Mitochondrial and plastid genomes of the colonial green alga Gonium pectorale give insights into the origins of organelle DNA architecture within the volvocales.

Volvocalean green algae have among the most diverse mitochondrial and plastid DNAs (mtDNAs and ptDNAs) from the eukaryotic domain. However, nearly all of the organelle genome data from this group are restricted to unicellular species, like Chlamydomonas reinhardtii, and presently only one multicellular species, the ∼4,000-celled Volvox carteri, has had its organelle DNAs sequenced. The V. carteri organelle genomes are repeat rich, and the ptDNA is the largest plastome ever sequenced. Here, we present the complete mtDNA and ptDNA of the colonial volvocalean Gonium pectorale, which is comprised of ∼16 cells and occupies a phylogenetic position closer to that of V. carteri than C. reinhardtii within the volvocine line. The mtDNA and ptDNA of G. pectorale are circular-mapping AT-rich molecules with respective lengths and coding densities of 16 and 222.6 kilobases and 73 and 44%. They share some features with the organelle DNAs of V. carteri, including palindromic repeats within the plastid compartment, but show more similarities with those of C. reinhardtii, such as a compact mtDNA architecture and relatively low organelle DNA intron contents. Overall, the G. pectorale organelle genomes raise several interesting questions about the origin of linear mitochondrial chromosomes within the Volvocales and the relationship between multicellularity and organelle genome expansion.

Introduction

Some of the most diverse and bizarre organelle genomes of all eukaryotes come from the Volvocales, which is a large order of predominantly freshwater green algae, belonging to chlorophycean class of the Chlorophyta. Volvocalean mitochondrial and plastid DNAs (mtDNAs and ptDNAs) show an impressive array of architectures, nucleotide landscapes, and coding compositions (Table 1)– and see Leliaert et al. [1] and Lee and Hua [2] for additional compilations. Moreover, certain volvocalean species, particularly those within the “Reinhardtinia clade” sensu Nakada et al. [3], have proven to be excellent systems for testing contemporary hypotheses on the evolution of organelle genome expansion and linearization [4], [5], [6].

Table 1

Completely sequenced organelle genomes from volvocalean green algae.

Species	Clade (lineage)	Organelle genome architecture
		MappingConformation	Size(kb)	AT content(%)	Coding(%)	Protein-codinggenes	Introns	GenBank/DDBJAccession
MITOCHONDRIAL DNA
Chlamydomonas reinhardtii	Reinhardtinia(volvocine)	Linear	16–19	55	67–82	7	0–3	EU306617–23
Chlamydomonas moewusii	Xenovolvoxa	Circular	23	65	54	7	9	AF008237
Chlorogonium elongatum	Caudivolvoxa	Circular	23	62	53	7	6	Y13643–4,Y07814
Dunaliella salina	Caudivolvoxa	Circular	28	66	42	7	18	GQ250045
Gonium pectorale	Reinhardtinia(volvocine)	Circular	16	61	73	7	1	AP012493
Polytomella capuana	Reinhardtinia	Linear	13	43	82	7	0	EF645804
Polytomella parva	Reinhardtinia	Linear	16	59	66	7	0	AY062933-4
Polytomella sp.SAG 63–10	Reinhardtinia	Linear	16	58	66	7	0	GU108480-1
Volvox carteri	Reinhardtinia(volvocine)	Circular	35	66	<40	7	3	EU760701,GU084821
PLASTID DNA
Chlamydomonas reinhardtii	Reinhardtinia(volvocine)	Circular	204	66	44	66	7	FJ423446
Dunaliella salina	Caudivolvoxa	Circular	269	68	35	66	>35	GQ250046
Gonium pectorale	Reinhardtinia(volvocine)	Circular	223	70	44	66	3	AP012494
Volvox carteri	Reinhardtinia(volvocine)	Circular	525	57	<20	66	9	GU084820

Note: Values rounded to whole numbers. Clade names are based on Nakada et al. [3]. Percent coding includes all annotated protein-, rRNA-, and tRNA-coding regions as well as non-standard ORFs, such as the rtl gene in the C. reinhardtii mtDNA. Gene number includes standard protein-coding genes, but does not include intronic or nonstandard ORFs, like rtl. Duplicate genes and introns were counted only once. Genome statistics for P. parva and P. piriformis are based on the concatenation of the two mitochondrial chromosomes; those for V. carteri should be considered as approximations as the mtDNA and ptDNA contain assembly gaps due to unresolved repeats. For C. reinhardtii, the mitochondrial genome size, intron number, and coding content can vary because of optional introns.

Most of our understanding of volvocalean mitochondrial and plastid genomes is limited to unicellular species, such as the model organisms Chlamydomonas reinhardtii and C. globosa (previously misidentified as C. incerta; see Nakada et al. [7]) [5], [8], the colorless and wall-less Polytomella capuana, P. parva, and P. piriformis [6], [9], [10], and the halotolerant β-carotene-rich Dunaliella salina [11]. Surprisingly little is known about the organelle genomes of colonial and multicellular volvocaleans, which are found within the volvocine lineage of the Reinhardtinia clade (Figure S1). Volvocine algae are preeminent models for studying the evolution of multicellularity [12], [13], and span the gamut of cellular complexity, from simple 4-celled species (e.g., Tetrabaena), to 8-64-celled colonial forms (e.g., Gonium), all the way to highly complex spheroidal taxa, with more than 500 cells (e.g., Volvox) [14], [15]. It is estimated that multicellular volvocine species last shared a common unicellular ancestor ∼200 million years ago [15].

Of the 8 different volvocalean algae for which complete mtDNA and/or ptDNA sequences are available [11], only one is multicellular: Volvox carteri, which is comprised of ∼4,000 cells. The organelle genomes of this species are distended with repetitive noncoding DNA, and similar palindromic repeats are located in both the mitochondrial and plastid compartments [16]. Moreover, the V. carteri ptDNA, at ∼525 kb, is among the largest plastomes ever observed (from any eukaryote) [4], dwarfing that of C. reinhardtii, which is 204 kb [17]. Although smaller than its plastid counterpart, the ∼35 kb mtDNA of V. carteri is still larger than any the other completely sequenced volvocalean mitochondrial genome. It is hypothesized that the expanded organelle genomes of V. carteri are a consequence of a low organelle mutation rate and/or a small effective population size [4].

The V. carteri mtDNA assembles as a circular molecule, contrasting the linear (or linear fragmented) architectures of all other well-studied Reinhardtinia-clade mitochondrial genomes, including those of C. reinhardtii, Polytomella spp., and the multicellular Pandorina morum [5], [11], [18]. These linear mtDNAs have evolved complex terminal structures [5], [10], called mitochondrial telomeres, which form long palindromic repeats at the genome ends. The origin and number of times that linear mitochondrial chromosomes have evolved within the Reinhardtinia is unknown, but it has been argued that they arose only once [4]. If true, this would imply that in a recent ancestor of V. carteri, the mtDNA reverted from a linear to a circular form.

To learn about organelle genome architecture within multicellular volvocine algae and to gain insight into ptDNA expansion and the origin of linear mtDNAs, we sequenced the mitochondrial and plastid genomes of Gonium pectorale–an 8- or 16-celled freshwater colonial alga, occupying a phylogenetic position closer to that of V. carteri than C. reinhardtii within the volvocine line [14], [15], [19] (Figure S1).

Materials and Methods

The organelle genomes described here come from Gonium pectorale K3-F3-4 (mating type minus), which was one of the F3 backcross strains to K41 (mating type plus) (originating from K41×K32 [F1 strains of Kaneko3×Kaneko4]) [20], [21] and is available as NIES-2863 from the Microbial Culture Collection at National Institute for Environmental Studies, Tsukuba, Japan (http://mcc.nies.go.jp/). G. pectorale was grown in 200–300 mL VTAC medium [22], [23] at 20°C on a 14∶10 h light-dark cycle, under cool-white fluorescent lamps (165–175 µmol m−2 s−1 intensity). Total DNA was extracted based on the protocol of Miller et al. [24].

Sequencing libraries were prepared from G. pectorale K3-F3-4 genomic DNA using the GS FLX Titanium Rapid Library Preparation Kit (F. Hoffmann-La Roche, Basel, Switzerland) and the TruSeq DNA Sample Prep Kit (Illumina Inc., San Diego, CA, USA), and were run on a GS FLX (F. Hoffmann-La Roche) and a MiSeq sequencer (Illumina Inc.), respectively. The GS FLX reads were assembled with Newbler v2.6. A fosmid library (23,424 clones) was constructed from G. pectorale K3-F3-4 genomic DNA using fosmid vector pKS300, which was developed in-house. End sequencing of the fosmid library and the BAC library of G. pectorale Kaneko3 (18,048 clones, Genome Institute (CUGI), Clemson Univ., Clemson, SC, USA) was carried out using a BigDye terminator kit ver3 (Life Technologies, Carlsbad, California, USA) and was run on automated ABI 3730 capillary sequencers (Life Technologies). The GS FLX contig sequences, which were derived from mitochondrial and chloroplast genomes, and the BAC/fosmid end-sequences were assembled using the Phrap/Consed systems. Gap closing and re-sequencing of low-quality regions in the assembly were performed by shotgun sequencing of the corresponding BAC/fosmid clones, PCR, primer walking, and direct sequencing of fosmid clones. The MiSeq sequence reads were mapped against the assembly sequences using the BWA program [25] after passing through the quality filter. The errors on each GS FLX assembly sequence were also corrected. The assembling delineated one circular mtDNA and two ptDNA isoforms (A and B), a common feature of plastid genomes with inverted repeats [26], [27] (Figure S2). The annotated G. pectorale mtDNA and ptDNA (isoform A) sequences are deposited in the DDBJ database under accession numbers AP012493 and AP012494, respectively.

Phylogenetic analyses were performed under maximum likelihood (ML) using RAxML [28] and PhyML 3.0 [29] with 100 bootstrap replicates. Maximum parsimony (MP) bootstrap analyses (based on 10 random replications of the full heuristic search with the tree bisection–reconnection branch-swapping algorithm) were performed in PAUP 4.0b10 [30] with 1,000 replications. MtDNA protein phylogeny was based on the deduced nad5, cox1, and cob amino acid sequences (Table S1), which were aligned using Clustal X [31]. Intron phylogenies were based on the deduced and aligned amino acid sequences of the nad5 and psaB intronic open reading frames (ORFs), which gave data matrices of 205 and 256 amino acids with 9 and 14 operational taxonomic units (OTUs), respectively (Tables S2, S3). Intron secondary structure maps were constructed as previously described [32].

Results and Discussion

The Gonium pectorale mtDNA: A Compact Circular Mapping Chromosome

The mitochondrial genome of G. pectorale has a conservative architecture: it is small (16 kb), circular-mapping, AT rich (61%), compact (73% coding), contains very few repeats, and has only a single intron (Figure 1, Table 1, Figure S3). It lacks the eccentricities that often characterize the mtDNAs of other volvocalean species, such as a high GC content (e.g., P. capuana), a linear or linear-fragmented conformation (e.g., P. parva), a large intron density (e.g., D. salina), non-standard genes (e.g., C. reinhardtii), and/or a bloated repeat-rich structure (e.g., V. carteri) [11]. The G. pectorale mtDNA is gene poor, encoding 7 proteins, 2 rRNAs, and 3 unique tRNAs, representing methionine, glutamine, and tryptophan (Figure 1). Two copies of trnM were identified adjacent to one another in the genome. Both have similar sequences and cloverleaf structures, and appear to have a role in elongation rather than initiation, as suggested for the trnM of other volvocalean algae. When ignoring non-standard genes and duplicate tRNAs, the G. pectorale mitochondrial gene repertoire mirrors those from all other available volvocalean algae, with the exception of Polytomella species, which lack trnW and trnQ. The G. pectorale mitochondrial large and small subunit (LSU and SSU) rRNA genes, like those from other available Reinhardtinia algae, are fragmented and scrambled throughout the genome into 8 and 4 coding modules, respectively. In V. carteri the eighth LSU module has been invaded by palindromic repeats, splitting it into two segments (L8a and L8b) [16]; in G. pectorale, however, the L8 module is intact.

Figure 1

Genetic map of the Gonium pectorale mitochondrial genome.

Note, the G. pectorale mtDNA is a circular-mapping molecule. Transfer RNA-coding regions are designated by the single-letter abbreviation of the amino acid they specify.

The sole intron of the G. pectorale mtDNA, located in nad5, is of group ID affiliation [33] (Figure S3) and encodes a putative intronic endonuclease. Other volvocaleans contain a nad5 group I intron (with the same insertion site), but none are from the Reinhardtinia clade. Our phylogenetic analyses of various volvocalean intronic ORFs (Figure S4) suggest that the G. pectorale nad5 intron either was acquired through horizontal transmission from a volvocalean closely related to Chlamydomonas moewusii or Chlorogonium elongatum or that it was present in the ancestor of the Volvocales and preserved in G. pectorale.

Linear mitochondrial chromosomes are widespread throughout the Reinhardtinia clade, occurring in all explored taxa [34], with the exception of V. carteri, which has a circular mtDNA map, but rare possible linear forms of the genome have been observed [4], [16] (Table 1). Our de novo and mapping assemblies of the G. pectorale mtDNA gave an unambiguous circular-mapping chromosome (see Materials and Methods), and although such a map could represent a circularly permuted, linear-type structure, various features of the G. pectorale mitochondrial genome support the idea that it is circular. For instance, all twelve of the G. pectorale mtDNA genes have the same transcriptional polarity–a trait that is also found in V. carteri and available volvocalean species with circular mitochondrial genomes. Conversely, in all of the sequenced linear mtDNAs from the Volvocales, the genes are divided into two transcriptional polarities, proceeding outward towards the ends of the chromosome [6].Furthermore, our Southern blot analysis of the G. pectorale mtDNA, cut with restriction enzymes, demonstrates that it is a circular molecule (Figure S5).

Our evidence for a circular mitochondrial genome in G. pectorale raises interesting questions about the origin of linear mtDNAs within the Reinhardtinia clade. There is little doubt that the ancestral volvocalean mtDNA was circular, and it is argued that there was a single shift from a circular to a linear mtDNA structure in the ancestor that gave rise to Reinhardtinia algae [6]. Within the Reinhardtinia clade, V. carteri and G. pectorale belong to a monophyletic colonial or multicellular volvocalean group from which unicellular members are separated [14], [15] (Figure 2), but the multicellular volvocalean Pandorina morum has a linear mtDNA [18]. Moreover, V. carteri and P. morum belong to the monophyletic Volvocaceae from which G. pectorale is excluded [14], [15], [19] (Figure S1). Thus, the appearance of circular mitochondrial genome maps in both V. carteri and G. pectorale suggests that the mtDNAs of these species independently reverted from a linear to a circular conformation in the two separate ancestors of G. pectorale and V. carteri (Figure S1) or alternatively that there were multiple origins of linear mitochondrial genomes in the Reinhardtinia clade, in the ancestors of Polytomella, C. reinhardtii, and P. morum (Figure S1). Studies of mtDNA structure from other volvocine species, such as Tetrabaena and Yamagishiella, are needed to further investigate these hypotheses.

Figure 2

MtDNA protein phylogeny of seven species belonging to Reinhardtinia clade and three outgroup species from the Volvocales.

The tree was constructed under the RAxML (with WAG+I+4G model) method using the concatenated sequences of the deduced nad5, cox1, and cob amino acid sequences. Left, middle, and right bootstrap values (≥50%) obtained using the RAxML, PhyML (with LG+I+4G model), and MP analysis, respectively. The amino acid sequences of the three proteins were aligned by Clustal X [29], and ambiguously aligned and highly variable regions were removed to construct a multiprotein data matrix of 909 amino acids from the 10 operational taxonomic units (Table S1).

The Gonium pectorale ptDNA Shows Moderate Genome Expansion

Volvocalean plastid genomes are big and that of G. pectorale, at 222.6 kb, is no exception. Of the approximately 300 complete (or almost complete) ptDNAs in GenBank, as of 1 August 2012, fewer than ten have a length >200 kb, all but one of which are from chlorophyte green algae, including the volvocaleans C. reinhardtii (204 kb), D. salina (269 kb) and V. carteri (∼525 kb) [4], [11], [17]. The large size of volvocalean ptDNAs is not a product of an inflated gene number, but a consequence of having an abundance of noncoding nucleotides, often represented by repetitive elements and introns. This is also true for the G. pectorale ptDNA, which is 56% (∼125 kb) noncoding. Almost all of these noncoding nucleotides are AT rich (average = 71%) and found in intergenic regions.

The coding regions also have a high AT content (68%) and encompass a total of 98 unique genes, encoding 67 proteins, 3 rRNAs, 27 tRNAs, and a single misc RNA (tscA) (Figure 3). Six of these genes (psbA, rrnL, rrnS, rrnF, trnA, and trnI) are duplicated, being located in a pair of 14.8 kb inverted repeats, which divide the G. pectorale ptDNA into a large (99.6 kb) and a small (93.5 kb) single-copy region (Figure 3). This gene complement and inverted-repeat arrangement is almost identical to those of C. reinhardtii and V. carteri (Figure 3, Figure S6).

Figure 3

Genetic map of the Gonium pectorale plastid genome.

Note, the G. pectorale ptDNA is a circular-mapping molecule. Transfer RNA-coding regions are designated by the single-letter abbreviation of the amino acid they specify.

Although some volvocalean algae harbour many ptDNA introns (Table 1)–V. carteri has 9 and D. salina has >35–G. pectorale harbours just three: one located in psaB, which appears to be of group IA affiliation [33] (Figure S3), and encodes a putative endonuclease-like protein, and two short group II introns (117 and 176 bp) found upstream of psaA exons 2 and 3 (Figure 3). Phylogenetic analysis of the G. pectorale intron (Figure S7) show that it is closely related to the psaB group I intron of the chlorophycean (but non-volvocalean) green alga Stigeoclonium helveticum [35]; moreover, both introns have the same insertion site within the psaB gene. V. carteri also has a psaB intron, but it is of group II affiliation [4]. In fact, there is not a single homologous pair of either group I or group II introns among the G. pectorale, V. carteri, and C. reinhardtii plastid genomes (Figure S6), suggesting that rapid horizontal intron transfer and loss occurred within the colonial Volvocales.

The G. pectorale plastid genome, like its V. carteri and C. reinhardtii counterparts, contains hundreds of short repetitive elements, distributed throughout the intergenic regions, as demonstrated by the dotplot similarity matrix (Figure S8). Many of the V. carteri ptDNA repeats are palindromes, and can be folded into hairpin structures [16]. The same is true for the G. pectorale ptDNA, which contains ∼135 short (13 nt) palindromic repeats (including eight in the coding regions) with the motif: 5′- TCCCCNNNGGGGA-3′ (Figure S9). This is fewer repeats than found in the V. carteri ptDNA, which contains over a thousand palindromic elements.

The G. pectorale ptDNA is slightly more expanded (by ∼19 kb) than that of C. reinhardtii, but much smaller than those of the unicellular D. salina (269 kb, ∼65% noncoding) and the multicellular V. carteri (∼525 kb, >80% noncoding) (Table 1). What has led to such a wide spectrum of ptDNA expansion within the Volvocales? One contemporary–and controversial [36], [37]–hypothesis for the evolution of genome size, called the mutational hazard hypothesis [38], argues that genome expansion is a product of a low effective population size (N) (which results in increased random genetic drift) and/or a low mutation rate (μ), which reduces the burden of harbouring excess DNA. The V. carteri ptDNA is estimated to have a very low Nμ [4], about twenty times lower than that of the C. reinhardtii ptDNA [39], which may explain why it is so bloated. We do not know the value of Nμ for the G. pectorale ptDNA–this will require sequencing the plastid genomes of several additional G. pectorale isolates. However, given that this species is ∼10 times larger than C. reinhardtii (16 cells vs a single cell) and a hundred times smaller than V. carteri (16 cells vs 4,000 cells), and that all three of these algae are found in a similar environment (freshwater ponds)–unlike D. salina, which is marine–one might expect the effective population size of G. pectorale to be similar or marginally smaller than that of C. reinhardtii, and much larger than that of V. carteri. If true, this may have contributed to G. pectorale having a ptDNA architecture comparable to that of C. reinhardtii but much different than that of V. carteri. Under this hypothesis, it can therefore be predicted that as more volvocine organelle DNAs are sequenced, species with large cell numbers and presumably low effective population sizes will have more bloated genomes than those with small cell numbers and large effective population sizes.

Simplified diagram for phylogenetic relationships of selected taxa of the unicellular, colonial and multicellular vovlocaleans.

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Diagrams of possible isoforms of ptDNA of Gonium pectorale. A. Two isoforms as found in other ptDNAs with a typical inverted repeat. B. Two additional isoforms that were not rejected based on assembling of our sequence data.

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Secondary structures of group I introns within the Gonium pectorale organelle DNAs. A. Mitochondrial nad5 group ID intron. B. Chloroplast psaB group IA intron.

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Phylogeny of Gonium pactorale nad5 group I intronic ORF. The tree was constructed under the RAxML (with WAG+4G model) method using 8 additional, related amino acid sequences selected based on the topology of the distance tree provided by blastp research of NCBI (http://www.ncbi.nlm.nih.gov/). Numbers on the left, middle and right at branches represent bootstrap values (≥50%) obtained using the RAxML, PhyML (with LG+4G model), and MP analysis, respectively. The amino acid sequences were aligned by Clustal X, and ambiguously aligned and highly variable regions were removed to construct a data matrix of 205 amino acids from the 9 operational taxonomic units (Table S2).

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Southern blot analysis of Gonium pectorale mtDNA with four restriction enzymes that cut the genome once (SacI and StuI) or twice (SacII and EcoRI). Genome map coordinates are based on the G. pectorale mtDNA DDBJ accession (AP012493). SacI and StuI digestions each gave single genome-sized bands (∼16 kb), and the SacII and EcoRI reactions each gave two bands. These data are consistent with the G. pectorale mtDNA being a circular molecules. Probe DNA was amplified by PCR with two specific primers (Gopec-mito-F 5′-CGGGCAAAGCATAATTAGTGTAG-3′ and Gopec-mito-R 5′-ACGAACAAGAGGAAGACCTAAC-3′).

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Venn diagram comparing the gene repertoires of three volvocalean chloroplast genomes (AP012494, GU084820 and FJ423446). 102 genes (single asterisk) shared by the three genomes include 12 genes distributed in IRA and IRB and trnI (cau), which was previously annotated as one of the triplicated trnM in C. reinhardtii and V. carteri. Double asterisks represent one of the duplicated genes in G. pectorale and C. reinhardtii. Triple asterisks exhibit one of the duplicated genes in C. reinhardtii. Note that all intronic ORFs in G. pectprale (1#) and V. carteri (6#) are unique for each genome and considered “non-coding” in the text.

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Phylogeny of Gonium pactorale psaB group I intronic ORF. The tree was constructed under the RAxML (with WAG+4G model) method using 13 additional, related amino acid sequences selected based on the topology of the distance tree provided by blastp research of NCBI (http://www.ncbi.nlm.nih.gov/). Numbers on the left, middle and right at branches represent bootstrap values (≥50%) obtained using the RAxML, PhyML (with LG+G model), and MP analysis, respectively. The amino acid sequences were aligned by Clustal X, and ambiguously aligned and highly variable regions were removed to construct a data matrix of 256 amino acids from the 14 operational taxonomic units (Table S3).

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Dotplot similarity matrix of the Gonium pectorale plastid genome. The X- and Y-axes each represent the G. pectorale plastid genome (222.6 kb). Dots in the nucleotide similarity matrix represent regions of sequence similarity. The matrix was generated using JDotter, with a sliding-window size of 50. The inverted repeats are highlighted in red in the matrix.

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Distribution of short (13 nt) palindromic repeats (including seven [red arrows] in five coding regions [blues arrows]) with the motif: 5′- TCCCCNNNGGGGA-3′ in ptDNA of Gonium pectorale. The repeats were examined by using Serial Cloner 2.5 (http://serialbasics.free.fr/Serial_Cloner.html).

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Amino acid alignment and origin of the data used for Figure 2.

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Amino acid alignment and origin of the data used for Figure S4.

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Amino acid alignment and origin of the data used for Figure S7.

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