Whole-genome duplications spurred the functional diversification of the globin gene superfamily in vertebrates

It has been hypothesized that two successive rounds of whole-genome duplication (WGD) in the stem lineage of vertebrates provided genetic raw materials for the evolutionary innovation of many vertebrate-specific features. However, it has seldom been possible to trace such innovations to specific functional differences between paralogous gene products that derive from a WGD event. Here, we report genomic evidence for a direct link between WGD and key physiological innovations in the vertebrate oxygen transport system. Specifically, we demonstrate that key globin proteins that evolved specialized functions in different aspects of oxidative metabolism (hemoglobin, myoglobin, and cytoglobin) represent paralogous products of two WGD events in the vertebrate common ancestor. Analysis of conserved macrosynteny between the genomes of vertebrates and amphioxus (subphylum Cephalochordata) revealed that homologous chromosomal segments defined by myoglobin + globin-E, cytoglobin, and the α-globin gene cluster each descend from the same linkage group in the reconstructed proto-karyotype of the chordate common ancestor. The physiological division of labor between the oxygen transport function of hemoglobin and the oxygen storage function of myoglobin played a pivotal role in the evolution of aerobic energy metabolism, supporting the hypothesis that WGDs helped fuel key innovations in vertebrate evolution.     

Functional diversification of sea lamprey globins in evolution and development

Agnathans have a globin repertoire that markedly differs from that of jawed (gnathostome) vertebrates. The sea lamprey (Petromyzon marinus) harbors at least 18 hemoglobin, two myoglobin, two globin X, and one cytoglobin genes. However, agnathan hemoglobins and myoglobins are not orthologous to their cognates in jawed vertebrates. Thus, blood-based O₂ transport and muscle-based O₂ storage proteins emerged twice in vertebrates from a tissue-globin ancestor. Notably, the sea lamprey displays three switches in hemoglobin expression in its life cycle, analogous to hemoglobin switching in vertebrates. To study the functional changes associated with the evolution and ontogenesis of distinct globin types, we determined O₂ binding equilibria, type of quaternary assembly, and nitrite reductase enzymatic activities of one adult (aHb5a) and one embryonic/larval hemoglobin (aHb6), myoglobin (aMb1) and cytoglobin (Cygb) of the sea lamprey. We found clear functional differentiation among globin types expressed at different developmental stages and in different tissues. Cygb and aMb1 have high O₂ affinity and nitrite reductase activity, while the two hemoglobins display low O₂ affinity and nitrite reductase activity. Cygb and aHb6 but not aHb5a show cooperative O₂ binding, correlating with increased stability of dimers, as shown by gel filtration and molecular modeling. The high O₂-affinity and the lack of cooperativity confirm the identity of the sea lamprey aMb1 as O₂ storage protein of the muscle. The dimeric structure and O₂-binding properties of sea lamprey and mammalian Cygb were very similar, suggesting a conservation of function since their divergence around 500 million years ago.     

Gene duplication as a major force in evolution

Gene duplication is an important mechanism for acquiring new genes and creating genetic novelty in organisms. Many new gene functions have evolved through gene duplication and it has contributed tremendously to the evolution of developmental programmes in various organisms. Gene duplication can result from unequal crossing over, retroposition or chromosomal (or genome) duplication. Understanding the mechanisms that generate duplicate gene copies and the subsequent dynamics among gene duplicates is vital because these investigations shed light on localized and genomewide aspects of evolutionary forces shaping intra-specific and inter-specific genome contents, evolutionary relationships, and interactions. Based on whole-genome analysis of Arabidopsis thaliana, there is compelling evidence that angiosperms underwent two whole-genome duplication events early during their evolutionary history. Recent studies have shown that these events were crucial for creation of many important developmental and regulatory genes found in extant angiosperm genomes. Recent studies also provide strong indications that even yeast (Saccharomyces cerevisiae), with its compact genome, is in fact an ancient tetraploid. Gene duplication can provide new genetic material for mutation, drift and selection to act upon, the result of which is specialized or new gene functions. Without gene duplication the plasticity of a genome or species in adapting to changing environments would be severely limited. Whether a duplicate is retained depends upon its function, its mode of duplication, (i.e. whether it was duplicated during a whole-genome duplication event), the species in which it occurs, and its expression rate. The exaptation of preexisting secondary functions is an important feature in gene evolution, just as it is in morphological evolution.     

Phylogenetic diversification of the globin gene superfamily in chordates

Phylogenetic reconstructions provide a means of inferring the branching relationships among members of multigene families that have diversified via successive rounds of gene duplication and divergence. Such reconstructions can illuminate the pathways by which particular expression patterns and protein functions evolved. For example, phylogenetic analyses can reveal cases in which similar expression patterns or functional properties evolved independently in different lineages, either through convergence, parallelism, or evolutionary reversals. The purpose of this article is to provide a robust phylogenetic framework for interpreting experimental data and for generating hypotheses about the functional evolution of globin proteins in chordate animals. To do this, we present a consensus phylogeny of the chordate globin gene superfamily. We document the relative roles of gene duplication and whole-genome duplication in fueling the functional diversification of vertebrate globins, and we unravel patterns of shared ancestry among globin genes from representatives of the three chordate subphyla (Craniata, Urochordata, and Cephalochordata). Our results demonstrate the value of integrating phylogenetic analyses with genomic analyses of conserved synteny to infer the duplicative origins and evolutionary histories of globin genes. We also discuss a number of case studies that illustrate the importance of phylogenetic information when making inferences about the evolution of globin gene expression and protein function. Finally, we discuss why the globin gene superfamily presents special challenges for phylogenetic analysis, and we describe methodological approaches that can be used to meet those challenges.     

Neuroglobin and cytoglobin: genes, proteins and evolution

Hemoglobin and myoglobin are oxygen transport and storage proteins of most vertebrates. Neuroglobin (Ngb) and cytoglobin (Cygb)--two recent additions to the vertebrate globin superfamily--have still disputed functions. Combining the data from all available resources, we investigate the evolution of these novel globins. Both Ngb and Cygb show little sequence variation in vertebrate evolution, suggesting conserved structures and functions, and an important role in the animal's metabolism. Exon-intron patterns remained unchanged in Ngb and Cygb, with the exception of the addition of a 3' exon to Cygb early in mammalian evolution. In phylogenetic analyses, Ngb forms a common branch with globin X, another recently identified globin with undefined function in lower vertebrates, and with some invertebrate nerve globins. This shows an early divergence of this branch in animal evolution. Cygb is related to myoglobin, and associated with an eye-specific globin from birds. The pattern of globin evolution shows that proteins with clear respiratory roles evolved independently from intracellular globins with uncertain functions. This result suggests either multiple independent functional changes or a yet undefined respiratory role of tissue globins like Ngb and Cygb.     

Chromosomal mapping,differential origin and evolution of the S100 gene family

S100 proteins are calcium-binding proteins, which exist only in vertebrates and which constitute a large protein family. The origin and evolution of the S100 family in vertebrate lineages remain a challenge. Here, we examined the synteny conservation of mammalian S100A genes by analysing the sequence of available vertebrate S100 genes in databases. Five S100A gene members, unknown previously, were identified by chromosome mapping analysis. Mammalian S100A genes are duplicated and clustered on a single chromosome while two S100A gene clusters are found on separate chromosomes in teleost fish, suggesting that S100A genes existed in fish before the fish-specific genome duplication took place. During speciation, tandem gene duplication events within the cluster of S100A genes of a given chromosome have probably led to the multiple members of the S100A gene family. These duplicated genes have been retained in the genome either by neofunctionalisation and/or subfunctionalisation or have evolved into non-coding sequences. However in vertebrate genomes, other S100 genes are also present i.e. S100P, S100B, S100G and S100Z, which exist as single copy genes distributed on different chromosomes, suggesting that they could have evolved from an ancestor different to that of the S100A genes.     

Gene expression and tissue distribution of cytoglobin and myoglobin in the Amphibia and Reptilia: possible compensation of myoglobin with cytoglobin in skeletal muscle cells of anurans that lack the myoglobin gene

Cytoglobin (Cygb), a recently discovered vertebrate cytoplasmic heme-binding globin, is considered to be in a clade with vertebrate myoglobin (Mb), which is exclusively distributed in the cytoplasm of cardiac and skeletal muscles as an oxygen storage protein. GenBank databases (NCBI and JGI) and gene synteny analyses showed the absence of the Mb gene (mb) in two anuran amphibians, Xenopus laevis and X. tropicalis. Here we conducted comparative studies on the gene expression and tissue distribution of Cygb and Mb in anuran and reptilian tissues. Cygb and Mb genes were cloned from a reptile, iguana (Iguana iguana). Two types of cygb (cygb-1 and -2) were cloned, with lengths of 1066 and 1034 bp, and 196 and 193 amino acid residues, respectively. Their nucleotide and amino acid sequence identities were 90 and 87%, respectively. The Mb gene covered 1416 bp with an open reading frame of 465 bp, giving rise to a 154 amino acid protein. The distal ligand-binding histidine at E7, the proximal heme-binding histidine at F8, and the phenylalanine residue at CD1 were conserved in Mb and Cygb. The nucleotide and amino acid sequence identity of I. iguana cygb-1 and cygb-2 against X. laevis cygb were approximately 67% and 65%, respectively. RT-PCR demonstrated that X. laevis cygb was uniquely expressed in the heart and skeletal muscles, and faintly in the liver and spleen, which was quite contrasted with Iguana and the other vertebrates, where mb is exclusively expressed in the heart and skeletal muscles. Immunohistochemical analyses showed the distribution of Cygb in the cytoplasm of skeletal muscle cells. Interestingly, Cygb in the heart was localized in the nuclei. Considering the absence of mb in the Anura, we hypothesize that Cygb in muscle cells of anurans compensates for the lack of Mb for the storage and intracellular transportation of oxygen.     

Evolution of developmental roles of Pax2/5/8 paralogs after independent duplication in urochordate and vertebrate lineages

Background

Gene duplication provides opportunities for lineage diversification and evolution of developmental novelties. Duplicated genes generally either disappear by accumulation of mutations (nonfunctionalization), or are preserved either by the origin of positively selected functions in one or both duplicates (neofunctionalization), or by the partitioning of original gene subfunctions between the duplicates (subfunctionalization). The Pax2/5/8 family of important developmental regulators has undergone parallel expansion among chordate groups. After the divergence of urochordate and vertebrate lineages, two rounds of independent gene duplications resulted in the Pax2, Pax5, and Pax8 genes of most vertebrates (the sister group of the urochordates), and an additional duplication provided the pax2a and pax2b duplicates in teleost fish. Separate from the vertebrate genome expansions, a duplication also created two Pax2/5/8 genes in the common ancestor of ascidian and larvacean urochordates.

Results

To better understand mechanisms underlying the evolution of duplicated genes, we investigated, in the larvacean urochordate Oikopleura dioica, the embryonic gene expression patterns of Pax2/5/8 paralogs. We compared the larvacean and ascidian expression patterns to infer modular subfunctions present in the single pre-duplication Pax2/5/8 gene of stem urochordates, and we compared vertebrate and urochordate expression to infer the suite of Pax2/5/8 gene subfunctions in the common ancestor of olfactores (vertebrates + urochordates). Expression pattern differences of larvacean and ascidian Pax2/5/8 orthologs in the endostyle, pharynx and hindgut suggest that some ancestral gene functions have been partitioned differently to the duplicates in the two urochordate lineages. Novel expression in the larvacean heart may have resulted from the neofunctionalization of a Pax2/5/8 gene in the urochordates. Expression of larvacean Pax2/5/8 in the endostyle, in sites of epithelial remodeling, and in sensory tissues evokes like functions of Pax2, Pax5 and Pax8 in vertebrate embryos, and may indicate ancient origins for these functions in the chordate common ancestor.

Conclusion

Comparative analysis of expression patterns of chordate Pax2/5/8 duplicates, rooted on the single-copy Pax2/5/8 gene of amphioxus, whose lineage diverged basally among chordates, provides new insights into the evolution and development of the heart, thyroid, pharynx, stomodeum and placodes in chordates; supports the controversial conclusion that the atrial siphon of ascidians and the otic placode in vertebrates are homologous; and backs the notion that Pax2/5/8 functioned in ancestral chordates to engineer epithelial fusions and perforations, including gill slit openings.     

The alphaD-globin gene originated via duplication of an embryonic alpha-like globin gene in the ancestor of tetrapod vertebrates

Gene duplication is thought to play an important role in the co-option of existing protein functions to new physiological pathways. The globin superfamily of genes provides an excellent example of the kind of physiological versatility that can be attained through the functional and regulatory divergence of duplicated genes that encode different subunit polypeptides of the tetrameric hemoglobin protein. In contrast to prevailing views about the evolutionary history of the alpha-globin gene family, here we present phylogenetic evidence that the alpha(A)- and alpha(D)-globin genes are not the product of a single, tandem duplication of an ancestral globin gene with adult function in the common ancestor of extant birds, reptiles, and mammals. Instead, our analysis reveals that the alpha(D)-globin gene of amniote vertebrates arose via duplication of an embryonic alpha-like globin gene that predated the radiation of tetrapods. The important evolutionary implication is that the distinct biochemical properties of alpha(D)-hemoglobin (HbD) are not exclusively derived characters that can be attributed to a post-duplication process of neofunctionalization. Rather, many of the distinct biochemical properties of HbD are retained ancestral characters that reflect the fact that the alpha(D)-globin gene arose via duplication of a gene that had a larval/embryonic function. These insights into the evolutionary origin of HbD illustrate how adaptive modifications of physiological pathways may result from the retention and opportunistic co-option of ancestral protein functions.     

Protein phylogenies provide evidence of a radical discontinuity between arthropod and vertebrate immune systems

 Protein phylogenies were used to test the hypothesis that aspects of the innate immune system of vertebrates have been conserved since the last common ancestor of vertebrates and arthropods. The phylogeny of lysozymes showed evidence of conservation of function, but phylogenies of seven other protein families did not. Natural resistance-associated macrophage protein, nitric oxide synthetase, and serine protease families all showed a pattern of gene duplication within vertebrates after their divergence from arthropods, giving rise to immune system-expressed genes in vertebrates. Insect hemolin, a member of the immunoglobulin superfamily, was found not to be closely related to members of that family having an immune system role in vertebrates; rather, it appeared most closely related to both arthropod and vertebrate molecules expressed in the nervous system. Thus, hemolin seems to have evolved its role independently in insects, probably through duplication of a neuroglian-like ancestor. Furthermore, vertebrate immune system-expressed serpins, chitinases, and pentraxins were found to lack orthologous relationships with arthropod members of the same families also functioning in immunity. Therefore members of these families have evolved immune system functions independently in the two phyla. It is now widely recognized that the specific immune system of vertebrates has no counterpart in invertebrates; these phylogenetic analyses suggest that there is a similar evolutionary discontinuity with respect to innate immunity as well. Received: 10 May 1997 / Revised: 10 September 1997     

Impact of gene gains,losses and duplication modes on the origin and diversification of vertebrates

The study of the evolutionary origin of vertebrates has been linked to the study of genome duplications since Susumo Ohno suggested that the successful diversification of vertebrate innovations was facilitated by two rounds of whole-genome duplication (2R-WGD) in the stem vertebrate. Since then, studies on the functional evolution of many genes duplicated in the vertebrate lineage have provided the grounds to support experimentally this link. This article reviews cases of gene duplications derived either from the 2R-WGD or from local gene duplication events in vertebrates, analyzing their impact on the evolution of developmental innovations. We analyze how gene regulatory networks can be rewired by the activity of transposable elements after genome duplications, discuss how different mechanisms of duplication might affect the fate of duplicated genes, and how the loss of gene duplicates might influence the fate of surviving paralogs. We also discuss the evolutionary relationships between gene duplication and alternative splicing, in particular in the vertebrate lineage. Finally, we discuss the role that the 2R-WGD might have played in the evolution of vertebrate developmental gene networks, paying special attention to those related to vertebrate key features such as neural crest cells, placodes, and the complex tripartite brain. In this context, we argue that current evidences points that the 2R-WGD may not be linked to the origin of vertebrate innovations, but to their subsequent diversification in a broad variety of complex structures and functions that facilitated the successful transition from peaceful filter-feeding non-vertebrate ancestors to voracious vertebrate predators.     

The phylogeny of human globin genes investigated by the maximum parsimony method

Gene phylogenetic trees were constructed by the maximum parsimony method for various sets of ninety six globin chain amino acid sequences spanning plant and animal kingdoms. The method, executed by several computer programs, constructed ancestor and descendant globin messengers on tree topologies which required the least number of nucleotide replacements to account for the evolution of the globins. The human myoglobin-hemoglobin divergence was traced to a gene duplication which occurred either in the first vertebrates or earlier yet in the common ancestor of chordates and annelids, the alpha-beta divergence to a gene duplication in the common ancestor of teleosts and tetrapods, the gamma divergence from typical beta chains to a gene duplication in basal therian mammals, and the delta separation from beta to a duplication in the basal catarrhine primates. Evidence was provided by the globin phylogenies for the hominoid affinities of the gibbon and the close phyletic relationship of the African apes to man. Over the period of teleos-tetrapod divergence the globin messengers evolved at an average rate of 18.5 nucleotide replacements per 100 codons per 10⁸ years, a faster rate than most previous estimates. Very fast and very slow rates were encountered in different globin lineages and at different stages of descent, reducing the effectiveness of globins as molecular clocks. Rates increased with gene duplication and decreased after selection discovered useful specializations in the products of genes which had previously been freer to accept mutations. The early eutherian radiation was characterized by rapid rates of globin evolution, but the later hominoid radiation by extremely slow rates. This pattern was related to more complicated grades of internal organization evolving in human ancestors. The types of nucleotide replacements in the globin messengers over the long course of globin evolution did not seem indicative of any special mutational mechanisms.     

The evolution of teleost pigmentation and the fish‐specific genome duplication

Teleost fishes have evolved a unique complexity and diversity of pigmentation and colour patterning that is unmatched among vertebrates. Teleost colouration is mediated by five different major types of neural‐crest derived pigment cells, while tetrapods have a smaller repertoire of such chromatophores. The genetic basis of teleost colouration has been mainly uncovered by the cloning of pigmentation genes in mutants of zebrafish Danio rerio and medaka Oryzias latipes. Many of these teleost pigmentation genes were already known as key players in mammalian pigmentation, suggesting partial conservation of the corresponding developmental programme among vertebrates. Strikingly, teleost fishes have additional copies of many pigmentation genes compared with tetrapods, mainly as a result of a whole‐genome duplication that occurred 320–350 million years ago at the base of the teleost lineage, the so‐called fish‐specific genome duplication. Furthermore, teleosts have retained several duplicated pigmentation genes from earlier rounds of genome duplication in the vertebrate lineage, which were lost in other vertebrate groups. It was hypothesized that divergent evolution of such duplicated genes may have played an important role in pigmentation diversity and complexity in teleost fishes, which therefore not only provide important insights into the evolution of the vertebrate pigmentary system but also allow us to study the significance of genome duplications for vertebrate biodiversity.     

Conservation of globin genes in the “living fossil” Latimeria chalumnae and reconstruction of the evolution of the vertebrate globin family

The (hemo-)globins are among the best-investigated proteins in biomedical sciences. These small heme-proteins play an important role in oxygen supply, but may also have other functions. In addition to well known hemoglobin and myoglobin, six other vertebrate globin types have been identified in recent years: neuroglobin, cytoglobin, globin E, globin X, globin Y, and androglobin. Analyses of the genome of the “living fossil” Latimeria chalumnae show that the coelacanth is the only known vertebrate that includes all eight globin types. Thus, Latimeria can also be considered as a “globin fossil”. Analyses of gene synteny and phylogenetic reconstructions allow us to trace the evolution and the functional changes of the vertebrate globin family. Neuroglobin and globin X diverged from the other globin types before the separation of Protostomia and Deuterostomia. The cytoglobins, which are unlikely to be involved in O₂ supply, form the earliest globin branch within the jawed vertebrates (Gnathostomata), but do not group with the agnathan hemoglobins, as it has been proposed before. There is strong evidence from phylogenetic reconstructions and gene synteny that the eye-specific globin E and muscle-specific myoglobin constitute a common clade, suggesting a similar role in intracellular O₂ supply. Latimeria possesses two α- and two β-hemoglobin chains, of which one α-chain emerged prior to the divergence of Actinopterygii and Sarcopterygii, but has been retained only in the coelacanth. Notably, the embryonic hemoglobin α-chains of Gnathostomata derive from a common ancestor, while the embryonic β-chains – with the exception of a more complex pattern in the coelacanth and amphibians – display a clade-specific evolution. Globin Y is associated with the hemoglobin gene cluster, but its phylogenetic position is not resolved. Our data show an early divergence of distinct globin types in the vertebrate evolution before the emergence of tetrapods. The subsequent loss of globins in certain taxa may be associated with changes in the oxygen-dependent metabolism. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Multiple Chromosomal Rearrangements Structured the Ancestral Vertebrate Hox-Bearing Protochromosomes

While the proposal that large-scale genome expansions occurred early in vertebrate evolution is widely accepted, the exact mechanisms of the expansion—such as a single or multiple rounds of whole genome duplication, bloc chromosome duplications, large-scale individual gene duplications, or some combination of these—is unclear. Gene families with a single invertebrate member but four vertebrate members, such as the Hox clusters, provided early support for Ohno's hypothesis that two rounds of genome duplication (the 2R-model) occurred in the stem lineage of extant vertebrates. However, despite extensive study, the duplication history of the Hox clusters has remained unclear, calling into question its usefulness in resolving the role of large-scale gene or genome duplications in early vertebrates. Here, we present a phylogenetic analysis of the vertebrate Hox clusters and several linked genes (the Hox “paralogon”) and show that different phylogenies are obtained for Dlx and Col genes than for Hox and ErbB genes. We show that these results are robust to errors in phylogenetic inference and suggest that these competing phylogenies can be resolved if two chromosomal crossover events occurred in the ancestral vertebrate. These results resolve conflicting data on the order of Hox gene duplications and the role of genome duplication in vertebrate evolution and suggest that a period of genome reorganization occurred after genome duplications in early vertebrates.     

Evolutionary Genomics and Adaptive Evolution of the Hedgehog Gene Family (Shh,Ihh and Dhh) in Vertebrates

The Hedgehog (Hh) gene family codes for a class of secreted proteins composed of two active domains that act as signalling molecules during embryo development, namely for the development of the nervous and skeletal systems and the formation of the testis cord. While only one Hh gene is found typically in invertebrate genomes, most vertebrates species have three (Sonic hedgehog – Shh; Indian hedgehog – Ihh; and Desert hedgehog – Dhh), each with different expression patterns and functions, which likely helped promote the increasing complexity of vertebrates and their successful diversification. In this study, we used comparative genomic and adaptive evolutionary analyses to characterize the evolution of the Hh genes in vertebrates following the two major whole genome duplication (WGD) events. To overcome the lack of Hh-coding sequences on avian publicly available databases, we used an extensive dataset of 45 avian and three non-avian reptilian genomes to show that birds have all three Hh paralogs. We find suggestions that following the WGD events, vertebrate Hh paralogous genes evolved independently within similar linkage groups and under different evolutionary rates, especially within the catalytic domain. The structural regions around the ion-binding site were identified to be under positive selection in the signaling domain. These findings contrast with those observed in invertebrates, where different lineages that experienced gene duplication retained similar selective constraints in the Hh orthologs. Our results provide new insights on the evolutionary history of the Hh gene family, the functional roles of these paralogs in vertebrate species, and on the location of mutational hotspots.     

Ancient large-scale genome duplications: phylogenetic and linkage analyses shed light on chordate genome evolution

Paralogous genes from several families were found in four human chromosome regions (4p16, 5q33-35, 8p12-21, and 10q24-26), suggesting that their common ancestral region underwent several rounds of large- scale duplication. Searches in the EMBL databases, followed by phylogenetic analyses, showed that cognates (orthologs) of human duplicated genes can be found in other vertebrates, including bony fishes. In contrast, within each family, only one gene showing the same high degree of similarity with all the duplicated mammalian genes was found in nonvertebrates (echinoderms, insects, nematodes). This indicates that large-scale duplications occurred after the echinoderms/chordates split and before the bony vertebrate radiation. It has been suggested that two rounds of gene duplication occurred in the vertebrate lineage after the separation of Amphioxus and craniate (vertebrates + Myxini) ancestors. Before these duplications, the genes that have led to the families of paralogous genes in vertebrates must have been physically linked in the craniate ancestor. Linkage of some of these genes can be found in the Drosophila melanogaster and Caenorhabditis elegans genomes, suggesting that they were linked in the triploblast Metazoa ancestor.