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Mol Biol Evol. 2012 July; 29(7): 1735–1745.
Published online 2012 January 24. doi:  10.1093/molbev/mss018
PMCID: PMC3375472

Evolution of the Globin Gene Family in Deuterostomes: Lineage-Specific Patterns of Diversification and Attrition

Abstract

In the Metazoa, globin proteins display an underlying unity in tertiary structure that belies an extraordinary diversity in primary structures, biochemical properties, and physiological functions. Phylogenetic reconstructions can reveal which of these functions represent novel, lineage-specific innovations, and which represent ancestral functions that are shared with homologous globin proteins in other eukaryotes and even prokaryotes. To date, our understanding of globin diversity in deuterostomes has been hindered by a dearth of genomic sequence data from the Ambulacraria (echinoderms + hemichordates), the sister group of chordates, and the phylum Xenacoelomorpha, which includes xenoturbellids, acoelomorphs, and nemertodermatids. Here, we report the results of a phylogenetic and comparative genomic analysis of the globin gene repertoire of deuterostomes. We first characterized the globin genes of the acorn worm, Saccoglossus kowalevskii, a representative of the phylum Hemichordata. We then integrated genomic sequence data from the acorn worm into a comprehensive analysis of conserved synteny and phylogenetic relationships among globin genes from representatives of the eight lineages that comprise the superphylum Deuterostomia. The primary aims were 1) to unravel the evolutionary history of the globin gene superfamily in deuterostomes and 2) to use the estimated phylogeny to gain insights into the functional evolution of deuterostome globins. Results of our analyses indicate that the deuterostome common ancestor possessed a repertoire of at least four distinct globin paralogs and that different subsets of these ancestral genes have been retained in each of the descendant organismal lineages. In each major deuterostome group, a different subset of ancestral precursor genes underwent lineage-specific expansions of functional diversity through repeated rounds of gene duplication and divergence. By integrating results of the phylogenetic analysis with available functional data, we discovered that circulating oxygen-transport hemoglobins evolved independently in several deuterostome lineages and that intracellular nerve globins evolved independently in chordates and acoelomorph worms.

Keywords: acorn worm, Ambulacraria, chordates, gene family evolution, hemoglobin, neuroglobin

Introduction

The structural unity of animal globins is reflected in the highly conserved “globin fold,” a characteristic three-dimensional folding of six to eight α-helices that enclose a noncovalently bound heme group in a hydrophobic pocket. This diagnostic structural feature is highly conserved among homologous globins from all kingdoms of life in spite of extensive divergence in amino acid sequence (Lesk and Chothia 1980; Lecomte et al. 2005). In animals, globins are expressed in a diverse range of tissues and cell types including muscle and nerve cells as well as circulating red blood cells, and in many invertebrates, extracellular globins are dissolved in vascular, coelomic, and/or perienteric fluids (Weber and Vinogradov 2001). This diversity in anatomical and cellular sites of expression is associated with a correspondingly wide variety of quaternary structures. Animal globins function as single-domain monomers, single-domain multisubunit proteins, or multidomain multisubunit proteins with 2–18 covalently linked globin domains per chain (Vinogradov 1985; Riggs 1991; Vinogradov et al. 1993; Terwilliger 1998; Weber and Vinogradov 2001; Projecto-Garcia et al. 2010). Some of the most remarkable elaborations of quaternary structure are found in the extracellular globins of invertebrates. The monomeric myoglobin (Mb; ~17 kDa) and the tetrameric hemoglobin (Hb; ~64 kDa) of gnathostome vertebrates are dwarfed by the multisubunit “hexagonal bilayer” globins of annelid and vestimentiferan worms (~3,600 kDa), which are composed of 144 globin chains and 36 linker peptides (Lamy et al. 1996), and the multidomain multisubunit globins of bivalve molluscs (~800–12,000 kDa) (Terwilliger and Terwilliger 1978).

The diversity of primary and quaternary structures displayed by animal globins underlies an equally rich diversity of function, ranging from reversible oxygen binding that is central to the familiar oxygen-transport and storage functions of vertebrate Hb and Mb, as well as functions related to facilitated oxygen diffusion, oxygen sensing, the scavenging of reactive oxygen and nitrogen species, redox signaling, and enzymatic functions involving NO oxygenase and reductase activities (Weber and Vinogradov 2001; Pesce et al. 2002; Fago et al. 2004; Hankeln et al. 2005; Burmester and Hankeln 2009). Phylogenetic analysis is required to determine which of these functions represent novel lineage-specific innovations, and which represent ancestral functions that are shared with homologous globin proteins in other eukaryotes and even prokaryotes (Hardison 1996, 1998; Vinogradov et al. 2005, 2006, 2007; Vinogradov and Moens 2008; Kakar et al. 2010). The primary functions of many animal globins have yet to be elucidated, and insights into their evolutionary origins and phylogenetic affinities may suggest hypotheses about their physiological roles. Although the majority of vertebrate globins are products of gene duplication or whole-genome duplication events that occurred in the stem lineage of vertebrates (Hoffmann et al. 2011, 2012; Storz et al. 2011), two monomeric globins, neuroglobin (Ngb) and globin X (GbX), have a more ancient history, as they derive from duplications that predate the origin of deuterostomes (Roesner et al. 2005; Ebner et al. 2010). The great antiquity and broad phyletic distribution of these two globins suggest that they may be performing physiological functions related to very fundamental aspects of cellular metabolism. The case of GbX is particularly interesting because it has been recently reported that GbX is a membrane-associated protein that may have an antioxidant function (Blank et al. 2011). Thus, identification of Ngb and GbX orthologs in nonchordate deuterostome taxa may provide additional insights into the physiological functions of these proteins.

Despite the significant advances in our understanding of globin gene family evolution in chordates (Ebner et al. 2003, 2010; Hoffmann, Opazo, et al. 2010, 2011, 2012; Hoffmann, Storz, et al. 2010; Storz et al. 2011), our understanding of globin diversity in deuterostomes as a whole has been hindered by a dearth of genomic sequence data for the Ambulacraria (echinoderms + hemichordates), the sister group of chordates, and the phylum Xenacoelomorpha, which includes xenoturbellids, acoelomorphs, and nemertodermatids. Globins were originally thought to have a very limited phyletic distribution among nonchordate deuterostomes (Vinogradov et al. 2006; Vinogradov and Moens 2008). A number of oxygen-transport globins have been functionally characterized in echinoderms, including sea cucumbers (class Holothuroidea) and brittle stars (class Ophiuroidea; Terwilliger and Read 1972; Bonaventura et al. 1976; Suzuki 1989; Mauri et al. 1991, McDonald et al. 1992; Baker and Terwilliger 1993; Kitto et al. 1998, Christensen et al. 2003), and bioinformatic searches in the genome of the sea urchin (class Echinoidea) revealed the presence of a 34-exon globin gene that encodes a single polypeptide with 16 covalently linked globin domains (Bailly and Vinogradov 2008). Here, we report the results of a phylogenetic and comparative genomic analysis of the globin gene set of deuterostomes. As a first step, we characterized the globin gene repertoire of the acorn worm, Saccoglossus kowalevskii, a representative of the phylum Hemichordata. We then integrated genomic sequence data from the acorn worm into a comprehensive analysis of conserved synteny and phylogenetic relationships among globin genes from representatives of the eight main lineages that comprise the superphylum Deuterostomia. The primary aims were 1) to unravel the evolutionary history of the globin gene superfamily in deuterostomes and 2) to use the estimated phylogeny to gain insights into the functional evolution of deuterostome globins. Results of our analyses indicate that the deuterostome common ancestor possessed a repertoire of at least four distinct globin paralogs and that different subsets of these ancestral genes have been retained in each of the descendant organismal lineages that survive to the present day. The phyletic distribution of globin genes among deuterostome taxa is clearly attributable to a winnowing of ancestral diversity such that different sets of structurally distinct paralogs have been retained in different lineages, and each of these paralogs seeded different lineage-specific expansions of functional diversity through repeated rounds of gene duplication and divergence.

Materials and Methods

Bioinformatic Analyses

We used bioinformatic tools to assemble a data set combining the complete globin gene repertoires of 18 deuterostome taxa, including 16 chordates (13 vertebrates, 2 urochordates, and 1 cephalochordate) plus 1 hemichordate and 1 echinoderm as representatives of Ambulacraria. The vertebrate species included five teleost fish (fugu, Takifugu rubripes; medaka, Oryzias latipes; pufferfish, Tetraodon nigroviridis; three-spined stickleback, Gasterosteus aculeatus; and zebrafish, Danio rerio), one amphibian (western clawed frog, Xenopus tropicalis), one squamate reptile (green anole lizard, Anolis carolinensis), three birds (chicken, Gallus gallus; turkey, Meleagris gallopavo; and zebra finch, Taeniopygia guttata), and three mammals (human, Homo sapiens; gray short-tailed opossum, Monodelphis domestica; and platypus, Ornithorhynchus anatinus). The nonvertebrate species included two sea squirts (Ciona intestinalis and C. savignyi, Urochordata), amphioxus (Branchiostoma floridae, Cephalochordata), the purple sea urchin (Strongylocentrotus purpuratus, Echinodermata), and the acorn worm (S. kowalevskii, Hemichordata). The acorn worm data were obtained from the Acorn Worm Genome Project hosted by the Human Genome Sequence Center from Baylor College of Medicine. Because the sea urchin possesses a globin gene that encodes 16 putative globin domains (Bailly and Vinogradov 2008), we considered each of the domains as separately alignable globin sequences in all analyses.

In addition to retrieving the complete globin gene repertoires of the species listed above, we also included individual globin sequences from several additional deuterostome taxa. For echinoderms, we included sequences from two subunits of the coelomic red blood cell Hbs of the sea cucumber, Caudina arenicola. For cartilaginous fish, we obtained α- and β-Hb sequences from the red stingray (Dasyatis akajei) and gummy houndshark (Mustelus antarcticus), as well as Mb sequences from the gummy houndshark and the Port Jackson shark (Heterodontus portusjacksoni). For birds, we included the globin E sequence from the preliminary release of the mallard duck (Anas platyrhynchos) genome, available from the Ensembl-Pre database. Most of the gnathostome vertebrates included in this study possess multiple paralogous copies of α- and β-like globin genes. Since the monophyly of the α- and β-globin gene families has been well established (Goodman et al. 1975, 1987), we only included a representative subset of the α- and β-globins from each species in our analyses. In the genomic analyses of the various chordate taxa and the sea urchin, our bioinformatic searches identified the same complement of globin genes that had been annotated previously. Because no information was available about the globin repertoire of the acorn worm genome, we used a representative set of the previously characterized deuterostome globins to seed bioinformatic searches for homologous sequences in the genome of this organism using the BLASTP, PSI-BLAST, and TBLASTN programs (Altschul et al. 1990, 1997).

Current estimates of deuterostome systematic relationships place xenoturbellids, acoelomorph worms, and nemertodermatids in the phylum Xenacoelomorpha, which is regarded as sister to Ambulacraria (Bourlat et al. 2006; Philippe et al. 2011). Thus, in order to include representatives from each of the main deuterostome lineages, we obtained two globin sequences from the acoel Symsagittifera roscoffensis, three globin sequences from the nemertodermatid Nemertoderma westbladi, plus 1 sequence from the xenoturbellid Xenoturbella bocki. The acoel globins derive from the acoel genome project, whereas the nemertodermatid and xenorurbellid globins were identified bioinformatically in the Trace Archive from the National Center for Biotechnology Information. The full list of sequences and the corresponding accession numbers are presented in supplementary table S1 (Supplementary Material online). Finally, based on the results from Hoogewijs et al. (2012), we added nine globin sequences from plants, which were included as outgroup sequences in phylogenetic analyses. The list of plant sequences and the corresponding accession numbers are presented in supplementary table S2 (Supplementary Material online).

Phylogenetic Analyses

We conducted our phylogenetic analyses in two stages. In the first stage, we aligned all sequences using MAFFT with automatic strategy selection (Katoh and Toh 2008), and we used Neighbor-Joining phylogenetic reconstructions as implemented in MEGA version 5 (Tamura et al. 2011) to identify and remove redundant sequences. In the second stage, we used the pruned data set to build multiple alignments using a variety of approaches: Kalign2 (Lassmann et al. 2009), the E-INS-i, G-INS-i, and L-INS-i strategies from Mafft v6.83 (Katoh and Toh 2008; Katoh et al. 2009), Muscle (Edgar 2004), PROMALS3d (Pei et al. 2008), and T-coffee (Notredame et al. 2000). In addition, we also tested a profile alignment approach in which the globin sequences from acorn worm, sea urchin, sea cucumber, and C. savignyi, plus those of the plant globins were added to a previously assembled alignment of globin sequences from vertebrates, amphioxus, and C. intestinalis (Hoffmann et al. 2011). We then employed MUMSA (Lassmann and Sonnhammer 2005, 2006) to rank the multiple alignments and then used the suiteMSA package ver 1.2.06 (Anderson et al. 2011) to visually compare between the two alignments with the highest MUMSA scores and selected the alignment from the profile alignment approach for structural reasons. Subsequently, we used the selected alignment to estimate phylogenetic relationships using Bayesian methods as implemented in MrBayes version 3.1.2 (Ronquist and Huelsenbeck 2003) under a mixed model of amino acid substitution. We set two independent runs of six simultaneous chains for 10,000,000 generations, sampling every 2,500 generations, and using default priors. Once convergence was verified, support for the nodes and parameter estimates were derived from a majority rule consensus of the last 2,500 trees. All trees were rooted with plant Hbs.

Results and Discussion

The Globin Gene Repertoire of Hemichordates

Our bioinformatic surveys of genomic and transcriptomic databases for the acorn worm revealed the presence of 17 putative globin genes that ranged in size from 151 to 243 codons (table 1). Sixteen of these sequences were derived from the acorn worm draft genome and one was derived from an mRNA record. Because the latter sequence (ACY92586) was identical to one of the genome-derived records (NP_001161601), we excluded the mRNA-derived sequence from all further analyses. The 16 acorn worm globin sequences were found on eight different genomic scaffolds, and bioinformatic predictions indicated that 11 of them have the 3-exon–2-intron structure typical of vertebrate-specific globins, 1 has a 4-exon–3-intron structure, and the remaining 4 are intron-less genes.

Table 1.
Globin Genes Identified in the Acorn Worm Genome Assembly.

The 16 acorn worm globins identified appear to be bona fide globins based on several criteria. The coding sequence lengths were within the range expected for functional globin proteins and were similar in size to the globins of sea squirts (Ebner et al. 2003) and amphioxus (Ebner et al. 2010). Moreover, the amino acid sequence alignment revealed that functionally important residues are conserved in all acorn worm globin sequences (fig. 1). The conserved sites include the CD1 phenylalanine, and the proximal (F8) and distal (E7) histidines, which are involved in heme-group coordination and/or Fe2+-ligand stabilization, respectively.

Fig. 1.
Structural alignment of acorn worm globins to Human Mb, Cygb, α-Hb, β-Hb, and Ngb sequences. The α-helical structure is shown on top of the alignments. The functionally important phenylalanine in position CD1 and the distal and ...

A Phylogenetic Tree of Deuterostome Globins

Current estimates of the deuterostome phylogeny (see the inset tree in fig. 2) place the group Ambulacraria, which includes echinoderms and hemichordates, as sister to the phylum Xenacoelomorpha, which includes acoels, nemertodermatids, and xenoturbellids (Bourlat et al. 2006; Philippe et al. 2011). To reconstruct the evolutionary history of the deuterostome globin gene superfamily, we combined the complete set of globins from the acorn worm with a diverse array of globins from echinoderms in the subphylum Echinozoa (the purple sea urchin [class Echinoidea] and sea cucumber [class Holothuroidea]) plus representatives of each of the major chordate and xenacoelomorph lineages.

Fig. 2.
Bayesian phylogram describing relationships among globin genes from representative deuterostome taxa, rooted with plant Hbs. Numbers next to the nodes correspond to Bayesian posterior probabilities. Circulating oxygen-transport globins are shown in red, ...

The initial data set of 180 sequences included an overrepresentation of vertebrate globins relative to those from nonvertebrate deuterostomes, as well as redundant sequences (the full list of sequences and the corresponding accession numbers are presented in supplementary tables S1 and S2, Supplementary Material online). In order to achieve a more balanced taxonomic representation without sacrificing phylogenetic coverage of globin diversity, we first generated a multiple sequence alignment using all sequences and we then built a neighbor joining tree (supplementary fig. S1, Supplementary Material online). The resultant tree allowed us to identify redundant and highly similar sequences, which we then removed to prune our initial data set to 110 sequences. We then aligned these sequences with eight alternative strategies (see Material and Methods), ranked the resulting alignments base on their MUMSA score, and compared the two alignments with the highest MUMSA scores (supplementary table S3, Supplementary Material online) using the suiteMSA package ver 1.2.06 (Anderson et al. 2011). For structural reasons, we selected the one from the profile alignment approach for all subsequent phylogenetic analyses (supplementary data file S1, Supplementary Material online).

Bayesian phylogenies arranged all deuterostome globins other than amphioxus Gb8 and the acorn worm intron-less globins (acorn worm Gb11, Gb12, Gb13, and Gb14) into four well-supported clades (fig. 2), each of them defined by the presence of both chordate and ambulacrarian globins. The first of these groups, clade 1, contains all vertebrate-specific globins (i. e., Cygb [including cyclostome Hbs], Mb, α- and β-Hb, GbE, and GbY), plus two separate groups of amphioxus globins, all urochordate globins, a clade of acorn worm globins, which is sister to a clade that includes nemertodermatid and xenoturbellid globins, and the two Hb subunit isoforms of the sea cucumber, which are sister to acoel Gb1. The second group, clade 2, contains acorn worm Gbs 7–10, 16, acoel Gb2, and vertebrate GbX and is sister to the third group, clade 3, which contains acorn worm Gb6 and Gb15, and amphioxus Gbs 3, 6, 1214. Finally, the fourth group, clade 4, includes the sea urchin subdomains, plus vertebrate Ngb and amphioxus Gb4. Relationships among these four clades are well resolved, with the Ngb-containing clade 4 sister to the remaining three clades.

These phylogenies placed the acorn worm globins in four separate clades and placed the six xenacoelomorph globins in three separate clades (fig. 2). Acorn worm Gb6 and Gb15 were placed in globin clade 3, together with amphioxus Gbs 3, 6, and 1214 with strong support, and the clade containing acorn worms Gbs 710 and Gb16 was placed in globin clade 2, together with acoel Gb2 and vertebrate GbX sequences also with strong support. Acorn worm Gbs 15 were placed in globin clade 1, in a clade sister to the globins from Nemertoderma and Xenoturbella, whereas the intron-less acorn worm globins (acorn worm Gbs1114) were placed in a deeply diverged clade with uncertain phylogenetic affinities. Finally, the remaining acoel globin, acoel Gb1, which is functionally similar to vertebrate Ngb (Bailly X, unpublished data) is also placed in globin clade 1, but sister to the two Hb subunit isoforms of the sea cucumber. In all cases, xenacoelomorph globins are placed sister to ambulacrarian globins, as expected under the systematic arrangement shown in the inset of figure 2.

The phylogenetic arrangement in figure 2 is consistent with previous studies in arranging vertebrate globins into those that derive from vertebrate-specific duplications (Cygb, GbE, GbY, Mb, and the Hbs from jawed and jawless vertebrates) and those that derive from duplications that predate the divergence between deuterostomes and protostomes (GbX and Ngb; Roesner et al. 2005; Hoffmann, Opazo, et al. 2010; Dröge and Makałowski 2011; Hoffmann et al. 2011, 2012; Hoogewijs et al. 2012; Storz et al. 2011). Furthermore, as in Hoogewijs et al. (2012), our results also identify the clade containing vertebrate Ngb as the deepest split among deuterostome globins.

Patterns of Conserved Synteny Corroborate the Phylogenetic Reconstructions

Because globins are short proteins, they provide a limited number of informative sites for resolving phylogenetic relationships. We therefore used comparisons of conserved synteny to provide a second line of evidence concerning the duplicative origins of deuterostome globins. This analysis of conserved synteny revealed that the genomic locations of the acorn worm globins are concordant with the inferred phylogenies. The five acorn worm globins that formed a clade sister to vertebrate GbX (Gbs 710, 16) are arranged in tandem along scaffold_38908, which also includes acorn worm Gb15. Four of the five acorn worm genes in globin clade 1 (Gbs 25) are also arranged in tandem on scaffold_9907, and the remaining acorn worm globins are located in separate scaffolds (table 1).

Genomic comparisons of conserved synteny provide additional support for globin clades 2–4 in the phylogeny of deuterostome globins (fig. 3). For example, in the case of globin clade 4, vertebrate Ngb, amphioxus Gb4, and the multidomain globin gene of the purple sea urchin (pGb) are each found adjacent to an ortholog of the human gene POMT2 (fig. 3A), which is consistent with results reported by Ebner et al. (2010). In the case of globin clade 2, the five acorn worm globins that are coorthologs of vertebrate GbX are arranged in a cluster that is flanked by orthologs of the human SRP14 gene, which is also located in the flanking region of GbX in frog, zebrafish, stickleback, medaka, and fugu (fig. 3B). Synteny comparisons for globin clade 3 are complicated by the fact that the four amphioxus globin genes in this clade are found on separate scaffolds (Ebner et al. 2010), and the same applies to the two acorn worm globins in this clade. Interestingly, both acorn worm Gb6 and amphioxus Gb3 are found next to an ortholog of the human SMOC2 gene (fig. 3C). Previous synteny comparisons had identified similarities in genomic context between vertebrate GbX and amphioxus Gb3, suggesting they might be orthologs (Ebner et al. 2010).

Fig. 3.
Patterns of conserved synteny in the genomic regions that harbor paralogous genes from deuterostome globin clades 2–4. (A) Synteny comparisons of the Ngb region in acorn worm, amphioxus, and vertebrates (as represented by human). (B) Synteny comparisons ...

Posttranslational Modifications in Globin Clades 2 and 3

Several acorn worm globins contain N- and C-terminal extensions as observed for nematode, fruitfly, and amphioxus globins as well as vertebrate Cygb and GbX (Burmester and Hankeln 1999; Burmester et al. 2002, 2006; Roesner et al. 2005; Hoogewijs et al. 2008; Ebner et al. 2010). Interestingly, recent evidence suggests that vertebrate GbX is a membrane-bound protein, and that the myristoylation and palmitoylation sites in the N-terminal extension of this protein are critical for its correct subcellular localization (Blank et al. 2011). Both N-myristoylation and S-palmitoylation are posttranslational modifications characteristic of membrane-associated proteins (Resh 1999; Farazi et al. 2001; Linder and Deschenes 2007), and thus, the presence of potential N-myristoylation and S-palmitoylation sites might provide clues to the subcellular localization and functional role of the analyzed proteins. Using computational prediction tools (Myristoylator [Bologna et al. 2004], MYR Predictor [Maurer-Stroh et al. 2002], and CSS-Palm [Ren et al. 2008]), we identified a putative N-terminal myristoylation site (at Gly2) in ten of the acorn worm globins and a palmitoylation site (at Cys3) in nine of them (supplementary table S4, Supplementary Material online). Moreover, all the vertebrate GbX sequences were predicted to have both myristoylation and palmitoylation sites, as was the case for the majority of the globins included in clades 2 and 3. These results suggest that these genes might encode membrane-associated globins with physiological roles similar to that of vertebrate GbX.

The Globin Repertoire of the Deuterostome Common Ancestor

For the purpose of reconstructing the complete complement of globin genes in the deuterostome common ancestor, we reasoned that monophyletic groups containing sequences from representatives of both Ambulacraria (echinoderms and hemichordates) and Chordata (vertebrates, cephalochordates, and urochordates) must trace their ancestry back to the stem lineage of deuterostomes. According to this criterion, we inferred that the last common ancestor of extant deuterostomes possessed a repertoire of at least four paralogous globin genes. These four globin genes may well be older than the divergence of Protostomia and Deuterostomia, and some of them may have originated prior to the split between animals and plants. It should be noted that this inferred number of ancestral globin genes represents a minimal estimate, as the addition of complete genome sequence data from additional deuterostomes might result in the discovery of additional globin lineages that could be traced to the deuterostome common ancestor. In fact, the phylogenetic relationships within globin clades 1 and 3 could reflect the presence of additional globin genes in the common ancestor of deuterostomes, and a similar case can be made for the divergent amphioxus Gb8 sequence and for the intron-less acorn worm globins.

Gains and Losses of Deuterostome Globin Genes

The phyletic distribution of the four defined globin lineages and the phylogenetic relationships within each of the corresponding clades reveal a complex history of lineage-specific gene duplications and deletions. None of the five deuterostome phyla for which we have whole-genome sequence data (Cephalochordata, Echinodermata, Hemichordata, Urochordata, and Vertebrata) appear to have retained representatives of all four ancestral deuterostome globins. The species included in this study exhibit tremendous variability in the size and membership composition of their globin gene repertoires. For example, the globin repertoires of the sea urchin and sea squirts descend from two distinct paralogs that were present in the deuterostome common ancestor, and each paralogous gene lineage diversified independently in the two separate organismal lineages via repeated rounds of duplication and divergence. The limited data available suggest that the globins from the sea cucumber also derive from several rounds of lineage-specific duplication and divergence. However, there are several Hb subunits from this species for which we have no sequence data (Kitto et al. 1998). Thus, additional genomic sequence data will be required to obtain more refined insights into the origins of the sea cucumber globins.

In contrast to the patterns observed in sea urchins and sea squirts, representatives of hemichordates, cephalochordates, and vertebrates have retained orthologous copies of at least three distinct genes that were present in the deuterostome common ancestor. These ancestral gene sets diversified independently in each of the different organismal lineages. Perhaps the most striking pattern is the one observed in globin clade 4, which contains vertebrate Ngbs. In this case, orthologs of Ngb have been secondarily lost in urochordates and hemichordates and have been retained as single copy genes in cephalochordates and in vertebrates. Remarkably, the Ngb ortholog in sea urchin has undergone multiple rounds of internal domain duplication so that it encodes a polypeptide with 16 covalently linked globin domains (Bailly and Vinogradov 2008).

Convergent Evolution of Deuterostome Globins

Phylogenetic analyses of vertebrate globins have revealed the origins of novel functional properties and have also revealed cases in which evolution has fashioned structurally distinct design solutions to similar physiological problems (Berenbrink et al. 2005; Berenbrink 2007; Opazo et al. 2008; Hoffmann, Storz, et al. 2010; Hoffmann, Opazo, et al. 2010; Storz et al. 2011). Our results demonstrate that within deuterostomes, circulating intracellular globins with oxygen-transport functions evolved at three times independently, in echinoderms, in cyclostome vertebrates, and in gnathostome vertebrates. Similarly, the functional Ngb of acoel, acoel Gb1, is clearly not orthologous to chordate Ngbs, indicating that these nerve globins evolved independently. In fact, acoel Gb1 is placed sister to the oxygen-carrying sea cucumber Hbs, in globin clade 1. A similar case of recruitment of an Hb-type globin as a nerve globin has been reported for the mollusc Spisula solidissima (Dewilde et al. 2006). In the case of circulating oxygen-transport globins, the underlying structural mechanisms of cooperative oxygen binding also evolved independently. Noncirculating globins, such as the gnathostome Cygb, GbE, GbX, and Ngb proteins are present in clades 1, 2, and 4, and circulating intracellular Hbs with cooperative oxygen binding are found on three different lineages within clade 1 (in gnathostomes [jawed vertebrates], cyclostomes [lampreys and hagfish], and sea cucumbers). In most gnathostomes, the cooperativity of tetrameric α2β2 Hb stems from an oxygenation-linked change in the Fe2+-F8(His) bond that is propagated to the α1β2 contacts. The resultant transition in quaternary structure between the oxy and the deoxy conformations involves a reordering of hydrogen bonds at the intersubunit contacts formed by the C, G, and H helices (Perutz et al. 1987). By contrast, in the oxygen-transport Hbs of cyclostomes, cooperativity stems from an oxygenation-linked dissociation of homo- and/or heterodimers into oxy-state monomers (e.g., Brittain and Wells 1986; Brittain et al. 1989; Fago and Weber 1995; Qiu et al. 2000; Fago et al. 2001). This ligand-dependent dissociation is mediated by intersubunit contacts involving the E and F helices such that the heme groups are in almost direct contact in the deoxy state. Similar modes of ligand-dependent dissociation/association dynamics are observed in the intracellular and extracellular Hbs of invertebrates (Kitto et al. 1998; Riggs 1998; Weber and Vinogradov 2001). For example, in the intracellular Hbs of the sea cucumber, Caudina arenicola, cooperativity stems from ligand-linked association into deoxygenated tetramers and higher level polymers (Bonaventura and Kitto 1973; Mitchell et al. 1995; Kitto et al. 1998). Similar to the Hbs of cyclostomes and those in many invertebrate taxa, the intersubunit contacts in the coelomic red blood cell Hbs of Caudina are formed by the E and F helices. However, crystal structures of Caudina Hbs indicate that the intersubunit contacts involve a unique set of residues (Mitchell et al. 1995; Kitto et al. 1998).

Results of our phylogenetic survey also shed light on the mechanism involved in the evolution of multidomain globins. Whereas Ngb is a single-domain monomer in all chordates examined to date, the Ngb ortholog in the purple sea urchin has been elaborated into a chimeric 16-domain protein through a combination of gene fusion and internal domain duplication (Bailly and Vinogradov 2008). As genome sequencing projects reveal globin gene structures for an increasing diversity of deuterostome and protostome taxa, it should be possible to evaluate the relative roles of internal domain duplication, domain shuffling, and gene fusion in the evolution of complex multidomain globins.

Supplementary Material

Supplementary tables S1–S4, figure S1, and data file S1 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).

Supplementary Data:

Acknowledgments

We thank the Acorn Worm Genome Sequencing Consortium for making their data publicly available and the Human Genome Sequence Center from Baylor College of Medicine for providing access to the Acorn Worm Genome Project. We also thank Y. Satta and two anonymous reviewers for helpful comments and suggestions. F.G.H. acknowledges grant support from the National Science Foundation (EPS-0903787), J.F.S. acknowledges grant support from the National Institutes of Health/NHLBI (R01 HL087216 and HL087216-S1) and the National Science Foundation (IOS-0949931), J.C.O. acknowledges grant support from the Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT 11080181), D.H. is supported by a Marie Curie IEF Fellowship from the European Commission, and T.H. acknowledges support by a Johannes Gutenberg University internal funding grant (SRFN).

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