The Essential Role of "Minor" Phyla in Molecular Studies of Animal Evolution

SYNOPSIS. Molecular studies have revealed many new hypothesesof metazoan evolution in recent years. Previously, using morphologicalmethods, it was difficult to relate "minor" animal groups representingmicroscopic metazoans to larger, more well known groups suchas arthropods, molluscs, and annelids. Molecular studies suggestthat acanthocephalans evolved from rotifers, that priapulidsshare common ancestry with all other molting animals (Ecdysozoa),and that flatworms, gnathostomulids and rotifers form a sistergroup to the remaining non-molting protostomes (Lophotrochozoa),together forming Spiralia. The lophophorate phyla (phoronids,brachiopods and bryozoans) appear as protostomes, allied withannelids and molluscs rather than with deuterostomes. Thesefindings present a very different view of metazoan evolution,and clearly show that small and simple animals do not necessarilyrepresent ancestral or primitive taxa.     

Evolution of the Multicellular Animals

SYNOPSIS. Molecular sequence analysis is providing new insightsinto the study of metazoan relationships. The use of ribosomalRNA sequences is revising many of the metazoan phylogenies thathave been established traditionally with anatomical and embryologicaldata. Four new findings that seem to be well supported by moleculardata, both from the authors' laboratories and from others, aredescribed and discussed. First, the arthropods are members ofa deep primary clade within the protostomes and are not thesister taxa of either the annelids or the mollusks. Second,the lophophorate animals are clearly protostomes and are containedwithin a lophotrochozoan superclade including the mollusks,annelids, and many other phyla. Third, the arthropods togetherwith all other molting animals comprise a second monophyleticsuperclade within the protostomes, the ecdysozoa. Fourth, theplatyhelminthes are contained within the lophotrochozoan superclade.     

Patterns of internal gene duplication in the course of metazoan evolution

Internal duplication can enhance the function of a gene or provide raw material for the emergence of a new function in a gene. Therefore, it is interesting to see whether the frequency of internal duplication has increased during metazoan evolution. The growing number of sequenced eukaryotic genomes provides an excellent opportunity to study the change in the pattern of internal duplication in the course of metazoan evolution. We studied repeated segments in proteins in the proteomes of 11 eukaryotes. We found that the frequency of internal duplication in Caenorhabditis elegans and Drosophila melanogaster (two protostomes) is higher than that in fungi but lower than that in chordates. Moreover, the frequencies of internal duplication for the chordates studied are largely similar. We classified orthologous proteins of chordates into three antiquity groups and found that more recently derived proteins in the metazoan lineage have higher repetitiveness than older ones. Our analysis suggests that lineage-specific internal duplication in protein evolution increases with organismal complexity before the emergence of chordates but not so afterward. Proteins with repeated regions might have been preferred before the protostome-chordate split. This finding supports the suggestion that exon-shuffling occurred more frequently after the first multicellular organism appeared and might have contributed to the metazoan radiation.     

Considerations for Reconstructing Metazoan History: Signal, Resolution, and Hypothesis Testing

SYNOPSIS. Certain issues of metazoan phytogeny remain difficultto resolve even with advances in phylogenetic theory and moleculartools. This lack of resolution might be due to limited phylogeneticsignal in regions of the metazoan tree. For example, 18S rDNAdata are consistent with a rapid radiation early in the historyof bilaterian animals. Despite this lack of resolution, ourunderstanding of metazoan evolutionary history can be furtheredby extracting phylogenetic signal from data with more appropriateevolutionary models and by explicitly testing alternative hypotheses.I examined 18S rDNA data across a diverse number of phyla toidentify potential problems with using genetic data to reconstructmetazoan history. Although the 18S performs well at the mostgeneral level of metazoan phytogeny, it has shortcomings particularlyamong the non-arthropod protostomes. Examples of parametricbootstrap simulation are given to illustrate how we may beginto address methodological issues when there is limited phylogeneticsignal. These simulations suggest that we are more likely tounderstand the bilaterian radiation event by increasing thelength of nucleotide sequences collected than by employing fastergenes.     

Animal phylogeny and the ancestry of bilaterians: inferences from morphology and 18S rDNA gene sequences

SUMMARY Insight into the origin and early evolution of the animal phyla requires an understanding of how animal groups are related to one another. Thus, we set out to explore animal phylogeny by analyzing with maximum parsimony 138 morphological characters from 40 metazoan groups, and 304 18S rDNA sequences, both separately and together. Both types of data agree that arthropods are not closely related to annelids: the former group with nematodes and other molting animals (Ecdysozoa), and the latter group with molluscs and other taxa with spiral cleavage. Furthermore, neither brachiopods nor chaetognaths group with deuterostomes; brachiopods are allied with the molluscs and annelids (Lophotrochozoa), whereas chaetognaths are allied with the ecdysozoans. The major discordance between the two types of data concerns the rooting of the bilaterians, and the bilaterian sister-taxon. Morphology suggests that the root is between deuterostomes and protostomes, with ctenophores the bilaterian sister-group, whereas 18S rDNA suggests that the root is within the Lophotrochozoa with acoel flatworms and gnathostomulids as basal bilaterians, and with cnidarians the bilaterian sister-group. We suggest that this basal position of acoels and gnathostomulids is artifactal because for 1000 replicate phylogenetic analyses with one random sequence as outgroup, the majority root with an acoel flatworm or gnathostomulid as the basal ingroup lineage. When these problematic taxa are eliminated from the matrix, the combined analysis suggests that the root lies between the deuterostomes and protostomes, and Ctenophora is the bilaterian sister-group. We suggest that because chaetognaths and lophophorates, taxa traditionally allied with deuterostomes, occupy basal positions within their respective protostomian clades, deuterostomy most likely represents a suite of characters plesiomorphic for bilaterians.     

Phylogenomic analyses of lophophorates (brachiopods, phoronids and bryozoans) confirm the Lophotrochozoa concept

Based on embryological and morphological evidence, Lophophorata was long considered to be the sister or paraphyletic stem group of Deuterostomia. By contrast, molecular data have consistently indicated that the three lophophorate lineages, Ectoprocta, Brachiopoda and Phoronida, are more closely related to trochozoans (annelids, molluscs and related groups) than to deuterostomes. For this reason, the lophophorate groups and Trochozoa were united to Lophotrochozoa. However, the relationships of the lophophorate lineages within Lophotrochozoa are still largely unresolved. Maximum-likelihood and Bayesian analyses were performed based on a dataset comprising 11,445 amino acid positions derived from 79 ribosomal proteins of 39 metazoan taxa including new sequences obtained from a brachiopod and a phoronid. These analyses show that the three lophophorate lineages are affiliated with trochozoan rather than deuterostome phyla. All hypotheses claiming that they are more closely related to Deuterostomia than to Protostomia can be rejected by topology testing. Monophyly of lophophorates was not recovered but that of Bryozoa including Ectoprocta and Entoprocta and monophyly of Brachiozoa including Brachiopoda and Phoronida were strongly supported. Alternative hypotheses that are refuted include (i) Brachiozoa as the sister group of Mollusca, (ii) ectoprocts as sister to all other Lophotrochozoa including Platyzoa, and (iii) ectoprocts as sister or to all other protostomes except chaetognaths.     

Sequence of the Pearl Oyster Carbonic Anhydrase-Related Protein and Its Evolutionary Implications

Carbonic anhydrases are conserved in vertebrates and invertebrates, and a noncatalytic carbonic anhydrase-related protein VIII (CARP VIII) has been found in deuterostomes and the phylum Placozoa. I isolated a cDNA encoding a noncatalytic CARP from the mantle of the pearl oyster Pinctada fucata. The polypeptide (CARP-1) predicted from the nucleotide sequence shares 44-60% identity with known CARP VIII sequences, and its phylogenetic analysis showed that P. fucata formed a single group with deuterostome invertebrates. However, since CARP VIII sequences are not identified in protostomes, these results suggest that CARP-1 may have originated in molluscs independently from deuterostome CARP VIII sequences.     

苔藓动物的起源与系统发生研究进展—形态学和分子生物学的证据

苔藓动物是后生动物中的一个重要类群。然而,和其它主要后生动物类群相比,长期以来对它的系统学研究却相对滞后。其起源,系统发生地位以及与其它后生物门类之间、其内部各高级分类群间的谱系发生关系一直存在争议。一般认为它是介于原口动物和后口动物之间的过渡类群。但是,近年来的分子系统学研究已经证实了它的原口归属。古生物学资料表明,虽然苔藓动物的大多数类群在奥陶纪已经分化出来,但它在寒武纪却缺乏任何化石记录。另外,苔藓动物起源的时间和方式、其内部各类群间的系统发生关系特别是现生类群和化石类群之间的关系等诸多问题的解决,还有待于大量的形态学和不同的分子数据的进一步积累,并结合其地层分布等各种相关资料进行综合研究。     

Analysis of the complete mitochondrial DNA sequence of the brachiopod terebratulina retusa places Brachiopoda within the protostomes

Brachiopod phylogeny is still a controversial subject. Analyses using nuclear 18SrRNA and mitochondrial 12SrDNA sequences place them within the protostomes but some recent interpretations of morphological data support a relationship with deuterostomes. In order to investigate brachiopod affinities within the metazoa further, we compared the gene arrangement on the brachiopod mitochondrial genome with several metazoan taxa. The complete (15 451 bp) mitochondrial DNA (mtDNA) sequence of the articulate brachiopod Terebratulina retusa was determined from two overlapping long polymerase chain reaction products. All the genes are encoded on the same strand and gene order comparisons showed that.only one major rearrangement is required to interconvert the T. retusa and Katharina tunicata (Mollusca: Polvplacophora) mitochondrial genomes. The partial mtDNA sequence of the prosobranch mollusc Littorina saxatilis shows complete congruence with the T. rehtusa gene arrangement with regard to the ribosomal and protein coding genes. This high similarity in gene arrangement is the first to be reported within the protostomes. Sequence analyses of mitochondrial protein coding genes also support a close relationship of the brachiopod with molluscs and annelids, thus supporting the clade Lophotrochozoa. Though being highly informative, sequence analyses of the mitochondrial protein coding genes failed to resolve the branching order within the lophotrochozoa.     

The sperm proteins from amphioxus mirror its basal position among chordates and redefine the origin of vertebrate protamines

The sperm nuclear basic proteins (SNBPs) that participate in chromatin condensation in spermatozoa belong to 3 groups: histone (H), protamine-like (PL), and protamine (P) type. They share a common origin with histone H1 resulting from the segregation of PL components, corresponding to different regions of an H1 precursor molecule (N-terminal, winged-helix, C-terminal domains), becoming independent and following a subsequent process of parallel vertical evolution (H <--> PL <--> P). In the present work, we describe the sequence and primary structure of the main SNBP component in the sperm of the cephalochordate Branchiostoma floridae (amphioxus), revealing that it represents the deuterostome counterpart of the PL-III SNBP component from molluscs corresponding to the H1 N-terminal region. Until now, this has been a missing piece needed to complete the evolutionary history of SNBPs in metazoan genomes. The discovery of this PL lineage in deuterostomes definitively validates the parallel vertical evolution of SNBPs across metazoans, giving further support to the "basal" position of amphioxus among chordates, with respect to tunicates. Sequence analyses suggest that later on in evolution, the appearance of positively selected arginine-rich protamines, derived from the H1 C-terminal region, led to the extinction of this PL lineage in the genomes of early protostomes and deuterostomes. Given that tunicates are now viewed as a sister group of vertebrates, the lysine to arginine transition responsible for the origin of vertebrate protamines must be set a step back from tunicates.     

Evolution of deuterostomy – and origin of the chordates

The chordates are usually characterized as bilaterians showing deuterostomy, i.e. the mouth developing as a new opening between the archenteron and the ectoderm, serial gill pores/slits, and the complex of chorda and neural tube. Both numerous molecular studies and studies of morphology and embryology demonstrate that the neural tube must be considered homologous to the ventral nerve cord(s) of the protostomes, but the origin of the ‘new’ mouth of the deuterostomes has remained enigmatic. However, deuterostomy is known to occur in several protostomian groups, such as the chaetognaths and representatives of annelids, molluscs, arthropods and priapulans. This raises the question whether the deuterostomian mouth is in fact homologous with that of the protostomes, viz. the anterior opening of the ancestral blastopore divided through lateral blastopore fusion, i.e. amphistomy. A few studies of gene expression show identical expression patterns around mouth and anus in protostomes and deuterostomes. Closer studies of the embryology of ascidians and vertebrates show that the mouth/stomodaeum differentiates from the anterior edge of the neural plate. Together this indicates that the chordate mouth has moved to the anterior edge of the blastopore, so that the anterior loop of the ancestral circumblastoporal nerve cord, which is narrow in the protostomes, has become indistinguishable. In the vertebrates, the mouth has moved further around the anterior pole to the ‘ventral’ side. The conclusion must be that the chordate mouth (and that of the deuterostomes in general) is homologous to the protostomian mouth and that the latest common ancestor of protostomes and deuterostomes developed through amphistomy, as suggested by the trochaea theory.     

Identification of chaetognaths as protostomes is supported by the analysis of their mitochondrial genome

Determining the phylogenetic position of enigmatic phyla such as Chaetognatha is a longstanding challenge for biologists. Chaetognaths (or arrow worms) are small, bilaterally symmetrical metazoans. In the past decades, their relationships within the metazoans have been strongly debated because of embryological and morphological features shared with the two main branches of Bilateria: the deuterostomes and protostomes. Despite recent attempts based on molecular data, the Chaetognatha affinities have not yet been convincingly defined. To answer this fundamental question, we determined the complete mitochondrial DNA genome of Spadella cephaloptera. We report three unique features: it is the smallest metazoan mitochondrial genome known and lacks both atp8 and atp6 and all tRNA genes. Furthermore phylogenetic reconstructions show that Chaetognatha belongs to protostomes. This implies that some embryological characters observed in chaetognaths, such as a gut with a mouth not arising from blastopore (deuterostomy) and a mesoderm derived from archenteron (enterocoely), could be ancestral features (plesiomorphies) of bilaterians.     

The partial mitochondrial genome of the Cephalothrix rufifrons (Nemertea, Palaeonemertea): characterization and implications for the phylogenetic position of Nemertea

A continuous 10.1kb fragment of the Cephalothrix rufifrons (Nemertea, Palaeonemertea) mitochondrial genome was sequenced and characterized to further assess organization of protostome mitochondrial genomes and evaluate the phylogenetic potential of gene arrangement and amino acid characters. The genome is A-T rich (72%), and this biased base composition is partly reflected in codon usage. Inferred tRNA secondary structures are typical of those reported for other metazoan mitochondrial DNAs. The arrangement of the 26 genes contained in the fragment exhibits marked similarity to those of many protostome taxa, most notably molluscs with highly conserved arrangements and a phoronid. Separate and simultaneous phylogenetic analyses of inferred amino acid sequences and gene adjacencies place the nemertean within the protostomes among coelomate lophotrochozoan taxa, but do not find a well-supported sister taxon link.     

Molecular data indicate the protostome affinity of brachiopods

Although the phylogenetic position of brachiopods has always been subject to debate, many authors place them as a sister group to deuterostomes on the basis of morphological and developmental characters. However, molecular phylogeny consistently places them among protostomes. More precisely, brachiopods are predicted to branch inside the lophotrochozoan assemblage, together with annelids, molluscs, nemerteans, flatworms, and others. That result has been criticized on the basis of (1) prior knowledge of brachiopod morphology and (2) the known limitations of molecular phylogenies. Here I review recent data of molecular origin, particularly those displaying qualitative properties close to those of morphological characters. The complement of Hox genes present in all metazoa tested to date has proved to be a powerful tool for broad phylogenetic reconstruction. The mitochondrial genome also provides qualitative characters, showing discrete events of gene rearrangements. After discussing the data and the way they should be interpreted in the perspective of several hypotheses for metazoan phylogeny, I conclude that they argue strongly in favor of the protostome (and lophotrochozoan) affinity of the brachiopods. There is therefore a need for a reinterpretation of brachiopod morphological and developmental characters. I also identify some research axes on brachiopod morphology.     

Origin and evolution of animal life cycles

The ‘origin of larvae’ has been widely discussed over the years, almost invariably with the tacit understanding that larvae are secondary specializations of early stages in a holobenthic life cycle. Considerations of the origin and early radiation of the metazoan phyla have led to the conclusion that the ancestral animal (= metazoan) was a holopelagic organism, and that pelago-benthic life cycles evolved when adult stages of holopelagic ancestors became benthic, thereby changing their life style, including their feeding biology. The literature on the larval development and phylogeny of animal phyla is reviewed in an attempt to infer the ancestral life cycles of the major animal groups. The quite detailed understanding of larval evolution in some echinoderms indicates that ciliary filter-feeding was ancestral within the phylum, and that planktotrophy has been lost in many clades. Similarly, recent studies of the developmental biology of ascidians have demonstrated that a larval structure, such as the tail of the tadpole larva, can easily be lost, viz. through a change in only one gene. Conversely, the evolution of complex structures, such as the ciliary bands of trochophore larvae, must involve numerous genes and numerous adaptations. The following steps of early metazoan evolution have been inferred from the review. The holopelagic ancestor, blastaea, probably consisted mainly of choanocytes, which were the feeding organs of the organism. Sponges may have evolved when blastaea-like organisms settled and became reorganized with the choanocytes in collar chambers. The eumetazoan ancestor was probably the gastraea, as suggested previously by Haeckel. It was holopelagic and digestion of captured particles took place in the archenteron. Cnidarians and ctenophores are living representatives of this type of organization. The cnidarians have become pelago-benthic with the addition of a sessile, adult polyp stage; the pelagic gastraea-like planula larva is retained in almost all major groups, but only anthozoans have feeding larvae. Within the Bilateria, two major lines of evolution can be recognized: Protostomia and Deuterostomia. In protostomes, trochophores or similar types are found in most spiralian phyla; trochophore-like ciliary bands are found in some rotifers, whereas all other aschelminths lack ciliated larvae. It seems probable that the trochophore was the larval type of the ancestral, pelago-benthic spiralian and possible that it was ancestral in all protostomes. Most of the non-chordate deuterostome phyla have ciliary filter-feeding larvae of the dipleurula type, and this strongly indicates that the ancestral deuterostome had this type of larva.     

The cambrian evolutionary ‘explosion’ recalibrated

The sudden appearance in the fossil record of the major animal phyla apparently records a phase of unparalleled, rapid evolution at the base of the Cambrian period, 545 Myr ago. This has become known as the Cambrian evolutionary ‘explosion’, and has fuelled speculation about unique evolutionary processes operating at that time. The acceptance of the palaeontological evidence as a true reflection of the evolutionary narrative has been criticised in two ways: from a reappraisal of the phylogenetic relationships of the early fossils, and from predicitions of molecular divergence times, based on six appropriate metazoan genes. Phylogenetic analysis of the arthropods implies an earlier, Precambrian history for most clades, and hence an extensive period of cladogenesis unrecorded by fossils. A similar argument can be applied to molluscs, lophophorates and deuterostomes. Molecular evidence implies divergence between clades to at least 1000 Myr ago. The apparent paradox between the sudden appearance of recognisable metazoans and their extended evolutionary history might be explained by a sudden Cambrian increase in body size, which was accompanied by skeletisation. A new paradigm suggests that the ‘explosion’ in the record may have been decoupled from the evolutionary innovation.     

Early Evolution of Histone Genes: Prevalence of an ‘Orphon’ H1 Lineage in Protostomes and Birth-and-Death Process in the H2A Family

The study of histone evolution has experienced a rebirth, for two main reasons: the identification of new essential histone variants responsible for regulating chromatin dynamics and the subsequent contradictions posed by this variability as it pertains to their long-term evolution process. Although different evolutionary models (e.g., birth-and-death evolution, concerted evolution) may account for the observed divergence of histone genes, conclusive evidence is lacking (e.g., histone H1) or totally nonexistent (e.g., histone H2A). While most of the published work has focused on deuterostomes, very little is known about the diversification and functional differentiation mechanisms followed by histone protein subtypes in protostomes, for which histone variants have only been recently described. In this study, we identify linker and core histone genes in three clam species. Our results demonstrate the prevalence of an 'orphon' H1 lineage in molluscs, a group in which the protostome H1 and sperm nuclear basic proteins are on the verge of diversification. They share an early monophyletic origin with vertebrate-specific variants prior to the differentiation between protostomes and deuterostomes. Given the intringuing evolutionary features of the histone H1 family, we have evaluated the relative importance of gene conversion, point mutation, and selection in maintaining the diversity found among H2A subtypes in eukaryotes. We show evidence for the first time that the long-term evolution of this family is not subject to concerted evolution but, rather, to a gradual evolution following a birth-and-death model under a strong purifying selection at the protein level.