blue rectangles, respectively.

Nucleotide positions at which the human and mammalian consenses differ from

each other are shown in red font colour.

Table 1. Distribution of mtDNA mutations by recurrence.

non-syn rRNA tRNA syn Length in base pairs 8812 2513 1486 4212a Number of observed substitutions (per site)

413 (0.047)

173 (0.069)

110 (0.074)

1037 (0.246)

Transition/transversion ratio 12.4 23.7 12.8 34.8b Invariable sites 8506 2404 1409 3427 Sites with single hit 241 80 57 617 Sites with 1 recurrent hit 47 21 12 111 Sites with 2 recurrent hits 9 2 4 40 Sites with 3 recurrent hits 3 0 3 12 Sites with ≥4 recurrent hits 6 6 1 5 No of variable sites (proportion)

306 (0.035)

109 (0.043)

77 (0.052)

785 (0.307)

a – including 2039 sites that are allowed to carry synonymous transversions b – effectively, Ts/Tv=16.8, when taking into account the number of sites that are allowed to vary.

Table 2. Distribution of mutations as a function of derived-allele frequency in 277 mtDNAs.

frequency non-syn syn non-

syn/syn tRNA rRNA rRNA/syn<1% 221 514 0.43 49 63 0.12 1-5% 67 197 0.34 24 28 0.14 5-10% 14 39 0.38 1 8 0.21 >10% 5 32 0.16* 3 10 0.31*

* p<0.05

Table 3. Ratios of numbers and rates of non-synonymous over synonymous sites, nucleotide diversity, Tajima’s D, Fu and Li’s D and F, and number of changes from and to

valine and threonine for the 13 protein-coding mitochondrial genes according to continental affiliation.

Continent Africa S Africa W Africa B.F.a D.R.b Pygmy E Africa Non-African SW Asia Oceania E Asia America S Europe N Europe Global No. of samples 129 16 70 33 37 25 15 148 23 12 18 15 20 38 277

ND1 MN/MS 13/46 3/14 11/26 5/15 8/20 1/9 1/11 15/39 3/8 2/6 2/9 4/4 2/5 3/8 19/74 dN/(dS+const.) 0.058 0.052 0.073 0.073 0.071 0.037 0.014 0.116 0.102 0.080 0.028 0.193 0.089 0.119 0.074 ND2 MN/MS 17/42 2/10 13/25 6/12 11/20 3/10 3/13 19/53 8/10 4/5 5/16 3/6 1/8 4/16 30/84 dN/(dS+const.) 0.129 0.079 0.118 0.086 0.140 0.108 0.064 0.138c 0.196 0.239 0.107 0.079 0.056 0.121 0.136 COI MN/MS 11/58 3/14 9/37 4/15 8/33 2/19 3/15 10/60 2/17 0/4 0/11 1/7 1/9 5/15 18/100 dN/(dS+const.) 0.022 0.050 0.060 0.060 0.060 0.042 0.055 0.028c 0.030 0 0 0.018 0.024 0.06 0.043 COII MN/MS 7/31 0/10 4/16 1/8 3/15 3/9 0/5 6/27 0/5 1/4 2/5 2/2 1/3 0/9 10/49 dN/(dS+const.) 0.045 0 0.035 0.008 0.053 0.093 0 0.025c 0 0.044 0.070 0.080 0.030 0 0.073 ATP8 MN/MS 8/9 5/6 4/8 0/5 4/7 1/3 1/5 6/8 2/1 0/1 1/1 1/0 0/0 1/2 12/14 dN/(dS+const.) 0.050 0.102 0.049 0 0.014 0.039 0.020 0.061c 0.055 0 0.101 0.168 0 0.008 0.054 ATP6 MN/MS 25/23 5/4 15/16 9/5 10/13 4/9 7/3 23/23 8/6 3/7 7/5 4/0 2/2 6/6 42/39 dN/(dS+const.) 0.174 0.191 0.187 0.212 0.168 0.079 0.300 0.236c 0.245 0.134 0.352 0.367 0.066 0.148 0.259 COIII MN/MS 8/38 2/10 3/20 0/11 3/18 3/14 2/14 11/37 2/9 0/8 2/10 0/3 2/6 3/11 16/59 dN/(dS+const.) 0.024 0.055 0.014 0 0.025 0.027 0.019 0.062c 0.033 0 0.037 0 0.057 0.124 0.037 ND3 MN/MS 5/9 1/1 3/6 2/4 3/4 2/2 2/2 5/10 1/3 1/3 1/5 2/1 1/1 2/1 7/18 dN/(dS+const.) 0.118 0.037 0.075 0.058 0.088 0.197 0.103 0.109c 0.087 0.055 0.045 0.103 0.045 0.108 0.157 ND4L MN/MS 1/10 0/3 1/9 0/6 1/7 0/4 0/4 1/10 0/1 0/1 0/3 0/0 0/2 1/3 2/16 dN/(dS+const.) 0.004 0 0.009 0 0.015 0 0 0.005 0 0 0 0 0 0.018 0.005 Table 3. continued

ND4 MN/MS 9/67 2/14 6/46 0/20 6/38 3/19 0/20 14/56 4/15 2/8 3/13 3/5 1/12 2/16 21/103 dN/(dS+const.) 0.023 0.030 0.015 0 0.026 0.034 0 0.029c 0.049 0.041 0.037 0.101 0.027 0.016 0.024 ND5 MN/MS 35/75 5/17 25/38 12/21 21/26 9/23 11/25 38/60 8/18 7/6 8/10 4/10 3/12 8/17 56/112 dN/(dS+const.) 0.181 0.154 0.193 0.161 0.220 0.106 0.220 0.098c 0.094 0.171 0.135 0.084 0.046 0.086 0.145

ND6 MN/MS 9/26 2/7 7/16 5/8 3/16 1/7 0/8 7/23 1/7 1/2 2/7 1/4 1/4 1/4 14/42 dN/(dS+const.) 0.059 0.060 0.061 0.079 0.047 0.054 0 0.033c 0.014 0.048 0.067 0.064 0.031 0.015 0.047 Cytb MN/MS 31/41 5/12 18/31 12/14 12/26 3/8 8/8 29/41 5/11 1/10 8/11 2/9 5/4 8/10 47/65 dN/(dS+const.) 0.145 0.080 0.163 0.213 0.126 0.111 0.193 0.199c 0.138 0.035 0.127 0.054 0.240 0.351 0.166

No. recurrent synonymous sites 81 2 28 5 14 4 2 71 8 3 4 2 1 1 241

No. recurrent non- synonymous sites 35 2 14 5 6 1 3 30 3 0 2 0 0 3 106

π (x10-3) 3.79 3.59 3.22 2.69 3.68 4.26 3.16 1.85 1.78 1.93 1.99 1.66 1.01 1.41 3.06 ±S.D. ±0.12 ±0.23 ±0.17 ±0.18 ±0.23 ±0.16 ±0.37 ±0.08 ±0.14 ±0.23 ±0.15 ±0.19 ±0.18 ±0.09 ±0.10

θ (x10-3) 10.55 4.12 7.54 4.34 7.01 3.99 4.61 10.02 3.75 2.54 3.79 2.11 2.24 3.44 15.36 ±S.D. ±2.45 ±1.49 ±1.96 ±1.31 ±2.08 ±1.30 ±1.69 ±2.28 ±1.25 ±1.00 ±1.34 ±0.79 ±0.78 ±1.03 ±3.14

Tajima’s D -2.139d -0.552 -2.014d -1.455 -1.806d 0.265 -1.385 -2.681g -2.117d -1.121 -2.012d -0.933 -2.249e -2.198e -2.528g

Fu&Li’s D -4.203f -0.668 -4.186f -2.524d -2.744d 0.963 -1.015 -8.572f -3.300f -1.407 -2.847f -1.272 -3.138f -4.440f -7.738f

Fu&Li’s F -3.883f -0.735 -3.956f -2.560d -2.871d 0.870 -1.291 -6.894f -3.437f -1.519 -3.024f -1.357 -3.349f -4.339f -5.872f

To Thr&Val 78 10 36 13 20 7 12 74 10 6 14 9 3 14 158

From Thr&Val 47 2 34 10 22 2 3 52 9 2 13 3 5 11 99 aRimaibe (N=10), Foulbe (10), Mossi (10), all from Burkina Faso, plus 2 Mandenka and 1 Ghanan (subset of West Africa)

bIndividuals of African descent from the Dominican Republic (subset of West Africa)

cSignificant (P<0.0001) difference between African and non-African distribution of dN/(dS+constant) values. dP<0.05 eP<0.01 fP<0.02 gP<0.001

Table 4. The rate of non-synonymous/synonymous changes in relation to continental geography and haplogroup diversity.

Clade Geography Diversity (ρ)a MN MS MN/MS S.D.

L1c West and Central Africa 12.7 25 65 0.38 L0d South Africa 9.1 15 42 0.36 M7 Southeast Asia 7.3 8 21 0.38

M8(CZ) Northeast Asia 6.7 12 31 0.39 U Europe 6.6 40 111 0.36

“Old” haplogroups >6 0.37 0.01K Europe 2.6 10 23 0.43 C Northeast Asia 2.6 8 13 0.62 D1 Native American 2.5 5 16 0.31

L1b1 West Africa 2.2 10 11 0.91 L2a1 West Africa 1.8 14 16 0.88 H1 Europe 1.2 11 19 0.58

“Young” haplogroups <3 0.62 0.24 a average number of synonymous substitutions to the ancestral sequence of the haplogroup. One or two of the most frequent haplogroups for a) Africa, b) Europe, c) East Asia and Native Americans are displayed for the upper and lower range of sequence diversity. Using the average mutation rate of synonymous transitions the threshold of 6 synonymous transitions for the “older” haplogroups means coalescent times of the haplogroups >40 thousand years, while the threshold of <3 synonymous transitions for the “younger” haplogroups means <20 thousand years of divergence.

Table 5. Significant differences (p<0.05) in mutational direction in synonymous sites

Amino codon change number of observed

Acid (count in CRS) changes1 to/from p p'

Ala GCG (8) to GCA (80) 9/5 3x10-7 2x10-4 GCU (43) to GCC (123) 19/8 2x10-6 0.002 Asn AAU (32) to AAC (132) 13/13 7x10-4 0.042 Asp GAU (15) to GAC (51) 12/5 2x10-4 0.004 Gln CAG (8) to CAA (82) 7/9 0.001 0.018 Glu GAG (24) to GAA (64) 18/10 4x10-4 0.046 Gly GGG (34) to GGA (67) 44/37 0.005 GGU (24) to GGC (87) 11/12 0.014 Ile AUU (124) to AUC (196) 38/26 0.002 Leu CUG (45) to CUA (276) 39/59 1x10-7 0.006 UUG (19) to CUG (45) 4/3 0.099 UUA (73) to CUA (276) 28/18 4x10-8 2x10-4 CUU (65) to CUC (167) 25/18 1x10-4 0.032 UUG (19) to UUA (73) 14/11 5x10-4 0.048 Lys AAG (10) to AAA (85) 6/10 0.004 Met AUG (40) to AUA (167) 31/29 5x10-6 0.008 Phe UUU (77) to UUC (139) 24/17 0.006 Pro CCG (7) to CCA (52) 14/10 1x10-5 3x10-4 CCU (41) to CCC (119) 22/14 3x10-4 0.008 Ser AGU (14) to AGC (39) 7/3 0.02 UCG (7) to UCA (83) 5/10 0.02 UCU (32) to UCC (99) 14/7 9x10-5 0.008 Thr ACU (52) to ACC (155) 27/16 6x10-7 0.002 ACG (10) to ACA (134) 24/11 9x10-16 9x10-13 Try UGG (11) to UGA (93) 18/36 5x10-4 0.031 Tyr UAU (46) to UAC (89) 22/17 0.01 Val GUU (31) to GUC (48) 15/6 0.008

Total NNG (223) to NNA (1256) 229/237 4.6x10-52 5.3E-19 NNU (596) to NNC (1444) 249/162 2.8x10-34 1.8E-27 inverse ND6 NNG (62) to NNA (35) 14/19 0.03 NNU (72) to NNC (6) 10/6 1.1x10-3 p is a Chi-square probability assuming equal rates of codon exchange and estimates the difference from the expected number of changes given the codon frequencies in the reference mtDNA sequence (ANDREWS et al. 1999). p' is binomial probability taking into account additional transitional biases observed over the whole mitochondrial genome favouring transitions G to A over A to G and T to C over C to T by factors of 2.33 and 1.93, per respective nucleotides. 1 - number of changes corresponds to mutations (including multiple hits per site) inferred in phylogenetic analysis (Figure 1)

Table 6. Number and location of recurrent mutations in the mtDNA coding region (577-16023). Number of recurrences nucleotide positions

14 709~r 10 13708(A to T) 8 1888~r, 8251 7 11914, 10398(A <-> T) 5 1438~r, 5460(A to T), 13105(I to V) 4 1598~r, 1719~r, 3010~r, 13928C(S to T), 13966(T to A), 15930~t, 5147, 13368,

15217 3 3394(Y to H), 5821~t, 12172~t, 14110(F to L), 15110(A to T), 15924~t, 5231,

6182, 6221, 7055, 8790, 9545, 9554, 9950, 12007, 12501, 13359, 15514 2 930~r, 1503~r, 2768~r, 3434(Y to C), 4025(T to M), 4048(D to N), 5046(V to I),

5773~t, 8027(A to T), 10084(I to T), 12236~t, 12950(N to S), 13759(A to T), 14687~t, 15758(I to V), 15927~t, 3666, 3915, 4562, 4580, 4688, 4703, 5417, 5471, 5585-nc, 6260, 6446, 6680, 6752, 7076, 7388, 8020, 8152, 8155, 8392, 8964, 8994, 9254, 9266, 9509, 9755, 9824, 9932, 10685, 10790, 11260, 11944, 12354, 12477, 12810, 14007, 14034, 14148, 14182, 14905, 15115, 15301, 15784, 15884-nc

Amino acid changes are indicated in parentheses, ~r – change in rRNA, ~t – change in tRNA sequences, nc – change in non-coding position. The following positions showed a single recurrence, having mutated twice: 593~t, 597~t, 719~r, 813~r, 827~r, 1018~r, 1193~r, 1243~r, 1694~r, 1811~r, 1822~r, 2245~r, 2332~r, 2352~r, 2416~r, 2706~r, 2757~r, 2772~r, 2789~r, 2885~r, 3203~r, 3206~r, 3505(T to A), 4500(S to P), 4596(V to I), 4824(T to A), 4917(N to D), 5442(F to L), 5910(A to T), 6253(M to T), 6261(A to T), 6480(V to I), 7389 (Y to H), 7444(Ter to K), 7569~t, 7673(I to V), 7805(V to I), 7853(V to I), 8329G~t, 8387(V to M), 8393(P to S), 8566(I to V), 8584(A to T), 9095(L to P), 9139(A to T), 9438(G to S), 9477(V to I), 9861(F to L), 9966(V to I), 10031~t, 10143(G to S), 10321(V to A), 10463~t, 11016(S to N), 11025(L to P), 12142~t, 12248~t, 12346(H to Y), 12358(T to A), 12397(T to A), 12940(A to T), 13135(A to T), 13145(S to N), 13651(T to A), 13879(S to P), 13889(C to Y), 14129(T to I), 14180(Y to C), 14315(S to N), 14798(F to L), 15287(F to L), 15314(A to T), 15317(A to T), 15323(A to T), 15326(T to A), 15479(F to L), 15907~t, 15928~t, 15939~t, 15951~t, 3483, 3591, 3693, 3777, 3834, 3852, 4038, 4117, 4200, 4248, 4655, 4715, 4823, 4883, 4907, 4916, 4937, 5054C, 5162, 5237, 5393, 5580-nc, 5581-nc, 5656-nc,6026, 6179, 6392, 6431, 6455, 6827, 7184, 7337, 7424, 7861, 8050, 8104, 8227, 8269, 8277-nc,8383, 8485, 8697, 8856, 9150, 9180, 9299, 9305, 9365, 9377, 9449, 9716, 9758, 9899, 10238, 10389, 10586, 10589, 10688, 11002, 11257, 11299, 11332, 11350, 11353, 11383, 11404, 11437, 11452, 11812, 11854, 12372, 12432, 12540, 12609, 12630, 12720, 12771, 13020, 13104, 13116, 13215, 13263, 13470, 13590, 13680, 13827, 13980, 14094, 14212, 14233, 14323, 14364, 14470, 14560, 14581, 14620, 14668, 15043, 15061, 15106, 15148, 15172, 15289, 15313, 15346, 15394, 15454, 15466, 15550, 15607, 15670, 15697, 15883, 15886-nc.

Table 7. Codon sites found to be under positive selection in the mitochondrion encoded protein genes

in primates.

gene codon No Nucleotide position Nh Post. Prob. 1 ND2 218 5121-3 0 0.9732 2 ND2 265 5262-4 2 0.9603 3 ATP6 10 8554-6 0 0.9521 4 ATP6 188 9088-0 0 0.9809 5 ND3 9 10083-5 3 0.9628 6 ND3 44 10188-0 0 0.9823 7 ND3 107 10377-9 0 0.9666 8 ND3 114 10398-0 8 0.9542 9 ND4L 9 10494-6 0 0.9641

10 ND4 55 10922-4 0 0.9897 11 ND4 424 12029-1 0 0.9738 12 ND5 109 12661-3 0 0.9514 13 ND5 202 12940-2 2 0.983 14 ND5 459 13711-3 1 0.9525 15 ND5 492 13810-2 0 0.9617 16 ND6 11 14641-3 0 0.954

The test of positive selection (dN/dS >> 1) was applied to 13 protein coding genes of mtDNA by the

phylogenetic tree involving 7 primate species: 1. Homo sapiens, 2. Pan troglodytes, 3. Gorilla gorilla,

4. Papio hamadryas, 5. Hylobates lar, 6. Pongo pygmaeus, and 7. Macaca sylvanus) using PAML

(YANG 2002). A model of neutral selection on codon sites was rejected in favor of a model allowing for

3 ratios across sites with K=3 site classes (p ≈ 0, df = 5). The 3-ratio model identified the listed 16 sites

to be under significant (posterior probabilities > 0.95) positive selection (dN/dS = 2.02). Nucleotide

positions and gene names are given as in human reference sequence (GenBank accession #

NC_001807.3). Nh - Number of non-synonymous mutations per respective codon observed in the tree

of 277 human mtDNA sequences (Figure 1).

Table 8. Predominant amino acid replacement types in mitochondrial genes by intra and interspecies

comparisons.

No3 Ala<>Thr Ile<>Val Ile<>Thr Phe<>Leu Asn<>Ser Met<>Thr Ser<>Thr IleHuman-human1 414 0.273 0.145 0.08 0.07 0.058 0.029 0.01 0

Human-chimpanzee2 167 0.18 0.132 0.078 0.042 0.066 0.072 0.042* 0Chimpanzee-

orangutan 452 0.119* 0.071* 0.082 0.066 0.031 0.053 0.04* 0

Cat-dog 96 0.055* 0.107 0.047 0.025* 0.02* 0.03 0.07* 0Pig-cow 479 0.054* 0.09 0.035* 0.033 0.027 0.05 0.044* 0

1 proportions of specified amino acid changes as reported in the tree of 277 human sequences (Figure

1).

2 proportions of changes between rCRC (NC_001807.3) and Pan troglodytes (D38113).

Orangutan (D38115); pig (AJ002189); dog (AY729880); cat (NC_001700); cow (V00654) sequences.

3 Total number of observed amino acid replacements

* Difference from the human-human pattern; χ2 probability p<0.01

Only the most frequent (>5% in at least one comparison) amino acid replacement types are reported.

825T~r

8655s

10688s

10810s

Af14 Af31 Af32 Af52 Af36Af04 Af101Af82 Af83 Af85 Af79 Ad15 Ad16 Ad17 Ad18 Ad19 Ad20 Af81 Af29 Ad24Ad23Ad22Ad21

L2

9221s

10115s

13590s

L2a

2789~r

7175s

7274s

7771s

11914s

13803s

14566s

709~r

6752s

9932s15939~t

9083(L-P)

9438(G-S)

15803(V-M)

11767s

14118s

10790s

12507s

11647s

3834s

6383s

6431s

9077(I-T)

8727s

9299s

13443s

7664(A-T)

8721s

13068s

13827s

7076s

3010~r

12172~t

12354s13948(P-S)

10481s

14142s

10903s15924~t

7316s

5814A~t#

8944(M-V)

12804s

14162(A-V)

2083~r

9968As14007s

513-nc

534-nc

4038s

7388s

12408s

15880s

L2b

1706~r

2358~r

4158s

4370~t

4767(M-V)

5027s

5331A(L-I)

5814~t#

6713s

8080s

8387(V-M)

12948s14059(I-V)

6026s10828s

13924(P-S)

3253~t7569~t

9168s12557(T-I)

5261s10685s

10724s

15777(S-N)

6614s6806s

8503s

8790s

9350s

13966(T-A)

14407(L-F)

6061(I-T)8020s

11314s15394s

11944s

L2c5471s

8329G~t

10586s

13440s

4048(D-N)

6445(T-M)

9509s

12142~t

680~r709~r

3200A~r

13928C(S-T)

13958C(G-A)

15849(T-I)

1442~r

2332~r

7624As

12236~t

15110(A-T)

15217s

479-nc719~r1211~r3537s

3591s

3825s

4562s

5069T(M-I)

5585-nc

6014s

7885s

8383s

8413s

9139(A-T)

9377s

9971s

11935s

12189~t

12950(N-S)

13020s

13708(A-T)

14299s

14687~t

15103Gs

15289s

15497(G-S)

15697s

15734(A-T)

15889~t

870~r

2159~r

2332~r

3254A~t

3434(Y-C)

6231s

8856s

9130A(L-M)

9554s

9941s

10700s

10955s

11353s

11944s

12630s

13239s

14845s15263(P-S)

15458(S-P)

15703s15777C(S-T)

4259(T-I)

4596(V-I)

10530(V-M)

13135(A-T)

13708(A-T)

13711(A-T)

15514s

16000~t

L2d

1040~r

Af07 Af18

513-nc709~r

4500(S-P)

5128(N-S)7805(V-I)

8227s10265s10389s11914s

12354s

12438s12903s

15217s15883s15930~t

9855(I-V)

13470s

1413~r

1694~r

4949s5483Gs

8478(S-L)

8487(P-L)

10783Cs

15454s

Af45

13105(I-V)

L3b

3450s

5773~t

6221s

9449s

10086(N-D)

13914As

15311(I-V)

15824(T-A)15940d~t

11002s 9079(N-D)

12441s

13434s

15664s

Af70 Af71 Af68 Af73

3394(Y-H) 5371G(S-W)

10084(I-T)

14180(Y-C)

15110(A-T)

15217s

15394s

Af74

3420s

Af76

12070s

15758(I-V)

10373s

Ad26 Ad27Ad25

8393(P-S)

11800s

15883s

1193~r

3441s

5211(L-F)

5581-nc

9477(V-I)

15299s

4200s9053(S-N)

9067(M-V)15550s

2093~r

5250s

7301s14161s

5821~t

6182s

6722s

8676s

9365s

9731As

12280~t

12534s

14260s

15314(A-T)

15479(F-L)

15514s

Af30

3504s

3729s

4619s

11503s

13368s

15434(L-F)

Ad28

L3d

5147s

7424s

8618(I-T)

13886(L-P)

14284s

921~r

6680s

Af72Af77 Af50

11383s

12188~t14034s

14634(M-V)15110(A-T)

1503~r

3474s4048(D-N)

5162s

7648s8749s

9452s

10837s

11887s

12406(V-I)

13884s

15106s

9022(A-T)

1193~r

6480(V-I)

13752(I-M)

15799s

15928~t

1719~r

3498Gs

4688s

8251s

10899(N-S)

11404s

15061s

Af19 Af23 Af26 Af28

2416~r15208s

3394(Y-H)

11518s

12873s

14272s

14584s

15115s

15865s

Ad29 Ad30

5054Cs

7765s

8485s

9151(I-V)

11401s

5246s

5605~t

6842s

L4g

7256s

7521~t

13650s

3516As

5442(F-L)9042s

9347s

10589s

10664s

10915s13276(M-V)

L0a

5460(A-T)

8428s

11176s

Af05 Af33 Af37Af40 Af20Af16 As46

L0a2

5147s

5711~t

6257s

8281-8289d-nc

8460(N-S)

11172(N-S)

597~t

5741-nc

10389s

9545s

9554s

3083~r

9214(H-R)

11143s

14755s

4916s

14371s

5911(A-V)

14007s

Ad1

L0d

1438~r

4232(I-T)

8152s

8251s

12121Gs15930~t

Af09 Af25Af55 Af27 Af24 Af17 Af22 Af54

719~r

3438s

6266s

456-nc

4197s

9150s

9438(G-S)

12235~t

13129(P-S)

15951~t

593~t

3666s

456-nc

597~t

4225(M-V)

5153s

6179s

8392s

8545(A-T)

12172~t

12234~t

12810s

14040s

14221s

3981s

4025(T-M)

4044s

11854s

15766s

4038s

4204s

4937s

6249(A-T)

6644s

7473~t

8284-nc

8420(T-A)

9230s

9305s

13827s

14007s

15346s

3618s

7283s

14315(S-N)

14659s

1243~r

8508(N-S)

8566(I-V)

9950s

12142~t

12696s

12798s

8383s

12501s

13542s

15692(M-V)

15697s

6692s

8790s

9037(M-V)

9887s

14280(S-P)

14560s

15449(F-L)

L1b

Af87 Af88 Af89 Af90 Af91 Af53

3666s

7055s

7389(Y-H)

13789(Y-H)

14178(I-V)

709~r

1738~r

5036s

5046(V-I)

5655~t

6548s

6827s

6989s

7867s

8248s

12519s

13880A(S-Y)

14203s

14769(N-S)

5393s

12609s7785(I-T)

2768~r

9755s

14110(F-L)

9716s15346s

4123(I-V)

7444(Ter-K)

8329G~t

8937s

11452s

14180(Y-C)

14759G(R-G)

15301s

As24

7868(L-F)

9270(L-F)

11659s14503s

14756(M-V)

Ad3Ad2 Ad4 Af102 Af97 Af98

6407s15927~t

14182s

4598s

8264s

9178(V-M)

11830s 4080s 7967s

3372s

5237s

11269s

12172~t13281s

1598~r

1888~r

14298G(I-L)

5216s

14131s

2245~r

Ad5 Ad6 Ad7 Ad8

13980s

7915s

5147s

5988s

7419(E-K)

Af01 Af02 Af06

6629s7673(I-V)

7960s9266s9545s

13741(T-A)13879(S-P)

10454~t

456-nc

1503~r

4596(V-I)4937s

4959(A-T)

5054s

11337(N-S)

11854s

12540s

12732s

12940(A-T)

14182s

14913(S-L)

15191s

4706s

8348~t

9494s

10927s

11002s

11928(N-S)

Ad9

513-nc

1598~r

2220~r

5162s

6150(V-I)

6253(M-T)

6962s

7076s

7337s

8784s

8877s

10031~t10792s

11164s

11252(I-V)

11654(T-A)

11959s

12477s

12540s15929~t

Af38 Af94

467-nc

2308~r

5984s

6182s

8928s

9311s

10398(A-T)

11167s

11257s

12930Ts

14034s

15663(I-T)

13966(T-A)

10321(V-A)

2395d~r5951s

8027(A-T)

9072s

13485s

14000A(L-Q)

14911s

Af96 Af34 Af35

2755~r

2863~r

3513s

3927s

4506(I-V)

7202s

9647s

12768s

6752s

Af93

7660s

7693s

9272s

2674~r

4634s

5054Cs

9336(M-V)

12477s15289s

L1c

Af39 Af95Ad10

4454A~t

8087s14088s

3796T(T-S)

3843s

14148s

L0d1L0d2

1048~r

4312~t

6185s

9755s

11914s

12007s

Ad11

6221As

7055s

15907~t

15978~t

3106~r

4345~t

6260s

7498~t

7789s8668(W-R)

8680s

9966(V-I)

12019s12501s

L1b1

L1c2

L1c1L1c3

L0k

4586s

9818s

2245~r

5603~t

11641s

15136s

15431(A-T) 4907s

5460(A-T)

7257(I-V)

10920(P-L)

12070s

13020s

5231s

8566(I-V)

12720s

14308s

498d-nc3756s

6815s

8113As

12720s

15466s

15941~t

2706~r

13759(A-T)

7828s

11437s

15550s

7154s

14560s

710~r

1438~r

2352~r

3308(M-T)

3693s

15115s

6071s

10586s

12810s

5196s

6917s

15226s

2416~r

8206s

3594s4104s

3918s

6260s

8104s

12609s

+8

+10

+3

+6

+6

5826~t

6663(I-V)

0

+4

+2

+7

+5

+7

+15+14

+16

+11

+5

+4

+5

3693s

L0

L1

8994s

1291~r

4025(T-M)

4562s

4688s

4824(T-A)

5231s

5553~t

8237(I-V)

8281-8289d-nc

8280A-nc8619s

9054s

9824s

9861(F-L)10084(I-T)

12681s

14338s14393(V-A)

15025s

15160s

11899s

12049s13149s

8155s 13116s 8965(I-V)

5046(V-I)

L2a2 L3d1

L3d3

L0a2b

L0a2a

14912G(S-A)

850~r1243~r2836~r

4542(H-Y)5811~t6938s8911s

9755s10499s10876s10939s11296s11299s11653s13590s

13819(F-S)

14020s14182s14371s14374s

9136(I-V)

13928C(S-T)

11302s

WWW S MPW MPMPMPMPMP SSSSSSSS WWWW WW WWWWW BP BP C W BP BP BPBP BPBPBP W

MPWWWWWW E MPMP MPMPMP WWWWWW WWWWWW C S E WWW WWWWWWW WWWW SS SSSE

SW SW

62 11+

108 16+

42 11+

15 7+

86 12+

32 8+

74 14+

112 16+

3849s

11332s

15886-nc

32 9+

3 2+ L1c1a57 13+

31 9+

160 22+

BPMP

24 8+

39 7+

522-523d-nc

2142+AG~r

2395d~r

5899+5C-nc

8278+6C-nc 523+CA-nc

522-523d-nc

769~r

1018~r65 8+

+7

L3

W MP MP

Af08Af10 Af21 Af84Af80Ad14Ad13

12693s

15784s

6216s 10143(G-S)

15735(A-V)

5090s

7663s

15258(D-G)

5581-nc

8251s

13651(T-A)

10245s

13759(A-T)

13928C(S-T)15326(T-A)

731~r

6480(V-I)

10685s

12753s

15749s

3589G(L-V)11692s

13888(C-R)

Af86 Ad32

L3f1

1822~r

3396s

4218s

5601~t

7819As

8527s

8932(P-S)

9950s

11440s

14769(N-S)

15514s15940d~t

1438~r

3200~r

3505(T-A)

8799s

9139(A-T)

12397(T-A)

13167s

5777~t

L3h

1719~r

4388~t

5300s

7861s

9509s

9575s

11590s

Ad33 Af46 Af51

2831~r

3777s

4859s

7055s

8767(T-A)

9827s

10044~t

10289s11563s

11963(V-I)

516-nc

709~r4117s

5316(A-T)

5492s

8047s

8485s

9080(N-S)

12432s

13708(A-T)

13926s

14110(F-L)

8387(V-M) 1888~r

7493~t

10463~t

11029s

14370s

Ad31

4393A~t

6431s

15565s

Af44 Af92 Af48

15061s

5393s

12795s

13368s

15317(A-T)

Af99 Af100

456+TTC-nc

709~r851~r

1822~r4496s

5111s5656-nc6297s8188s

8582(A-V)8754s

11025(L-P)12303~t13722s

13752As

14212s

14239s

14581s

14971s

1850~r

5978s

7424s

12780s

8856s

11770s

13105(I-V)

Ad12

629~t

2283~r

3210~r

3434(Y-C)

4755s

5237s

8251s

8417(L-F)

11317s

11812s

12400(T-A)

12542(A-V)

13708(A-T)

13981(P-S)

14794s16017~t

L0a1

2758~r

7146(A-T)

L5L0f1719~r

2772~r

2789~r

6446s

7148s

10143(G-S)

11287s

13708(A-T)

14109s

15852(I-T)

978~r

2792C~r

3852s

4194s

4562s

4964s

5321s

9300(A-T)

9581s

9620s

9755s

10790s

11299s

13470s

13680s

13928C(S-T)

14620s

2885~r

8468s

13105(V-I)

L5a

8155s

3423s

12432s

12950(N-S)

7972s

930~r

4137s

5147s

6182s

9305s

9329s

11881s

12236~t

14905s

15217s

13506s

15301s

+3

+9

+4

+5

+6

-3

+3

3866(I-T)

5096s

L2a1

13966(T-A)

15884-nc

E W EE

E S WS WW W W WWW E E

12 3+

513-514d-nc

523+CA-nc

523+CA-nc

523+CA-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

523+CA-nc

523+CA-nc

522-523d-nc

522-523d-nc

522-523d-nc

11 6+

8 3+

32 9+

21 7+

L3d3a3 2+

27 7+

Figure 1

Af69

11974s

7389(Y-H)

13116s

8964s

813~r

6671s

3705s

6378s12346(H-Y)

593~t

8392s

9196s

13149Ts

M2a

M1

6446s

6680s

12403(L-F)14110(F-L) M3

447G-nc1780~r

8502(N-S)

4924(S-N)

4965(S-G)

5252s7961s

9758s

9965s

12810s

4580s

482-nc

4703s

D

D4 D5

4883s5178A(L-M)

3010~r8414(L-F)

14668s10397s

D4a

3206~r

511-nc

2177~r4721s

12007s

13293s

15930~t

1760~r

3381s

9266s

10954s

11260s

11827s15148s

709~r

1888~r

3921s

8697s

11016(S-N)

12477s

14323s15287(F-L)

9509s14687~t

4059As

5585-nc

6827s9095(L-P)

9180s

12007s

732~r

4697s

4973s

7738s

9233s

12025s

12236~t

14110(F-L)14315(S-N)

14323s

14356s

15862s

5773~t7040s

13359s

15601s

4131s

9052(S-G)13359s

9861(F-L)

1402C~r4655s9296s

14605s

469G-nc4248s

8473s

D1

2092~r

3483s

6446s

7112s

10874s

729~r

8251s

12381s

14494s

14687~t

3663s

11248s

5821~t

5471s

15313s15719(Y-H)

10816Ts

11914s

13059s

14016s14094s

15514(K-N)

1107~r

9180s

3552As9545s

11914s

13263s

14318(N-S)

7861s

10682s11902s

13968s

5821~t

6338s

10754s

11839s

C

10400s14783s15043s

M8

4715s

7196As8584(A-T)

15487Ts

Z

6752s

9090s15784s

773~r4363~t

4715s

6590s

8227s

10208s

12996s

14668s

493-nc

7337s

M7a

M7

M7b

M7c

6455s9824s

2626~r2772~r

4386~t

4958s

12771s14364s

4047s

11017s

11368s

14148s

14185Ts15886-nc

4071s

4048(D-N)

4164s

5351s

6680s

7684s

12405s

12811(Y-H)

3483s

6551s

8155s

3606s

Q

4117s5843~t

8790s

13500s

Q1Q3

8964s

14025s

2768~r4335~t

7169s

13368s15939~t

5417s9254s

12248~t

10790s11260s

1391~r

4707(L-F)

6221s

7388s

10801s

13368s

14404s

M42

1598~r

4508s

8251s

9156s

2281C~r

3426s

5021s

6104s

8050s

8101s

9314s

9353s

11452s

7675s

8805s

8838s10688s

11016(S-N)

11701s

13145(S-N)

13660(N-D)13980s

M

M8a

14470s

5100s

6179s

9758s

11560s

1719~r15930~t

L3e

10819s

13800As

4092s

14311s

10679s

2352~r

14212s

L3e1L3e2

6587s

10370s

14152s

5774A~t

6221s

9254s

11024s

14569s

15670s15942~t

750~r

5262(A-T)

L3e4

L3e32880~r8392s

5584-nc

3915s

8688Cs

11149s

11257s

13749s

14173s

2000~r

4655s6524s

9554s

10667s10816(K-N)

12248~t13101Cs

13197s

14905s

4823s

4907s

10181s

14869s

1799~r2262~r

3852s

7196s

8251s15924~t

2145~r

4907s

11350s

2483~r

4167s4500(S-P)

6092s

8823s

9377s

1694~r

11377s

5186s

489-nc

8793s

12771s

15172s

4916s

+8

+9

Af15 Af43 As18 As16 As10 As32 As15 As47 As02 As01 As07 As08 As27 As05 Am01 Am07 Am08 Am09 Am02 Am10 Am11 Am14 As74 As09As33 Am03 As30Am04 Am06 As06 As45 As04 Oc03 Oc04 Oc09 Oc14 Oc01 Oc17 Oc12 Oc13As71Af49 Af47 Ad34 Af75 Ad37 Af78Af64 Af63 Af65 Af66 Af67 Ad35 Ad36

Am17

A

663~r1736~r4248s

8794(H-Y)

5093s

6641s

12007s

As73 Oc11

1598~r

1888~r

4917(N-D)#5291s

8251s

8269s11491s

14384C(A-G)

15040s

Oc06

709~r1811~r2792~r10358s

13594(S-G)

W

709~r

1243~r

8994s

11674s

11947s

5231s

12372s

5471s

5474s

5498s

5580 nc-

5821~t

10685s

13857s

As12 As23 Eu51

5580 nc-

6599s

7864s

14148s

1406~r

13215s

13263s

13344s

15784s

Eu01

N1

10238s12501s

I

4529Ts

8251s

10034~t

15043s

15924~t

Eu74 As34 As40

813~r

6182s

8281-8289d-nc

8706T(M-I)

12961(S-G)13889(C-Y)

14233s 4050s

8050s

X2

1719~r

Am05 Eu55

6221s6371s

502 nc-8269s

14470s

14818Cs

499 nc-

11932s

15034s15894~t

3645s

3915s

6116s

7804s15287(F-L)

2849~r

8281-8289d-nc

B

R12

9644s

14305s

15884-nc

10118s

709~r

9293s

14890s

Oc10

4011s4200s

4695(F-L)4703s5417s

8906(H-R)

9386s

9881s

11404s12172~t

14989s15562s

As03

F

3970s

6392s

10310s

513-nc

6653s8020s8575s

8603(F-S)

9899s10097Cs15930~t

B4a

5465s

9123s

10238s

As72

709~r1888~r9254s

15292s

B5b

709~r

As28 As26Am12 Am16Am15

499-nc827~r

4820s13590s

15535s

B4b

4977s

6473s

9950s11177(P-S)

3434 Y C( - )

9095(L-P)

14485s

6119s

14049s

15265s

3760G(S-A)

7813s

R

12705s

Eu40 Eu47Eu32

709~r

7094s

13680s2757~r

10211s

Eu08 Eu04 Eu54

V

4580s15904~t

7444(Ter-K)8910s

14121s

As37

6248s

8994s

9449s

14440s

As38

827~r

Af41

2355~r4997s

11353s15674(S-P)

2442~r3847s

13188s HV

Eu05

H1

3010~r

Eu06 Eu15Eu17 Eu18

9986s

14224s

3305-nc9545s

11152s

14374s

2281~r

6253(M-T)

15589As

Eu02

8014s

9923s

Eu93 Eu09 Eu10

477-nc

H

Eu12

7891s8277-nc

15172s

Eu39 Eu41

4733s6755s

13731s 723~r

As42

11516s

Eu95 Eu43 Eu44 Eu80

1462~r

4883s

6314s

Af13

3666s

714~r

3903s

7762s

8119s14407G(L-F)

9150s

Eu87

8994s

Eu92

8371s

2706~r7028s

T

J

489-nc

12612s

709~r1888~r

4917(N-D)#

8697s10463~t

13368s14905s

15607s15928~t

T1 T2

J1c

462-nc3010~r

14798(F-L)

Eu57 Eu52 Eu53

482-nc508-nc

3394(Y-H)

7184s9554s

2387~r10192(S-F)#

3203~r

4976s

5836~t10529s11080s

Eu91

5198s

15916~t

Eu23 Eu46 Eu35 Eu82Eu84

12633As

9899s

1888~r4580s4994s

3125~r

3804s

11812s14233s

Eu27

783~r

9524s10822s11914s

12346(H-Y)15479(F-L)

6260s

Eu42 Eu33

1888~r4823s6935s

Eu79As11

930~r5147s

8281-8289d-nc

15884C nc-

6734s

I1

11719s 4216 #(Y-H)11251s

15452A(L-I)

12414s

N

9540s

10873s15301s

+6

1888~r

12618s

13879(S-P)

14581s

15313s

12501s15106s

7645s

11260s

U

As17

U1a

Af42

2218~r

7581~t

12879s

13104s

14070s

15954C-nc

505-nc

3591s

8602(F-L)

9746s

9833s

13422s

8857(G-S)

15115s15217s

Eu26

6761s

8790s

U3

Eu36

4703s

6518s

9266s

14139s

15454s

Eu83

499-nc3010~r

15190s

U2e

3116~r

3720s

5390s

5426s

6045s

6152s

10876s

11197s

11732s

13020s

13734s

15907~t

508-nc

12354s

Eu81

1585~r

9360s

9752s

9932s

11383s

499-nc

5999s

As14

3531s

3834s9299s

11350s

12615s

13111s14094s

15951~t

4646s6047s

As29

6164s

11204(F-L)

12888s13899s

14620s

Eu29

4811s6146s

8818s

9070G(S-A)

10907(F-L)

11009s

11332s

14866s

9698s

3480s

10550s

11299s

14167s

14798(F-L)

K

Eu94 Eu76 Eu77Eu03Eu30As36 Oc05

497-nc

3777s7278(F-L)

7729s8604s9800s

13326s13665s

15908~t

3206~r

6293s

8521s11485s

6975s7388s

8410s9448(Y-C)

1189~r

4856s

5592~t

8901s

3348s

5081s

5130s

7076s

5773~t

11025(L-P)

10786s

U5

U5b

3197~r

13617s

7768s14182s

14793(H-R)

Eu28 Eu88 Eu38 Eu56Eu37 Eu96 Eu22

5387s5585-nc6023s

7569~t11914s15412s

3714s

10619s

10768s

14034s

12582s

7843s7978s8104s9148s

11107s

455d-nc9548s13351s

5339s12801s

13889(C-Y)15466s

981~r

U5a9667(N-S)

Eu45 Eu59

2757~r5656-nc10283s

12616s

13359s

1721~r4732(N-S)

13637(Q-R)

Eu78Eu85 Eu89

3212~r

9845s

5508s

13105(I-V)

5301(I-V)

9109(I-V)

7853(V-I) 11447(V-M)

7853(V-I)

15236(I-V)

4025(T-M)

15758(I-V)

14484 M V #( - )15812(V-M)

13105(I-V)

4560(V-M)

5046(V-I)

13402(I-V)

13780(I-V)

9966(V-I)

3547(I-V)

8864(V-A)

14553(V-I)

10321(V-A)

9234(M-V)

15212(I-V)

13321(I-V)

7697(V-I)

14514(M-V)4944(I-V)

15758(I-V)

13117(I-V)

13105(I-V)

4561 V-A)(

9477(V-I)

6366(V-I)

15791(I-V)

7257(I-V)

10589s

12678s12136s

U8a

3738s

5240s

6392s

6455s

7055s

9365s

10733s13145(S-N)

Eu86

1503~r3732s

7340s9932s

11470s

Oc16

1438~r3203~r3882s

4122s

4384~t8578(P-S)

8859s

15355s

U9

6026s

K1 K29716s

+6

4991s

14364s

15148s

1811~r

12308~t

11467s

12372s

P2

12950C(N-T)

8396(T-A)

10845(T-I)

14979(I-T)

6261(A-T)

12635(I-T)

13651(T-A)

9064(A-T)

4734(T-A)

5319(T-A)

5910(A-T)

12715(T-A)

15317(A-T)

15473(T-A)

8659(T-A)

15323(A-T) 13928C(S-T)

3316(A-T)

3797G(T-S)

15080(T-A)12362(T-I)

10084 I( -T)

11084(T-A)

5460(A-T)

15326(T-A)

12940(A-T)

5460(A-T)

3506(T-I)

11150(A-T)

8684(T-I)

13967(T-M)14060(I-T)

14207(T-I)

4824(T-A)

5910(A-T)

8027(A-T)

3505(T-A)

5460(A-T)

10398(T-A)

5074(I-T)

13966(T-A)

6267(A-T)

13135(A-T)

8027(A-T)

13708(A-T)

13759(A-T)

13928C(S-T)

10398(T-A)

10978s16000T~t

9972(I-V)

5095(I-T)

15110(A-T)

15314(A-T)

9116(I-T)

14766(T-I)

14129(T-I)

9007(T-A)

12341(T-I)

15323 A-T)(

5460(A-T) 13966(T-A)

8803(T-A)3796(T-A)

10398(T-A)

13708(A-T)

7258(I-T)6261(A-T)

12397(T-A)8854(A-T)

8701(A-T)10398(A-T)

10506(T-A)

13934(T-M)

15848(T-A)

13708(A-T)14129(T-I)

5460(A-T)

15693(M-T)

9055(A-T)

13708(A-T)

8400(M-T)

10398(T-A)

9531(T-A)

9682(M-T)

4129(T-A)

15218(A-T)

JT

930~r

(pre-HV)1

N9a

L3i

15884-nc

E W SWE WWWWWWWWW W E E EESWSWSWSWSW EEEE S SS SS S S NSEEE SW EEEE M MM P PP A A

S A SWE NA SW S SWSW N SS EEEE PA SW E S N SWNN SW NNN SW NNN AS NN NNNN N S NNN

E SW N SSW NS SS SS SS N N NN SW N S S N N W

H7

4793s

Eu58 Eu24 Eu48

13359s

15409s

4688s7205s 7184s

13707s

13477(A-T)3335(I-T)

5348s

S

H2

Eu34

1438~r

4769s

8843(I-T)13708(A-T)

N S N S N N N S N SW NN

H6a

Eu07

9380s

1005~r3915s

13215s

As41

4727s

As25 As13

8940s 14978s

7805(V-I)

11253(I-T)

Eu14

H5a

456-nc4336~t#

Eu49 Eu50 Eu11

11281s

5839~t

10344C(I-L)

549-nc

721~r

8167s

Eu90

N S S SW SWSW

U8

pre-HV

1438~r

12026(I-V)

As35

6776s

SW

H3

15670s

C

N C

GH HHHH H H

HHH

HHHH

HH

H

30 7+

25 7+

17 5+

46 5+

32 10+

49 11+45 10+

48 9+

49 11+

42 6+

45 6+

22 5+

18 4+

9 1+

37 11+

16 6+

16 6+

7 2+

16 6+

14 6+

18 5+

B2

1598~r

4895s5744-nc

6767s

8584(A-T)8829s

9950s

11437s

12361(T-A)

15850s

R11

15508s15223s

15662(I-V)15851(I-V)

15927~t

709~r

10398(T-A)

8277 nc-

10031~t

11061(S-F)

12950(N-S)13681(T-A)

F4

5263(A-V)

12630s

15670s

8278+2C-nc

D5a

7673(I-V)11944s

6596s

Z2

3394(Y-H)

5442(F-L)4850s

11665s

12091s

S1

12358(T-A)

12358(T-A)

N9S

5417s8404s

573+C-nc

573+C-nc

573+2C-nc

573+3C-nc

573+4C-nc

573+5C-nc

573+5C-nc

573+5C-nc

573+5C-nc

960d~r

960d~r

960+C~r

2232+A~r

3159+T~r

5899+5C-nc

8285+4C-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

522-523d-nc

523+CA-nc

523+CA-nc

523+CA-nc

523+CA-nc

523+2(CA)-nc

523+CA-nc

523+CA-nc

523+CA-nc

W3

P

15607s

X2b

X1719~r

8393(P-S)13708(A-T)

15927~t

X2e

15310s

D4b2

8020s1382C~r

8964s9824s

5231s

13104s

D4k3

U4

6386s 18 4+13 4+

15 5+

16 6+

11 4+8 5+

14 4+11 5+

45 13+

14 7+

17 4+

Figure 1, continued

961~r

15067s

10398(T-A)

15833s

573+4C-nc

595+C-nc

Eu25 Eu75

11914s15924~t

G

G

G

G

A

G

U

C

U

U

U

U

U

U UU

U

U

CC

C

C

C

A

AA A

A

A

A

C

C

C

C

Thr

G

G

G

G

C

C

C C

A

A

A A

U

U

U

U

U

A

AAAA

A

A

A

A

U U

GG

G

G

G

G

A

G

U

C

U

U

U

U

U

U

U

U

U

U

CC

C

C

C

A

A

A

A

A

A

A

CC

C C

LeuG

G

C

CC

C

A

A

A

AU

U

UU

U

A

AA

A

A

A

A

A AA

A

U

U

AG

AG

15924

15930

15927

15953

15888

UAA

A

G

G

G

G

C

C

CC

A

UU

UU

U

3304

3230

Cys C

C

U

U U

C

C

A

A

U

U

U

C

G

G

G

GC

A

A

A

A

UU

U

C

G

G

G

G

C

A

AA

A

U

U U

C

G

GG

G

C

A

AA

A

U

U

U

C

G

G

G

G C

A

A

A

A

U

U

U5826

5761

58145773

5821

His

G

GG

U

U

U

GGG

A

G

G

G

G

A

A

C

U

C

U

C

U

C

U

C

U

CU

C

U

CU

CU

C

U

C

U

C

U

C

U

N

A

A

A

A

A

A

C

A

A

AA

U

A

AA

AA

A A A

A

A

A

A

C12138

12206

12172

natural variation

confirmed pathologicalvariation

100% conserved in 31mammalian tRNAs

>90% conserved in 31mammalian tRNAs

difference in the humanand mammalian consensus

Figure 2