Elaine C. Seaver

Broad phylogenomic sampling improves resolution of the animal tree of life.

Long-held ideas regarding the evolutionary relationships among animals have recently been upended by sometimes controversial hypotheses based largely on insights from molecular data. These new hypotheses include a clade of moulting animals (Ecdysozoa) and the close relationship of the lophophorates to molluscs and annelids (Lophotrochozoa). Many relationships remain disputed, including those that are required to polarize key features of character evolution, and support for deep nodes is often low. Phylogenomic approaches, which use data from many genes, have shown promise for resolving deep animal relationships, but are hindered by a lack of data from many important groups. Here we report a total of 39.9 Mb of expressed sequence tags from 29 animals belonging to 21 phyla, including 11 phyla previously lacking genomic or expressed-sequence-tag data. Analysed in combination with existing sequences, our data reinforce several previously identified clades that split deeply in the animal tree (including Protostomia, Ecdysozoa and Lophotrochozoa), unambiguously resolve multiple long-standing issues for which there was strong conflicting support in earlier studies with less data (such as velvet worms rather than tardigrades as the sister group of arthropods), and provide molecular support for the monophyly of molluscs, a group long recognized by morphologists. In addition, we find strong support for several new hypotheses. These include a clade that unites annelids (including sipunculans and echiurans) with nemerteans, phoronids and brachiopods, molluscs as sister to that assemblage, and the placement of ctenophores as the earliest diverging extant multicellular animals. A single origin of spiral cleavage (with subsequent losses) is inferred from well-supported nodes. Many relationships between a stable subset of taxa find strong support, and a diminishing number of lineages remain recalcitrant to placement on the tree.

Assessing the root of bilaterian animals with scalable phylogenomic methods

A clear picture of animal relationships is a prerequisite to understand how the morphological and ecological diversity of animals evolved over time. Among others, the placement of the acoelomorph flatworms, Acoela and Nemertodermatida, has fundamental implications for the origin and evolution of various animal organ systems. Their position, however, has been inconsistent in phylogenetic studies using one or several genes. Furthermore, Acoela has been among the least stable taxa in recent animal phylogenomic analyses, which simultaneously examine many genes from many species, while Nemertodermatida has not been sampled in any phylogenomic study. New sequence data are presented here from organisms targeted for their instability or lack of representation in prior analyses, and are analysed in combination with other publicly available data. We also designed new automated explicit methods for identifying and selecting common genes across different species, and developed highly optimized supercomputing tools to reconstruct relationships from gene sequences. The results of the work corroborate several recently established findings about animal relationships and provide new support for the placement of other groups. These new data and methods strongly uphold previous suggestions that Acoelomorpha is sister clade to all other bilaterian animals, find diminishing evidence for the placement of the enigmatic Xenoturbella within Deuterostomia, and place Cycliophora with Entoprocta and Ectoprocta. The work highlights the implications that these arrangements have for metazoan evolution and permits a clearer picture of ancestral morphologies and life histories in the deep past.

Insights into bilaterian evolution from three spiralian genomes

Current genomic perspectives on animal diversity neglect two prominent phyla, the molluscs and annelids, that together account for nearly one-third of known marine species and are important both ecologically and as experimental systems in classical embryology. Here we describe the draft genomes of the owl limpet (Lottia gigantea), a marine polychaete (Capitella teleta) and a freshwater leech (Helobdella robusta), and compare them with other animal genomes to investigate the origin and diversification of bilaterians from a genomic perspective. We find that the genome organization, gene structure and functional content of these species are more similar to those of some invertebrate deuterostome genomes (for example, amphioxus and sea urchin) than those of other protostomes that have been sequenced to date (flies, nematodes and flatworms). The conservation of these genomic features enables us to expand the inventory of genes present in the last common bilaterian ancestor, establish the tripartite diversification of bilaterians using multiple genomic characteristics and identify ancient conserved long- and short-range genetic linkages across metazoans. Superimposed on this broadly conserved pan-bilaterian background we find examples of lineage-specific genome evolution, including varying rates of rearrangement, intron gain and loss, expansions and contractions of gene families, and the evolution of clade-specific genes that produce the unique content of each genome.

Growth patterns during segmentation in the two polychaete annelids, Capitella sp. I and Hydroides elegans: comparisons at distinct life history stages.

Summary Many animals generate new body segments sequentially from a posterior growth zone, and this is generally thought to be the case for the annelids. Most annelids, including polychaetes, have an indirect life cycle and generate their earliest segments during larval life. We have characterized the nature of the growth zone in two polychaetes, Hydroides elegans and Capitella sp. I, during both larval and juvenile stages of segment formation by examining cell division patterns with 5‐bromo‐2′‐deoxyuridine incorporation. Cell division patterns show commonalities between the two species, even though they have distinct body plans and life history characteristics. In both polychaetes, larval segments arise from a field of dividing cells located in lateral regions of the body, rather than from a localized posterior growth zone. Circumferential expansion of the forming segmental tissue is particularly pronounced in Capitella sp. I. Post‐metamorphic segments, in contrast, originate from a classical posterior growth zone, with the exception of four posterior thoracic segments of H. elegans, which appear to arise from an area in the middle of the body, indicating plasticity of segment‐generating mechanisms present in different annelid life histories. The distinct nature of larval versus juvenile growth zones in H. elegans and Capitella sp. I raises the question of the mechanistic relationship between these two growth zones. The results of this study increase our understanding of the cellular origins of segments in annelids, and serve as a basis for interpretation of molecular expression patterns associated with segment formation in polychaetes.

Genomic Organization and Expression Demonstrate Spatial and Temporal Hox Gene Colinearity in the Lophotrochozoan Capitella sp. I

Hox genes define regional identities along the anterior–posterior axis in many animals. In a number of species, Hox genes are clustered in the genome, and the relative order of genes corresponds with position of expression in the body. Previous Hox gene studies in lophotrochozoans have reported expression for only a subset of the Hox gene complement and/or lack detailed genomic organization information, limiting interpretations of spatial and temporal colinearity in this diverse animal clade. We studied expression and genomic organization of the single Hox gene complement in the segmented polychaete annelid Capitella sp. I. Total genome searches identified 11 Hox genes in Capitella, representing 11 distinct paralog groups thought to represent the ancestral lophotrochozoan complement. At least 8 of the 11 Capitella Hox genes are genomically linked in a single cluster, have the same transcriptional orientation, and lack interspersed non-Hox genes. Studying their expression by situ hybridization, we find that the 11 Capitella Hox genes generally exhibit spatial and temporal colinearity. With the exception of CapI-Post1, Capitella Hox genes are all expressed in broad ectodermal domains during larval development, consistent with providing positional information along the anterior–posterior axis. The anterior genes CapI-lab, CapI-pb, and CapI-Hox3 initiate expression prior to the appearance of segments, while more posterior genes appear at or soon after segments appear. Many of the Capitella Hox genes have either an anterior or posterior expression boundary coinciding with the thoracic–abdomen transition, a major body tagma boundary. Following metamorphosis, several expression patterns change, including appearance of distinct posterior boundaries and restriction to the central nervous system. Capitella Hox genes have maintained a clustered organization, are expressed in the canonical anterior–posterior order found in other metazoans, and exhibit spatial and temporal colinearity, reflecting Hox gene characteristics that likely existed in the protostome–deuterostome ancestor.

Somatic and germline expression of piwi during development and regeneration in the marine polychaete annelid Capitella teleta.

BackgroundStem cells have a critical role during adult growth and regeneration. Germline stem cells are specialized stem cells that produce gametes during sexual reproduction. Capitella teleta (formerly Capitella sp. I) is a polychaete annelid that reproduces sexually, exhibits adult growth and regeneration, and thus, is a good model to study the relationship between somatic and germline stem cells.ResultsWe characterize expression of the two C. teleta orthologs of piwi, genes with roles in germline development in diverse organisms. Ct-piwi1 and Ct-piwi2 are expressed throughout the life cycle in a dynamic pattern that includes both somatic and germline cells, and show nearly identical expression patterns at all stages examined. Both genes are broadly expressed during embryonic and larval development, gradually becoming restricted to putative primordial germ cells (PGCs) and the posterior growth zone. In juveniles, Ct-piwi1 is expressed in the presumptive gonads, and in reproductive adults, it is detected in gonads and the posterior growth zone. In addition, Ct-piwi1 is expressed in a population of putative PGCs that persist in sexually mature adults, likely in a stem cell niche. Ct-piwi1 is expressed in regenerating tissue, and once segments differentiate, it becomes most prominent in the posterior growth zone and immature oocytes in regenerating ovaries of regenerating segments.ConclusionsIn C. teleta, piwi genes may have retained an ancestral role as genetic regulators of both somatic and germline stem cells. It is likely that piwi genes, and associated stem cell co-regulators, became restricted to the germline in some taxa during the course of evolution.

Developmental expression of foxA and gata genes during gut formation in the polychaete annelid, Capitella sp. I

SUMMARY Most bilaterian animals have evolved a through gut that is regionally specialized along the anterior–posterior axis. In the polychaete annelid, Capitella sp. I, the alimentary canal is subdivided into a buccal cavity, pharynx, esophagus, midgut, and hindgut. Members of the Fox and GATA families of transcription factors have conserved functions in patterning ectodermal and endodermal gut components. We have isolated and characterized expression of one FoxA gene (CapI‐foxA) and four GATA genes (CapI‐gataB1, CapI‐gataB2, CapI‐gataB3, and CapI‐gataA1) from Capitella sp. I. Both gene families are expressed in the developing gut of this polychaete. CapI‐foxA, an ortholog of the FoxA subgroup, is expressed in vegetal hemisphere micromeres of cleavage‐stage embryos, in multiple blastomeres within and surrounding the blastopore during gastrulation, and throughout morphogenesis of the pharynx, esophagus, and hindgut. The CapI‐gataB genes group within the vertebrate GATA4/5/6 subfamily, appear to be products of lineage‐specific gene duplication, and are expressed in specific domains of endomesoderm. CapI‐gataB1 is expressed in endoderm precursors and throughout developing midgut endoderm, and is particularly prominent at anterior and posterior midgut boundaries. CapI‐gataB2 is co‐expressed with CapI‐gataB1 in midgut endoderm, and is also expressed in visceral mesoderm. CapI‐gataB3 is limited to and coexpressed with CapI‐gataB2 in visceral mesoderm. CapI‐gataA1 groups within the vertebrate GATA1/2/3 subfamily and is expressed primarily in ectodermal tissues of the brain, ventral nerve cord, lateral trunk, and both pharyngeal and esophageal regions of the foregut. Collectively, the CapI‐foxA and CapI‐gata genes show patterns of expression that span almost the entire length of the developing alimentary canal, consistent with a role in gut development.

ParaHox gene expression in the polychaete annelid Capitella sp. I

Hox and ParaHox genes are transcriptional regulators vital for many aspects of embryonic development in bilaterian animals and are considered to have originated from one ancestral proto-Hox/ParaHox cluster. Hox genes are clustered in the genome of both protostomes and deuterostomes, and there is a specific relationship between the position of a gene in the cluster and the position of its expression along the animal body axis (colinearity). It is not clear whether the ParaHox genes Gsx, Xlox, and, Cdx generally exhibit a similar phenomenon since developmental expression for all three ParaHox genes within a single species has not yet been described for any protostome animal. Here we show the spatial and temporal localization for all three ParaHox genes in the polychaete Capitella sp. I, a member of one of the morphologically most diverse and understudied groups within the Metazoa, the Lophotrochozoa. Our data demonstrate that although both CapI-Xlox and CapI-Cdx are regionally expressed in the gut, the three Capitella sp. I ParaHox genes as a group do not perfectly fit predictions of temporal or spatial colinearity. Instead, there is a conservation of expression across species associated with development of particular tissues, and the relative order of initiation of ParaHox gene expression likely reflects the relative order of species-specific tissue development during ontogenesis.

β-Catenin is required for the establishment of vegetal embryonic fates in the nemertean, Cerebratulus lacteus

Downstream components of the canonical Wnt signaling pathway that result in the nuclear localization of beta-catenin are involved in diverse developmental processes including the formation of the mesendoderm, the regulation of axial properties and asymmetric cell divisions in a wide array of metazoans. The nemertean worm, Cerebratulus lacteus, represents a member of the understudied lophotrochozoan clade that exhibits a highly stereotyped spiral cleavage program in which ectodermal, endodermal, and mesodermal origins are known from intracellular fate mapping studies. Here, the embryonic distribution of beta-catenin protein was studied using injection of synthetic mRNA, encoding GFP-tagged beta-catenin, into fertilized eggs. During the early cleavage stages beta-catenin was destabilized/degraded in animal hemisphere blastomeres and became localized to the nuclei of the four vegetal-most cells at the 64-cell stage, which give rise to definitive larval and adult endoderm. Functional assays indicate that beta-catenin plays a key role in the development of the endoderm. Morpholino knockdown of endogenous beta-catenin, as confirmed by Western analysis, resulted in the failure to gastrulate, absence of the gut and an animalized phenotype in the resulting larvae, including the formation of ectopic (anterior) apical organ tissue with elongated apical tuft cilia and no indications of dorsoventral polarity. Similarly, over-expression of the cytoplasmic domain of cadherin or a beta-catenin-engrailed repressor fusion construct prevented endoderm formation and generated the same animalized phenotype. Injections of mRNA encoding either a stabilized, constitutively activated form of beta-catenin or a dominant negative form of GSK3-beta converted all or nearly all cells into endodermal fates expressing gut-specific esterase. Thus, beta-catenin appears to be both necessary and sufficient to promote endoderm formation in C. lacteus, consistent with its role in endoderm and endomesoderm formation in anthozoan cnidarians, ascidians, and echinoderms. Consistent with the results of other studies, beta-catenin may be viewed as playing a role in the development of posterior/vegetal larval fates (i.e., endoderm) in C. lacteus. However, unlike the case found in polychaete annelid and soil nematode embryos, there is no evidence for a role of beta-catenin in regulating cell fates and asymmetric cell divisions along the entire anterior-posterior axis.

Vasa and nanos are coexpressed in somatic and germ line tissue from early embryonic cleavage stages through adulthood in the polychaete Capitella sp. I

Members of the vasa and nanos gene families are involved in germ line development in a number of diverse animals. As a polychaete annelid model for studies of the germ line, Capitella sp. I has several advantages including the presence of dedicated gonads, individuals that reproduce multiple times, and the presence of males, females, and hermaphrodites. Germ line development has not been characterized in Capitella sp. I, nor is the mechanism of germ line specification generally well understood in annelids. We have cloned vasa and nanos orthologues from Capitella sp. I and found that both CapI-vasa and CapI-nanos transcripts are expressed in developing gametes of sexually mature adults. Characterization of both these genes during embryonic, larval, and juveniles stages reveals expression in multiple somatic tissues for CapI-vasa and CapI-nanos with largely overlapping but not identical expression patterns. In early cleavage stages, both transcripts are broadly expressed; following gastrulation, expression is observed in the presumptive brain, mesodermal bands, and developing foregut. Using CapI-nanos and CapI-vasa as markers, we have identified putative primordial germ cells (PGCs) in larvae, which are initially present as small bilateral clusters in segment 4 and as a single cluster at late larval stages. In adults, a single large cluster of putative PGCs is present in segments 5 and 6. In addition to highlighting differences in expression profiles for these two genes among lophotrochozoans, we present a hypothesis concerning the origin and development of PGCs in Capitella sp. I.