Evolution of Endurance Running Genes Across Primates

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Natalia T. Grube CUNY Hunter College

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Evolution of endurance running genes across primates

Natalia Grube

Submitted in partial fulfillment of the requirements for the degree of Master of Arts, Department of Anthropology, Hunter College The City University of New York

2019

April 12, 2019 Michael E. Steiper Signature of First Reader [Professor Michael E. Steiper]

April 2, 2019 Herman Pontzer Date Signature of Second Reader [Professor Herman Pontzer]

Table of Contents I. Introduction ...... 3 II. Hypothesis ...... 12 III. Materials & Methods ...... 13 IV. Results ...... 21 V. Discussion ...... 25 VI. Conclusion ...... 28 VII. References ...... 29

Figures

1. 1000 genomes allele frequency ...... 9 2. Phylogeny of branch test model ...... 12 3. MAFFT Alignment ...... 18 4. ACE Tree...... 23 5. ACTN3 Tree ...... 25

Supplementary

1. ACE Protein alignment ...... 36 2. ACTN3 Protein alignment ...... 47

2 I. Introduction

Bipedalism has conferred major phenotypic changes that distinguish hominins from other primates and is considered one of the most significant adaptations to occur in human evolution

(Harcourt-Smith and Aiello 2004; Aiello and Dean 1990). The origin of habitual bipedalism, derived from fossil evidence, indicates that Australopithecus, a group of early hominin species present ~1-4.5 mya, were obligate bipeds (Aiello and Dean 1990; Ward 2002; Bramble and

Lieberman 2004). The essentially modern human body shape, consistent with obligate striding bipedalism, is first apparent in early Homo erectus (~2 mya) (Bramble and Lieberman 2004).

Structural adaptations, such as specialized limb, foot and postcranial adaptations, are evidence for improved walking performance in open habitats and ability to cope with structural balancing whilst engaged in locomotion (Harcourt-Smith and Aiello 2004; Bramble and Lieberman 2004;

Jungers 1988). Running, in addition to walking, has been considered a mode of locomotion that has also influenced human evolution (Bramble and Lieberman 2004).

Humans are comparatively poor sprinters compared to other quadruped cursors (Bramble and Lieberman 2004; Garland 1983). Humans, compared to other mammals, are unusually specialized for endurance running (ER) – the ability to run many kilometers over extended periods of time using aerobic respiration. Human specific phenotypes to have evolved solely for the function of ER, this hypothesis emerges as the ER hypothesis (Bramble and Lieberman 2004;

Carrier et al. 1984; Lieberman et al. 2009; Mattson 2012).

Bramble and Lieberman (2004) identify many morphological features in Homo that are favorable for long distance running, the most significant traits are outlined in Table 1.

3 Table 1 Morphological and Genetic traits linked to ER

Morphological features Functional Role Genus Reference

Long Achilles tendon Energetics Homo * Bramble et al. 2004

Energetics Homo * Bramble et al. 2004, Longitudinal Arch of the Foot Venkadesan et al. 2009 Long Legs Energetics H. erectus Bramble et al. 2004

Close-packed calcaneocuboid joint Energy Storage H. habilis Bramble et al. 2004

Nuchal Ligament Stabilization Homo * Bramble et al. 2004, Lieberman et al. 2009 Enlarged posterior and anterior semicircular Stabilization H. erectus Bramble et al. 2004 canals Stabilized Sacroiliac Joint Trunk Stabilization H. erectus Bramble et al. 2004

Expanded Gluteus Maximus Stabilization H. erectus Bramble et al. 2004

Genetic Variants

ACTN3 R577X ER Homo * Liebenberg, 2006 ACE I polymorphism ER Homo * Ostrander et al. 2009

* Evidence for this trait are predicted to have evolved in Homo. However, this is not definitive, and more research is needed.

Anatomical structures that are argued to have been relevant for the energetic cost of

running are the Achilles tendon, longitudinal arch of the foot, and evolution of long legs

(Bramble and Lieberman 2004; Alexander 1991). The biomechanics of running differ from those

of walking. Long spring-like tendons acquired in humans, in contrast to apes, are improve the

efficiency of running (but not walking) by minimizing the mechanical work required (Bramble

and Lieberman 2004). Connecting the heel to major plantar flexors of the foot, the Achilles

tendon is substantially more developed in Homo than in australopithecines (Bramble and

Lieberman 2004; Latimer and Lovejoy 1989). The Achilles tendon is argued to be the most

important of the spring-like tendons regarding ER and is suggested to have evolved in the genus

Homo (Bramble and Lieberman 2004). Along with the Achilles tendon the longitudinal arch of

4 the foot is yet an additional spring important for running. The longitudinal arch is supported by the plantar arch and functions as a spring, conserving energy when individuals are engaged in running (Bramble and Lieberman 2004; Ker et al. 1987).

Research on the evolution of the transverse arch, which is critical for stiffness in the foot of Homo and has a more functional role in bipedalism, evolved well before the emergence of the longitudinal arch and other adaptations for ER (Venkadesan et al. 2017). Model reconstruction of early hominin foot adaptations reveals that A. afarensis, the fossil foot Burtele, and H. naledi all have a transverse arch and likely possessed a human-like gait (Venkadesan et al. 2017). In addition, structural mechanisms that are critical for establishing the longitudinal arch are reduced or absent in H. florensis (Harcourt-Smith 2002; Jungers et al. 2009). The absence or reduction of longitudinal arch in these species indicates poor ER capabilities, and because the longitudinal arch is present in H. sapiens this morphological trait most likely evolved in early Homo

(Venkadesan et al. 2017).

During running, rather than walking, humans increase speed by increasing stride length.

Increased stride length is due in part to the adaptation of long legs. Relative leg length is much greater in H. erectus (1.8 mya) than A. afarensis, showing that leg length relative to body mass greatly increased in the genus Homo (Bramble and Lieberman 2004), which suggest that walking is not the sole factor contributing to the elongation of limb length in legs. Evidence for the impact of ER on hominin evolution is highlighted when comparing the performance criteria of distance between various mammals and humans (Bramble and Lieberman 2004). There are many quadrupedal cursors that can easily sprint faster than humans as mentioned above. However, sustained running speeds over long distances are only reserved for few mammalian cursors, such as the African wild dogs (Lycaon), hyenas, and horses (Holekamp et al. 2000; Bramble and

5 Lieberman 2004; Carrier et al. 1984). Endurance strategies in many of these species have been argued to be adaptations that are critical for hunting and scavenging (Carrier et al. 1984;

Lieberman et al. 2009; Liebenberg 2006).

Hominin ER may have first been practiced in the context of scavenging (Liebenberg

2006; Bramble and Lieberman 2004). Indeed, if ER made early hominins more successful at scavenging, which in turn conferred a fitness advantage, then selection would act to increase genotypes associated with endurance running. Increases in this phenotype over two million years would have “preadapted” Homo for persistence hunting establishing early Homo as dominant hunters in Africa (Bramble and Lieberman 2004; McRae 2011; Liebenberg 2006). Additionally, it is important to consider how the environment may have shaped ER traits. Humans are unique from other primates and mammals in that we have evolved morphological adaptations to help with the increased thermogenic effects of running rather than in walking (Aiello and Wells 2002;

Cheuvront and Haymes 2001). Morphological adaptations such as hairlessness and an increase in eccrine sweat glands allow humans to keep cool while engaging in locomotion such as running especially if their engaging in long distance running or running during hot periods of the day

(Jablonski 2008). Since humans are not constrained by high environmental temperatures like other mammalian cursors (Lieberman et al. 2009), our early ancestors were more likely to scavenge or engage in persistence hunting during periods of the day when there was high heat.

Although this trait has not been extensively studied in non-human primates, humans are the only primates that engage in this behavior, making it a unique trait (Lieberman et al. 2009; Bramble and Lieberman 2004). Phenotypic adaptations in humans that would have been essential to the energetic, mechanical and thermoregulatory challenges posed by running (i.e. increase in eccrine

6 sweat glands, hairlessness) are adaptations not found in any other primate species except hominins (Table 1).

Lieberman and Bramble (2009) have proposed three hypothesis for the timing of the evolution of ER capabilities : 1) ER capabilities evolved after the divergence of the Pan and hominin lineages (6 mya), 2) ER evolved during the transition between australopithecine and early Homo or 3) ER capabilities evolved more recently in Homo, perhaps with H. erectus, H. heidelbergensis or H. sapiens (Lieberman et al. 2009; Bramble and Lieberman 2004).

Factors contributing to the evolution of endurance running in humans are going to be governed by both genetic and environment, whereby the environment acts as a selective pressure. For example, human legs have 50% of both slow oxidative (type I) muscle fibers and fast glycolytic (type II) muscle fibers (Lieberman et al. 2009; McArdle et al. 2010). Increases in percentage of slow twitch (type I) muscle fibers in trained endurance athletes (Gollnick et al.

1973) highlights physiological ability of skeletal muscle acclimatization in humans during times of intense endurance training. Lieberman and Bramble (2004) propose that the high percentage of slow twitch muscle fibers necessary for endurance running may have originated in humans from a null mutation in the -actinin 3, ACTN3 gene.

ACTN3 encodes for a sarcomeric enzyme that functions in skeletal muscle fibers and is responsible for the production of powerful muscle contractions (Yang et al. 2003; Ma et al. 2013;

Mills et al. 2001). ACTN3 functions solely in fast glycolytic (type II) muscle fibers. This gene is located on human chromosome 11 and is 16,487 basepairs (bp) in length (MacArthur and North

2004; Mills et al. 2001; MacArthur et al. 2007). A common nonsense mutation in this gene is known as the R577X allele, which leads to deficiency in the production of -actinin 3 in type II muscle fibers (MacArthur et al. 2007; MacArthur et al. 2008; Ma et al. 2013). This null

7 mutation, which leads to a lack of enzyme production in type II muscle fibers (which function best for well-trained power athletes (Lieberman et al. 2009; Thayer et al. 2000), is linked to endurance performance in elite athletes (Maffulli 2013; Ma et al. 2013; Yang et al. 2003;

MacArthur and North 2004). Knockout mouse studies on the R577X mutation has found that

ACTN3 deficient individuals display better recovery from fatigue, longer twitch half relaxation time (Chan et al. 2008), a shift in muscle metabolism toward a more efficient aerobic pathway and an increase in endurance performance (MacArthur et al. 2008; MacArthur et al. 2007).

Clearly, it is reasonable to hypothesize a connection between the evolution of the ACTN3 gene and ER (Bramble and Lieberman 2004).

The allele frequency of the specific R577X mutation in human populations is variable. A search in the 1000 Genome Browser shows that the frequency of the 577X allele is over all at a lower frequency compared to the R577 allele. The frequency of the 577X allele in 1000 genomes population data set is 40% while the other allele is represented in 60% of human populations

(The Genomes Project Consurtium, 2015). Population genetic data of the rs1815739 (R577X snp) in 1000 genomes maps the frequency of the ACTN3 R577X variant, with the X allele encoding nucleotide position T which transcribes into a stop codon (X), and the C nucleotide encoding an arginine associated with typical allele (R), is consistent with frequency data published in Yang et al. (2003). Yang et al. (2003) report that the frequency of this allele in human populations is also variable with 25% in Asian populations, 18% in Europeans and less than 1% in African Bantu populations. Frequency variation of the R577X is suggested by Yang et al. (2003) to confer differential fitness in humans under certain environmental conditions.

This may mean that the ACTN3 X allele frequency shifted in human populations that inhabited different environmental areas. Notably this allele frequency is higher in non-African

8 populations (Figure 1). It is tempting to speculate that this allele conferred a greater advantage to migrating human populations and increased in frequency as these populations settled out of

Africa. In the context of early human migration, long distance migration may have been a selective pressure acting on endurance abilities in humans and may be why we see an increase frequency of this allele in African subpopulations such as Europeans, Asians etc.

In addition to this, genotyping of this allele in other primates has found no instance of the R577X allele outside humans suggesting that this allele arose in the genus Homo (Mills et al. 2001;

McRae 2011).

McRae (2011) provides a thorough analysis on the evolution of the ACTN3 gene in human populations. Phylogenetic analysis of the 577X allele in 48 individuals from different populations (African, Asian and Europeans) yields that the X allele arose prior to human migration out of Africa 60,000 years ago (Watson et al. 1997) or 86,000 (64,500-107,500) years ago (McRae 2011). However, the ER hypothesis suggests that ER is a commonality to all humans. If this trait is unique to our species and may have conferred a fitness advantage, we would expect to see high frequency of alleles associated with endurance in every human population. Interestingly, this is not reflected in the allele frequency of the 577X endurance allele. The allele frequency of ACTN3 shows that endurance running allele is not a commonality

9 in all humans and is therefore inconsistent with the endurance running hypothesis, which suggests that ER is a trait that is universal across all humans. Alternatively, this can also be a form of balancing selection, in which this genetic polymorphism is maintained in a population because it incurs a fitness advantage in different environments (Hedrick 1998).

Similar to ACTN3, the ACE gene also has an allele variant that correlates with endurance athletic performance (Puthucheary et al. 2011; Montgomery et al. 1998; Tsianos et al. 2004;

Ostrander et al. 2009). ACE is found on human chromosome 17 and is 21,310 bp in length. ACE functions as a key enzyme in the renin angiotensin system, a system involved in homeostasis of blood pressure and fluid balance. ACE’s main function is to convert angiotensin I into a vasoactive angiotensin II protein (Gard 2010; Tiret et al. 1992; Puthucheary et al. 2011).

Angiotensin II causes vasoconstriction of blood vessels causing blood pressure to increase

(Sparks et al. 2015). Two alleles of ACE are related to physical performance; the “insertion” allele (I) and the “deletion” (D) allele.

The ACE insertion (I) allele, has an Alu insertion of 287 bp which results in the production of lower levels of ACE serum and therefore lower levels of angiotensin II. This allele is linked to improved performance of endurance events in elite athletes (Montgomery et al. 1998;

Rigat et al. 1992; Ma et al. 2013). Conversely, the ACE deletion (D) allele, does not have the 287 bp insertion and has increased levels of ACE serum and therefore increased levels of angiotensin

II. This has been shown to be associated with short-burst power events like sprinting in varying athletic populations (Ostrander et al. 2009; Tsianos et al. 2004; Montgomery et al. 1998). The frequency of ACE I allele, allele linked to endurance, is higher than ACE D allele (Zerbino et al.

2017). Frequency of the endurance allele (ACE I) in 1000 genome population genetics data shows that endurance allele is at a higher frequency in every human population except European

10 populations (Figure 1). The ACE I allele occurs in linkage disequilibrium with rs4343(A) SNP while the alternative genotype, ACE D allele occurs in linkage disequilibrium with rs4343(G)

SNP (Abdollahi et al. 2008). The frequency of this allele in human populations is not consistent with the ER hypothesis which suggest that ER is trait that is universal across all modern humans.

Balancing selection may also be contributing to the allele frequency of the null mutation in

African sub populations. I speculate that the ACTN3 R577X frequency variation of the null allele is maintained because it may have an adaptive advantage in different environments, i.e. as we migrated out of Africa.

Studies of the origin and evolution of the ACE gene suggest that the Alu polymorphism occurred after the human-chimpanzee split, between 230,000 to 1 million years ago (Rieder et al.

1999). Interestingly, Tung et al (2007) identified a similar Alu polymorphism in wild and captive baboon populations at ACE. This study found complex variation of the homeostatic enzyme in baboons and shows that ACE serum production in baboons varies with age (Tung et al. 2007).

Patterns of within human variation for ACE (I) and ACTN3 577X is not consistent with the endurance running hypothesis as this hypothesis implies that this trait evolved adaptively in humans and therefore we should see genotypes associated with ER phenotype across all human populations or again it may be something more complex like balancing selection, where the I/D allele is being maintained in African sub populations. However, two alleles associated with endurance running in humans (ACE I, ACTN3 577X) do not reflect this. In order to discern how the evolution of these endurance variants have been acting on or shaping human evolution it is necessary to look at the phylogenetic differences of these two genes comparing the human lineage to other primate species (Figure 2).

I. Hypothesis

Here I examine the pattern of molecular evolution at ACE and ACTN3 in humans and nonhuman primates to determine whether these genes are evolving differently along the human lineage than in other primates. I do this by phylogenetically comparing humans to a sample of non-human primates. I hypothesize that a pattern of differential evolution is acting on ACE and

ACTN3 in humans when compared to non-human primates, because ER is a uniquely human trait as evidenced by structural adaptations that have shaped the human body. To address the endurance running hypothesis, I will discern how these two genes associated with ER have evolved across primate species. First, I tested whether ACE and ACTN3 have been evolving adaptively across 13 and 14 primate species, respectively. Second, I tested whether patterns of molecular evolution at ACE and ACTN3 differ between humans and the non-human primates. If

12 ER, a uniquely human trait, was a critical adaptation in human evolution I hypothesize that both genes should show differential signs of selection in the human lineage when compared to other primate lineages.

II. Materials & Methods

Sequences

The methods outlined here apply to both genes analyzed: ACE and ACTN3. Primate sequences were obtained from the UCSC genome browser (genome.ucsc.edu). Human sequences for both

ACE and ACTN3 where obtained using the Hg38 assembly. Primate genome assemblies and gene sequence coordinates are listed in Table 2 and 3, isomer versions listed in Table 4. These were converted from the human (Hg38) assembly using BLAT tool in the UCSC genome browser and/or obtained via the “convert” option in other genomes (Table 4). To compare biological relatedness of nucleotide sequences BLAST NCBI local alignment tool was used

(blast.ncbi.nlm.nih.gov/Blast.cgi). In Table 4, query coverage refers to the proportion or effective size of your sequence being compared. If query coverage is low, fewer data (nucleotides) are being compared. “Ident” percentage indicates the extent to which two nucleotide sequences are identical. Finally, sequences were oriented such that they were all in the same complement

(Table 4).

Table 2 UCSC Genome coordinates for ACE Species UCSC Name Genome Coordinates Assembly Name Genome Reference chr17:63476071- Homo sapiens hg38 (2013) Consortium Human 63499380 Reference 38 JH650321:1020752- Max-Planck Institute Pan paniscus panPan1 (2012) 1043588 panpan1 Pan chr17:62231070- CSAC Pan_troglodytes- panTro4 (2011) troglodytes 62253620 2.1.4 chr5:20252640- Wellcome Trust Sanger Gorilla gorilla gorGor3 (2011) 20275207 Institute gorGor 3.1 chr17:53171716- WUSTL version Pongo abelii ponAbe2 (2007) 53194843 Pongo_abelii-20.2 Nomascus chr19:28194570- Gibbon Genome nomLeu3 (2013) leucogenys 28216633 Sequencing Consortium chr16:55330602- Baylor College of Papio anubis papAnu2 (2012) 55353025 Medicine Chlorocebus chr16:57783684- Vervet Genomics chLSab2 (2014) sabaeus 57808466 Consortium Macaca chr16:61001460- Beijng Genomics rheMac3 (2010) mulatta 61023953 Institute, Shenzhen Macaca chr16:60765336- macFas5 (2013) Washington University fascicularis 60788878 Saimiri JH378264:2207726- saiBol1 (2011) Broad SaiBol 1.0 boliviensis 2230521 Callithrix chr5:82198255- WUSTL version calJac3 (2009) jacchus 82223146 Callithrix jacchus-3.2 Broad Institute and Baylor Microcebus scaffold_823:267076- micMur2 (2015) College of Medicine murinus 292521 Mmure_2.0 Otolemur GL873537:16342081- otoGar3 (2011) Broad Institute garnettii 16363733

Table 3 UCSC Genome coordinates for ACTN3 Species UCSC Genome Coordinates Assembly Name Name Homo sapiens hg38 (2013) chr11:66,546,841- Genome Reference Consortium 66,563,329 Human Reference 38 Pan paniscus panPan1 JH650582:5325817- Max-Planck Institute panpan1 (2012) 5344203 Pan troglodytes panTro4 chr11:64500519-64519619 CSAC Pan_troglodytes-2.1.4 (2011) Gorilla gorilla gorGor3 chr11:63435027-63457197 Wellcome Trust Sanger Institute (2011) gorGor 3.1 Pongo abelii ponAbe2 chr11:9372153-9392812 WUSTL version Pongo_abelii-20.2 (2007) Nomascus nomLeu3 chr4:86941346-86962137 Gibbon Genome Sequencing leucogenys (2013) Consortium Papio anubis papAnu2 chr14:7549310-7568677 Baylor College of Medicine (2012) Chlorocebus chLSab2 chr1: 7706848-7726633 Vervet Genomics Consortium sabaeus (2014) Macaca mulatta rheMac3 chr14:8123186-8142604 Beijng Genomics Institute, Shenzhen (2010) Macaca macFas5 chr14:7930475-7950146 Washington University fascicularis (2013) Saimiri saiBol1 JH378199:2888607- Broad SaiBol 1.0 boliviensis (2011) 2906030 Tarsius syrichta TarSyr2 KE931117v1:189364- Washington University (2013) 209094 Microcebus micMur2 KQ058516v1:1357388- Broad Institute and Baylor College of murinus (2015) 1372390 Medicine Mmure_2.0 Otolemur otoGar3 GL873653:3550804- Broad Institute garnettii (2011) 3568359

Table 4 NCBI Blast data ACTN3 Homo sapiens actinin alpha 3 gene/pseudogene, transcript variant 1, coding, mRNA, NM_001104 Species Query Cover Ident Reverse BLAT Convert Pan paniscus 97% 99% x Pan troglodytes 95% 99% x Gorilla gorilla 98% 98% x Pongo abelii 98% 95% x Nomascus leucogenys 98% 95% x Papio anubis 97% 93% x Chlorocebus sabaeus 97% 93% x x Macaca mulatta 94% 92% x x Macaca fascicularis 97% 92% x x Saimiri boliviensis 89% 87% x Tarsius syrichta 58% 79% x Microcebus murinus 67% 81% x Otolemur garnettii 55% 80% x x

ACE Homo sapiens angiotensin I converting enzyme (ACE), transcript variant 1, mRNA, NM_000789 Species Query Cover Ident Reverse BLAT Convert Pan paniscus 98% 98% x Pan troglodytes 85% 98% x Gorilla gorilla 91% 98% x x* Pongo abelii 97% 96% x Nomascus leucogenys 92% 95% x x* Papio anubis 98% 93% x Chlorocebus sabaeus 91% 93% x x Macaca mulatta 93% 93% x Macaca fascicularis 97% 93% x Saimiri boliviensis 90% 88% x Microcebus murinus 40% 81% x x Callithrix jacchus 83% 88% x Otolemur garnettii 21% 79% x x

*Data was retrieved in 2015. To retrieve Gorgor3 Nomleu2 sequence where converted from Hg38->Hg19->primate using UCSC genome Lift-over tool (https://genome.ucsc.edu/cgi-bin/hgLiftOver)

16 Table 5 NCBI CDS coordinates ACTN3 , chromosome 11 Exons Start Position End Position CDS 1 66546938 66547084 544 690 2 66551239 66551353 4845 4959 3 66551528 66551647 5134 5253 4 66554045 66554131 7651 7737 5 66554536 66554623 8142 8229 6 66555130 66555208 8736 8814 7 66555286 66555367 8892 8973 8 66556145 66556230 9751 9836 9 66557133 66557225 10739 10831 10 66557699 66557929 11305 11535 11 66558027 66558174 11633 11780 12 66559236 66559386 12842 12992 13 66559968 66560076 13574 13682 14 66560171 66560311 13777 13917 15 66560573 66560755 14179 14361 16 66561227 66561361 14833 14967 17 66561458 66561637 15064 15243 18 66562022 66562168 15628 15774 19 66562257 66562322 15863 15928 20 66562796 66562954 16402 16560 21 66563035 66563193 16641 16799 ACE, chromosome 17 Exons Start Position End Position CDS 1 63477095 63477343 35 283 2 63477931 63478098 871 1038 3 63479007 63479100 1947 2040 4 63479769 63479912 2709 2852 5 63480337 63480528 3277 3468 6 63481091 63481188 4031 4128 7 63481566 63481738 4506 4678 8 63482466 63482689 5406 5629 9 63483029 63483173 5969 6113 10 63483460 63483558 6400 6498 11 63483849 63483971 6789 6911 12 63484330 63484541 7270 7481 13 63485236 63485372 8176 8312 14 63486557 63486715 9497 9655 15 63486986 63487073 9926 10013 16 63488648 63488791 11588 11731 17 63488941 63489132 11881 12072 18 63490954 63491051 13894 13991 19 63491209 63491381 14149 14321 20 63493436 63493659 16376 16599 21 63493922 63494066 16862 17006 22 63494372 63494470 17312 17410 23 63496394 63496516 19334 19456 24 63496798 63496985 19738 19925 25 63497137 63497366 20077 20306

Alignment (MAFFT)

Codon sequences were aligned using the MAFFT alignment tool (Katoh and Standley 2013), with the “add frags” option (http://mafft.cbrc.jp/alignment/server/add). MAFFT “add frags” allows for fragmentary sequences to be aligned to an alignment of longer sequences (Figure 3).

Thus, CDS regions of the genes were aligned against whole gene sequences. Once CDS regions where aligned with gene sequences, CDS regions for all fourteen-primate species were manually trimmed for both ACE and ACTN3 genes. Bioinformatics sequence manipulation translate tool

(bioinformatics.org/sms2/translate) was used to translate CDS regions to ensure no stop codons where within the sequence and that the alignments were in an open reading frame. If there was an error within a sequence it was manually corrected for (see results). The final alignment for

ACE was 3951 bp., for ACTN3, 2709 bp in length (Supplementary Figures 1 and 2).

Figure 3: MAFFT add-fragments figure retrieved from (https://mafft.cbrc.jp/alignment/server/add_fragments.html)

ACE Sequence alignment

After aligning the exons, I translated each exon to find issues with the translation (e.g. gaps in reading frame) that required manual adjustment to preserve an open reading frame. Of the 13 primate species analyzed, eight species required manual adjustment. In all these cases, it seemed likely that these problems reflected sequencing errors or un-sequenced bases, not actual base

changes. Most of these adjustments are due to fragmented DNA sequence, meaning incomplete base pairs from the genome sequence for certain coding regions for the gene that were unable to translate into proteins. For Pan paniscus stop codons in exon 20 of the sequence were replaced with N’s. Pan troglodytes exons 7, 24, and 25 were completely removed and replaced with N’s due to fragmentary DNA sequences. Additionally, exon 1 and 4 in Pan troglodytes base pairs were replaced with N’s due to stop codons. Exon 22 had three gaps in Pongo abelii sequence, this sequence was aligned with the rest of the primates and thus is more likely prone to alignment error than a three base pair deletion. To translate this sequence exon 22 of Pongo abelii was removed. Exon 1 of Pongo abelii, due to DNA fragmentation was replaced with N’s. Macaca mulatta also had a fragmented exon 1 it was replaced by N’s. Callithrix jaccus within exon 4 was out of frame. A region (actcaccaacatcctggct) has an extra T pushing the exon out of frame, this was probably due to an error because the rest of the gene translates. Therefore, the T in the above region was removed (actcaccaacatcctggc-). Exon 20 of Callitrhix jaccus had N’s added to the beginning of sequence to keep in alignment, additionally exon 5 (gacgatctggagcacctc→ gacgatctggagcacct-) and 13 (accgcacatcccaggtggtgtggaaac → accgcacatcccaggtggtgtggaa) each had a base pair removed to keep alignment in frame. Microcebus murinus exon 3 was completely removed due to DNA fragmentation, additionally exon 25 sequence was edited to keep in frame

(ggccccaccgcaggcccccagttttggc → ggccccaccgcaggcccagttttggc), this caused translation errors and subsequent removal of Microcebus murinus gene entirely. Tarsius syrichta gene was removed entirely due to it being a totally fragmented and incomplete sequence. Once sequences were adjusted, the final alignment was 3951 base pairs in length with 13 primate species excluding Tarsius syrichta and Microcebus murinus.

ACTN3 Sequence alignment results

Of the fourteen primate sequences analyzed only one species had their sequence manually adjusted. Pan paniscus exon 12 was replaced with N’s to translate sequence. The entire gene for

Callithrix jacchus was removed due to poor alignment. The final alignment of ACTN3 gene was

2709 base pairs in length with 14 primate species excluding Callithrix jacchus.

Selection Analysis using PAML

To determine whether selection has acted on the ACE and ACTN3 proteins, I compared the rate of synonymous and nonsynonymous substitution rates at these genes using the PAML package, i.e using Ka/Ks or dN/dS tests (Yang 2007). The CODEML program (part of PAML) detects adaptive molecular evolution based under various models of codon substitution using likelihood ratio tests (Yang 2007). CODEML was implemented to detect selection on coding regions separately for both ACE and ACTN3 in my alignment of 13 and14 primate sequences, respectively. For each test, the F3x4 codon model was used and a phylogenetic user tree was provided. This phylogenetic tree was based on the currently accepted primate phylogeny

(Perelman et al. 2011). CODEML was implemented to detect selection on coding regions separately for both ACE and ACTN3 in my alignment of 13 and 14 primate sequences, respectively. Two different models were used to analyze patterns of adaptive molecular evolution acting on both the ACE and ACTN3 genes. In the null model, a single  (the ratio of nonsynonymous/synonymous substitution rates = dn/ds or Ka/Ks) is estimated for all of the branches of the tree (Yang 1997). This null model is specified by m=0 and Nsites = 0 omega = 1.

The null model was compared to an alternative model, the branch model. In the branch model 

is allowed to vary among branches in the phylogeny and is used to detect positive selection acting on a particular lineages (Yang and Nielsen 1998). In this study, my hypothesis predicts that there is some change or differentiation in adaptive evolution among the human branch. Thus,

I am testing whether humans have different  compared to the rest of the primate branches because ER is a unique trait to hominins. The branch model was therefore named the “human selection model” in which  varied among the human branch for both ACE and ACTN3 genes

(see Figure 2). The likelihoods for the null and human selection model were compared using a likelihood ratio test (LRT) for both ACE and ACTN3. In a LRT, a significant difference between models is assessed by chi-squared distribution test with 1 degree of freedom. When a significant difference is detected, the model with the higher likelihood is favored. If a significant difference is not detected, the simpler model is preferred, i.e. the model with fewer parameters (Yang,

2007).

III. Results

ACE

BLAST NCBI results

Identity scores for all sequences range from 79%- 98% (Table 4.), generally indicating that ACE primate sequences are similar to the ACE human sequence and one another. This is expected amongst primates since all species tested belong to the same order, especially in a gene with is inferred to have a generally conserved function across this group. Query coverage for Otolemur garnettii (21%) and Microcebus murinus (40%) was low and this is most likely due to sequences not being complete.

PAML Analysis

Before examining patterns of selection acting on the human lineage, I analyzed patterns of adaptive evolution across the entire primate phylogeny as described in the null model. Coding regions for ACE were analyzed for adaptive molecular evolution across all primate sequences

(n=13). PAML analysis yielded signatures of evolution that are consistent with purifying selection (dn/ds =  < 1) for ACE ( = 0.12). Subsequently, we examined whether ACE in the human lineage, adapted to endurance, is evolving differently from other primate lineages who do not harbor the trait of endurance running i.e. human selection model (branch test). When the entire coding region for ACE was tested, the human selection model incorporating a different  value for the human lineage, is not a better fit to the data than a model fitting only one  to all lineages (Figure 4, Table 6). The  for the human lineage, for ACE was lower ( = 0.10) than the remaining primate lineages ( = 0.12) though this finding was not statistically significant (p =

0.78). Thus, evolution at the ACE is consistent with negative purifying selection across humans and all other primates studied. Selection acting on the human branch is not different than the rest of the primate branches, therefore the null model is favored for ACE. Summary statistics for LRT are shown in Table 6.

ACTN3

BLAST NCBI

Identity scores for all sequence range from 79%- 99% (Table 4.), generally indicating that

ACTN3 primate sequences are similar to the ACTN3 human sequen1ce and one another. This is expected amongst primates since all species tested belong to the same order. Query coverage for

Otolemur garnettii (55%) and Microcebus murinus (67%) was the lowest, this is most likely due to sequences not being fully complete.

PAML Analysis

Before examining patterns of selection acting on the human lineage, we analyzed patterns of adaptive evolution across the entire primate phylogeny as described in the null model. Coding regions for ACTN3 were analyzed for adaptive molecular evolution across all primate groups.

PAML analysis yielded that signatures of evolution are consistent with strong purifying selection for ACTN3 ( = 0.03). Subsequently, we examined whether ACTN3 in the human lineage, adapted to endurance, is evolving differently from other primate lineages that do not harbor the trait of endurance running, I call this analysis the human model (branch test). When the entire coding region was tested for ACTN3, the human model incorporating a different  value for the human lineage is a better fit to the data than the null model fitting only one  to all lineages (p =

0.0009) (Table 7, Figure 5). The  for the human lineage for ACTN3 was higher ( = 0.3; Table

7) than the remaining primate lineages ( = 0.03). Thus, both models predict that coding regions for ACTN3 are consistent with negative purifying selection in all primate lineages. However, the human selection model (branch test), allowing dn/ds to vary in humans, fits significantly better than model that constrained dn/ds to a single measure across all primate lineages. These results indicate that humans are under significantly weaker purifying selection than the rest of the primate lineages.

IV. Discussion

The pattern of molecular evolution at ACE and ACTN3 gene provides insight on how both genes evolved across humans and other primate species. Both genes are under purifying selection in humans and other primates inferring that the biological structures of both genes are conserved across primate species (Loewe 2008). Negative selection, also called purifying selection, is a form of selection that prevents amino acid changes to presumably optimal protein structures. Negative selection ensures that beneficial variants are maintained in an evolving population. ACE is evolving the same across all primate species in our primate sample. In contrast, ACTN3 is under differential selection in humans when compared to our phylogenetic

sample of primate species. Given this finding, I chose to further examine the human ACTN3 protein sequence.

The peptide chain of ACTN3 for Homo sapiens has five amino acid differences compared to other primate sequences (Table 8).

Notably the 523-amino acid position occurs in strong linkage disequilibrium with 577X allele (Mills et al. 2001). When expressed in heterozygotes (523R/577X) there is no deleterious effect (Mills et al. 2001). To determine the origin of the 577X allele (which occurs in strong linkage disequilibrium with 523R allele), Mills et al. (2001) genotyped over sixty non-human primates whom were all homozygous for “wild-type” alleles in exon 15 (523Q) and 16 (577R), suggesting that the 523R and 577X polymorphism originated after the human-chimpanzee divergence six million years ago. This evidence shows that there is no support for a R577X as a balanced polymorphism in any mammal species tested in this study other than humans. It is not known when the other amino acid changes occurred in time or how, if at all, they affect ACTN3 function. The ACTN3 peptide is 901 amino acids long, and of these 901 amino acids, the

secondary structure of ACTN3 is not fully known (Franzot et al. 2005). My analysis highlighted

4 other changes in the amino acid structure. At position 342, humans have an isoleucine (I) instead of a valine (V). Both amino groups have hydrophobic side chains and therefore may be a conservative non-synonymous substitution. At position 446 humans have a hydrophobic leucine instead of a nonpolar serine. Nonpolar molecules are considered hydrophobic therefore this may also represent a conservative non-synonymous substitution. The non-synonymous substitutions at position 628 and 776 for humans, have polar neutral side chains, cysteine (C) and glutamine

(Q) respectively, instead of arginine (R), an electrically charged side chain, compared to the rest of the primate species. It is not known if these substitutions confer a radical change in the amino acid structure of ACTN3 protein product in humans compared to other primate species. More research on the protein structure of ACTN3 will help to discern how these non-synonymous substitutions impact the function of ACTN3 polypeptide.

Conversely, ACE confers no sign of differential selection in humans. Selective pressure acting on ACE is the same across primate species. ACE serves a critical and functional role in the renin system. Selection acting on ACE is likely conserved across primate species because of its overarching hierarchal role in maintaining blood pressure and homeostasis within the body.

Ka/Ks methods are useful and powerful for detecting adaptive evolution across genetic sites.

However, detecting selection on a small number of amino acid sites within a sequence may be hard to detect by methods such as PAML (Anisimova et al. 2002). Prediction of positively selected sites is unreliable when sequences are very similar and the number of lineages is small

(Anisimova et al. 2002). When using methods such as PAML, comparing models (LRT’s) is deemed as a reliable method to ensure robustness of statistical analysis in addition to increasing the number of lineages sequenced (Anisimova et al. 2002). Our analysis used comparative

models (LRT’s) to discern how evolution was acting on ACE and ACTN3 in addition to using fourteen primate lineages to ensure robustness of statistical analysis (see methods).

V. Conclusion

Morphological evidence has shown that endurance running may have been a critical adaptation for early human evolution, migration and hunting (Lieberman et al. 2009; Bramble and Lieberman 2004). Genetic adaptations, in addition to morphological adaptations, must have acted on the genomes of humans to increase the frequency of alleles associated with endurance running. While the frequency of endurance alleles in human population are variable (McRae

2011), my study does not offer support for adaptive evolution at the ACE and ACTN3 genes along the human lineage. My results show that there is differential selection operating on

ACTN3 gene in human compared to non-human primates, however. The effect is likely due to the five amino acid substitutions along the human lineage. The degree which these amino acid changes affect the peptide structure and function of the ACTN3 gene in humans is unknown and should be investigated further. ER is a complex polygenic trait. Adaptive genetic changes that favored ER along the human lineages remain to be discovered.

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S1. ACE PROTEIN ALIGNMENT

NomLeu3_ACE ------chlSab2_ACE MGA------ASGRRGPG------PLLQPLLL------macFas5_ACE MGA------ASGRQGPG------PLLQPLLL------rheMac3_ACE ------papAnu2_ACE MGA------AWGRRGPA------PLLQPLLL------gorGor3_ACE MGA------ASGRRGPG------LLLPLPL------panPan1_ACE ------hg38_ACE_ MGA------ASGRRGPG------LLLPLPL------panTro4_ACE ------ponAbe2_ACE MGA------ASGRRGPG------LLLLLLPLS------calJac3_ACE MGA------ASGHRGPGP------PLLPSLPLPLPPLLL saiBol1_ACE ------otoGar3_ACE MGA------SSGLRASGP------PPVLSLLPL----LQL

NomLeu3_ACE ------chlSab2_ACE LLLLPPQPALALD-PGLQPGNFSADEAGAQLFAQSYNSSAEQVLFQSVAA macFas5_ACE LLLLPPQPALALD-PGLQPGNFSADEAGAQLFAQSYNSSAEQVLFQSVAA rheMac3_ACE ------papAnu2_ACE LLLLPPQPALALD-PGLQPGNFSADEAGAQLFAQSYNSSAEQVLFQSVAA gorGor3_ACE LLLLPPQPALALD-PGLQPGNFSADETGAQLFAQSYNSSAEQVLFQSVAA panPan1_ACE ------hg38_ACE_ LLLLPPQPALALD-PGLQPGNFSADEAGAQLFAQSYNSSAEQVLFQSVAA panTro4_ACE ------X-PGLQPGNFFADEAGAQLFAQSYNSSAEQV--QSVAA ponAbe2_ACE LLLPPPQPSLALD-PGLQPGNFSADEAGAQLFAQSYKXXXXXXXXXXXXX calJac3_ACE LLLLPPPPALALD-PGLQPGNFPADEAGAQLFAQSYNSSAEHVLFQRIAA saiBol1_ACE ------otoGar3_ACE LLLPPSPAAQALD-PGLLPGNFSADEAGAQLFAQSYNSSAEQVLFQSTAA

NomLeu3_ACE ------EEAA chlSab2_ACE SWAHDTNITAENARR------QEEAA macFas5_ACE SWAHDTNITAENARR------QEEAA rheMac3_ACE ------EEAA papAnu2_ACE SWAHDTNITAENARR------QEEAA gorGor3_ACE SWAHDTNITAENARR------QEEAA panPan1_ACE ------EEAA hg38_ACE_ SWAHDTNITAENARR------QEEAA panTro4_ACE SWAHDTNITAENARR------QEEAA ponAbe2_ACE XXXXXXXXXXXXXXX------XEEAA calJac3_ACE SWAHDTNITAENARR------QEKAA saiBol1_ACE ------EEAA otoGar3_ACE SWAYSTNITEENAQR------QEEAV

NomLeu3_ACE LLSQEFAEAW---GQKAKELY---EPMWQNFTDPELRKIIG--AVRTLGS chlSab2_ACE LLSQEFAEAW---GQKAKELY---EPIWQNFTDPELRKIIG--AVGTLGS

macFas5_ACE LLSQEFAEAW---GQKAKELY---EPIWQNFTDPELRKIIG--AVGTLGS rheMac3_ACE LLSQEFAEAW---GQKAKELY---EPIWQNFTDPELRKIIG--AVGTLGS papAnu2_ACE LLSQEFAEAW---GQKAKELY---EPIWQNFTDPELRKIIG--AVGTLGS gorGor3_ACE LLSQEFAEAW---GQKAKELY---EPIWQNFTDPQLRRIIG--AVRTLGS panPan1_ACE LLSQEFAEAW---GQKAKELY---EPVWQNFTDPQLRRIIG--AVRTLGS hg38_ACE_ LLSQEFAEAW---GQKAKELY---EPIWQNFTDPQLRRIIG--AVRTLGS panTro4_ACE LLSQEFAEAW---GQKAKELY---EPVWQNFTDPQLRRIIG--AVRTLGS ponAbe2_ACE LLSQEFAEAW---GQKAKELY---EPIWQNFTDPELRKIIG--AVRTLGS calJac3_ACE LVTQEFAEAW---GQKAKELY---EPIWQNFTDPELRKIIG--AVRTLGP saiBol1_ACE LLTQEFAEAW---GQKAKELY---EPIWQNFTDPELRKIIG--AVRTLGP otoGar3_ACE LLNQ--AEAW---GQKAKELY---GPIWQNFTDPKLRKVIR--AVCTLGP

NomLeu3_ACE ANLPLAKRQQYNALLSNMSRIYSTAKVCLPNKTATCWSLDPDLTNI--LA chlSab2_ACE ANLPLAKRQQYNALLSNMSRIYSTAKVCLPNKTATCWSLDPDLTNI--LA macFas5_ACE ANLPLAKRQQYNALLSNMSRIYSTAKVCLPNKTATCWSLDPDLTNI--LA rheMac3_ACE ANLPLAKRQQYNALLSNMSRIYSTAKVCLPNKTATCWSLDPDLTNI--LA papAnu2_ACE ANLPLAKRQQYNALLSNMSRIYSTAKVCLPNKTATCWSLDPDLTNI--LA gorGor3_ACE ANLPLAKRQQYNALLSNMSRIYSTTKVCLPNKTATCWSLDPDLTNI--LA panPan1_ACE ANLPLAKRQQYNALLSNMSRIYSTAKVCLPNKTATCWSLDPDLTNI--LA hg38_ACE_ ANLPLAKRQQYNALLSNMSRIYSTAKVCLPNKTATCWSLDPDLTNI--LA panTro4_ACE ANLPLAKRQQYNALLSNMSRIYSTAKVCLPNKTATCWSLDP---XI--LA ponAbe2_ACE ANLPLAKRQQYNALLSNMSRIYSTASLPPHRLPPAVLNQDPDLTNI--LA calJac3_ACE ANLPLAKRQQYNTLLSNMSRTYSTAKVCLPNKTATCWSLDPELTNI--LA saiBol1_ACE ANLPLAKRQQYNTLLSNMSRTYSTAKVCLPNKTATCWSLDPELTNI--LA otoGar3_ACE ANLPLAKQQQYVSLQTNMSRIYSTTKVCFPNKTATCWSLDPELTNI--LA

NomLeu3_ACE SSRSYAMLLFAWEGWHNAAGIPLKPLY----EDFTALSNEAY-KQD---- chlSab2_ACE SSRSYAMLLFAWEGWHNAAGIPLKPLY----EDFTALSNEAY-KQDGFTD macFas5_ACE SSRSYAMLLFAWEGWHNAAGIPLKPLY----EDFTALSNEAY-KQDGFTD rheMac3_ACE SSRSYAMLLFAWEGWHNAAGIPLKPLY----EDFTALSNEAY-KQDGFTD papAnu2_ACE SSRSYAMLLFAWEGWHNAAGIPLKPLY----EDFTALSNEAY-KQDGFTD gorGor3_ACE SSRSYAMLLFAWEGWHNAAGIPLKPLY----KDFTALSNEAY-KQDGFTD panPan1_ACE SSRSYAMLLFAWEGWHNAAGIPLKPLY----EDFTALSNEAY-KQDGFTD hg38_ACE_ SSRSYAMLLFAWEGWHNAAGIPLKPLY----EDFTALSNEAY-KQDGFTD panTro4_ACE SSRSXAMLLFAWEGWHNAAGIPLKPLY----EDFTALSNEAY-KQDGFTD ponAbe2_ACE SSRSYAMLLFAWEGWHNAAGIPLKPLY----EDFTALSNEAY-KQDGFTD calJac3_ACE SSRSYAMLLFAWEGWHNAAGIPLKPLY----EDFTALSNEAY-KQDXXXX saiBol1_ACE SSRSYAMLLFAWEGWHNAAGIPLKPLY----EDFTALSNEAS-KQDGFSD otoGar3_ACE SSRSYARLLFAWEGWHDTVGIPLKALY----QDFTTISNEAY-RQDGFSD

NomLeu3_ACE ------DLYQQLEPLYLNLHAFVRRALHRRYGDRYIN chlSab2_ACE TGAYWRSWYNSPTFEDDLEHLYQQLEPLYLNLHAFVRRALHRRYGDRYIN macFas5_ACE TGAYWRSWYNSPTFEDDLEHLYQQLEPLYLNLHAFVRRALHRRYGDRYIN rheMac3_ACE TGAYWRSWYNSPTFEDDLEHLYQQLEPLYLNLHAFVRRALHRRYGDRYIN papAnu2_ACE TGAYWRSWYNSPTFEDDLEHLYQQLEPLYLNLHAFVRRALHRRYGDRYIN gorGor3_ACE TGAYWRSWYNSPTFEDDLEHLYQQLEPLYLNLHAFVRRALHRRYGDRYIN panPan1_ACE TGAYWRSWYNSPTFEDDLEHLYQQLEPLYLNLHAFVRRALHRRYGDRYIN

hg38_ACE_ TGAYWRSWYNSPTFEDDLEHLYQQLEPLYLNLHAFVRRALHRRYGDRYIN panTro4_ACE TGAYWRSWYNSPTFEDDLEHLYQQLEPLYLNLHAFVRRALHRRYGDRYIN ponAbe2_ACE TGAYWRSWYNSPTFEDDLEHLYQQLEPLYLNLHAFVRRALHRRYGDRYIN calJac3_ACE XXXXXRSWYESPTFEDDLEHLYHQLEPLYLNLHAFVRRALHRRYGDRYIN saiBol1_ACE TGAYWRSWYDSPTFEDDLEHLYHQLEPLYLNLHAFVRRALHRRYGDRYIN otoGar3_ACE TGAYWRSWYNSATFEEDLEHLYHQLEPLYLNLHAYVRRALHRRYGDRYIN

NomLeu3_ACE LRGP------IPAHLLGDMWAQSWENIYDMVVPFP--- chlSab2_ACE LRGP------IPAHLLGDMWAQSWENIYDMVVPFP--- macFas5_ACE LRGP------IPAHLLGDMWAQSWENIYDMVVPFP--- rheMac3_ACE LRGP------IPAHLLGDMWAQSWENIYDMVVPFP--- papAnu2_ACE LRGP------IPAHLLGDMWAQSWENIYDMVVPFP--- gorGor3_ACE LRGP------IPAHLLGDMWAQSWENIYDMVVPFP--- panPan1_ACE LRGP------IPAHLLGDMWAQSWENIYDMVVPFP--- hg38_ACE_ LRGP------IPAHLLGDMWAQSWENIYDMVVPFP--- panTro4_ACE LRGP------IPAHLLGDMWAQSWENIYDMVVPFP--- ponAbe2_ACE LRGP------IPAHLLGDMWAQSWENIYDMVVPFP--- calJac3_ACE LRGP------IPAHLLGDMWAQSWDNIYDMVVPFP--- saiBol1_ACE LRGP------IPAHLLGDMWAQSWDNIYDMVVPFP--- otoGar3_ACE LRGP------IPAHLLGDMWAQSWDNLYDMVVPFP---

NomLeu3_ACE ------DKPNLDVTSTMLQQGWNATHMFRVAEEF---FTSL chlSab2_ACE ------DKPNLDVTSTMLQQGWNATHMFRVAEEF---FTSL macFas5_ACE ------DKPNLDVTSTMLQQGWNATHMFRVAEEF---FTSL rheMac3_ACE ------DKPNLDVTSTMLQQGWNATHMFRVAEEF---FTSL papAnu2_ACE ------DKPNLDVTSTMLQQGWNATHMFRVAEEF---FTSL gorGor3_ACE ------DKPNLDVTSTMLQQGWNATHMFRVAEEF---FTSL panPan1_ACE ------DKPNLDVTSTMLQQGWNATHMFRVAEEF---FTSL hg38_ACE_ ------DKPNLDVTSTMLQQGWNATHMFRVAEEF---FTSL panTro4_ACE ------DKPNLDVTSTMLQQXXXXXXXXXXXXXX---XXXX ponAbe2_ACE ------DKPNLDVTSTMLQRGWNATHMFRVAEEF---FTSL calJac3_ACE ------DKPNLDVTSTMLQQGWNATHMFRVAEEF---FVSL saiBol1_ACE ------DKPNLDVTITMLQQGWNATHMFRVAEEF---FVSL otoGar3_ACE ------GKPNLDVTSTMVKQGWNATHMFRVAEEF---FTSL

NomLeu3_ACE ELSPMPPEFWEGSMLEKPADGREVVCHASAWDFYNRKDFRIKQCTRVTMD chlSab2_ACE ELSPMPPEFWEGSMLEKPADGREVVCHASAWDFYNRKDFRIKQCTRVTMD macFas5_ACE ELSPMPPEFWEGSMLEKPADGREVVCHASAWDFYNRKDFRIKQCTRVTMD rheMac3_ACE ELSPMPPEFWEGSMLEKPADGREVVCHASAWDFYNRKDFRIKQCTRVTMD papAnu2_ACE ELSPMPPEFWEGSMLEKPADGREVVCHASAWDFYNRKDFRIKQCTRVTMD gorGor3_ACE ELSPMPPEFWEGSMLEKPADGREVVCHASAWDFYNRKDFRIKQCTRITMD panPan1_ACE ELSPMPPEFWEGSMLEKPADGREVVCHASAWDFYNRKDFRIKQCTRVTMD hg38_ACE_ ELSPMPPEFWEGSMLEKPADGREVVCHASAWDFYNRKDFRIKQCTRVTMD panTro4_ACE XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXIKQCTRVTMD ponAbe2_ACE ELSPMPPEFWEGSMLEKPADGREVVCHASAWDFYNRKDFRIKQCTRVTMD calJac3_ACE GLSPMPPEFWAESMLEKPADGREVVCHASAWDFYNRKDFRIKQCTRVTMD saiBol1_ACE GLSPMPPEFWAESMLEKPADGREVVCHASAWDFYNRKDFRIKQCTRVTMD

otoGar3_ACE GLSPMPPEFWAESMLEKPADGREVVCHASAWDFYNRKDFRIKQCTQVTMD

NomLeu3_ACE QLSTVHHEMGHIQYYLQYKDLPVSLRGG--ANPGFHEAIGDVLALSVSTP chlSab2_ACE QLSTVHHEMGHIQYYLQYKDLPVSLRGG--ANPGFHEAIGDVLALSVSTP macFas5_ACE QLSTVHHEMGHIQYYLQYKDLPVSLRGG--ANPGFHEAIGDVLALSVSTP rheMac3_ACE QLSTVHHEMGHIQYYLQYKDLPVSLRGG--ANPGFHEAIGDVLALSVSTP papAnu2_ACE QLSTVHHEMGHIQYYLQYKDLPVSLRGG--ANPGFHEAIGDVLALSVSTP gorGor3_ACE QLSTVHHEMGHIQYYLQYKDLPVSLRGG--ANPGFHEAIGDVLALSVSTP panPan1_ACE QLSTVHHEMGHIQYYLQYKDLPVSLRGG--ANPGFHEAIGDVLALSVSTP hg38_ACE_ QLSTVHHEMGHIQYYLQYKDLPVSLRRG--ANPGFHEAIGDVLALSVSTP panTro4_ACE QLSTVHHEMGHIQYYLQYKDLPVSLRGG--ANPGFHEAIGDVLALSVSTP ponAbe2_ACE QLSTVHHEMGHIQYYLQYKDLPVSLRGG--ANPGFHEAIGDVLALSVSTP calJac3_ACE QLSTVHHEMGHVQYYLQYKDQPVSLREG--ANPGFHEAIGDVLALSVSTP saiBol1_ACE QLFTVHHEMGHVQYYLQYKDQPVSLRKG--ANPGFHEAIGDVLALSVSTP otoGar3_ACE QLSTVHHEMGHVQYYLQYKDQPVSLREG--ANPGFHEAIGDVLGLSVSTP

NomLeu3_ACE EHLYKIGLLD---RVTNDTESDINYLLKMALEKIAFLPFGYLVDQWRWGV chlSab2_ACE AHLHKIGLLD---RVTNDTESDINYLLKMALEKIAFLPFGYLVDQWRWGV macFas5_ACE AHLHKIGLLD---RVTNDTESDINYLLKMALEKIAFLPFGYLVDQWRWGV rheMac3_ACE AHLHKIGLLD---HVTNDTESDINYLLKMALEKIAFLPFGYLVDQWRWGV papAnu2_ACE AHLHKIGLLD---RVTNDTESDINYLLKMALEKIAFLPFGYLVDQWRWGV gorGor3_ACE EHLYKIGLLD---HVTNDTESDINYLLKMALEKIAFLPFGYLVDQWRWGV panPan1_ACE AHLHKIGLLD---NVTNDTESDINYLLKMALEKIAFLPFGYLVDQWRWGV hg38_ACE_ EHLHKIGLLD---RVTNDTESDINYLLKMALEKIAFLPFGYLVDQWRWGV panTro4_ACE AHLHKIGLLD---NVTNDTESDINYLLKMALEKIAFLPFGYLVDQWRWGV ponAbe2_ACE EHLYKIGLLD---CVTNDTESDINYLLKMALEKIAFLPFGYLVDQWRWGV calJac3_ACE AHLYKIGLLD---HVTNDTESDINYLLKMALEKIAFLPFGYLVDQWRWGV saiBol1_ACE AHLYKIGLLD---HVTNDTESDINYLLKMALEKIAFLPFGYLVDQWRWGV otoGar3_ACE AHLHKIGLLD---HVTNDTESDINYLLKMALDKIAFLPFGYLVDQWRWGV

NomLeu3_ACE FSGRTPPSRYNFDWWYLRTKYQGICPPVTRN------ETHFDA chlSab2_ACE FNGRTPPSHYNFDWWYLRTKYQGICPPVTRN------ETHFDA macFas5_ACE FNGRTPPSHYNFDWWYLRTKYQGICPPVTRN------ETHFDA rheMac3_ACE FNGRTPPSHYNFDWWYLRTKYQGICPPVTRN------ETHFDA papAnu2_ACE FNGRTPPSHYNFDWWYLRTKYQGICPPVTRN------ETHFDA gorGor3_ACE FSGRTPPSHYNFDWWYLRTKYQGICPPVTRN------ETHFDA panPan1_ACE FSGRTPNSRYNFDWWYLRTKYQGICPPVTRN------ETHFDA hg38_ACE_ FSGRTPPSRYNFDWWYLRTKYQGICPPVTRN------ETHFDA panTro4_ACE FSGRTPNSRYNFDWWYLRTKYQGICPPVTRN------ETHFDA ponAbe2_ACE FSGRTPPSRYNFDWWYLRTKYQGICPPVTRN------ETHFDA calJac3_ACE FRGRTPPSRYNFDWWYLRTKYQGICPPVTRN------ETHFDA saiBol1_ACE FSGRTPPSRYNFDWWYLRTKYQGICPPVTRN------ETHFDA otoGar3_ACE FSGHTPPSRYNSDWWYLRTKYQGICPPVVRN------ETHFDA

NomLeu3_ACE GAKFHVPNVTPYIRYFVSFVLQFQFHEALCKEAGYEGPLHQCDIYQSTKA chlSab2_ACE GAKFHVPNVTPYIRYFVSFVLQFQFHEALCKEAGYEGPLHQCDIYQSTKA macFas5_ACE GAKFHVPNVTPYIRYFVSFVLQFQFHEALCKEAGYEGPLHQCDIYQSTKA

rheMac3_ACE GAKFHVPNVTPYIRYFVSFVLQFQFHEALCKEAGYEGPLHQCDIYQSTKA papAnu2_ACE GAKFHVPNVTPYIRYFVSFVLQFQFHEALCKEAGYEGPLHQCDIYQSTKA gorGor3_ACE GAKFHVPNVTPYIRYFVSFVLQFQFHEALCKEAGYEGPLHQCDIYQSTKA panPan1_ACE GAKFHVPNVTPYIRYFVSFVLQFQFHEALCKEAGYEGPLHQCDIYQSTKA hg38_ACE_ GAKFHVPNVTPYIRYFVSFVLQFQFHEALCKEAGYEGPLHQCDIYRSTKA panTro4_ACE GAKFHVPNVTPYIRYFVSFVLQFQFHEALCKEAGYEGPLHQCDIYQSTKA ponAbe2_ACE GAKFHVPNVTPYIRYFVSFVLQFQFHEALCKEAGYEGPLHQCDIYQSTKA calJac3_ACE GAKFHVPNVTPYIRYFVSFVLQFQFHQALCQEAGYQGALHQCDIYQSTKA saiBol1_ACE GAKFHVPNVTPYIRYFVSFVLQFQFHQALCQEAGYQGALHQCDIYQSTKA otoGar3_ACE GAKFHIPNGTPYIRYFVSFILQFQFHQALCKEAGHQGPLHQCDIYKSTQA

NomLeu3_ACE -GAKLRKVLQAGSSRPWQEVLKDMVGLDALDAQPLLKYFQPVTQWLQEQN chlSab2_ACE -GAKLRKVLQAGSSRPWQEVLKDMVGSDALDAQPLLNYFQPVTQWLQEQN macFas5_ACE -GAKLRKVLQAGSSRPWQEVLKDMVGSDALDAQPLLNYFQPVTQWLQEQN rheMac3_ACE -GAKLRKVLQAGSSRPWQEVLKDMVGSDALDAQPLLNYFQPVTQWLQEQN papAnu2_ACE -GAKLRKVLQAGSSRPWQEVLKDMVGSDTLDAQPLLNYFQPVTQWLQEQN gorGor3_ACE -GAKLRKVLQAGSSRPWQEVLKDMVGLDALDAQPLLKYFQPVTQWLQEQN panPan1_ACE -GAKLRKVLQAGSSRPWQEVLKDMVGLDALDAQPLLKYFQPVTQWLQEQN hg38_ACE_ -GAKLRKVLQAGSSRPWQEVLKDMVGLDALDAQPLLKYFQPVTQWLQEQN panTro4_ACE -GAKLRKVLQAGSSRPWQEVLKDMVGLDALDAQPLLKYFQPVTQWLQEQN ponAbe2_ACE -GAKLRKVLQAGSSRPWQEVLKDMVGLDTLDAQPLLKYFQPVTQWLQEQN calJac3_ACE -GAKLRKVLQAGSSRPWQEVLKEMVGSDALDAQPLLDYFQPVTQWLQEQN saiBol1_ACE -GAKLRKVLQAGSSRPWQEVLKEMVGSDALDAQPLLDYFQPLTQWLQEQN otoGar3_ACE -GAKLQEVLRAGSSRPWQEVLKNMTGSDALDAQPLLDYFQPVSQWLQEQN

NomLeu3_ACE QQNGEVLGWPEYQWRPPLPDNYPEGIDLVTDEAEASKFVEEYDRTSQVVW chlSab2_ACE QQNGEVLGWPEYQWRPPLPDNYPEGIDLVTDEAEASKFVEEYDRTSQVVW macFas5_ACE RQNGEVLGWPEYQWRPPLPDNYPEGIDLVTDEAEASKFVEEYDRTSQVVW rheMac3_ACE RQNGEVLGWPEYQWRPPLPDNYPEGIDLVTDEAEASKFVEEYDRTSQVVW papAnu2_ACE RQNGEVLGWPEYQWRPPLPENYPEGIDLVTDEAEASKFVEEYDRTSQVVW gorGor3_ACE QQNGEVLGWPEYQWHPPLPDNYPEGVDLVTDEAEASKFVEEYDRTSQVVW panPan1_ACE QQNGEVLGWPEYQWHPPLPDNYPEGIDLVTDEAEASKFVEEYDRTSQVVW hg38_ACE_ QQNGEVLGWPEYQWHPPLPDNYPEGIDLVTDEAEASKFVEEYDRTSQVVW panTro4_ACE QQNGEVLGWPEYQWHPPLPDNYPEGIDLVTDEAEASKFVEEYDRTSQVVW ponAbe2_ACE QQNGEVLGWPEYQWHPPLPDNYPEGIDLVTDEAEASKFVEEYDRTSQVVW calJac3_ACE QKNGEVLGWPEYQWRPPLPDNYPEGIDLVTDEAEARKFLEEYDRTSQVVW saiBol1_ACE QKNSEVLGWPEYQWRPPLPNNYPEGIDLVTDEAEARKFVEEYDRTSQVVW otoGar3_ACE QQNNEILGWPEYQWRPPLPTNYPEGIDLITDEAEANKFVEEYDQVSQVVW

NomLeu3_ACE NEYAEANWNYNTNITTETSKILLQKNMQLANHTLKYGTQARRFDVNHLQN chlSab2_ACE NEYAEANWNYNTNITTETSKILLQKNMQIANHTLKYGTQAR--DVNHFQN macFas5_ACE NEYAEANWNYNTNITTETSKILLQKNMQIANHTLKYGTQAR--DVNHFQN rheMac3_ACE NEYAEANWNYNTNITTETSKILLQKNMQIANHTLKYGTQAR--DVNHFQN papAnu2_ACE NEYAEANWNYNTNITTETSKILLQKNMQIANHTLKYGTQAR--DVNHFQN gorGor3_ACE NEYAEANWNYNTNITTETSKILLQKNMQIANHTLKYGTQAR--DVNQLQN panPan1_ACE NEYAEANWNYNTNITTETSKILLQKNMQIANHTLKYGTQAR--DVNQLQN hg38_ACE_ NEYAEANWNYNTNITTETSKILLQKNMQIANHTLKYGTQARKFDVNQLQN

panTro4_ACE NEYAEANWNYNTNITTETSKILLQKNMQIANHTLKYGTQAR--DVNQLQN ponAbe2_ACE NEYAEANWNYNTNITTETSKILLQKNMQIANHTLKYGTQAR--DVNHLQN calJac3_ACE KDYAEANWNYYTNITRETSSVLLQKNMQIANYTLQYGTQAR--DVSHFQN saiBol1_ACE NEYAEANWNYNTNITRETSSILLQKNMQIANYTLQYGTQAR--DVSHFQN otoGar3_ACE NEFAEANWNYNTNITTEASQILLQKNLEIANHTLKWGIQARQFDVSTFQN

NomLeu3_ACE T--TIKRIIKKVQDLERAALPAQELEEYNKILLDMETTYSVATVCHTNGS chlSab2_ACE T--TIKRIIKKVQDLERAALPAQELEEYNKILLDMETTYSVATVCHTNGS macFas5_ACE T--TIKRIIKKVQDLERAALPAQELEEYNKILLDMETTYSVATVCHTNGS rheMac3_ACE T--TIKRIIKKVQDLERAALPAQELEEYNKILLDMETTYSVATVCHTNGS papAnu2_ACE T--TIKRIIKKVQDLERAALPAQELEEYNKILLDMETTYSVATVCHTNGS gorGor3_ACE T--TIKRIIKKVQDLERAALPAQELEEYNKILLDMETTYSVATVCHTNGS panPan1_ACE T--TIKRIIKKVQDLERAALPAQELEEYNKILLDMETTYSVATVCHTNGS hg38_ACE_ T--TIKRIIKKVQDLERAALPAQELEEYNKILLDMETTYSVATVCHPNGS panTro4_ACE T--TIKRIIKKVQDLERAALPAQELEEYNKILLDMETTYSVATVCHTNGS ponAbe2_ACE T--TIKRIIKKVQDLERAALPAQELEEYNKILLDMETTYSVATVCHTNGS calJac3_ACE T--TIKRILKKVQDLERAALPAQELEEYNKILLDMETTYSVATVCHTNGS saiBol1_ACE T--TIKRILKKVQDLERAALPAQELEEYNKILLDMETTYSVATVCHTNGS otoGar3_ACE T--TTKRVIKKVQDLDRAALPAKELEEYNKILLEMETTYSVATVCHTNGT

NomLeu3_ACE CLQLEPDLTNVMATSRKYEDLLWAWEGWRDKA---GRAILQFYPKYVELI chlSab2_ACE CLQLEPDLTNVMATSRKYEELLWAWEGWRDKA---GRAILQFYPKYVELI macFas5_ACE CLQLEPDLTNVMATSRKYEELLWAWEGWRDKA---GRAILQFYPKYVELI rheMac3_ACE CLQLEPDLTNVMATSRKYEELLWAWEGWRDKA---GRAILQFYPKYVELI papAnu2_ACE CLQLEPDLTNVMATSRKYEELLWAWEGWRDKA---GRAILQFYPKYVELI gorGor3_ACE CLQLEPDLTNVMATSRKYEDLLWAWEGWRDKA---GRAILQFYPKYVELI panPan1_ACE CLQLEPDLTNVMATSRKYEDLLWAWEGWRDKA---GRAILQFYPKYVELI hg38_ACE_ CLQLEPDLTNVMATSRKYEDLLWAWEGWRDKA---GRAILQFYPKYVELI panTro4_ACE CLQLEPDLTNVMATSRKYEDLLWAWEGWRDKA---GRAILQFYPKYVELI ponAbe2_ACE CLQLEPDLTNVMATSRKYEDLLWAWEGWRDKA---GRAILQFYPKYVELI calJac3_ACE CLQLEPDLTNVMATSRKYEELLWAWKGWRDKA---GRTILQFYPKYVEFI saiBol1_ACE CLQLEPDLTNVMATSRKYEELLWAWKGWRDKT---GRTILQFYPKYVEFI otoGar3_ACE CLQLEPDLTSMMATSRQYYELLWAWKSWRDKV---GRAILPSFPKYVELT

NomLeu3_ACE NQAAQLNGYVDAGDSWRSVYETPSLEQDLERLFQELQPLYLNLHAYVRRA chlSab2_ACE NQAARLNGYVDAGDSWRSMYETPSLEQDLERLFQELQPLYLNLHAYVRRA macFas5_ACE NQAARLNGYVDAGDSWRSMYETPSLEQDLERLFQELQPLYLNLHAYVRRA rheMac3_ACE NQAARLNGYVDAGDSWRSMYETPSLEQDLERLFQELQPLYLNLHAYVRRA papAnu2_ACE NQAARLNGYVDAGDSWRSMYETPSLEQDLERLFQELQPLYLNLHAYVRRA gorGor3_ACE NQAARLNGYVDAGDSWRSMYETPSLEQDLERLFQELQPLYLNLHAYVRRA panPan1_ACE NQAARLNGYVDAGDSWRSMYETPSLEQDLERLFQELQPLYLNLHAYVRRA hg38_ACE_ NQAARLNGYVDAGDSWRSMYETPSLEQDLERLFQELQPLYLNLHAYVRRA panTro4_ACE NQAARLNGYVDAGDSWRSMYETPSLEQDLERLFQELQPLYLNLHAYVRRA ponAbe2_ACE NQAARLNGYVDAGDSWRSMYETPSLEQDLERLFQELQPLYLNLHAYVRRA calJac3_ACE NKAARLNGYVDAGDSWRSMYEMPSLEQDLERLFQQLQPLYLNLHAYVRRA saiBol1_ACE NKAARLNGYVDAGDSWRSMYETPSLEQDLERLFQELQPLYLNLHAYVRRA otoGar3_ACE NKAARLNGYIDGGDSWRSMYEMPSLEQNLEELFQELQPLYLNLHAYVRRA

NomLeu3_ACE LHRHYGAQRINLEGPI---PAHLLGNMWAQTWSNIYDLVV-----PFPSA chlSab2_ACE LHHHYGAQHINLEGPI---PAHLLGNMWAQTWSNIYDLVV-----PFPSA macFas5_ACE LHRHYGAQHINLEGPI---PAHLLGNMWAQTWSNIYDLVV-----PFPSA rheMac3_ACE LHRHYGAQHINLEGPI---PAHLLGNMWAQTWSNIYDLVV-----PFPSA papAnu2_ACE LHRHYGAQHINLEGPI---PAHLLGNMWAQTWSNIYDLVV-----PFPSA gorGor3_ACE LHRHYGAQHINLEGPI---PAHLLGNMWAQTWSNIYDLVV-----PFPSA panPan1_ACE LHRHYGAQHINLEGPI---PAHLLGNMWAQTWSNIYDLVV-----PFPSA hg38_ACE_ LHRHYGAQHINLEGPI---PAHLLGNMWAQTWSNIYDLVV-----PFPSA panTro4_ACE LHRHYGAQHINLEGPI---PAHLLGNMWAQTWSNIYDLVV-----PFPSA ponAbe2_ACE LHRHYGAQHINLEGPI---PAHLLGNMWAQTWSNIYDLVV-----PFPSA calJac3_ACE LHRHYGAQHINLEGPI---PAHLLGNMWAQTWSNIYDLVV-----PFPSA saiBol1_ACE LHRHYGAQHINLEGPI---PAHLLGNMWAQTWSNIYDLVV-----PFPSA otoGar3_ACE LHRHYGAQHINLEGPI---PAHLLGNMWAQTWSNIYDLVV-----PFPSA

NomLeu3_ACE PSMDTTEAMLKQGWTPRR------MFKEADDFFTSLGLLPVPPEFWN chlSab2_ACE PSMDPTEAMLKQGWTPRR------MFKEADDFFTSLGLLPVPPEFWN macFas5_ACE PSMDPTEAMLKQGWTPRR------MFKEADDFFTSLGLLPVPPEFWN rheMac3_ACE PSMDPTEAMLKQGWTPRR------MFKEADDFFTSLGLLPVPPEFWN papAnu2_ACE PSMDPTEAMLKQGWTPRR------MFKEADDFFTSLGLLPVPPEFWN gorGor3_ACE PSMDATEAMLKQGWTPRR------MFKEADDFFTSLGLLPVPPEFWN panPan1_ACE PSMDTTEAMLKQGWTPRR------MFKEADDFFTSLGLLPVPPEFWN hg38_ACE_ PSMDTTEAMLKQGWTPRR------MFKEADDFFTSLGLLPVPPEFWN panTro4_ACE PSMDTTEAMLKQGWTPRR------MFKEADDFFTSLGLLPVPPEFWN ponAbe2_ACE PSMDTTEAMLKQGWTPRR------MFKEADDFFTSLGLLPVPPEFWN calJac3_ACE TLMDTTEAMLKQGWTPRR------MFKEADDFFTSLGLLPVPPEFWN saiBol1_ACE TLMDTTEAMLKQGWTPRR------MFKEADDFFTSLGLLPVPPEFWN otoGar3_ACE PSMDATEAMIKQGWTPRR------MFKEADNFFISLGLLPVPPEFWN

NomLeu3_ACE KSMLEKP-TDGREVVCHASAWDFYNGKDFRIKQCTTVNLEDLVVAHHEMG chlSab2_ACE KSMLEKP-TDGREVVCHASAWDFYNGKDFRIKQCTTVNLEDLVVAHHEMG macFas5_ACE KSMLEKP-TDGREVVCHASAWDFYNGKDFRIKQCTTVNLEDLVVAHHEMG rheMac3_ACE KSMLEKP-TDGREVVCHASAWDFYNGKDFRIKQCTTVNLEDLVVAHHEMG papAnu2_ACE KSMLEKP-TDGREVVCHASAWDFYNGKDFRIKQCTTVNLEDLVVAHHEMG gorGor3_ACE KSMLEKP-TDGREVVCHASAWDFYNGKDFRIKQCTTVNLEDLVVAHHEMG panPan1_ACE KSMLEKP-TDGREVVCHASAWDFYNGKDFRIKQCTTVNLEDLVVAHHEMG hg38_ACE_ KSMLEKP-TDGREVVCHASAWDFYNGKDFRIKQCTTVNLEDLVVAHHEMG panTro4_ACE KSMLEKP-TDGREVVCHASAWDFYNGKDFRIKQCTTVNLEDLVVAHHEMG ponAbe2_ACE KSMLEKP-TDGREVVCHASAWDFYNGKDFRIKQCTTVNLEDLVVAHHEMG calJac3_ACE KSMLEKP-TDGREVVCHASAWDFYNGKDFRXXXXXXXXXXXXXXXXXXXX saiBol1_ACE KSMLEKP-TDGREVVCHASAWDFYNGKDFRIKQCTTVNLEDLVVAHHEMG otoGar3_ACE KSMLEKP-TDGREVVCHASAWDFYNGKDFRIKQCTSVNMEDLVVAHHEMG

NomLeu3_ACE HIQYFMQYKDLPVALREGANPGFHEAIGDVLALSVSTPKHLHSLNLLSSE chlSab2_ACE HIQYFMQYKDLPVALREGANPGFHEAIGDVLALSVSTPKHLHSLNLLSSE macFas5_ACE HIQYFMQYKDLPVALREGANPGFHEAIGDVLALSVSTPKHLHSLNLLSSE rheMac3_ACE HIQYFMQYKDLPVALREGANPGFHEAIGDVLALSVSTPKHLHSLNLLSSE

papAnu2_ACE HIQYFMQYKDLPVALREGANPGFHEAIGDVLALSVSTPKHLHSLNLLSSE gorGor3_ACE HIQYFMQYKDLPVALREGANPGFHEAIGDVLALSVSTPKHLHSLNLLSSE panPan1_ACE HIQYFMQYKDLPVALREGANPGFHEAIGDVLALSVSTPKHLHSLNLLSSE hg38_ACE_ HIQYFMQYKDLPVALREGANPGFHEAIGDVLALSVSTPKHLHSLNLLSSE panTro4_ACE HIQYFMQYKDLPVALREGANPGFHEAIGDVLALSVSTPKHLHSLNLLSSE ponAbe2_ACE HIQYFMQYKDLPVALREGANPGFHEAIGDVLALSVSTPKHLHSLNLLSSE calJac3_ACE XXXXXXQYKDLPVALRDGANPGFHEAIGDVLALSVSTPKHLHSLNLLSSE saiBol1_ACE HIQYFMQYKDLPVALRDGANPGFHEAIGDVLALSVSTPKHLHSLNLLSSE otoGar3_ACE HIQYFMQYKDLPVALREGANPGFHEAIGDVLALSVSTPKHLHSLNLLSSE

NomLeu3_ACE GGSDEHDINFLMKMALDKIAFIPFSYLVDQWRWRVFDGSITKENYNQEWW chlSab2_ACE GGSHEHDINFLMKMALDKIAFIPFSYLVDQWRWRVFDGSITKENYNQEWW macFas5_ACE GGSHEHDINFLMKMALDKIAFIPFSYLVDQWRWRVFDGSITKENYNQEWW rheMac3_ACE GGSHEHDINFLMKMALDKIAFIPFSYLVDQWRWRVFDGSITKENYNQEWW papAnu2_ACE GGSHEHDINFLMKMALDKIAFIPFSYLVDQWRWRVFDGSITKENYNQEWW gorGor3_ACE GGSDEHDINFLMKMALDKIAFIPFSYLVDQWRWRVFDGSITKENYNQEWW panPan1_ACE GGSDEHDINFLMKMALDKIAFIPFSYLVDQWRWRVFDGSITKENYNQEWW hg38_ACE_ GGSDEHDINFLMKMALDKIAFIPFSYLVDQWRWRVFDGSITKENYNQEWW panTro4_ACE GGSDEHDINFLMKMALDKIAFIPFSYLVDQWRWRVFDGSITKENYNQEWW ponAbe2_ACE GGSDEHDINFLMKMALDKIAFIPFSYLVDQWRWRVFDGSITKENYNQEWW calJac3_ACE GDSFENNINFLMKMALEKIAFIPFSYLVDQWRWRVFDGSITKENYNQEWW saiBol1_ACE GDSFEHNINFLMKMALEKIAFIPFSYLVDQWRWRVFDGSITKENYNQEWW otoGar3_ACE GVGYENDINFLMKMALDKVAFIPFSYLVDQWRWRVFDGSISKENYNQEWW

NomLeu3_ACE SLRLKYQG------LCP---PVP---RTQGDFDPGAKFHVPSSVPY chlSab2_ACE SLRLKYQG------LCP---PVP---RTQGDFDPGAKFHIPSSVPY macFas5_ACE SLRLKYQG------LCP---PVP---RTQGDFDPGAKFHVPSSVPY rheMac3_ACE SLRLKYQG------LCP---PVP---RTQGDFDPGAKFHVPSSVPY papAnu2_ACE SLRLKYQG------LCP---PVP---RTQGDFDPGAKFHVPSSVPY gorGor3_ACE SLRLKYQG------LCP---PVP---RTQGDFDPGAKFHVPSSVPY panPan1_ACE SLRLKYQG------LCP---PVP---RTQGDFDPGAKFHIPSSVPY hg38_ACE_ SLRLKYQG------LCP---PVP---RTQGDFDPGAKFHIPSSVPY panTro4_ACE SLRLKYQG------LCP---PVP---RTQGDFDPGAKFHIPSSVPY ponAbe2_ACE SLXXXXXX------XXX---XXX---XXXXXXXXXXXXXXXXXXXX calJac3_ACE SLRLKYQG------LCP---PVP---RTQGDFDPGAKFHIPSSVPY saiBol1_ACE SLRLKYQG------LCP---PVP---RTQGDFDPGAKFHIPSSVPY otoGar3_ACE SLRLKYQG------LCP---PVA---RSQGDFDPGAKFHIPSSVPY

NomLeu3_ACE IRYFVSFII------QFQFHE------ALCQAAGHTGPLHKC chlSab2_ACE IRYFISFII------QFQFHE------ALCQAAGHTGPLHKC macFas5_ACE IRYFISFII------QFQFHE------ALCQAAGHTGPLHKC rheMac3_ACE IRYFISFII------QFQFHE------ALCQAAGHTGPLHKC papAnu2_ACE IRYFISFII------QFQFHE------ALCQAAGHTGPLHKC gorGor3_ACE IRYFVSFII------QFQFHE------ALCQAAGHTGPLHKC panPan1_ACE IRYFVSFII------QFQFHE------ALCQAAGHTGPLHKC hg38_ACE_ IRYFVSFII------QFQFHE------ALCQAAGHTGPLHKC panTro4_ACE IRYFVSFII------QFQFHE------ALCQAAGHTGPLHKC

ponAbe2_ACE XXYFVSFII------QFQFHE------ALCQAAGHKGPLHKC calJac3_ACE IRYFVSFII------QFQFHE------ALCQVAGHTGPLHKC saiBol1_ACE IRYFVSFII------QFQFHE------ALCQVAGHTGPLHKC otoGar3_ACE IRYFVSFVI------QFQFHE------ALCHAAGHEGPLHKC

NomLeu3_ACE DIYQSKEAGQRLATAMKLGFS-RPW------PEAMQLITGQPNMSA chlSab2_ACE DIYQSKEAGQRLATAMKLGFS-RPW------PEAMQLITGQPNMSA macFas5_ACE DIYQSKEAGQRLATAMKLGFS-RPW------PEAMQLITGQPNMSA rheMac3_ACE DIYQSKEAGQRLATAMKLGFS-RPW------PEAMQLITGQPNMSA papAnu2_ACE DIYQSKEAGQRLATAMKLGFS-RPW------PEAMQLITGQPNMSA gorGor3_ACE DIYQSKEAGQRLATAMKLGFS-RPW------PEAMQLITGQPNMSA panPan1_ACE DIYQSKEAGQRLATAMKLGFS-RPW------PEAMQLITGQPNMSA hg38_ACE_ DIYQSKEAGQRLATAMKLGFS-RPW------PEAMQLITGQPNMSA panTro4_ACE DIYQSKEAGQRLAXXXXXXXX-XXX------XXXXXXXXXXXXXXX ponAbe2_ACE DIYQSKEAGQRLATAMKLGFS-RPW------PEAMQLITGQPNMSA calJac3_ACE DIYQSKEAGQRLAAAMKLGFS-QPW------PEAMQLITGQPNMSA saiBol1_ACE DIYQSKEAGQRLAAAMKLGFS-QPW------PEAMRLITGQPNMSA otoGar3_ACE DIYQSKEAGKRLADAMKLGFS-KPW------PEAMKLITGQPNMSA

NomLeu3_ACE S-AMLSYFKPLLDWLRTENELHGEKLGWPQYNWTPNSARSEGPLPDSGHV chlSab2_ACE S-AMLSYFKPLLDWLRTENELHGEKLGWPQYNWTPNSARSEGPLPDSGRV macFas5_ACE S-AMLSYFKPLLDWLRTENELHGEKLGWPQYNWTPNSARSEGPLPDSGRV rheMac3_ACE S-AMLSYFKPLLDWLRTENELHGEKLGWPQYNWTPNSARSEGPLPDSGRV papAnu2_ACE S-AMLSYFKPLLDWLRTENELHGEKLGWPQYNWTPNSARSEGPLPDSGRV gorGor3_ACE S-AMLSYFKPLLDWLRTENELHGEKLGWPQYNWTPNSARSEGPLPDSGRV panPan1_ACE S-AMLSYFKPLLDWLRTENELHGEKLGWPQYNWTPNSARSEGPLPDSGRV hg38_ACE_ S-AMLSYFKPLLDWLRTENELHGEKLGWPQYNWTPNSARSEGPLPDSGRV panTro4_ACE X-XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX ponAbe2_ACE S-AMLSYFKPLLDWLRTENELRGEKLGWPQYNWTPNSARSEGPLPDSGRV calJac3_ACE S-AMLSYFKPLLDWLRAENELHGEKLGWPQYNWTPNSARSEGPLRDSGRV saiBol1_ACE S-AMLSYFKPLLDWLRAENELHGEKLGWPQYNWTPNSARSEGPFPDSGRV otoGar3_ACE S-AMMNYFKPLLDWLVTENGRHGEKLGWPQYNWTPDSARSEGPVPDTGRV

NomLeu3_ACE SFLGLDLDAQQARVGQWLLLFLGIALLVATLGLSQRLFSIRHRSLHRHPH chlSab2_ACE SFLGLDLDAQQARVGQWLLLFLGIALLVATLGLSQRLFSIRHQSLHRHPQ macFas5_ACE SFLGLDLDAQQARVGQWLLLFLGIALLVATLGLSQRLFSIRHQSLHQHPQ rheMac3_ACE SFLGLDLDAQQARVGQWLLLFLGIALLVATLGLSQRLFSIRHQSLHQHPQ papAnu2_ACE SFLGLDLDAQQARVGQWLLLFLGIALLVATLGLSQRLFSIRHQSLHRHPQ gorGor3_ACE SFLGLDLDAQQARVGQWLLLFLGIALLVATLGLSQRLFSIRHRSLHRHSH panPan1_ACE SFLGLDLDAQQARVGQWLLLFLGIALLVATLGLSQRLFSIRHRSLHRHSH hg38_ACE_ SFLGLDLDAQQARVGQWLLLFLGIALLVATLGLSQRLFSIRHRSLHRHSH panTro4_ACE XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX ponAbe2_ACE SFLGLDLDAQQARVGQWLLLFLGIALLVATLGLSQRLFSIRHHRLHRHPH calJac3_ACE SFLGLDLEPQQARVGQWLLLFLGIALLVATLGLSQRLFSIRHHSLRQPHH saiBol1_ACE SFLGLDLEPQQARVGQWLLLFLGIALLVATLGLSQRLFSIRRHSLRQPHH otoGar3_ACE NFLGLNLDAQQARVGQWVLLFLGVALLVATLGLTQRLFSIRHHSIRRPHR

NomLeu3_ACE GPQFGSEVELRHS-- chlSab2_ACE GPQFGSEVELRHS-- macFas5_ACE GPQFGSEVELRHS-- rheMac3_ACE GPQFGSEVELRHS-- papAnu2_ACE GPQFGSEVELRHS-- gorGor3_ACE GPQFGSEVELRHS-- panPan1_ACE GPQFDSEVELRHS-- hg38_ACE_ GPQFGSEVELRHS-- panTro4_ACE XXXXXXXXXXXXX-- ponAbe2_ACE EPQFGSEVELRHS-- calJac3_ACE GPQFGSEVELRHS-- saiBol1_ACE GPQFGSEVELRHS-- otoGar3_ACE GPQFGSEVELRHS--

S2. ACTN3 PROTEIN ALIGNMENT chlSab2_ACTN3 MMMVMQPEGLGAREGPFAGSGGGGEYMEQEEDWDRDLLLDPAWEKQQRKT macFas5_ACTN3 MMMVMQPEGLGAREGPFAGSGGGGEYMEQEEDWDRDLLLDPAWEKQQRKT papAnu2_ACTN3 MMMVMQPEGLGAREGPFAGSGGGGEYMEQEEDWDRDLLLDPAWEKQQRKT rheMac3_ACTN3 MMMVMQPEGLGAREGPFAGSGGGGEYMEQEEDWDRDLLLDPAWEKQQRKT gorGor3_ACTN3 MMMVMQPEGLGAGEGRFAGGGGGGEYMEQEEDWDRDLLLDPAWEKQQRKT panPan1_ACTN3 MMMVMQPEGLGAGEGRFAGGGGGGEYMEQEEDWDRDLLLDPAWEKQQRKT saiBol1_ACTN3 MMMVMQPEGLGAGEGPFAGGGGGGEYMEQEEDWDRDLLLDPAWEKQQRKT nomLeu3_ACTN3 MMMVMQPEGLGAREGRFAGGGGGGEYMEQEEDWDRDLLLDPAWEKQQRKT hg38_ACTN3 MMMVMQPEGLGAGEGRFAGGGGGGEYMEQEEDWDRDLLLDPAWEKQQRKT ponAbe2_ACTN3 MMMVMQPEGLGAGEGRFAGGGGGGEYMEQEEDWDRDLLLDPAWEKQQRKT panTro4_ACTN3 MMMVMQPEGLGAGEGRFAGGGGGGEYMEQEEDWDRDLLLDPAWEKQQRKT micMur2_ACTN3 MMMVMQPEGLGAGERPFMGGGGGGEYMEQEEDWDRDLLLDPAWEKQQRKT otoGar3_ACTN3 MMMVMQPEGLGVGERSFA---GGGEYMEQEEDWDRDLLLDPAWEKQQRKT tarSyr2_ACTN3 MMMVLQPEGLGAGVGPFAVGGGGGEYMEQEEDWDRDLLLDPAWEKQQRKT chlSab2_ACTN3 FTAWCNSHLRKAGTQIENIEDDFRNGLKLMLLLEVISGERLPRPDKGKMR macFas5_ACTN3 FTAWCNSHLRKAGTQIENIEDDFRNGLKLMLLLEVISGERLPRPDKGKMR papAnu2_ACTN3 FTAWCNSHLRKAGTQIENIEDDFRNGLKLMLLLEVISGERLPRPDKGKMR rheMac3_ACTN3 FTAWCNSHLRKAGTQIENIEDDFRNGLKLMLLLEVISGERLPRPDKGKMR gorGor3_ACTN3 FTAWCNSHLRKAGTQIENIEEDFRNGLKLMLLLEVISGERLPRPDKGKMR panPan1_ACTN3 FTAWCNSHLRKAGTQIENIEEDFRNGLKLMLLLEVISGERLPRPDKGKMR saiBol1_ACTN3 FTAWCNSHLRKAGTQIENIEEDFRNGLKLMLLLEVISGERLPRPDKGKMR nomLeu3_ACTN3 FTAWCNSHLRKAGTQIENIEEDFRNGLKLMLLLEVISGERLPRPDKGKMR hg38_ACTN3 FTAWCNSHLRKAGTQIENIEEDFRNGLKLMLLLEVISGERLPRPDKGKMR ponAbe2_ACTN3 FTAWCNSHLRKAGTQIENIEEDFRNGLKLMLLLEVISGERLPRPDKGKMR panTro4_ACTN3 FTAWCNSHLRKAGTQIENIEEDFRNGLKLMLLLEVISGERLPRPDKGKMR micMur2_ACTN3 FTAWCNSHLRKAGTQIENIEEDFRNGLKLMLLLEVISGERLPRPDKGKMR otoGar3_ACTN3 FTAWCNSHLRKAGTQIENIEEDFRNGLKLMLLLEVISGERLPRPDKGKMR

tarSyr2_ACTN3 FTAWCNSHLRKAGTQIENIEDDFRNGLKLMLLLEVISGERLPRPDKGKMR chlSab2_ACTN3 FHKIANVNKALDFIASKGVKLVSIGAEEIVDGNLKMTLGMIWTIILRFAI macFas5_ACTN3 FHKIANVNKALDFIASKGVKLVSIGAEEIVDGNLKMTLGMIWTIILRFAI papAnu2_ACTN3 FHKIANVNKALDFIASKGVKLVSIGAEEIVDGNLKMTLGMIWTIILRFAI rheMac3_ACTN3 FHKIANVNKALDFIASKGVKLVSIGAEEIVDGNLKMTLGMIWTIILRFAI gorGor3_ACTN3 FHKIANVNKALDFIASKGVKLVSIGAEEIVDGNLKMTLGMIWTIILRFAI panPan1_ACTN3 FHKIANVNKALDFIASKGVKLVSIGAEEIVDGNLKMTLGMIWTIILRFAI saiBol1_ACTN3 FHKIANVNKALDFIASKGVKLVSIGAEEIVDGNLKMTLGMIWTIILRFAI nomLeu3_ACTN3 FHKIANVNKALDFIASKGVKLVSIGAEEIVDGNLKMTLGMIWTIILRFAI hg38_ACTN3 FHKIANVNKALDFIASKGVKLVSIGAEEIVDGNLKMTLGMIWTIILRFAI ponAbe2_ACTN3 FHKIANVNKALDFIASKGVKLVSIGAEEIVDGNQKMTLGMIWTIILRFAI panTro4_ACTN3 FHKIANVNKALDFIASKGVKLVSIGAEEIVDGNLKMTLGMIWTIILRFAI micMur2_ACTN3 FHKIANVNKALDFIASKGVKLVSIGAEEIVDGNLKMTLGMIWTIILRFAI otoGar3_ACTN3 FHKIANVNKALDFIASKGVKLVSIGAEEIVDGNLKMTLGMIWTIILRFAI tarSyr2_ACTN3 FHKIANVNKALDFIASKGVKLVSIGAEEIVDGNLKMTLGMIWTIILRFAI chlSab2_ACTN3 QDISVEETSAKEGLLLWCQRKTAPYRNVNVQNFHTSWKDGLALCALIHRH macFas5_ACTN3 QDISVEETSAKEGLLLWCQRKTAPYRNVNVQNFHTSWKDGLALCALIHRH papAnu2_ACTN3 QDISVEETSAKEGLLLWCQRKTAPYRNVNVQNFHTSWKDGLALCALIHRH rheMac3_ACTN3 QDISVEETSAKEGLLLWCQRKTAPYRNVNVQNFHTSWKDGLALCALIHRH gorGor3_ACTN3 QDISVEETSAKEGLLLWCQRKTAPYRNVNVQNFHTSWKDGLALCALIHRH panPan1_ACTN3 QDISVEETSAKEGLLLWCQRKTAPYRNVNVQNFHTSWKDGLALCALIHRH saiBol1_ACTN3 QDISVEETSAKEGLLLWCQRKTAPYRNVNVQNFHTSWKDGLALCALIHRH nomLeu3_ACTN3 QDISVEETSAKEGLLLWCQRKTAPYRNVNVQNFHTSWKDGLALCALIHRH hg38_ACTN3 QDISVEETSAKEGLLLWCQRKTAPYRNVNVQNFHTSWKDGLALCALIHRH ponAbe2_ACTN3 QDISVEETSAKEGLLLWCQRKTAPYRNVNVQNFHTSWKDGLALCALIHRH panTro4_ACTN3 QDISVEETSAKEGLLLWCQRKTAPYRNVNVQNFHTSWKDGLALCALIHRH micMur2_ACTN3 QDISVEETSAKEGLLLWCQRKTAPYRNVNVQNFHTSWKDGLALCALIHRH otoGar3_ACTN3 QDISVEETSAKEGLLLWCQRKTAPYRNVNVQNFHTSWKDGLALCALIHRH tarSyr2_ACTN3 QDISVEETSAKEGLLLWCQRKTAPYRNVNVQNFHTSWKDGLALCALIHRH chlSab2_ACTN3 RPDLIDYAKLRKDDPIGNLNTAFEVAEKYLDIPKMLDAEDIVNTPKPDEK macFas5_ACTN3 RPDLIDYAKLRKDDPIGNLNTAFEVAEKYLDIPKMLDAEDIVNTPKPDEK papAnu2_ACTN3 RPDLIDYAKLRKDDPIGNLNTAFEVAEKYLDIPKMLDAEDIVNTPKPDEK rheMac3_ACTN3 RPDLIDYAKLRKDDPIGNLNTAFEVAEKYLDIPKMLDAEDIVNTPKPDEK gorGor3_ACTN3 RPDLIDYAKLRKDDPIGNLNTAFEVAEKYLDIPKMLDAEDIVNTPKPDEK panPan1_ACTN3 RPDLIDYAKLRKDDPIGNLNTAFEVAEKYLDIPKMLDAEDIVNTPKPDEK saiBol1_ACTN3 RPDLIDYAKLRKDDPIGNLNTAFEVAEKYLDIPKMLDAEDIVNTPKPDEK nomLeu3_ACTN3 RPDLVDYAKLRKDDPIGNLNTAFEVAEKYLDIPKMLDAEDIVNTPKPDEK hg38_ACTN3 RPDLIDYAKLRKDDPIGNLNTAFEVAEKYLDIPKMLDAEDIVNTPKPDEK ponAbe2_ACTN3 RPDLIDYAKLRKDDPIGNLNTAFEVAEKYLDIPKMLDAEDIVNTPKPDEK panTro4_ACTN3 RPDLIDYAKLRKDDPIGNLNTAFEVAEKYLDIPKMLDAEDIVNTPKPDEK micMur2_ACTN3 RPDLIDYAKLRKDDPVGNLNTAFEVAEKYLDIPKMLDAEDIVNTPKPDEK otoGar3_ACTN3 RPDLIDYAKLRKDDPVGNLNTAFEVAEKYLDIPKMLDAEDIVNTPKPDEK tarSyr2_ACTN3 RPDLIDYAKLRKDDPIGNLNTAFEVAEKYLDIPKMLDAEDIVNTPKPDEK

chlSab2_ACTN3 AIMTYVSCFYHAFAGAEQAETAANRICKVLAVNQENEKLMEEYEKLASEL macFas5_ACTN3 AIMTYVSCFYHAFAGAEQAETAANRICKVLAVNQENEKLMEEYEKLASEL papAnu2_ACTN3 AIMTYVSCFYHAFAGAEQAETAANRICKVLAVNQENEKLMEEYEKLASEL rheMac3_ACTN3 AIMTYVSCFYHAFAGAEQAETAANRICKVLAVNQENEKLMEEYEKLASEL gorGor3_ACTN3 AIMTYVSCFYHAFAGAEQAETAANRICKVLAVNQENEKLMEEYEKLASEL panPan1_ACTN3 AIMTYVSCFYHAFAGAEQAETAANRICKVLAVNQENEKLMEEYEKLASEL saiBol1_ACTN3 AIMTYVSCFYHAFAGAEQAETAANRICKVLAVNQENEKLMEEYEKLASEL nomLeu3_ACTN3 AIMTYVSCFYHAFAGAEQAETAANRICKVLAVNQENEKLMEEYEKLASEL hg38_ACTN3 AIMTYVSCFYHAFAGAEQAETAANRICKVLAVNQENEKLMEEYEKLASEL ponAbe2_ACTN3 AIMTYVSCFYHAFAGAEQAETAANRICKVLAVNQENEKLMEEYEKLASEL panTro4_ACTN3 AIMTYVSCFYHAFAGAEQAETAANRICKVLAVNQENEKLMEEYEKLASEL micMur2_ACTN3 AVMTYVSCFYHAFAGAEQAETAANRICKVLAVNQENEKLMEEYEKLASEL otoGar3_ACTN3 AVMTYVSCFYHAFAGAEQAETAANRICKVLAVNQENEKLMEEYEKLASEL tarSyr2_ACTN3 AVMTYVSCFYHAFAGAEQAETAANRICKVLAVNQENEKLMEEYEKLASEL chlSab2_ACTN3 LEWIRRTVPWLENRVGEPSMSAMQRKLEDFRDYRRLHKPPRVQEKCQLEI macFas5_ACTN3 LEWIRRTIPWLENRVGEPSMSAMQRKLEDFRDYRRLHKPPRVQEKCQLEI papAnu2_ACTN3 LEWIRRTIPWLENRVGEPSMSAMQRKLEDFRDYRRLHKPPRVQEKCQLEI rheMac3_ACTN3 LEWIRRTIPWLENRVGEPSMSAMQRKLEDFRDYRRLHKPPRVQEKCQLEI gorGor3_ACTN3 LEWIRRTVPWLENRVGEPSMSAMQRKLEDFRDYRRLHKPPRVQEKCQLEI panPan1_ACTN3 LEWIRRTVPWLENRVGEPSMSAMQRKLEDFRDYRRLHKPPRVQEKCQLEI saiBol1_ACTN3 LEWIRRTVPWLENRVGEPSMSAMQRKLEDFRDYRRLHKPPRVQEKCQLEI nomLeu3_ACTN3 LEWIRRTVPWLENRVGEPSMSAMQRKLEDFRDYRRLHKPPRVQEKCQLEI hg38_ACTN3 LEWIRRTVPWLENRVGEPSMSAMQRKLEDFRDYRRLHKPPRIQEKCQLEI ponAbe2_ACTN3 LEWIHRTVPWLENRVGEPSMSAMQRKLEDFRDYRRLHKPPRVQEKCQLEI panTro4_ACTN3 LEWIRRTVPWLENRVGEPSMSAMQRKLEDFRDYRRLHKPPRVQEKCQLEI micMur2_ACTN3 LEWIRRTVPWLENRVGEPSMSAMQRKLEDFRDYRRLHKPPRVQEKCQLEI otoGar3_ACTN3 LEWIRRTVPWLENRVGEPSMSAMQRKLEDFRDYRRLHKPPRVQEKCQLEI tarSyr2_ACTN3 LEWIRRTVPWLENRVGEPSMSAMQRKLEDFRDYRRLHKPPRVQEKCQLEI chlSab2_ACTN3 NFNTLQTKLRLSHRPAFMPSEGKLVSDIANAWRGLEQVEKGYEDWLLSEI macFas5_ACTN3 NFNTLQTKLRLSHRPAFMPSEGKLVSDIANAWRGLEQVEKGYEDWLLSEI papAnu2_ACTN3 NFNTLQTKLRLSHRPAFMPSEGKLVSDIANAWRGLEQVEKGYEDWLLSEI rheMac3_ACTN3 NFNTLQTKLRLSHRPAFMPSEGKLVSDIANAWRGLEQVEKGYEDWLLSEI gorGor3_ACTN3 NFNTLQTKLRLSHRPAFMPSEGKLVSDIANAWRGLEQVEKGYEDWLLSEI panPan1_ACTN3 NFNTLQTKLRLSHRPAFMPSEGKLVSDIANAWRGLEQVEKGYEDWLLSEI saiBol1_ACTN3 NFNTLQTKLRLSHRPAFMPSEGKLVSDIANAWRGLEQVEKGYEDWLLSEI nomLeu3_ACTN3 NFNTLQTKLRLSHRPAFMPSEGKLVSDIANAWRGLEQVEKGYEDWLLSEI hg38_ACTN3 NFNTLQTKLRLSHRPAFMPSEGKLVSDIANAWRGLEQVEKGYEDWLLSEI ponAbe2_ACTN3 NFNTLQTKLRLSHRPAFMPSEGKLVSDIANAWRGLEQVEKGYEDWLLSEI panTro4_ACTN3 NFNTLQTKLRLSHRPAFMPSEGKLVSDIANAWRGLEQVEKGYEDWLLSEI micMur2_ACTN3 NFNTLQTKLRLSHRPAFMPSEGKLVSDIANAWRGLEQVEKGYEDWLLSEI otoGar3_ACTN3 NFNTLQTKLRLSHRPAFMPSEGKLVSDIANAWRGLEQVEKGYEDWLLSEI tarSyr2_ACTN3 NFNTLQTKLRLSHRPAFMPSEGKLVSDIANAWRGLEQVEKGYEDWLLSEI chlSab2_ACTN3 RRLQRLQHLAEKFRQKASLHEAWTRGKEEMLSQRDYDSASLQEVRALLRR macFas5_ACTN3 RRLQRLQHLAEKFRQKASLHEAWTRGKEEMLSQRDYDSASLQEVRALLRR

papAnu2_ACTN3 RRLQRLQHLAEKFRQKASLHEAWTRGKEEMLSQRDYDSASLQEVRALLRR rheMac3_ACTN3 RRLQRLQHLAEKFRQKASLHEAWTRGKEEMLSQRDYDSASLQEVRALLRR gorGor3_ACTN3 RRLQRLQHLAEKFRQKASLHEAWTRGKEEMLSQRDYDSASLQEVRALLRR panPan1_ACTN3 RRLQRLQHLAEKFRQKASLHEAWTRGKEEMLSQRDYDSASLQEVRALLRR saiBol1_ACTN3 RRLQRLQHLAEKFRQKASLHEAWTRGKEEMLSQRDYDSASLQEVRALLRR nomLeu3_ACTN3 RRLQRLQHLAEKFRQKASLHEAWTRGKEEMLSQRDYDSASLQEVRALLRR hg38_ACTN3 RRLQRLQHLAEKFRQKASLHEAWTRGKEEMLSQRDYDSALLQEVRALLRR ponAbe2_ACTN3 RRLQRLQHLAEKFRQKASLHETWTRGKEEMLSSRDYDSASLQEVRALLRR panTro4_ACTN3 RRLQRLQHLAEKFRQKASLHEAWTRXXXXXXXXXXXXXXXXXXXXXXXXX micMur2_ACTN3 RRLQRLQHLAEKFRQKASLHEAWTRGKEDMLSQRDYESASLQEVRALLRR otoGar3_ACTN3 RRLQRLQHLAEKFRQKASLHEAWTRGKEDMLSQRDYESASLQEVRALLRR tarSyr2_ACTN3 RRLQRLQHLAEKFRQKASLHESWTRGKLDMLSQRDYESASLQEVRALLRR chlSab2_ACTN3 HEAFESDLAAHQDRVEHIAALAQELNELDYHEAASVNSRCQAICDQWDNL macFas5_ACTN3 HEAFESDLAAHQDRVEHIAALAQELNELDYHEAASVNSRCQAICDQWDNL papAnu2_ACTN3 HEAFESDLAAHQDRVEHIAALAQELNELDYHEAASVNSRCQAICDQWDNL rheMac3_ACTN3 HEAFESDLAAHQDRVEHIAALAQELNELDYHEAASVNSCCQAICDQWDNL gorGor3_ACTN3 HEAFESDLAAHQDRVEHIAALAQELNELDYHEAASVNSRCQAICDQWDNL panPan1_ACTN3 HEAFESDLAAHQDRVEHIAALAQELNELDYHEAASVNSRCQAICDQWDNL saiBol1_ACTN3 HEAFESDLAAHQDRVEHIAALAQELNELDYHEAASVNSRCQAICDQWDNL nomLeu3_ACTN3 HEAFESDLAAHQDRVEHIAALAQELNELDYHEAASVNSRCQAICDQWDNL hg38_ACTN3 HEAFESDLAAHQDRVEHIAALAQELNELDYHEAASVNSRCQAICDQWDNL ponAbe2_ACTN3 HEAFESDLAAHQDRVEHIAALAQELNELDYHEAASVNSRCQAICDQWDNL panTro4_ACTN3 XXXXXXXXXXXXXXXXXXXXXXXXXXELDYHEAASVNSRCQAICDQWDNL micMur2_ACTN3 HEAFESDLAAHQDRVEHIAALAQELNELDYHEAASVNSRCQAICDQWDNL otoGar3_ACTN3 HEAFESDLAAHQDRVEHIAALAQELNELDYHEATSVNSRCQAICDQWDNL tarSyr2_ACTN3 HEAFESDLAAHQDRVEHIAALAQELNELDYHEAASVNSRCQAICDQWDQL chlSab2_ACTN3 GTLTQKRRDALERMEKLLETIDQLQLEFARRAAPFNNWLDGAVEDLQDVW macFas5_ACTN3 GTLTQKRRDALERMEKLLETIDQLQLEFARRAAPFNNWLDGAVEDLQDVW papAnu2_ACTN3 GTLTQKRRDALERMEKLLETIDQLQLEFARRAAPFNNWLDGAVEDLQDVW rheMac3_ACTN3 GTLTQKRRDALERMEKLLETIDQLQLEFARRAAPFNNWLDGAVEDLQDVW gorGor3_ACTN3 GTLTQKRRDALERMEKLLETIDQLQLEFARRAAPFNNWLDGAVEDLQDVW panPan1_ACTN3 GTLTQKRRDALERMEKLLETIDQLQLEFARRAAPFNNWLDGAVEDLQDVW saiBol1_ACTN3 GTLTQKRRDALERMEKLLETIDQLQLEFARRAAPFNNWLDGAVEDLQDVW nomLeu3_ACTN3 GTLTQKRRDALERMEKLLETIDQLQLEFARRTAPFNNWLDGAVEDLQDVW hg38_ACTN3 GTLTQKRRDALERMEKLLETIDRLQLEFARRAAPFNNWLDGAVEDLQDVW ponAbe2_ACTN3 GTLTQKRRDALERMEKLLETIDQLQVEFARRAAPFNNWLDGAVEDLQDVW panTro4_ACTN3 GTLTQKRRDALERMEKLLETIDQLQLEFARRAAPFNNWLDGAVEDLQDVW micMur2_ACTN3 GTLTQKRRDALERMEKLLETIDQLQLEFARRAAPFNNWLDGAVEDLQDVW otoGar3_ACTN3 GTLTQKRRDALERMEKLLETIDQLQLEFARRAAPFNNWLDGAVEDLQDVW tarSyr2_ACTN3 GTLTQKRRDALERMEKLLETIDQLQLEFARRAAPFNNWLDGAVEDLQDVW chlSab2_ACTN3 LVHSVEETQSLLTAHDQFKATLPEADRERGAIMGIQGEIQKICQTYGLRP macFas5_ACTN3 LVHSVEETQSLLTAHDQFKATLPEADRERGAIMGIQGEIQKICQTYGLRP papAnu2_ACTN3 LVHSVEETQSLLTAHDQFKATLPEADRERGAIMGIQGEIQKICQTYGLRP rheMac3_ACTN3 LVHSVEETQSLLTAHDQFKATLPEADRERGAIMGIQGEIQKICQTYGLRP

gorGor3_ACTN3 LVHSVEETQSLLTAHDQFKATLPEADREQGAIMGIQGEIQKICQTYGLRP panPan1_ACTN3 LVHSVEETQSLLTAHDQFKATLPEADRERGAIMGIQGEIQKICQTYGLRP saiBol1_ACTN3 LVHSVEETQSLLTAHDQFKATLPEADRERGAIMGIQGEIQKICQTYGLRP nomLeu3_ACTN3 LVHSVEETQSLLTAHDQFKATLPEADRERGAIMGIQGEIQKICQTYGLRP hg38_ACTN3 LVHSVEETQSLLTAHDQFKATLPEADRERGAIMGIQGEIQKICQTYGLRP ponAbe2_ACTN3 LVHSVEETQSLLTAHDQFKATLPEADRERGAIMGIQGEIQKICQTYGLRP panTro4_ACTN3 LVHSVEETQSLLTAHDQFKATLPEADRERGAIMGIQGEIQKICQTYGLRP micMur2_ACTN3 LVHSVEETQSLLTAHEQFKATLPEADRERGAIMGIQAEIQKICQTYGLRP otoGar3_ACTN3 LVHSVEETQSLVTAHEQFKATLPEADRERGAIMGIQGEIQKICQTYGLRP tarSyr2_ACTN3 LVHSVEETQSLVTAHDQFKATLSEADRERGAILGIQSEIQKICQTYGLRP chlSab2_ACTN3 CSTNPYITLSPQDINTKWDMVRKLVPSRDQTLQEELARQQVNERLRRQFA macFas5_ACTN3 CSTNPYITLSPQDINTKWDMVRKLVPSRDQTLQEELARQQVNERLRRQFA papAnu2_ACTN3 CSTNPYITLSPQDINTKWDMVRKLVPSRDQTLQEELARQQVNERLRRQFA rheMac3_ACTN3 CSTNPYITLSPQDINTKWDMVRKLVPSRDQTLQEELARQQVNERLRRQFA gorGor3_ACTN3 CSTNPYITLSPQDINTKWDMVRKLVPSRDQTLQEELARQQVNERLRRQFA panPan1_ACTN3 CSTNPYITLSPQDINTKWDMVRKLVPSRDQTLQEELARQQVNERLRRQFA saiBol1_ACTN3 CSTNPYITLSPQDINTKWDMVRKLVPSRDQTLQEELARQQVNERLRRQFA nomLeu3_ACTN3 CSTNPYVTLSPQDINTKWDMVRKLVPSRDQTLQEELARQQVNERLRRQFA hg38_ACTN3 CSTNPYITLSPQDINTKWDMVRKLVPSCDQTLQEELARQQVNERLRRQFA ponAbe2_ACTN3 CSTNPYITLSPQDINTKWDMVRKLVPSRDQTLQEELARQQVNERLRRQFA panTro4_ACTN3 CSTNPYITLSPQDINTKWDMVRKLVPSRDQTLQEELARQQVNERLRRQFA micMur2_ACTN3 STTSPYISLSPQDINNKWDKIRKLVPSRDQTLQEELARQQVNERLRRQFA otoGar3_ACTN3 SSSNPYISLSPQDINNKWDKIRKLVPSRDQTLQEELARQQVNERLRRQFA tarSyr2_ACTN3 NITNPYINLTPQDINSKWDQVRKLVPSRDQTLQEELARQQVNERLRRQFA chlSab2_ACTN3 AQANAIGPWIQAKVEEVGRLAAGLAGSLEEQMAGLRQQEQNIINYKTNID macFas5_ACTN3 AQANAIGPWIQAKVEEVGRLAAGLAGSLEEQMAGLRQQEQNIINYKTNID papAnu2_ACTN3 AQANAIGPWIQAKVEEVGRLAAGLAGSLEEQMAGLRQQEQNIINYKTNID rheMac3_ACTN3 AQANAIGPWIQAKVEEVGRLAAGLAGSLEEQMAGLRQQEQNIINYKTNID gorGor3_ACTN3 AQANAIGPWIQAKVEEVGRLAAGLAGSLEEQMAGLRQQEQNIINYKTNID panPan1_ACTN3 AQANAIGPWIQAKVEEVGRLAAGLAGSLEEQMAGLRQQEQNIINYKTNID saiBol1_ACTN3 AQANAIGPWIQAKVEEVGRLAAGLAGSLEEQMAGLRQQEQNIINYKTNID nomLeu3_ACTN3 AQANAIGPWIQAKVEEVGRLAAGLAGSLEEQMAGLRQQEQNIINYKTNID hg38_ACTN3 AQANAIGPWIQAKVEEVGRLAAGLAGSLEEQMAGLRQQEQNIINYKTNID ponAbe2_ACTN3 AQANAVGPWIQAKVEEVGRLAAGLAGSLEEQMAGLRQQEQNIINYKTNID panTro4_ACTN3 AHANAIGPWIQAKVEEVGRLAAGLAGSLEEQMAGLLQQEQNIINYKTNID micMur2_ACTN3 AQANAVGPWIQTKVEEVGRLAAGLIGSLEDQMAGLRQQEQNIINYKSNID otoGar3_ACTN3 AQANAVGPWIQAKVEEVGRLAAGLIGSLEDQMAGLRQQEQNIINYKSNID tarSyr2_ACTN3 AQANAVGPWIQAKVEEVGRLAAGLIGSLEDQMAGLRQQEHNIINYKSNID chlSab2_ACTN3 RLEGDHQLLQESLVFDNKHTVYSMEHIRVGWEQLLTSIARTINEVENQVL macFas5_ACTN3 RLEGDHQLLQESLVFDNKHTVYSMEHIRVGWEQLLTSIARTINEVENQVL papAnu2_ACTN3 RLEGDHQLLQESLVFDNKHTVYSMEHIRVGWEQLLTSIARTINEVENQVL rheMac3_ACTN3 RLEGDHQLLQESLVFDNKHTVYSMEHIRVGWEQLLTSIARTINEVENQVL gorGor3_ACTN3 RLEGDHQLLQESLVFDNKHTVYSMEHIRVGWEQLLTSIARTINEVENQVL panPan1_ACTN3 QLEGDHQLLQESLVFDNKHTVYSMEHIRVGWEQLLTSIARTINEVENQVL

saiBol1_ACTN3 RLEGDHQLLQESLVFDNKHTVYSMEHIRVGWEQLLTSIARTINEVENQVL nomLeu3_ACTN3 RLEGDHQLLQESLVFDNKHTVYSMEHIRVGWEQLLTSIARTINEVENQVL hg38_ACTN3 RLEGDHQLLQESLVFDNKHTVYSMEHIRVGWEQLLTSIARTINEVENQVL ponAbe2_ACTN3 RLEGDHQLLQESLVFDNKHTVYSMEHIRVGWEQLLTSIARTINEVENQVL panTro4_ACTN3 RLEGDHQLLQESLVFDHKHTVYSMEHIRVGWEQLLTSIARTINEVENQVL micMur2_ACTN3 RLEGDHQLLQESLVFDNKHTVYSMEHIRVGWEQLLTSIARTINEVENQVL otoGar3_ACTN3 RLEGDHQLLQESLVFDNKHTVYSMEHIRVGWEQLLTSIARTINEVENQVL tarSyr2_ACTN3 RLEGDHQLLQENLVFDNKHTVYSMEHIRVGWEQLLTSIARTINEVENQVL chlSab2_ACTN3 TRDAKGLSQEQLNEFRASFNHFDRKRNGMMEPDDFRACLISMGYDLGEVE macFas5_ACTN3 TRDAKGLSQEQLNEFRASFNHFDRKRNGMMEPDDFRACLISMGYDLGEVE papAnu2_ACTN3 TRDAKGLSQEQLNEFRASFNHFDRKRNGMMEPDDFRACLISMGYDLGEVE rheMac3_ACTN3 TRDAKGLSQEQLNEFRASFNHFDRKRNGMMEPDDFRACLISMGYDLGEVE gorGor3_ACTN3 TRDAKGLSQEQLNEFRASFNHFDRKRNGMMEPDDFRACLISMGYDLGEVE panPan1_ACTN3 TRDAKGLSQEQLNEFRASFNHFDRKRNGMMEPDDFRACLISMGYDLGEVE saiBol1_ACTN3 TRDAKGLSQEQLNEFRASFNHFDRKRNGMMEPDDFRACLISMGYDLGEVE nomLeu3_ACTN3 TRDAKGLSQEQLNEFRASFNHFDRKRNGMMEPDDFRACLISMGYDLGEVE hg38_ACTN3 TRDAKGLSQEQLNEFRASFNHFDRKQNGMMEPDDFRACLISMGYDLGEVE ponAbe2_ACTN3 TRDAKGLSQEQLNEFRASFNHFDRKRNGMMEPDDFRACLISMGYDLGEVE panTro4_ACTN3 TRDAKGLSQEQLNEFRASFNHFDRKRNGMMEPDDFRACLISMGYDLGEVE micMur2_ACTN3 TRDAKGLSQEQLNEFRASFNHFDRKRNGMMEPDDFRACLISMGYDLGEVE otoGar3_ACTN3 TRDAKGLSQEQLNEFRASFNHFDRKRNGMMEPDDFRACLISMGYDLGEVE tarSyr2_ACTN3 TRDAKGLSQEQLNEFRASFNHFDRKRNGRMEPDDFRACLISMGYDLGEAE chlSab2_ACTN3 FARIMTMVDPNAAGVVTFQAFIDFMTRETAETDTAEQVVASFKILAGDKN macFas5_ACTN3 FARIMTMVDPNAAGVVTFQAFIDFMTRETAETDTAEQVVASFKILAGDKN papAnu2_ACTN3 FARIMTMVDPNAAGVVTFQAFIDFMTRETAETDTAEQVVASFKILAGDKN rheMac3_ACTN3 FARIMTMVDPNAAGVVTFQAFIDFMTRETAETDTAEQVVASFKILAGDKN gorGor3_ACTN3 FARIMTMMDPNAAGVVTFQAFIDFMTRETAETDTTEQVVASFKILAGDKN panPan1_ACTN3 FARIMTMVDPNAAGVVTFQAFIDFMTRETAETDTTEQVVASFKILAGDKN saiBol1_ACTN3 FARIMTMVDPNAAGVVTFQAFIDFMTRETAETDTAEQVVASFKILAGDKN nomLeu3_ACTN3 FARIMTMVDPNAAGVVTFQAFIDFMTRETAETDTTEQVVASFKILAGDKN hg38_ACTN3 FARIMTMVDPNAAGVVTFQAFIDFMTRETAETDTTEQVVASFKILAGDKN ponAbe2_ACTN3 FARIMTMVDPNAAGVVTFQAFIDFMTRETAETDTTEQVVASFKILAGDKN panTro4_ACTN3 FARIMTMVDPNAAGVVTFQAFIDFMTRETAETDTTEQVVASFKILAGDKN micMur2_ACTN3 FARIMTMVDPNAAGVVTFQAFIDFMTRETAETDTAEQVVASFKILAGDKK otoGar3_ACTN3 FARIMTMVDPNAAGVVTFQAFIDFMTRETAETDTAEQVVASFKILAGDKS tarSyr2_ACTN3 FARIMTMVDPNASGVVTFQAFIDFMTRETAETDTAEQVVASFKILAGDKN chlSab2_ACTN3 YITPEELRRELPAEQAEYCIRRMVPYKGSGAPAGALDYVAFSSALYGESD macFas5_ACTN3 YITPEELRRELPAEQAEYCIRRMVPYKGSGAPAGALDYVAFSSALYGESD papAnu2_ACTN3 YITPEELRRELPAEQAEYCIRRMVPYKGSGAPAGALDYVAFSSALYGESD rheMac3_ACTN3 YITPEELRRELPAEQAEYCIRRMVPYKGSGAPAGALDYVAFSSALYGESD gorGor3_ACTN3 YITPEELRRELPAKQAEYCIRRMVPYKGSGAPAGALDYVAFSSALYGESD panPan1_ACTN3 YITPEELRRELPAEQAEYCIRRMVPYKGSGAPAGALDYVAFSSALYGESD saiBol1_ACTN3 YITPEELRRELPAEQAEYCIRRMVPYKGSGAPAGALDYVAFSSALYGESD nomLeu3_ACTN3 YITPEELRRELPAEQAEYCIRRMVPYKGSGAPAGALDYVAFSSALYGESD

hg38_ACTN3 YITPEELRRELPAKQAEYCIRRMVPYKGSGAPAGALDYVAFSSALYGESD ponAbe2_ACTN3 YITPEELRRELPAELAEYCIRRMVPFKGSGAPAGALDYVAFSSALYGESD panTro4_ACTN3 YITPEELRRELPAEQAEYCIRRMVPYKGSGAPAGALDYVAFSSALYGESD micMur2_ACTN3 YITAEELRRELPAEQAEYCIRRMVPYKGSGAPAGALDYVAFSSALYGESD otoGar3_ACTN3 YITPEELRRELPAEQAEYCIRRMVSYKGSGAPPGALDYVAFSSALYGESD tarSyr2_ACTN3 YITPEELRRELPAEQAEYCIRRMVAYKGSGAPVGALDYVAFSSALYGESD chlSab2_ACTN3 L macFas5_ACTN3 L papAnu2_ACTN3 L rheMac3_ACTN3 L gorGor3_ACTN3 L panPan1_ACTN3 L saiBol1_ACTN3 L nomLeu3_ACTN3 L hg38_ACTN3 L ponAbe2_ACTN3 L panTro4_ACTN3 L micMur2_ACTN3 L otoGar3_ACTN3 L tarSyr2_ACTN3 L