Bruno Cossermelli Vellutini

Xenacoelomorpha is the sister group to Nephrozoa

The position of Xenacoelomorpha in the tree of life remains a major unresolved question in the study of deep animal relationships. Xenacoelomorpha, comprising Acoela, Nemertodermatida, and Xenoturbella, are bilaterally symmetrical marine worms that lack several features common to most other bilaterians, for example an anus, nephridia, and a circulatory system. Two conflicting hypotheses are under debate: Xenacoelomorpha is the sister group to all remaining Bilateria (= Nephrozoa, namely protostomes and deuterostomes) or is a clade inside Deuterostomia. Thus, determining the phylogenetic position of this clade is pivotal for understanding the early evolution of bilaterian features, or as a case of drastic secondary loss of complexity. Here we show robust phylogenomic support for Xenacoelomorpha as the sister taxon of Nephrozoa. Our phylogenetic analyses, based on 11 novel xenacoelomorph transcriptomes and using different models of evolution under maximum likelihood and Bayesian inference analyses, strongly corroborate this result. Rigorous testing of 25 experimental data sets designed to exclude data partitions and taxa potentially prone to reconstruction biases indicates that long-branch attraction, saturation, and missing data do not influence these results. The sister group relationship between Nephrozoa and Xenacoelomorpha supported by our phylogenomic analyses implies that the last common ancestor of bilaterians was probably a benthic, ciliated acoelomate worm with a single opening into an epithelial gut, and that excretory organs, coelomic cavities, and nerve cords evolved after xenacoelomorphs separated from the stem lineage of Nephrozoa.

Clustered brachiopod Hox genes are not expressed collinearly and are associated with lophotrochozoan novelties

Significance Hox genes pattern the anteroposterior axis of all animals that have left and right body sides. In many animals, Hox genes are clustered along the chromosomes and expressed in spatial and temporal order. This coordinated regulation is thought to have preserved the cluster through a developmental constraint. Our study of the genomic organization and the embryonic spatial and temporal expression of Hox genes in sessile marine animals called lampshells (brachiopods) shows that along with having a broken Hox cluster, they lack both temporal and spatial collinearity. Furthermore, we present molecular evidence that the hard tissues (chaetae and shells) of segmented worms, mollusks, and brachiopods share a common origin that dates back to the Early Cambrian. Temporal collinearity is often considered the main force preserving Hox gene clusters in animal genomes. Studies that combine genomic and gene expression data are scarce, however, particularly in invertebrates like the Lophotrochozoa. As a result, the temporal collinearity hypothesis is currently built on poorly supported foundations. Here we characterize the complement, cluster, and expression of Hox genes in two brachiopod species, Terebratalia transversa and Novocrania anomala. T. transversa has a split cluster with 10 genes (lab, pb, Hox3, Dfd, Scr, Lox5, Antp, Lox4, Post2, and Post1), whereas N. anomala has 9 genes (apparently missing Post1). Our in situ hybridization, real-time quantitative PCR, and stage-specific transcriptomic analyses show that brachiopod Hox genes are neither strictly temporally nor spatially collinear; only pb (in T. transversa), Hox3 (in both brachiopods), and Dfd (in both brachiopods) show staggered mesodermal expression. Thus, our findings support the idea that temporal collinearity might contribute to keeping Hox genes clustered. Remarkably, expression of the Hox genes in both brachiopod species demonstrates cooption of Hox genes in the chaetae and shell fields, two major lophotrochozoan morphological novelties. The shared and specific expression of Hox genes, together with Arx, Zic, and Notch pathway components in chaetae and shell fields in brachiopods, mollusks, and annelids provide molecular evidence supporting the conservation of the molecular basis for these lophotrochozoan hallmarks.

Embryonic, Larval, and Juvenile Development of the Sea Biscuit Clypeaster subdepressus (Echinodermata: Clypeasteroida)

Sea biscuits and sand dollars diverged from other irregular echinoids approximately 55 million years ago and rapidly dispersed to oceans worldwide. A series of morphological changes were associated with the occupation of sand beds such as flattening of the body, shortening of primary spines, multiplication of podia, and retention of the lantern of Aristotle into adulthood. To investigate the developmental basis of such morphological changes we documented the ontogeny of Clypeaster subdepressus. We obtained gametes from adult specimens by KCl injection and raised the embryos at 26C. Ciliated blastulae hatched 7.5 h after sperm entry. During gastrulation the archenteron elongated continuously while ectodermal red-pigmented cells migrated synchronously to the apical plate. Pluteus larvae began to feed in 3 d and were 20 d old at metamorphosis; starved larvae died 17 d after fertilization. Postlarval juveniles had neither mouth nor anus nor plates on the aboral side, except for the remnants of larval spicules, but their bilateral symmetry became evident after the resorption of larval tissues. Ossicles of the lantern were present and organized in 5 groups. Each group had 1 tooth, 2 demipyramids, and 2 epiphyses with a rotula in between. Early appendages consisted of 15 spines, 15 podia (2 types), and 5 sphaeridia. Podial types were distributed in accordance to Lovéns rule and the first podium of each ambulacrum was not encircled by the skeleton. Seven days after metamorphosis juveniles began to feed by rasping sand grains with the lantern. Juveniles survived in laboratory cultures for 9 months and died with wide, a single open sphaeridium per ambulacrum, aboral anus, and no differentiated food grooves or petaloids. Tracking the morphogenesis of early juveniles is a necessary step to elucidate the developmental mechanisms of echinoid growth and important groundwork to clarify homologies between irregular urchins.

Expression of segment polarity genes in brachiopods supports a non-segmental ancestral role of engrailed for bilaterians

The diverse and complex developmental mechanisms of segmentation have been more thoroughly studied in arthropods, vertebrates and annelids—distantly related animals considered to be segmented. Far less is known about the role of “segmentation genes” in organisms that lack a segmented body. Here we investigate the expression of the arthropod segment polarity genes engrailed, wnt1 and hedgehog in the development of brachiopods—marine invertebrates without a subdivided trunk but closely related to the segmented annelids. We found that a stripe of engrailed expression demarcates the ectodermal boundary that delimits the anterior region of Terebratalia transversa and Novocrania anomala embryos. In T. transversa, this engrailed domain is abutted by a stripe of wnt1 expression in a pattern similar to the parasegment boundaries of insects—except for the expression of hedgehog, which is restricted to endodermal tissues of the brachiopod embryos. We found that pax6 and pax2/5/8, putative regulators of engrailed, also demarcate the anterior boundary in the two species, indicating these genes might be involved in the anterior patterning of brachiopod larvae. In a comparative phylogenetic context, these findings suggest that bilaterians might share an ancestral, non-segmental domain of engrailed expression during early embryogenesis.

Embryonic chirality and the evolution of spiralian left-right asymmetries.

The group Spiralia includes species with one of the most significant cases of left–right asymmetries in animals: the coiling of the shell of gastropod molluscs (snails). In this animal group, an early event of embryonic chirality controlled by cytoskeleton dynamics and the subsequent differential activation of the genes nodal and Pitx determine the left–right axis of snails, and thus the direction of coiling of the shell. Despite progressive advances in our understanding of left–right axis specification in molluscs, little is known about left–right development in other spiralian taxa. Here, we identify and characterize the expression of nodal and Pitx orthologues in three different spiralian animals—the brachiopod Novocrania anomala, the annelid Owenia fusiformis and the nemertean Lineus ruber—and demonstrate embryonic chirality in the biradial-cleaving spiralian embryo of the bryozoan Membranipora membranacea. We show asymmetric expression of nodal and Pitx in the brachiopod and annelid, respectively, and symmetric expression of Pitx in the nemertean. Our findings indicate that early embryonic chirality is widespread and independent of the cleavage programme in the Spiralia. Additionally, our study illuminates the evolution of nodal and Pitx signalling by demonstrating embryonic asymmetric expression in lineages without obvious adult left–right asymmetries. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’.

Cleavage modification did not alter blastomere fates during bryozoan evolution

BackgroundStereotypic cleavage patterns play a crucial role in cell fate determination by precisely positioning early embryonic blastomeres. Although misplaced cell divisions can alter blastomere fates and cause embryonic defects, cleavage patterns have been modified several times during animal evolution. However, it remains unclear how evolutionary changes in cleavage impact the specification of blastomere fates. Here, we analyze the transition from spiral cleavage – a stereotypic pattern remarkably conserved in many protostomes – to a biradial cleavage pattern, which occurred during the evolution of bryozoans.ResultsUsing 3D-live imaging time-lapse microscopy (4D-microscopy), we characterize the cell lineage, MAPK signaling, and the expression of 16 developmental genes in the bryozoan Membranipora membranacea. We found that the molecular identity and the fates of early bryozoan blastomeres are similar to the putative homologous blastomeres in spiral-cleaving embryos.ConclusionsOur work suggests that bryozoans have retained traits of spiral development, such as the early embryonic fate map, despite the evolution of a novel cleavage geometry. These findings provide additional support that stereotypic cleavage patterns can be modified during evolution without major changes to the molecular identity and fate of embryonic blastomeres.

Brachiopods possess a split Hox cluster with signs of spatial, but not temporal collinearity

Temporal collinearity is often regarded as the force preserving Hox clusters in vertebrate genomes. Studies that combine genomic and gene expression data in invertebrates would allow generalizing this observation across all animals, but are scarce, particularly within Lophotrochozoa (e.g., snails and segmented worms). Here, we use two brachiopod species –Terebratalia transversa, Novocrania anomala– to characterize the complement, cluster and expression of their Hox genes. T. transversa has an ordered, split cluster with ten genes (lab, pb, Hox3, dfd, scr, lox5, antp, lox4, post2, post1), while N. anomala has nine (missing post1). Our in situ hybridization, qPCR and stage specific transcriptomic analyses show that brachiopod Hox genes are neither strictly temporally nor spatially collinear; only pb (in T. transversa), Hox3 and dfd (in both brachiopods) show staggered mesodermal expression. The spatial expression of the Hox genes in both brachiopod species correlates with their morphology and demonstrates cooption of Hox genes in the chaetae and shell fields, two major lophotrochozoan morphological novelties. The shared and specific expression of a subset of Hox genes, Arx and Zic orthologs in chaetae and shell-fields between brachiopods, mollusks, and annelids supports the deep conservation of the molecular basis forming these lophotrochozoan hallmarks. Our findings challenge that collinearity alone preserves lophotrochozoan Hox clusters, indicating that additional genomic traits need to be considered in understanding Hox evolution.Hox genes are often clustered in animal genomes and exhibit spatial and/or temporal collinearity. It is generally believed that temporal collinearity is the major force preserving Hox clusters. However, studies combining genomic and gene expression analyses of Hox genes are scarce, particularly within Spiralia and Lophotrochozoa (e.g. mollusks, segmented worms, and flatworms). Here, we use two brachiopod species --Terebratalia transversa and Novocrania anomala-- that respectively belong to the two major brachiopod lineages to characterize their Hox complement, the presence of a Hox cluster, and the temporal and spatial expression of their Hox genes. We demonstrate that the Hox complement consists of ten Hox genes in T. transversa (lab, pb, Hox3, dfd, scr, lox5, antp, lox4, post2 and post1) and nine in N. anomala (missing post1). Additionally, T. transversa has an ordered, split Hox cluster. Expression analyses reveal that Hox genes are neither temporally nor spatially collinear, and only the genes pb (in T. transversa), Hox3 and dfd (in both brachiopods) show staggered expression in the mesoderm. Remarkably, lab, scr, antp and post1 are associated with the development of the chaetae and shell-forming epithelium, as also observed in annelid chaetae and mollusk shell fields. This, together with the expression of Arx homeobox, supports the deep conservation of the molecular basis for chaetae formation and shell patterning in Lophotrochozoa. Our findings challenge the current evolutionary scenario that (temporal) collinearity is the major mechanism preserving Hox clusters, and suggest that Hox genes were involved in the evolution of lophotrochozoan novelties.

A novel approach using increased taxon sampling reveals thousands of hidden orthologs in flatworms

Gains and losses shape the gene complement of animal lineages and are a fundamental aspect of genomic evolution. Acquiring a comprehensive view of the evolution of gene repertoires is limited by the intrinsic limitations of common sequence similarity searches and available databases. Thus, a subset of the complement of an organism consists of hidden orthologs, those with no apparent homology with common sequenced animal lineages –mistakenly considered new genes– but actually representing rapidly evolving orthologs or undetected paralogs. Here, we describe Leapfrog, a simple automated BLAST pipeline that leverages increased taxon sampling to overcome long evolutionary distances and identify hidden orthologs in large transcriptomic databases. As a case study, we used 35 transcriptomes of 29 flatworm lineages to recover 3,427 hidden orthologs, some of them not identified by OrthoFinder, a common orthogroup inference algorithm. Unexpectedly, we do not observe a correlation between the number of hidden orthologs in a lineage and its ‘average’ evolutionary rate. Hidden orthologs do not show unusual sequence composition biases (e.g. GC content, average length, domain composition) that might account for systematic errors in sequence similarity searches. Instead, gene duplication and divergence of one paralog and weak positive selection appear to underlie hidden orthology in Platyhelminthes. By using Leapfrog, we identify key centrosome-related genes and homeodomain classes previously reported as absent in free-living flatworms, e.g. planarians. Altogether, our findings demonstrate that hidden orthologs comprise a significant proportion of the gene repertoire in flatworms, qualifying the impact of gene losses and gains in gene complement evolution.

Larval Evolution: I’ll Tail You Later…

Larval stages can be astonishingly different from their adult forms. A new study in acorn worms shows that the whole larval body is patterned only with a subset of anterior genes, revealing the intricate developmental bases that underlie the evolution of larval forms.

Conserved traits of spiralian development in the bryozoan Membranipora membranacea

Background: Spiral cleavage is a remarkably conserved pattern of embryogenesis present in animals of the clade Spiralia, such as annelids, molluscs, flatworms and nemerteans. However, not all spiralians display spiral cleavage. Recent phylogenies suggest that the spiral arrangement of embryonic blastomeres is an ancestral trait for the Spiralia and that it was secondarily modified in several spiralian lineages, such as gastrotrichs, brachiopods and bryozoans. To better understand the evolution of cleavage patterns in relation to blastomere fate maps and embryonic gene expression, we describe the cell lineage and molecular patterning in the embryogenesis of the bryozoan Membranipora membranacea. Results: M. membranacea develops through a unique stereotypic cleavage pattern with biradial symmetry and an embryo organized in identical quadrants with synchronous cell divisions. The quadrant identities are established as early as the 28-cell stage, when one vegetal blastomere (3D) activates the MAPK pathway, marking the future posterior region of the larva. Cells from this posterior quadrant divide asynchronously in subsequent stages leading to the morphological differentiation between quadrants. The first quartet of M. membranacea gives rise to the apical organ and ciliated band, the second and third quartet forms the oral/anal ectoderm, the fourth quartet the mesoderm and the vegetal blastomeres form the endoderm. We found that the early embryonic organization and the fate map of these early blastomeres in the bryozoan embryo are similar to a typical spiral-cleaving embryo. Furthermore we observe that correspondent blastomeres between the bryozoan and spiral-cleaving embryos share similar molecular identities, as revealed by the activity of MAPK and the expression of otx and foxa. The development of M. membranacea mainly differs from spiral-cleaving embryos in the downstream portions of the cell lineage, and in the origin of the mesoderm, which is formed by multiple fourth quartet blastomeres. Conclusions: The similarity between the fate map of M. membranacea and spiral-cleaving embryos indicates that the cleavage geometry of the bryozoan evolved decoupled from other spiralian developmental traits. In this case, the blastomere fates remained evolutionarily conserved despite the drastic modification in the cleavage pattern from spiral to biradial. These findings suggest that blastomere fates of spiral-cleaving embryos might not be linked to the stereotypic spiral cleavage pattern, but depend on other factors, such as the underlying molecular patterning. Our comparative analysis on the bryozoan reveals yet another facet of how early development evolves and helps to shed some light into the developmental diversity of spiralians.Stereotypic cleavage patterns play a crucial role in cell fate determination by precisely positioning early embryonic blastomeres. Although misplaced cell divisions can alter blastomere fates and cause embryonic defects, cleavage patterns have changed several times during animal evolution. Here, we analyze the evolutionary transition from spiral cleavage – a stereotypic pattern remarkably conserved in many protostomes – to the biradial cleavage of bryozoans. We characterize the cell lineage, MAPK signaling and expression of several developmental genes in the bryozoan Membranipora membranacea, and found that the fate and the genes expressed in the early bryozoan blastomeres are similar to their putative homologous blastomeres in spiral-cleaving embryos. The data indicate that cleavage geometry evolved independent from other developmental traits during the transition from spiral to biradial cleavage in the bryozoan lineage, revealing that stereotypic cleavage patterns can be evolutionarily modified without major changes to the molecular identity and fate of embryonic blastomeres.