Polyploidy in vertebrate ancestry: Ohno and beyond

Over 30 years ago, Susumu Ohno proposed that two rounds of polyploidy occurred early in vertebrate evolution. We re-examine this proposal using three recent lines of evidence. First, total gene number estimates from completely sequenced genomes suggest an increase in total gene number somewhere along the vertebrate or prevertebrate lineage, compatible with Ohno's model. Second, analyses of homeobox and other genes from amphioxus reveal very extensive gene duplication specifically on the vertebrate lineage. This refines the timing of putative polyploidy to after the divergence of amphioxus and vertebrates. Third, the existence of four-fold paralogy regions in the human genome is suggestive of two rounds of polyploidy, although other explanations are possible. We propose an experimental test, based on chromosomal localization of genes in amphioxus, that should resolve whether paralogy regions are indeed remnants of duplication in vertebrate ancestry.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 425–430.     

The amphioxus Hox cluster: deuterostome posterior flexibility and Hox14

SUMMARY The amphioxus ( Branchiostoma floridae ) Hox cluster is a model for the ancestral vertebrate cluster, prior to the hypothesized genome-wide duplications that may have facilitated the evolution of the vertebrate body plan. Here we describe the posterior (5') genes of the amphioxus cluster, and report the isolation of four new homeobox genes. Vertebrates possess 13 types of Hox gene (paralogy groups), but we show that amphioxus possesses more than 13 Hox genes. Amphioxus is now the first animal in which a Hox14 gene has been found. Our mapping and phylogenetic analysis of amphioxus "Posterior Class" Hox genes reveals that these genes are evolving at a faster rate in deuterostomes than in protostomes, a phenomenon we term Posterior Flexibility.     

An antecedent of the MHC-linked genomic region in amphioxus

The MHC genes on human chromosome 6 are located within one of the best-characterised paralogy regions of the human genome. Numerous genes mapping around this location, 6p21, have paralogues at one, two or three other chromosomal locations on HSA 1, 9 and 19. The similarity between these four chromosomal regions suggests the linkages may have adaptive significance, and/or they may be echoes of segmental or genome duplication in human ancestry. Here, we show that six amphioxus cosmids, containing genes orthologous to those from the human MHC-linked paralogy regions, map to a single amphioxus chromosome. The composition of the MHC-linked genomic region, therefore, pre-dates vertebrate origins.     

基因倍增和脊椎动物起源

有机体基因复制导致基因复杂性增加及其和脊椎动物起源的关系已经成为进化生物学研究的热点。20世纪70年代由Ohno提出后经Holland等修正的原始脊索动物经两轮基因组复制产生脊椎动物的假设目前已被广泛接受。脊椎动物起源和进化过程中发生过两轮基因组复制的主要证据有三点：（1）据估计脊椎动物基因组内编码基因数目大约相当于果蝇、海鞘等无脊椎动物的4倍;原口动物如果蝇和后口动物如头索动物文昌鱼的基因组大都只有单拷贝的基因,而脊椎动物的基因组则通常有4个同属于一个家族的基因。（2）无脊椎动物如节肢动物、海胆和头索动物文昌鱼都只有一个Hox基因簇,而脊椎动物除鱼类外,有7个具有Hox基因簇,其余都具有4个Hox基因簇。（3）基因作图证明,不但在鱼类和哺乳动物染色体广大片段上基因顺序相似,而且有证据显示哺乳动物基因组不同染色体之间存在相似性。据认为第一次基因倍增发生在脊椎动物与头索动物分开之后,第二次基因倍增发生在有颌类脊椎动物和无颌类脊椎动物分开以后。但是,基因是逐个发生倍增,还是通过基因组内某些DNA片段抑或整个基因组的加倍而实现的,目前还颇有争议。     

Coparalogy: physical and functional clusterings in the human genome

Two rounds of large-scale duplications are thought to have occurred in early vertebrate ancestry; this is now known as the "2R hypothesis." They have led to the constitution of subfamilies of paralogous genes. Chromosomal regions that contain present-day paralogs (paralogous regions or paralogons) have been identified in mammals. We show that sets of paralogons (PGs) can be assembled in a tentative "human genome paralogy map" that includes all autosomes and X. A total of 14 PGs, containing more than 1600 genes, were assembled in this paralogy map. Genes that belong to the same PG are coparalogs. We show that identification of coparalogy can be used (i) to broaden data on gene mapping, (ii) to identify physical gene clusters that derive from early cis-duplications, and (iii) to speculate on coevolution and coregulation of genes sharing a common structure or function (functional clusters). Thus, coparalogy analyses should parallel phylogenetic analyses and can help draw hypotheses on gene and genome evolution.     

Phylogenetic analysis of T-Box genes demonstrates the importance of amphioxus for understanding evolution of the vertebrate genome

The duplication of preexisting genes has played a major role in evolution. To understand the evolution of genetic complexity it is important to reconstruct the phylogenetic history of the genome. A widely held view suggests that the vertebrate genome evolved via two successive rounds of whole-genome duplication. To test this model we have isolated seven new T-box genes from the primitive chordate amphioxus. We find that each amphioxus gene generally corresponds to two or three vertebrate counterparts. A phylogenetic analysis of these genes supports the idea that a single whole-genome duplication took place early in vertebrate evolution, but cannot exclude the possibility that a second duplication later took place. The origin of additional paralogs evident in this and other gene families could be the result of subsequent, smaller-scale chromosomal duplications. Our findings highlight the importance of amphioxus as a key organism for understanding evolution of the vertebrate genome.     

Phylogenetic analyses alone are insufficient to determine whether genome duplication(s) occurred during early vertebrate evolution

The widely accepted notion that two whole-genome duplications occurred during early vertebrate evolution (the 2R hypothesis) stems from the fact that vertebrates often possess several genes corresponding to a single invertebrate homolog. However the number of genes predicted by the Human Genome Project is less than twice as many as in the Drosophila melanogaster or Caenorhabditis elegans genomes. This ratio could be explained by two rounds of genome duplication followed by extensive gene loss, by a single genome duplication, by sequential local duplications, or by a combination of any of the above. The traditional method used to distinguish between these possibilities is to reconstruct the phylogenetic relationships of vertebrate genes to their invertebrate orthologs; ratios of invertebrate-to-vertebrate counterparts are then used to infer the number of gene duplication events. The lancelet, amphioxus, is the closest living invertebrate relative of the vertebrates, and unlike protostomes such as flies or nematodes, is therefore the most appropriate outgroup for understanding the genomic composition of the last common ancestor of all vertebrates. We analyzed the relationships of all available amphioxus genes to their vertebrate homologs. In most cases, one to three vertebrate genes are orthologous to each amphioxus gene (median number=2). Clearly this result, and those of previous studies using this approach, cannot distinguish between alternative scenarios of chordate genome expansion. We conclude that phylogenetic analyses alone will never be sufficient to determine whether genome duplication(s) occurred during early chordate evolution, and argue that a "phylogenomic" approach, which compares paralogous clusters of linked genes from complete amphioxus and human genome sequences, will be required if the pattern and process of early chordate genome evolution is ever to be reconstructed.     

Revisiting the origin of the vertebrate Hox14 by including its relict sarcopterygian members

Bilaterian Hox genes play pivotal roles in the specification of positional identities along the anteroposterior axis. Particularly in vertebrates, their regulation is tightly coordinated by tandem arrays of genes [paralogy groups (PGs)] in four gene clusters (HoxA-D). Traditionally, the uninterrupted Hox cluster (Hox1-14) of the invertebrate chordate amphioxus was regarded as an archetype of the vertebrate Hox clusters. In contrast to Hox1-13 that are globally regulated by the "Hox code" and are often phylogenetically conserved, vertebrate Hox14 members were only recently revealed to be present in an African lungfish, a coelacanth, chondrichthyans and a lamprey, and decoupled from the Hox code. In this study we performed a PCR-based search of Hox14 members from diverse vertebrates, and identified one in the Australian lungfish, Neoceratodus forsteri. Based on a molecular phylogenetic analysis, this gene was designated NfHoxA14. Our real-time RT-PCR suggested its hindgut-associated expression, previously observed also in cloudy catshark HoxD14 and lamprey Hox14α. It is likely that this altered expression scheme was established before the Hox cluster quadruplication, probably at the base of extant vertebrates. To investigate the origin of vertebrate Hox14, by including this sarcopterygian Hox14 member, we performed focused phylogenetic analyses on its relationship with other vertebrate posterior Hox PGs (Hox9-13) as well as amphioxus posterior Hox genes. Our results confirmed the hypotheses previously proposed by other studies that vertebrate Hox14 does not have any amphioxus ortholog, and that none of 1-to-1 pairs of vertebrate and amphioxus posterior Hox genes, based on their relative location in the clusters, is orthologous.     

Amphioxus and ascidian Dmbx homeobox genes give clues to the vertebrate origins of midbrain development

The ancestral chordate neural tube had a tripartite structure, comprising anterior, midbrain-hindbrain boundary (MHB) and posterior regions. The most anterior region encompasses both forebrain and midbrain in vertebrates. It is not clear when or how the distinction between these two functionally and developmentally distinct regions arose in evolution. Recently, we reported a mouse PRD-class homeobox gene, Dmbx1, expressed in the presumptive midbrain at early developmental stages, and the hindbrain at later stages, with exclusion from the MHB. This gene provides a route to investigate the evolution of midbrain development. We report the cloning, genomic structure, phylogeny and embryonic expression of Dmbx genes from amphioxus and from Ciona, representing the two most closely related lineages to the vertebrates. Our analyses show that Dmbx genes form a distinct, ancient, homeobox gene family, with highly conserved sequence and genomic organisation, albeit more divergent in Ciona. In amphioxus, no Dmbx expression is observed in the neural tube, supporting previous arguments that the MHB equivalent region has been secondarily modified in evolution. In Ciona, the CiDmbx gene is detected in neural cells caudal to Pax2/5/8-positive cells (MHB homologue), in the Hox-positive region, but, interestingly, not in any cells rostral to them. These results suggest that a midbrain homologue is missing in Ciona, and argue that midbrain development is a novelty that evolved specifically on the vertebrate lineage. We discuss the evolution of midbrain development in relation to the ancestry of the tripartite neural ground plan and the origin of the MHB organiser.     

Phylogenetic analysis of Amphioxus genes of the proprotein convertase family, including aPC6C, a marker of epithelial fusions during embryology

The proprotein convertases (PCs) comprise a family of subtilisin-like endoproteases that activate precursor proteins (including, prohormones, growth factors, and adhesion molecules) during their transit through secretory pathways or at the cell surface. To explore the evolution of the PC gene family in chordates, we made a phylogenetic analysis of PC genes found in databases, with special attention to three PC genes of the cephalochordate amphioxus, the closest living invertebrate relative to the vertebrates. Since some vertebrate PC genes are essential for early development, we investigated the expression pattern of the C isoform of the amphioxus PC6 gene (aPC6C). In amphioxus embryos and larvae, aPC6C is expressed at places where epithelia fuse. Several kinds of fusions occur: ectoderm-to-ectoderm during neurulation; mesoderm-to-ectoderm during formation of the preoral ciliated pit; and endoderm-to-ectoderm during formation of the mouth, pharyngeal slits, anus, and external opening of the club-shaped gland. Presumably, at all these sites, aPC6C is activating proteins favoring association between previously disjunct cell populations.     

Evolution of CUT class homeobox genes: insights from the genome of the amphioxus, Branchiostoma floridae

CUT class homeobox genes, including CUX/CASP, ONECUT, SATB and COMPASS family genes, are known to exhibit diverse features in the homeodomain and the domain architecture. Furthermore, the intron/exon organization of CUX/CASP is different between vertebrates and protostomes, and SATB genes are only known for vertebrates, whereas COMPASS genes have only been found in protostomes. These observations suggest a complex evolutionary history for the CUT class homeobox genes, but the evolution of CUT class homeobox genes in the lineage to vertebrates remained largely unknown. To obtain clearer insights into this issue, we searched the genome of amphioxus, Branchiostoma floridae, a lower chordate, for CUT class homeobox genes by extensive BLAST survey and phylogenetic analyses. We found that the genome of Branchiostoma floridae encodes each single orthologue of CUX/CASP, ONECUT, and COMPASS, but not the SATB gene, and one atypical CUT gene likely specific to this species. In addition, the genomic structure of the amphioxus CUX/CASP gene turned out to be protostome-type, but not vertebrate-type. Based on these observations, we propose a model in which SATB is suggested to evolve at the expense of COMPASS and this change, together with the structural change in CUX/CASP, is supposed to take place in the lineage to vertebrates after divergence of the amphioxus and vertebrate ancestors. The present study provides an example of dramatic evolution among homeobox gene groups in the vertebrate lineage and highlights the ancient character of amphioxus, retaining genomic features shared by protostomes.     

Novel paralogy relations among human chromosomes support a link between the phylogeny of doublesex-related genes and the evolution of sex determination

Recent advances in the evolutionary genetics of sex determination indicate that DMRT1 may be a vertebrate equivalent of the Drosophila melanogaster master sex regulator gene, doublesex. The role of DMRT1 seems to be confined to some aspects of male sex differentiation, whereas in Drosophila, doublesex has wider developmental effects in both sexes. This suggests other homologs of doublesex may exist in the vertebrate genome and encode sex-specific functions not displayed by DMRT1. We identified and characterized five novel human DM genes, distinct from previously described family members. Human DM genes map to three well-defined regions of chromosomes 1, 9, and 19 (one gene on chromosome 19 having an additional homolog on chromosome X). We collated data indicating these chromosomal regions harbor multiple syntenic genes sharing highly specific paralogy relations, suggesting that they arose early during vertebrate evolution. The 9p21-p24.3 bands represent the ancestral copy and harbor closely linked DM genes that may reflect the overall diversity of the fruit fly DM gene family. The human genome contains a small number of potential doublesex homologs that may be involved in human sexual development. Identifying highly conserved chromosomal regions, such as distal 9p, is an important tool to trace complex ancient evolutionary processes inaccessible by other approaches.     

Evolution of vertebrate central nervous system is accompanied by novel expression changes of duplicate genes

The evolution of the central nervous system (CNS) is one of the most striking changes during the transition from invertebrates to vertebrates. As a major source of genetic novelties, gene duplication might play an important role in the functional innovation of vertebrate CNS. In this study, we focused on a group of CNS-biased genes that duplicated during early vertebrate evolution. We investigated the tempo-spatial expression patterns of 33 duplicate gene families and their orthologs during the embryonic development of the vertebrate Xenopus laevis and the cephalochordate Brachiostoma belcheri. Almost all the identified duplicate genes are differentially expressed in the CNS in Xenopus embryos, and more than 50% and 30% duplicate genes are expressed in the telencephalon and mid-hindbrain boundary, respectively, which are mostly considered as two innovations in the vertebrate CNS. Interestingly, more than 50% of the amphioxus orthologs do not show apparent expression in the CNS in amphioxus embryos as detected by in situ hybridization, indicating that some of the vertebrate CNS-biased duplicate genes might arise from non-CNS genes in invertebrates. Our data accentuate the functional contribution of gene duplication in the CNS evolution of vertebrate and uncover an invertebrate non-CNS history for some vertebrate CNS-biased duplicate genes.     

The single amphioxus Mox gene: insights into the functional evolution of Mox genes,somites, and the asymmetry of amphioxus somitogenesis

Mox genes are members of the "extended" Hox-cluster group of Antennapedia-like homeobox genes. Homologues have been cloned from both invertebrate and vertebrate species, and are expressed in mesodermal tissues. In vertebrates, Mox1 and Mox2 are distinctly expressed during the formation of somites and differentiation of their derivatives. Somites are a distinguishing feature uniquely shared by cephalochordates and vertebrates. Here, we report the cloning and expression of the single amphioxus Mox gene. AmphiMox is expressed in the presomitic mesoderm (PSM) during early amphioxus somitogenesis and in nascent somites from the tail bud during the late phase. Once a somite is completely formed, AmphiMox is rapidly downregulated. We discuss the presence and extent of the PSM in both phases of amphioxus somitogenesis. We also propose a scenario for the functional evolution of Mox genes within chordates, in which Mox was co-opted for somite formation before the cephalochordate-vertebrate split. Novel expression sites found in vertebrates after somite formation postdated Mox duplication in the vertebrate stem lineage, and may be linked to the increase in complexity of vertebrate somites and their derivatives, e.g., the vertebrae. Furthermore, AmphiMox expression adds new data into a long-standing debate on the extent of the asymmetry of amphioxus somitogenesis.     

Analysis of lamprey and hagfish genes reveals a complex history of gene duplications during early vertebrate evolution

It has been proposed that two events of duplication of the entire genome occurred early in vertebrate history (2R hypothesis). Several phylogenetic studies with a few gene families (mostly Hox genes and proteins from the MHC) have tried to confirm these polyploidization events. However, data from a single locus cannot explain the evolutionary history of a complete genome. To study this 2R hypothesis, we have taken advantage of the phylogenetic position of the lamprey to study the history of gene duplications in vertebrates. We selected most gene families that contain several paralogous genes in vertebrates and for which lamprey genes and an out-group are known in databases. In addition, we isolated members of the nuclear receptor superfamily in lamprey. Hagfish genes were also analyzed and found to confirm the lamprey gene analysis. Consistent with the 2R hypothesis, the phylogenetic analysis of 33 selected gene families, dispersed through the whole genome, revealed that one period of gene duplication arose before the lamprey-gnathostome split and this was followed by a second period of gene duplication after the lamprey-gnathostome split. Nevertheless, our analysis suggests that numerous gene losses and other gene-genome duplications occurred during the evolution of the vertebrate genomes. Thus, the complexity of all the paralogy groups present in vertebrates should be explained by the contribution of genome duplications (2R hypothesis), extra gene duplications, and gene losses.     

Phylogenetic history of paralogous gene quartets on human chromosomes 1, 2, 8 and 20 provides no evidence in favor of the vertebrate octoploidy hypothesis

Fourfold paralogy regions in the human genome have been considered historical remnants of whole-genome duplication events predicted to have occurred early in vertebrate evolution. Taking advantage of the well-annotated and high-quality human genomic sequence map as well as the ever-increasing accessibility of large-scale genomic sequence data from a diverse range of animal species, we investigated the prediction that the ancestral vertebrate genome was shaped by two rapid rounds of whole-genome duplication within a period of 10 million years. Both the map self-comparison approach and a phylogenetic analysis revealed that gene families identified as tetralogous on human chromosomes 1/2/8/20 arose by small-scale duplication events that occurred at widely different time points in animal evolution. Furthermore, the data discount the likelihood that tree topologies of the form ((A,B)(C,D)) are best explained by the octoploidy hypothesis. We instead propose that such symmetrical tree patterns are also consistent with local duplications and rearrangement events.     

Fourfold paralogy regions on human HOX-bearing chromosomes: Role of ancient segmental duplications in the evolution of vertebrate genome

BackgroundSusumu Ohno’s idea that modern vertebrates are degenerate polyploids (concept referred as 2R hypothesis) has been the subject of intense debate for past four decades. It was proposed that intra-genomic synteny regions (paralogons) in human genome are remains of ancient polyploidization events that occurred early in the vertebrate history. The quadruplicated paralogon centered on human HOX clusters is taken as evidence that human HOX-bearing chromosomes were structured by two rounds of whole genome duplication (WGD) events.ResultsEvolutionary history of human HOX-bearing chromosomes (chromosomes 2/7/12/17) was evaluated by the phylogenetic analysis of multigene families with triplicated or quadruplicated distribution on these chromosomes. Topology comparison approach categorized the members of 44 families into four distinct co-duplicated groups. Distinct gene families belonging to a particular co-duplicated group, exhibit similar evolutionary history and hence have duplicated simultaneously, whereas genes of two distinct co-duplicated groups do not share their evolutionary history and have not duplicated in concert with each other.ConclusionThe recovery of co-duplicated groups suggests that “ancient segmental duplications and rearrangements” is the most rational model of evolutionary events that have generated the triplicated and quadruplicated paralogy regions seen on the human HOX-bearing chromosomes.     

Comparison of Pax1/9 locus reveals 500-Myr-old syntenic block and evolutionary conserved noncoding regions

Identification of conserved genomic regions within and between different genomes is crucial when studying genome evolution. Here, we described regions of strong synteny conservation between vertebrate deuterostomes (tetrapods and teleosts) and invertebrate deuterostomes (amphioxus and sea urchin). The shared gene contents across phylogenetically distant species demonstrate that the conservation of the regions stemmed from an ancestral segment instead of a series of independent convergent events. Comparison of the syntenic regions allows us to postulate the primitive gene organization in the last common ancestor of deuterostomes and the evolutionary events that occurred to the 3 distinct lineages of sea urchin, amphioxus, and vertebrates after their separation. In addition, alignment of the syntenic regions led to the identification of 8 noncoding evolutionarily conserved regions shared between amphioxus and vertebrates. To our knowledge, this is the first report of conserved noncoding sequences shared by vertebrates and nonvertebrates. These noncoding sequences have high possibility of being elements that regulate neighboring genes. They are likely to be a factor in the maintenance of conserved synteny over long phylogenetic distance in different deuterostome lineages.     

Going nuclear: gene family evolution and vertebrate phylogeny reconciled

Gene duplications have been common throughout vertebrate evolution, introducing paralogy and so complicating phylogenetic inference from nuclear genes. Reconciled trees are one method capable of dealing with paralogy, using the relationship between a gene phylogeny and the phylogeny of the organisms containing those genes to identify gene duplication events. This allows us to infer phylogenies from gene families containing both orthologous and paralogous copies. Vertebrate phylogeny is well understood from morphological and palaeontological data, but studies using mitochondrial sequence data have failed to reproduce this classical view. Reconciled tree analysis of a database of 118 vertebrate gene families supports a largely classical vertebrate phylogeny.