Loss of ancestral genes in the genomic evolution of Ciona intestinalis

Comparison of the predicted protein sets encoded by the complete genomes of two vertebrates (human and pufferfish), the urochordate Ciona intestinalis, three nonchordate animals, and two fungi were used to reconstruct a set of gene families present in the common ancestor of chordates. These ancestral families were much more likely to be lost in Ciona than in either vertebrate. In addition, of 256 duplicate gene pairs that arose by duplication prior to the most recent common ancestor of vertebrates and insects, one of the duplicate genes was four times as likely to be lost in Ciona as in the vertebrates. These results show that the genome of Ciona is not representative of the ancestral chordate genome with respect to gene content but rather shows derived features that may reflect adaptation of the specific ecological niche of urochordates.     

An aboral-dorsalization hypothesis for chordate origin

Chordates originated from a common ancestor(s) shared with two other deuterostome groups, echinoderms and hemichordates, by creating a novel type of tadpole-like larva, which was characterized by a dorsal hollow neural tube and notochord. Recent molecular phylogeny supports the notion that echinoderms and hemichordates form a clade named the Ambulacraria and that, among the chordates, cephalochordates are more basal than urochordates and vertebrates. An aboral-dorsalization hypothesis is proposed to explain how the tadpole-type larva evolved. Embryological comparison of cephalochordates with nonchordate deuterostomes suggests that, because of limited space on the oral side of the ancestral embryo, morphogenesis to form the neural tube and notochord occurred on the aboral side of the embryo. Namely, the dorsalization of the aboral side of the ancestral embryo may have been a key developmental event that led to the formation of the basic chordate body plan.     

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.     

Origins of anteroposterior patterning and Hox gene regulation during chordate evolution

All chordates share a basic body plan and many common features of early development. Anteroposterior (AP) regions of the vertebrate neural tube are specified by a combinatorial pattern of Hox gene expression that is conserved in urochordates and cephalochordates. Another primitive feature of Hox gene regulation in all chordates is a sensitivity to retinoic acid during embryogenesis, and recent developmental genetic studies have demonstrated the essential role for retinoid signalling in vertebrates. Two AP regions develop within the chordate neural tube during gastrulation: an anterior 'forebrain-midbrain' region specified by Otx genes and a posterior 'hindbrain-spinal cord' region specified by Hox genes. A third, intermediate region corresponding to the midbrain or midbrain-hindbrain boundary develops at around the same time in vertebrates, and comparative data suggest that this was also present in the chordate ancestor. Within the anterior part of the Hox-expressing domain, however, vertebrates appear to have evolved unique roles for segmentation genes, such as Krox-20, in patterning the hindbrain. Genetic approaches in mammals and zebrafish, coupled with molecular phylogenetic studies in ascidians, amphioxus and lampreys, promise to reveal how the complex mechanisms that specify the vertebrate body plan may have arisen from a relatively simple set of ancestral developmental components.     

Urochordates are monophyletic within the deuterostomes

Understanding the phylogenetic relationships of the three major urochordate groups within the deuterostomes is central to understanding the evolution of the chordates. We have prepared a detailed phylogenetic analysis of urochordates based on comparisons of 10 new urochordate 18S ribosomal DNA sequences with other urochordate sequences in GenBank. Maximum parsimony, neighbor-joining, minimum evolution, and maximum likelihood analyses of this large urochordate data set are consistent with a topology in which the urochordates are monophyletic within the deuterostomes and there are four separate clades of urochordates. These four distinct clades--styelid + pyurid ascidians, molgulid ascidians, phlebobranch ascidians + thaliaceans, and larvaceans--are mostly consistent with traditional morphological hypotheses and classifications. However, we find that the ascidians may not be a monophyletic group (as they have been considered traditionally) but instead appear paraphyletic. Another disparity with traditional classification is that the thaliaceans do not form a separate urochordate clade but rather cluster with the phlebobranch ascidians. Larvaceans have long branch lengths, which can be problematic for molecular phylogenetic methods, and their position within the urochordates cannot be unequivocally determined with 18S rDNA. This is important because the tadpole morphology of larvacean and ascidian larvae is the key trait of interest that distinguishes urochordates as chordates. Nevertheless, the present data set resolves at least three clades of urochordates and suggests strongly that urochordates form a monophyletic clade within the deuterostomes.     

The Origin of the Chordates

The study of development and comparisons of the adult structures of the several groups of protochordate animals reveals something of their interrelationships and origin. The hemichordates are perhaps closer to the echinoderms than to the chordates, but these groups appear to have been derived from a bilaterally symmetrical dipleurula ancestor, not from a sessile pterobranch-like form. The origin of the chordates is speculative but the idea of a prototunicate stage is rejected. The tunicate is viewed as a highly modified end product, with fewer similarities to the ancestral form than amphioxus. Amphioxus is quite suggestive of the vertebrate, yet it is more like the tunicate in the details of its embryology and along with that rather extreme peripheral group is best thought of as constituting a subphylum, the Acraniata. The idea of the early vertebrate as a filter feeder must be rejected since it is assumed here that perfection of that function led to a sessile or inactive way of life (as in the acraniates or lamprey larva) and failed to lead to the active creature with highly developed sensory, neural, and locomotor systems identified here as the protovertebrate. Further, the muscular plastic pharynx and moveable mouth of the protovertebrate suggest feeding on larger organisms, predation, and the abandonment of ciliary water-current feeding.     

Enhancer evolution in chordates: Lessons from functional analyses of cephalochordate cis-regulatory modules

Chordates comprise three major groups, cephalochordates (amphioxus), tunicates (urochordates), and vertebrates. Since cephalochordates were the early branching group, comparisons between amphioxus and other chordates help us to speculate about ancestral chordates. Here, I summarize accumulating data from functional studies analyzing amphioxus cis-regulatory modules (CRMs) in model systems of other chordate groups, such as mice, chickens, clawed frogs, fish, and ascidians. Conservatism and variability of CRM functions illustrate how gene regulatory networks have evolved in chordates. Amphioxus CRMs, which correspond to CRMs deeply conserved among animal phyla, govern reporter gene expression in conserved expression domains of the putative target gene in host animals. In addition, some CRMs located in similar genomic regions (intron, upstream, or downstream) also possess conserved activity, even though their sequences are divergent. These conservative CRM functions imply ancestral genomic structures and gene regulatory networks in chordates. However, interestingly, if expression patterns of amphioxus genes do not correspond to those of orthologs of experimental models, some amphioxus CRMs recapitulate expression patterns of amphioxus genes, but not those of endogenous genes, suggesting that these amphioxus CRMs are close to the ancestral states of chordate CRMs, while vertebrates/tunicates innovated new CRMs to reconstruct gene regulatory networks subsequent to the divergence of the cephalochordates. Alternatively, amphioxus CRMs may have secondarily lost ancestral CRM activity and evolved independently. These data help to solve fundamental questions of chordate evolution, such as neural crest cells, placodes, a forebrain/midbrain, and genome duplication. Experimental validation is crucial to verify CRM functions and evolution.     

Characterization of microRNAs in cephalochordates reveals a correlation between microRNA repertoire homology and morphological similarity in chordate evolution

SUMMARY Cephalochordates, urochordates, and vertebrates comprise the three extant groups of chordates. Although higher morphological and developmental similarity exists between cephalochordates and vertebrates, molecular phylogeny studies have instead suggested that the morphologically simplified urochordates are the closest relatives to vertebrates. MicroRNAs (miRNAs) are regarded as the major factors driving the increase of morphological complexity in early vertebrate evolution, and are extensively characterized in vertebrates and in a few species of urochordates. However, the comprehensive set of miRNAs in the basal chordates, namely the cephalochordates, remains undetermined. Through extensive sequencing of a small RNA library and genomic homology searches, we characterized 100 miRNAs from the cephalochordate amphioxus, Branchiostoma japonicum , and B. floridae . Analysis of the evolutionary history of the cephalochordate miRNAs showed that cephalochordates possess 54 miRNA families homologous to those of vertebrates, which is threefold higher than those shared between urochordates and vertebrates. The miRNA contents demonstrated a clear correlation between the extent of miRNA overlapping and morphological similarity among the three chordate groups, providing a strong evidence of miRNAs being the major genetic factors driving morphological complexity in early chordate evolution.     

Evolution of Chordate Actin Genes: Evidence from Genomic Organization and Amino Acid Sequences

The origin and evolutionary relationship of actin isoforms was investigated in chordates by isolating and characterizing two new ascidian cytoplasmic and muscle actin genes. The exon–intron organization and sequences of these genes were compared with those of other invertebrate and vertebrate actin genes. The gene HrCA1 encodes a cytoplasmic (nonmuscle)-type actin, whereas the MocuMA2 gene encodes an adult muscle-type actin. Our analysis of these genes showed that intron positions are conserved among the deuterostome actin genes. This suggests that actin gene families evolved from a single actin gene in the ancestral deuterostome. Sequence comparisons and molecular phylogenetic analyses also suggested a close relationship between the ascidian and vertebrate actin isoforms. It was also found that there are two distinct lineages of muscle actin isoforms in ascidians: the larval muscle and adult body-wall isoforms. The four muscle isoforms in vertebrates show a closer relationship to each other than to the ascidian muscle isoforms. Similarly, the two cytoplasmic isoforms in vertebrates show a closer relationship to each other than to the ascidian and echinoderm cytoplasmic isoforms. In contrast, the two types of ascidian muscle actin diverge from each other. The close relationship between the ascidian larval muscle actin and the vertebrate muscle isoforms was supported by both neighbor-joining and maximum parsimony analyses. These results suggest that the chordate ancestor had at least two muscle actin isoforms and that the vertebrate actin isoforms evolved after the separation of the vertebrates and urochordates. Received: 20 June 1996 / Accepted: 16 October 1996     

A wide-range phylogenetic analysis of Zic proteins: implications for correlations between protein structure conservation and body plan complexity

We compared Zic homologues from a wide range of animals. Striking conservation was found in the zinc finger domains, in which an exon-intron boundary has been kept in all bilateralians but not cnidarians, suggesting that all of the bilateralian Zic genes are derived from a single gene in a bilateralian ancestor. There were additional conserved amino acid sequences, ZOC and ZF-NC. Combined analysis of the zinc finger, ZOC, and ZF-NC revealed the presence of two classes of Zic, based on the degree of protein structure conservation. The "conserved" class includes Zic proteins from the Arthropoda, Mollusca, Annelida, Echinodermata, and Chordata (vertebrates and cephalochordates), whereas the "diverged" class contains those from the Platyhelminthes, Cnidaria, Nematoda, and Chordata (urochordates). The result indicates that the ancestral bilateralian Zic protein had already acquired an entire set of conserved domains, but that this was lost and diverged in the platyhelminthes, nematodes, and urochordates.     

The evolutionary history of placodes: a molecular genetic investigation of the larvacean urochordate Oikopleura dioica

The evolutionary origin of vertebrate placodes remains controversial because divergent morphologies in urochordates, cephalochordates and vertebrates make it difficult to recognize organs that are clearly homologous to placode-derived features, including the olfactory organ, adenohypophysis, lens, inner ear, lateral line and cranial ganglia. The larvacean urochordate Oikopleura dioica possesses organs that morphologically resemble the vertebrate olfactory organ and adenohypophysis. We tested the hypothesis that orthologs of these vertebrate placodes exist in a larvacean urochordate by analyzing the developmental expression of larvacean homologs of the placode-marking gene families Eya, Pitx and Six. We conclude that extant chordates inherited olfactory and adenohypophyseal placodes from their last common ancestor, but additional independent proliferation and perhaps loss of placode types probably occurred among the three subphyla of Chordata.     

Developmental genetics in primitive chordates

Recent advances in the study of the genetics and genomics of urochordates testify to a renewed interest in this chordate subphylum, believed to be the most primitive extant chordate relatives of the vertebrates. In addition to their primitive nature, many features of their reproduction and early development make the urochordates ideal model chordates for developmental genetics. Many urochordates spawn large numbers of transparent and externally developing embryos on a daily basis. Additionally, the embryos have a defined and well-characterized cell lineage until the end of gastrulation. Furthermore, the genomes of the urochordates have been estimated to be only 5-10% of the size of the vertebrates and to have fewer genes and less genetic redundancy than vertebrates. Genetic screens, which are powerful tools for investigating developmental mechanisms, have recently become feasible due to new culturing techniques in ascidians. Because hermaphrodite ascidians are able to self-fertilize, recessive mutations can be detected in a single generation. Several recent studies have demonstrated the feasibility of applying modern genetic techniques to the study of ascidian biology.     

Complete nucleotide sequence of the mitochondrial genome of Doliolum nationalis with implications for evolution of urochordates

The evolutionary history of the diverse lifestyles adopted by urochordates has attracted intense interest because it may effect the evolutionary history of vertebrates. Here, we report the complete mitochondrial (mt) DNA sequence of the pelagic thaliacean doliolid Doliolum nationalis. The doliolid mt genome shares the unusual tRNAs of trnM(uau) and trnG(ucu) with other ascidians, such as Halocynthia and Ciona. On the other hand, the gene order of the doliolid mt genome is significantly different from that of any ascidian species or vertebrate reported to date. Phylogenetic analyses of the amino acid sequences of 12 protein-coding genes strongly support the sister-grouping of doliolids and the Phlebobranch ascidian Ciona, with the Stolidobranch ascidian alocynthia as the outgroup, thereby providing strong support for the paraphyly of ascidians, as has been suggested by 18S rDNA studies. Given the paraphyletic nature of ascidians, it seems likely that the common ancestor of ascidians and thaliaceans was sessile, as are the present-day ascidians, and that the thaliaceans subsequently evolved a pelagic lifestyle.     

The emergence of the chordate body plan: some puzzles and problems

Lacalli, T.C. 2010. The emergence of the chordate body plan: some puzzles and problems. —Acta Zoologica (Stockholm) 91 : 4–10 Rather than being sessile filter feeders, ancestral chordates are now thought to have evolved from more active benthic animals, possibly hemichordate‐like, that took to swimming, to generate something resembling modern amphioxus. This general picture conceals a number of specific problems that underline how little we understand the transition in detail. I will address three. First, and closest to resolution is the issue of dorsoventral inversion, which has implications for understanding how an internalized brain evolved. This is because the mouth, dorsal after inversion, has first to be moved out of the way. Its migration down the left side of the head during amphioxus development may be a recapitulation of this event. Two other puzzles, both further from resolution are: (1) the significance, if any, of the neurenteric canal, which may be telling us something important about the true nature of deuterostomy, specifically whether hemichordates and echinoderms are deuterostomes for a different reason than chordates, and (2) whether the functional digestive tract of chordates is a secondary replacement of an earlier structure whose fate remains unexplained. Resolving these latter two issues will require a better understanding of molecular level events during development in protochordates and their immediate invertebrate relatives.     

BfCBR: a cannabinoid receptor ortholog in the cephalochordate Branchiostoma floridae (Amphioxus)

A gene encoding an ortholog of vertebrate CB(1)/CB(2) cannabinoid receptors was recently identified in the urochordate Ciona intestinalis (CiCBR; [Elphick, M.R., Satou, Y., Satoh, N., 2003. The invertebrate ancestry of endocannabinoid signalling: an orthologue of vertebrate cannabinoid receptors in the urochordate Ciona intestinalis. Gene 302, 95-101.]). Here a cannabinoid receptor ortholog (BfCBR) has been identified in the cephalochordate Branchiostoma floridae. BfCBR is encoded by a single exon and is 410 amino acid residue protein that shares 28% sequence identity with CiCBR and 23% sequence identity with human CB(1) and human CB(2). The discovery of BfCBR and CiCBR and the absence of cannabinoid receptor orthologs in non-chordate invertebrates indicate that CB(1)/CB(2)-like cannabinoid receptors originated in an invertebrate chordate ancestor of urochordates, cephalochordates and vertebrates. Furthermore, analysis of the relationship of BfCBR and CiCBR with vertebrate CB(1) and CB(2) receptors indicates that the gene/genome duplication that gave rise to CB(1) and CB(2) receptors occurred in the vertebrate lineage. Identification of BfCBR, in addition to CiCBR, paves the way for comparative analysis of the expression and functions of these proteins in Branchiostoma and Ciona, respectively, providing an insight into the ancestral functions of cannabinoid receptors in invertebrate chordates prior to the emergence of CB(1) and CB(2) receptors in vertebrates.     

Regulatory genes in the ancestral chordate genomes

Changes or innovations in gene regulatory networks for the developmental program in the ancestral chordate genome appear to be a major component in the evolutionary process in which tadpole-type larvae, a unique characteristic of chordates, arose. These alterations may include new genetic interactions as well as the acquisition of new regulatory genes. Previous analyses of the Ciona genome revealed that many genes may have emerged after the divergence of the tunicate and vertebrate lineages. In this paper, we examined this possibility by examining a second non-vertebrate chordate genome. We conclude from this analysis that the ancient chordate included almost the same repertory of regulatory genes, but less redundancy than extant vertebrates, and that approximately 10% of vertebrate regulatory genes were innovated after the emergence of vertebrates. Thus, refined regulatory networks arose during vertebrate evolution mainly as preexisting regulatory genes multiplied rather than by generating new regulatory genes. The inferred regulatory gene sets of the ancestral chordate would be an important foundation for understanding how tadpole-type larvae, a unique characteristic of chordates, evolved.     

The deuterostome ancestor

Hemichordates, the phylum of bilateral animals closest to chordates, can illuminate the evolutionary origins of various chordate traits to determine whether these were already present in a shared ancestor (the deuterostome ancestor) or were evolved within the chordate line. We find that an anteroposterior map of gene expression domains, representing 42 genes of neural patterning, is closely similar in hemichordates and chordates, though it is restricted to the neural ectoderm in chordates whereas in hemichordates, which have a diffuse nervous system, it encircles the whole body. This map allows an accurate alignment of the anterioposterior axes of members of the two groups. We propose that this map dates back at least to the deuterostome ancestor. The map of dorsoventral expression domains, organized along a Bmp-Chordin developmental axis, is also similar in the two groups in terms of many gene expression domains and for the placement of the gill slits, heart, and post-anal tail. The two groups, however, differ in two major respects along this axis. The nervous system and epidermis are not segregated into distinct territories in hemichordates, as they are in chordates, and furthermore, the mouth is on the Chordin side in hemichordates but the Bmp side in chordates. The dorsoventral dimension has undergone extensive modification in the chordate line, including centralization of the nervous system, segregation of epidermis, derivation of the notochord, perhaps from the gut midline, and relocation of the mouth. Based on the shared domain maps, speculations can be made for the remodeling of the body axis in the chordate line.     

Organizing chordates with an organizer

Understanding how the chordate body plan originated and evolved is still controversial. The discovery by Spemann and Mangold in 1924 of the vertebrate organizer and its inductive properties in patterning the AP and DV axis was followed by a long gap until the 1960s when scientists started characterizing the molecular events responsible for such inductions. However, the evolutionary origin of the organizer itself remained obscure until very recently; did it appear together with the origin and radiation of vertebrates, or was it a chordate affair? A recent study by Yu and collaborators, 1 which analyses the expression of several organizer-specific genes in amphioxus together with recent phylogenetic data that reversed the position of invertebrate extant chordates (e.g. urochordates and cephalochordates), indicates that the organizer probably appeared in early chordates. It likely had separate signalling centres generating BMP and Wnt signalling gradients along the DV and AP axis. The organizer was then lost in the urochordate lineage, most probably as an adaptation to a rapid and determinate development. BioEssays 29:619–624, 2007. © 2007 Wiley Periodicals, Inc.     

Cambrian cinctan echinoderms shed light on feeding in the ancestral deuterostome

Reconstructing the feeding mode of the latest common ancestor of deuterostomes is key to elucidating the early evolution of feeding in chordates and allied phyla; however, it is debated whether the ancestral deuterostome was a tentaculate feeder or a pharyngeal filter feeder. To address this, we evaluated the hydrodynamics of feeding in a group of fossil stem-group echinoderms (cinctans) using computational fluid dynamics. We simulated water flow past three-dimensional digital models of a Cambrian fossil cinctan in a range of possible life positions, adopting both passive tentacular feeding and active pharyngeal filter feeding. The results demonstrate that an orientation with the mouth facing downstream of the current was optimal for drag and lift reduction. Moreover, they show that there was almost no flow to the mouth and associated marginal groove under simulations of passive feeding, whereas considerable flow towards the animal was observed for active feeding, which would have enhanced the transport of suspended particles to the mouth. This strongly suggests that cinctans were active pharyngeal filter feeders, like modern enteropneust hemichordates and urochordates, indicating that the ancestral deuterostome employed a similar feeding strategy.