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

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: 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.

2 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 [email protected], and P.J.O., Institute of Functional Genomics, University of Regensburg,

Josef-Engert-Str. 9, 93053 Regensburg, Germany, e-mail: [email protected].

3 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

4 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

5 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

6 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

7 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

8 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)

9 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

10 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

11 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

12 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).

13 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

Leu (MCFARLAND et al. 2004), as illustrated in the case of tRNA 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,

14 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

15 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

16 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

17 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

18 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.

19 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.

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24 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

25 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.

26 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.

27 Table 1. Distribution of mtDNA mutations by recurrence.

non-syn rRNA tRNA syn Length in base pairs 8812 2513 1486 4212a Number of observed 413 173 110 1037 substitutions (per site) (0.047) (0.069) (0.074) (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 306 109 77 785 (proportion) (0.035) (0.043) (0.052) (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. non- frequency non-syn syn 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 c dN/(dS+const.) 0.129 0.079 0.118 0.086 0.140 0.108 0.064 0.138 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 c dN/(dS+const.) 0.022 0.050 0.060 0.060 0.060 0.042 0.055 0.028 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 c dN/(dS+const.) 0.045 0 0.035 0.008 0.053 0.093 0 0.025 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 c dN/(dS+const.) 0.050 0.102 0.049 0 0.014 0.039 0.020 0.061 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 c dN/(dS+const.) 0.174 0.191 0.187 0.212 0.168 0.079 0.300 0.236 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 c dN/(dS+const.) 0.024 0.055 0.014 0 0.025 0.027 0.019 0.062 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 c dN/(dS+const.) 0.118 0.037 0.075 0.058 0.088 0.197 0.103 0.109 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 c dN/(dS+const.) 0.023 0.030 0.015 0 0.026 0.034 0 0.029 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 c dN/(dS+const.) 0.181 0.154 0.193 0.161 0.220 0.106 0.220 0.098 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 c dN/(dS+const.) 0.059 0.060 0.061 0.079 0.047 0.054 0 0.033 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 c dN/(dS+const.) 0.145 0.080 0.163 0.213 0.126 0.111 0.193 0.199 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) c Significant (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.

a Clade Geography Diversity (ρ) 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 0.01 K Europe 2.6 1023 0.43 C Northeast Asia 2.6 8 130.62 D1 Native American 2.5 5 160.31 L1b1 West Africa 2.2 1011 0.91 L2a1 West Africa 1.8 1416 0.88 H1 Europe 1.2 1119 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

number of Amino codon change observed changes1 Acid (count in CRS) 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 Ile Human-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* 0 Chimpanzee- 452 0.119* 0.071* 0.082 0.066 0.031 0.053 0.04* 0 orangutan Cat-dog 96 0.055* 0.107 0.047 0.025* 0.02* 0.03 0.07* 0 Pig-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. 0 160+ 22

3516As Figure 1 1048~r 5442(F-L) 9042s 4312~t 9347s 6185s 10589s 9755s 10664s 11914s 10915s 12007s 13276(M-V) 108+ 16 L0 +8 14560s 2885~r 1438~r 8468s 4586s 4232(I-T) +5 9818s 8152s 8251s 522-523d-nc +4 12121Gs 3666s 2758~r 15431(A-T) 4907s 15930~t 7055s 7146(A-T) 5460(A-T) 7389(Y-H) +9 7257(I-V) 13789(Y-H) 8155s 8994s +2 14178(I-V) 13105(V-I) 2245~r 10920(P-L) 112+ 16 13506s 5603~t 12070s +5 15301s 11641s 498d-nc 13020s L1 3423s 15136s 3756s 6071s 12432s 6815s 710~r 10586s +3 8113As 1438~r 5460(A-T) 12810s 850~r 12720s 2352~r 8428s L5 1243~r 15466s 3308(M-T) L0f 11176s 2836~r 15941~t 3693s +7 12950(N-S) 1719~r 4542(H-Y) 15115s + 2395d~r 2772~r +10 5811~t 62 11 +6 6938s 5951s 2789~r 522-523d-nc 8911s L0d 8027(A-T) 7972s 2706~r +4 6446s 5231s 9136(I-V) 7154s 9072s 13759(A-T) 7148s 8566(I-V) 9755s 13485s 10143(G-S) L5a 12720s 10499s 709~r 14000A(L-Q) 11287s 930~r 14308s 10876s 1738~r 14911s 13708(A-T) +6 +7 4137s + 10939s 5036s + 14109s 42 11 11296s 86 12 5147s 5046(V-I) 15852(I-T) 11299s 719~r 3981s 6182s L0a 5147s 5655~t L1c 5096s 11653s 3438s 4025(T-M) 10321(V-A) 6917s 9305s 5711~t 6548s 13590s 6266s 4044s 11302s 9329s 6257s 6827s 11899s 13819(F-S) 11854s 15226s 11881s +3 L0a1 8281-8289d-nc 6989s +15 13928C(S-T) 15766s 12236~t 8460(N-S) L0d1 7867s 12049s +14 3866(I-T) 14020s 7828s 3618s 14905s 978~r 3849s 11172(N-S) 14182s 8248s 13149s +11 11437s 7283s 15217s 2792C~r 5911(A-V) 32+ 9 14371s 1243~r L0d2 12519s 3796T(T-S) 15550s 14315(S-N) 3843s 6221As 3852s 11332s 14374s 7389(Y-H) 13880A(S-Y) 7055s 5393s L0a2 14659s L1c2 14148s -3 4194s 14007s 11143s 523+CA-nc 8508(N-S) 456-nc 522-523d-nc 14203s 5196s 15907~t 12795s 15886-nc 3372s 513-nc 4562s 14755s +6 8566(I-V) 597~t 4038s 14769(N-S) 15978~t 456+TTC-nc 4964s 13368s 5237s 2395d~r 6692s 523+CA-nc 21+ 7 L0k 456-nc 9950s 4225(M-V) 4204s +16 L1c1 4454A~t 513-514d-nc 15317(A-T) 11269s 8790s 1598~r 1291~r 5321s 522-523d-nc 12142~t 8383s 5153s 4937s 5899+5C-nc 8087s L0a2a 2220~r 4025(T-M) 709~r 9300(A-T) 12172~t 4197s 12696s 12501s 9037(M-V) 6179s 6249(A-T) 6629s 14088s L1c3 14182s 9545s L1b 5162s 4562s 851~r 9581s 13281s 9150s 12798s 13542s 9887s 8392s 6644s 2768~r 7673(I-V) 57+ 13 1822~r 9554s 6150(V-I) 4688s 629~t 9620s 13966(T-A) 32+ 9438(G-S) 15692(M-V) 14280(S-P) 8545(A-T) 7473~t 15+ 7 7960s 4496s 6253(M-T) 9266s 4824(T-A) 3106~r 2283~r 9755s 15061s 4598s 12235~t 15697s 14560s 12172~t 8284-nc L1c1a 2755~r 5111s 6407s 4916s L0a2b L1b1 6962s 9545s 5231s 467-nc 4345~t 3210~r 10790s 8264s 13116s 2245~r 13129(P-S) 15449(F-L) 12234~t 8420(T-A) 9755s 5393s 2863~r 5656-nc 15927~t 7076s 13741(T-A) 2308~r 6260s 3434(Y-C) 11299s 14371s 14110(F-L) 5553~t 3513s 6297s 9178(V-M) 522-523d-nc 15951~t 12810s 9230s 4123(I-V) 13879(S-P) 5984s 7498~t 4755s 13470s 1598~r 5216s 13980s 5147s 7337s 8237(I-V) 8188s 14040s 9305s 7444(Ter-K) 7785(I-T) 12609s 6182s 3927s 7789s 5237s 597~t 8784s 456-nc8281-8289d-nc 13680s 14221s 11974s 8329G~t 5988s 4706s 8928s 4506(I-V) 8668(W-R) 8582(A-V) 11830s 4080s 5741-nc 14131s 7967s 593~t 8877s 523+CA-nc 8280A-nc 8251s 13928C(S-T) 13827s 8937s 15346s 9716s 7915s 7419(E-K) 8348~t 7202s 8754s 3083~r 10389s 3666s 10031~t 1503~r 8619s 9311s 8680s 8417(L-F) 11025(L-P) 14620s 14007s 11452s 9494s 9647s 9214(H-R) 1888~r 10792s 4596(V-I) 9054s 10398(A-T) 9966(V-I) 11317s 12303~t 15346s14180(Y-C) 7868(L-F) 10927s 12768s 14298G(I-L) 11164s 4937s 9824s 11167s 12019s 11812s 13722s 14759G(R-G) 9270(L-F) 11002s 11257s 12501s 11252(I-V) 4959(A-T) 9861(F-L) 12400(T-A) 13752As 15301s 11659s 11928(N-S) 2674~r 11654(T-A) 5054s 10084(I-T) 12930Ts 7660s 12542(A-V) 14212s 14503s 4634s 11959s 11337(N-S) 12681s 14034s 7693s 13708(A-T) 14239s 14756(M-V) 5054Cs 12477s 10454~t 11854s 14338s 15663(I-T) 9272s 13981(P-S) 14581s 12540s 12540s 14393(V-A) 9336(M-V) 14794s 14971s 12477s 15929~t 12732s 6752s 16017~t 15884-nc 15025s 5826~t 15289s 12940(A-T)15160s 1850~r 8856s 14182s 13966(T-A) 5978s 11770s 14913(S-L) 7424s 13105(I-V) 523+CA-nc 15191s 12780s Af44 Af92 Af48 Af16 Af40 Ad1 Ad2 Ad3 Af20 As46 Ad4 Af05 Af33 Af37 Af102 Af97 Af98 Af09 Af55 Af25 Af27 Af24 Af17 Af22 Af54 Af87 Af88 Af89 Af90 Af91 Af53 As24 Ad5 Ad6 Ad7 Ad8 Ad9 Af01 Af02 Af06 Ad10 Af38 Af94 Af39 Af95 Af96 Af34 Af35 Af93 Ad11 Ad12 Af99 Af100 E W E E W W W S W MP MP MP MP MP MP S S S S S S S S W W W W W W SW W W W W W BP BP C W BPBP BP BP BP BP BP BP W W MP MP MP SW +3 825T~r 8655s 10688s 10810s 3594s 2416~r 4104s 8206s +5 +5 7256s 7521~t 9221s 13650s 10115s 13590s 3918s 769~r 74+ 14 6260s 8104s +7 1018~r L2 12609s 11944s 3693s 65+ 8 1442~r L2d +4 522-523d-nc L3 2789~r 2332~r 7175s 7624As 479-nc 9855(I-V) 13105(I-V) 1822~r 1719~r 7274s 719~r 13470s 3396s 4388~t 12236~t 5147s 7771s 1211~r 3450s 4218s 5300s 15110(A-T) 3537s 7424s 11914s 5773~t 5601~t 7861s 15217s 870~r 3591s 8618(I-T) 13803s L4g 5821~t 6221s 7819As 9509s 2159~r 3825s 13886(L-P) 14566s 522-523d-nc 6182s 9449s 8527s 9575s 1706~r 2332~r 4562s 513-nc 14284s 32+ 8 680~r 6722s 10086(N-D) 8932(P-S) 11590s 2358~r 709~r 3254A~t 5069T(M-I) 709~r + 8676s 13914As 39 7 9950s L2a 4158s 3200A~r 3434(Y-C) 5585-nc 4500(S-P) 12693s 709~r 9365s 15311(I-V) L3d 11440s 4370~t 13928C(S-T) 6231s 6014s 5128(N-S) 3504s 921~r L3h 15784s 6752s 9731As 15824(T-A) 14769(N-S) 4767(M-V) 13958C(G-A) 8856s 7885s 7805(V-I) 3729s 9932s 12280~t 15940d~t 15514s 2831~r 516-nc 12+ 3 15939~t 5027s 15849(T-I) 9130A(L-M) 8278+6C-nc 4619s 3394(Y-H) 523+CA-nc 6680s 1719~r 12534s + 1193~r 15940d~t 3777s 523+CA-nc + 5331A(L-I) 83+ 9554s 8383s 8227s 24 8 11503s 11518s 3498Gs L2a1 31 9 14260s 12873s 6480(V-I) 27+ 7 4688s 4859s 709~r 6216s 10143(G-S) 5581-nc 3010~r 534-nc 5814~t# 9941s 8413s 10265s L3b 13368s 4117s L2c 10389s 15314(A-T) 3420s 10373s 14272s 13752(I-M) L3d1 8251s 7055s 15735(A-V) 6663(I-V) 4038s 9083(L-P) L2a2 11767s 6713s 1040~r 10700s 9139(A-T) 15434(L-F) L3f1 5316(A-T) 8080s 9377s 11914s 15479(F-L) 14584s 15799s 10899(N-S) 8767(T-A) 3589G(L-V)731~r 10245s 7388s 9438(G-S) 14118s 5471s 10955s 2142+AG~r 5046(V-I) 5492s 522-523d-nc 513-nc 12354s 15514s 11002s 9079(N-D) 15115s 15928~t 5054Cs 1503~r 11404s 9827s 5090s 11692s 13759(A-T) 12408s 15803(V-M) 8387(V-M) 8329G~t 4048(D-N)11353s 9971s 2093~r 4200s 8047s 6480(V-I) 10903s 12438s 12441s 7765s 3474s 15061s 10044~t 7663s 13888(C-R) 13928C(S-T) 15880s 7664(A-T) 12948s 10586s 11944s 11935s 5777~t 1438~r 8485s 10685s 15924~t 8727s 6383s + 6445(T-M) 12903s 5250s 9053(S-N) 12070s 8393(P-S) 13434s 4048(D-N) 5246s + 10289s 15326(T-A) 11647s 8721s 14059(I-V) 32 9 13440s 12630s 12189~t 15865s 9022(A-T) 8485s 11 6 3200~r 9080(N-S) 12753s 9299s 6431s 4259(T-I) 9509s 15217s 7301s 9067(M-V) 15664s 11383s 5605~t 11563s 13068s 13239s12950(N-S) 15758(I-V) 11800s 9151(I-V)5162s 3505(T-A) 15258(D-G) 15749s 9077(I-T) 4596(V-I) 12142~t 14161s 15550s 12188~t 6842s 11963(V-I) 12432s 8251s 12172~t 10481s 13443s 13020s 15883s 15883s 11401s 7648s 2416~r L3d3 8799s 13827s 14845s 14034s 4393A~t 13708(A-T) 13651(T-A) 12354s 14142s 5814A~t# 2083~r 522-523d-nc L2b 6614s 10530(V-M) 15930~t 8749s 15208s 15263(P-S)13708(A-T) 14634(M-V) 32+ 6431s 9139(A-T) 13926s 13948(P-S) 8944(M-V) 9968As 3834s 8964s 6806s 13135(A-T) 1413~r 3394(Y-H) 5371G(S-W) 9452s 10790s 7076s 6026s 15458(S-P)14299s 15110(A-T) 15565s 12397(T-A) 14110(F-L) 14912G(S-A) 12804s 10828s 13708(A-T) 1694~r 1193~r 14007s 12507s 8503s 15703s 14687~t 10084(I-T) 10837s 13167s 13924(P-S) 13711(A-T) 4949s 3441s 14162(A-V) 15777C(S-T) 15103Gs 14180(Y-C) 11887s L3d3a 8387(V-M) 1888~r 8790s 6061(I-T) 15514s 5483Gs 5211(L-F) 12406(V-I) 522-523d-nc 3253~t 15289s 15110(A-T) 7493~t 9350s 8020s 16000~t 8478(S-L) 5581-nc 7316s 7569~t 5261s 15497(G-S) 15217s 13884s 10463~t 13966(T-A)11314s 8487(P-L) 9477(V-I) 9168s 10685s 15697s 15394s 15106s 11029s 15394s 10783Cs 8155s 13116s 8965(I-V) 12557(T-I) 10724s14407(L-F) 15734(A-T) 15299s 15454s 14370s 15777(S-N) 15889~t

Af10 Af21 Af08 Ad13 Ad14 Af80 Af84 Af82 Af83 Af85 Af79 Ad15 Ad16 Af14 Af31 Af04 Af32 Af52 Af36 Af101 Ad17 Ad18 Ad19 Ad20 Ad21 Af81 Ad22 Af29 Ad23 Ad24 Af07 Af18 Af45 Ad25 Af74 Af70 Af71 Af76 Ad26 Ad27 Af68 Af73 Af30 Ad28 Af77 Af50 Ad29 Af72 Af69 Ad30 Af19 Af23 Af26 Af28 Ad31 Af86 Ad32 Ad33 Af46 Af51 E S S W W W W W W W W W W E MP MP MP MP MP MP W W W W W W W W W W C S E W W WWW W WWW W W W W E W W W SS S S S W W W W E E Figure 1, continued

10819s 489-nc

2352~r 7645s +8 14212s 10679s 30+ 7 10400s 11260s 14783s 13800As 14905s L3e 15043s 13967(T-M) 6587s 750~r 10370s 46+ 5 14060(I-T) 5262(A-T) 14152s 15758(I-V) L3e2 6446s 447G-nc 4883s M 4715s 8793s 4823s 482-nc 4916s 1760~r 9509s 522-523d-nc 6455s 4117s 2000~r 5584-nc 6680s 1780~r 5178A(L-M) 7196As 12771s 13105(I-V) 2483~r 1694~r 5186s 4580s 14687~t 930~r 9824s 5843~t 4655s 5252s 3381s + 8584(A-T) L3e1 4167s 4560(V-M) 12403(L-F) 511-nc 5773~t 25 7 + 8790s 6524s 7961s 9266s 49 11 L3i 1799~r 4907s 4500(S-P) 11377s L3e4 14110(F-L) 2177~r 4059As 7040s 15487Ts 12940(A-T) +9 9554s 8396(T-A) 10954s 3010~r D 45+ 10 2262~r 10181s 6092s M3 4721s 4734(T-A) 13359s 1107~r M7 4071s 13500s 1598~r 5774A~t 10667s 8502(N-S) 11260s 8414(L-F) 4092s 3852s 14869s 8823s 6261(A-T) 5319(T-A) 15473(T-A) 5301(I-V) 3552As M8 2626~r 32+ 10 4508s 6221s 10816(K-N) M1 9758s 11827s 14668s 6752s 6179s 2772~r 14311s 7196s 9377s 12810s 12007s 15148s 5585-nc 15601s + 10397s 9545s 8684(T-I) 4850s 8251s 9254s 12248~t 813~r 732~r 17 4 9090s 4386~t 4048(D-N) 5460(A-T) Q 2768~r 8251s 15670s 12635(I-T) 5910(A-T) 11914s 14470s 5442(F-L) 9156s 11024s 13101Cs 6671s 15784s 4958s 8964s 4335~t 15924~t 13293s 6827s 4697s 522-523d-nc D4 2092~r 13263s 4164s 11665s 13197s 12950C(N-T) D5 14025s 14484(-) M V # 13651(T-A) 709~r 9095(L-P) 4973s 9861(F-L) 3206~r 17+ 5 14318(N-S) 12771s 5351s 12091s 15812(V-M) 522-523d-nc 14569s M2a 15930~t 9180s 7738s 18+ 5 14364s 5460(A-T) Q3 M42 2880~r 1888~r 1382C~r 14979(I-T) 1438~r Z M8a 15172s 15670s 9233s 5231s 523+CA-nc D1 5821~t 6680s 8392s 593~t 3921s 12007s 8020s 9180s C 3394(Y-H) Q1 3506(T-I) 573+4C-nc 7675s 15942~t 12025s 13104s 8964s 3483s 3316(A-T) 15930~t 1719~r 7684s L3e3 14207(T-I) 8392s 8697s 523+CA-nc 12026(I-V) M7c 4025(T-M) 5417s 2281C~r 8805s 12236~t 9824s D4a 6446s 10816Ts M7a 7853(V-I) 1391~r 9196s 4703s 9064(A-T) 3663s 5471s 7169s 9254s 3426s 8838s 2145~r 10845(T-I) 12715(T-A) 7112s 11914s 522-523d-nc 7697(V-I) Z2 12405s 4707(L-F) 12248~t 13149Ts 11016(S-N) 11248s 15313s 522-523d-nc 13368s 5021s 10688s 4907s 13105(I-V) 14110(F-L) 10874s 14687~t 13059s D5a 1888~r 12501s 12811(Y-H) 6221s 15884-nc 12477s 15719(Y-H) 773~r 4047s 15939~t 6104s 11016(S-N) 11350s 3705s 14315(S-N) D4k3 15323(A-T) 13928C(S-T) 14016s 595+C-nc 12618s 15106s 7388s 522-523d-nc 14323s 522-523d-nc 4363~t 5899+5C-nc 8050s 11150(A-T) 6378s 14323s 14094s 573+5C-nc 3797G(T-S) 13321(I-V) 493-nc 4715s 10801s 3915s 522-523d-nc 15287(F-L) 522-523d-nc 11017s M7b 10790s 8101s 11701s 12346(H-Y) 14356s D4b2 15514(K-N) 6596s 2232+A~r 7861s 13879(S-P) 522-523d-nc 6590s 13368s 8688Cs 4924(S-N) 729~r 11084(T-A) 11260s 9314s 13145(S-N) 15317(A-T) 469G-nc 7673(I-V) 5821~t 10682s 14581s 7337s 3483s 522-523d-nc 14404s 11149s 573+2C-nc 8251s 8227s 11368s 9353s 13660(N-D) 4965(S-G) 15862s 4248s 11944s 6338s 11902s 15313s 10084( I -T) 5100s 6551s 3606s 11257s 9965s 4131s 1402C~r 12381s 10208s 14148s 13980s 8473s 7853(V-I)13968s 11447(V-M) 9758s 8155s 15236(I-V) 11452s 13749s 9052(S-G) 4655s 14494s 12362(T-I) 14185Ts 9296s 8659(T-A) 10754s15080(T-A) 11560s 15326(T-A) 14173s 13359s 12996s 15886-nc 14605s 9109(I-V) 11839s 14668s

Af49 Af47 Ad34 Af64 Af63 Af65 Af66 Af67 Ad35 Ad36 Af75 Ad37 Af78 Af15 Af43 As18 As16 As10 As32 As15 As47 As02 As01 As07 As08 As27 As05 Am01 Am07 Am08 Am09 Am02 Am10 Am11 Am14 As74 As33 As09 Am04 Am03 Am06 As30 As71 As06 As45 As04 Oc03 Oc04 Oc09 Oc14 Oc01 Oc17 Oc12 Oc13 E E W W W W W W W W W W W EESW SW SW SW SW SW E E E E E E S SSN S S SSE E E C S N SW EMME E E M PPP A A 8701(A-T) 10398(A-T) +6 9540s 10873s 15301s 48+ 9

663~r 15884C- nc 6221s N 12705s 8404s 5417s 1736~r 8251s 1719~r 6371s 42+ 6 4248s 10238s 13966(T-A) 4824(T-A) 8281-8289d-nc 9644s R 11467s S N9 709~r 12501s 14470s 3970s 15607s 8794(H-Y) 49+ 11 14305s 12308~t 1243~r 6392s 709~r 5231s 15884-nc 12372s 3505(T-A) B 10310s 45+ 6 1811~r 12358(T-A) N1 X 5465s 709~r 522-523d-nc 5046(V-I) 13928C(S-T) A 2792~r 12372s 9123s 499-nc 1598~r 709~r P 10358s 1598~r 5460(A-T) 573+4C-nc 1719~r 4991s 1811~r U 3197~r 14+ 7 10238s 827~r 8584(A-T) 8277- nc 9477(V-I) 12358(T-A) 1888~r 8994s 4529Ts 14364s 4820s 8829s 8278+2C-nc R12 13617s 13594(S-G) 8251s F 15148s 9698s + 4917(N-D)# 11674s 13590s 9950s 10031~t 522-523d-nc 523+CA-nc 4703s 499-nc 45+ 13 22 5 5291s 10034~t 1438~r 11947s X2 B4a 15535s 10398(T-A)10398(T-A) 4011s 3116~r 6518s 5999s 8269s 12414s 10398(T-A) 4200s 3203~r 3480s U8 14793(H-R) U5 7768s 522-523d-nc 12361(T-A) 11061(S-F) +6 3720s 9266s + N9a 11491s 13780(I-V) 8393(P-S) 4695(F-L) 3882s 6386s 9055(A-T) 3738s 18 4 14182s 709~r 15223s 5390s 10506(T-A) + 14384C(A-G) 16+ 6 15043s 13708(A-T) B4b 12950(N-S) 4703s 4122s 522-523d-nc 4646s 5240s 13 4 1888~r 15508s 13681(T-A) 5426s 13934(T-M) 6047s 10550s 15218(A-T) U5a 15040s 15924~t 15927~t 15310s 5417s 8859s 2218~r 6392s W 9254s 15662(I-V) 6045s 14139s 14620s 11299s U5b S1 961~r 1406~r + 3547(I-V) 8027(A-T) 14890s 7581~t U9 6455s 9667(N-S) 7843s 455d-nc 5339s 522-523d-nc 1721~r 15 5 15292s 15851(I-V) 5263(A-V) 6152s 15454s 11332s 14167s 7978s 5471s 13263s 4977s 8906(H-R) 12879s 573+5C-nc 7055s 9548s 12801s 2757~r 4732(N-S) 522-523d-nc 15784s I X2b 15927~t 12630s 10876s 15693(M-T) 14798(F-L) 8104s 5656-nc 5474s 6734s 6473s 9386s 13104s 3531s 18+ 4 9365s 981~r 12582s 13351s 13889(C-Y) 13637(Q-R) 5093s 3645s 523+CA-nc 15670s 11197s 9148s 15466s 10283s 5498s 5580- nc 9950s R11 9881s 14070s 3834s 10733s 9531(T-A) 3714s 5910(A-T) 3915s P2 11732s U3 1189~r K 11107s 12616s 5580- nc X2e 11177(P-S) 10398(T-A) 15954C-nc 9299s 9716s 13145(S-N) 10619s 3212~r 4129(T-A) 6641s 6599s W3 6116s 2849~r 813~r I1 13020s 522-523d-nc U4 10398(T-A) 13359s 5821~t 11404s 11350s 10768s 9845s 8027(A-T) 7864s 7804s 5074(I-T) 11+ 5 B5b 499-nc 523+CA-nc 14+ 4 12136s 10685s F4 12172~t 13734s 12615s 14034s 12678s 12007s 13402(I-V) 15287(F-L) 6182s 502- nc 499- nc B2 9972(I-V) 3010~r 4811s K2 13857s 13708(A-T) U1a 15907~t 13111s 6146s 497-nc K1 U8a 15791(I-V) 14148s 8281-8289d-nc 8269s 10978s 15190s 523+2(CA)-nc 9682(M-T) 5508s 13215s 6267(A-T) 3434(-) Y C 6026s 14094s 8285+4C-nc 15067s 14818Cs 6119s 3760G(S-A) 16000T~t 13759(A-T) 573+5C-nc 11914s 523+CA-nc 573+3C-nc 3348s 523+CA-nc 7257(I-V) 13344s 8706T(M-I) 11932s 513-nc 15951~t 9070G(S-A) 9095(L-P)14049s 5095(I-T) 14989s U2e 1585~r 15924~t 4856s 4561( V-A) 960d~r 10589s 12961(S-G) 13135(A-T) 1503~r 14485s 15265s 7813s 573+5C-nc 15562s 8857(G-S) 4944(I-V) 10907(F-L) 5773~t 5592~t 5081s 5387s 13889(C-Y) 15034s 522-523d-nc 3159+T~r 508-nc 3777s 3732s 4895s 6653s 709~r 14514(M-V) 9360s 11009s 6975s 11025(L-P) 8901s 5130s 5585-nc 14233s 4050s 15894~t 8020s 505-nc 12354s 5460(A-T) 7278(F-L) 7340s 5744-nc 4384~t 15115s 9752s 14866s 7729s 7388s 7076s 6023s 8050s 8575s 3591s 15848(T-A) 6164s 9932s 13117(I-V) 6767s 8578(P-S) 15217s 9932s 8818s 8604s 8410s 3206~r10786s 6366(V-I) 9966(V-I) 8602(F-L)6761s 9800s 11470s 13708(A-T) 11437s 8603(F-S) 9293s 11383s 11204(F-L) 9448(Y-C) 6293s 7569~t 9746s 8790s 13326s 15850s 9899s 10118s 13708(A-T) 12888s 11485s 8400(M-T) 11914s 10097Cs 15355s 9833s 13665s 14129(T-I) 13899s 15758(I-V) 13105(I-V) 8521s 15412s 15930~t 13422s 15908~t Am17 Oc06 As73 Oc11 As12 As23 Eu51 Eu01 Eu74 As34 As40 Am05 Eu55 As72 Am12 Am15 Am16 As28 As26 As03 Oc10 Oc16 As17 Af42 Eu26 Eu83 Eu36 Eu81 As14 As29 Eu29 Eu94 Eu25 As36 Eu76 Eu30 Eu03 Eu75 Oc05 Eu77 Eu86 Eu28 Eu88 Eu37 Eu38 Eu56 Eu96 Eu22 Eu45 Eu59 Eu85 Eu89 Eu78 S A E A SW SW H N S SW SW N H E C S S E E E A P SW E S N N N SW SW N N N SW N N N S A NNN NNN N H S H H N N N

11719s 16+ 6 4216(Y-H) # 11251s pre-HV 14766(T-I) 2442~r + 15452A(L-I) 37+ 11 3847s 16 6 13188s HV 2706~r 489-nc 709~r 573+C-nc 4580s 10398(T-A) JT 522-523d-nc 7028s 1888~r 6248s 15904~t + 12612s 4917(N-D)# 15110(A-T) 91 (pre-HV)1 8994s 13708(A-T) 8697s 15314(A-T) 9449s H 10463~t 2355~r 3010~r 10211s 14440s V 709~r 1438~r 6776s 4793s 456-nc 9380s 8940s 14978s 13368s 4997s 573+C-nc J 7094s 72+ 4769s 4336~t# 14905s 11353s 2757~r 462-nc 7444(Ter-K) 13680s 15674(S-P) 8910s H1 H3 H7 H6a 3010~r 15607s 9986s 2281~r 8014s 477-nc 522-523d-nc 12341(T-I) 714~r 3305-nc 14798(F-L) 14121s 8371s 7891s 4733s 5348s 15928~t + 960d~r 9923s 3796(T-A) 15323( A-T) 3903s H2 H5a 4727s 9545s + 16 6 14129(T-I) 6253(M-T) 9150s 8277-nc 6755s 5839~t 522-523d-nc 14 6 827~r 14224s 8803(T-A) 11516s 7762s 11253(I-T) 15589As 15172s 13731s 13359s 4688s 15833s 11152s 12633As 11812s 9116(I-T) 8864(V-A) 723~r 8119s 8843(I-T) 3335(I-T) J1c 5198s 13477(A-T) 7205s 10344C(I-L) 14374s 85+ T 14233s 14407G(L-F) 9007(T-A) 960+C~r 13708(A-T) 7184s 482-nc + 15409s 11 4 8994s 1462~r 13707s 11281s 1005~r 508-nc T1 3915s 3394(Y-H) 3203~r 15916~t 3125~r 9899s T2 4883s 3666s 6260s 930~r 783~r 549-nc 7805(V-I) 7184s 4976s 14553(V-I) 3804s 5147s 5460(A-T) 13966(T-A) 9234(M-V) 522-523d-nc 6261(A-T) 721~r 13215s 9554s 5836~t 7258(I-T) 1888~r 6314s 4580s 12397(T-A) 8281-8289d-nc 9524s 8167s 10529s 10321(V-A) 8854(A-T) 522-523d-nc 4994s 1888~r 10822s 11080s 15212(I-V) 2387~r 4823s 11914s 6935s 10192(S-F)# 12346(H-Y) 15479(F-L)

Af41 As38 As37 Eu08 Eu04 Eu54 Eu32 Eu92 Eu05 Eu06 Eu17 Eu18 Eu02 Eu93 Eu95 Eu09 Eu10 As42 Eu12 Eu39 Eu41 Eu87 Eu43 Eu44 Eu80 Af13 Eu34 As35 Eu58 Eu24 Eu48 Eu49 Eu50 Eu11 Eu90 Eu07 As41 Eu15 Eu14 As25 As13 Eu40 Eu47 Eu57 Eu52 Eu53 Eu91 Eu23 Eu84 Eu46 Eu35 Eu82 Eu42 Eu33 As11 Eu27 Eu79 E SW SW N S H S N SSS S S N N N N SW N S S N HHN W N SW H S HHH G N S SW S S SW SW S H H H H N S N H N N S N SW N N Figure 2

5761 U 5826 12206 A U C 12138 G C G C C G U A U G A U C G 5773 A U 5814 C G A 5821 A U A G C C A U A U A G A G C G U C A U A C U G U G A A U C G C A G C A U C C C A U U C C U U A U Cys A C U U G A G G G U A A His A U A A C A A C C C U U A AG A A U U A G natural variation AC G C C G A A U confirmed pathological U G U A U variation C A U A U U G A 100% conserved in 31 U A 12172 C A mammalian tRNAs G A U G 15953 >90% conserved in 31 A 3304 15888 mammalian tRNAs A G C 3230 U A G C N C G difference in the human U A and mammalian consensus C G U A U A A U U A A U U U U G C G C U A U U U U C A A A C A U C A A A A C C G U C U C A U A U G G A C A G U A A G G C C A U A C Thr U C G C G A G U U U A A G UAA C A G 15930 U G C Leu U C G A G C G U A A A A C G U AC A C 15927 A U G C A U U A 15924 A U C A A C U A C A U G U U A U A A