Single-specimen systematics resolves the phylogeny and diversity conundrum of enigmatic crustacean y-larvae.

Resolving the evolutionary history of organisms is a major goal in biology. Yet for some taxa the diversity, phylogeny, and even adult stages remain unknown. The enigmatic crustacean "y-larvae" (Facetotecta) are one particularly striking example. Here, we use extensive video-imaging and single-specimen molecular sequencing of >200 y-larval specimens to comprehensively explore for the first time their evolutionary history and diversity. This integrative approach revealed five major clades of Facetotecta, four of which encompass a considerable larval diversity. Whereas morphological analyses recognized 35 y-naupliar "morphospecies", molecular species-delimitation analyses suggested the existence of between 88 and 127 species. The phenotypic and genetic diversity between the morphospecies suggests that a more elaborate classification than the current one-genus approach is needed. Morphology and molecular data were highly congruent at shallower phylogenetic levels, but no morphological synapomorphies could be unambiguously identified for major clades, which mostly comprise both planktotrophic and lecithotrophic y-nauplii. We argue that lecithotrophy arose several times independently whereas planktotrophic y-nauplii, which are structurally more similar across clades, most likely display the ancestral feeding mode of Facetotecta. We document a remarkably complex and highly diverse phylogenetic backbone for a taxon of larval marine crustaceans, the full life cycle of which remains a mystery.

A phylogenomic approach to resolving interrelationships of polyclad flatworms, with implications for life-history evolution.

Platyhelminthes (flatworms) are a diverse invertebrate phylum useful for exploring life-history evolution. Within Platyhelminthes, only two clades develop through a larval stage: free-living polyclads and parasitic neodermatans. Neodermatan larvae are considered evolutionarily derived, whereas polyclad larvae are hypothesized to be ancestral due to ciliary band similarities among polyclad and other spiralian larvae. However, larval evolution has been challenging to investigate within polyclads due to low support for deeper phylogenetic relationships. To investigate polyclad life-history evolution, we generated transcriptomic data for 21 species of polyclads to build a well-supported phylogeny for the group. The resulting tree provides strong support for deeper nodes, and we recover a new monophyletic clade of early branching cotyleans. We then used ancestral state reconstructions to investigate ancestral modes of development within Polycladida and more broadly within flatworms. In polyclads, we were unable to reconstruct the ancestral state of deeper nodes with significant support because early branching clades show diverse modes of development. This suggests a complex history of larval evolution in polyclads that likely includes multiple losses and/or multiple gains. However, our ancestral state reconstruction across a previously published platyhelminth phylogeny supports a direct developing prorhynchid/polyclad ancestor, which suggests that a larval stage in the life cycle evolved along the polyclad stem lineage or within polyclads.

Worms and gills, plates and spines: the evolutionary origins and incredible disparity of deuterostomes revealed by fossils, genes, and development.

Deuterostomes are the major division of animal life which includes sea stars, acorn worms, and humans, among a wide variety of ecologically and morphologically disparate taxa. However, their early evolution is poorly understood, due in part to their disparity, which makes identifying commonalities difficult, as well as their relatively poor early fossil record. Here, we review the available morphological, palaeontological, developmental, and molecular data to establish a framework for exploring the origins of this important and enigmatic group. Recent fossil discoveries strongly support a vermiform ancestor to the group Hemichordata, and a fusiform active swimmer as ancestor to Chordata. The diverse and anatomically bewildering variety of forms among the early echinoderms show evidence of both bilateral and radial symmetry. We consider four characteristics most critical for understanding the form and function of the last common ancestor to Deuterostomia: Hox gene expression patterns, larval morphology, the capacity for biomineralization, and the morphology of the pharyngeal region. We posit a deuterostome last common ancestor with a similar antero-posterior gene regulatory system to that found in modern acorn worms and cephalochordates, a simple planktonic larval form, which was later elaborated in the ambulacrarian lineage, the ability to secrete calcium minerals in a limited fashion, and a pharyngeal respiratory region composed of simple pores. This animal was likely to be motile in adult form, as opposed to the sessile origins that have been historically suggested. Recent debates regarding deuterostome monophyly as well as the wide array of deuterostome-affiliated problematica further suggest the possibility that those features were not only present in the last common ancestor of Deuterostomia, but potentially in the ur-bilaterian. The morphology and development of the early deuterostomes, therefore, underpin some of the most significant questions in the study of metazoan evolution.

Chromosome-level genome assembly, annotation, and phylogenomics of the gooseneck barnacle Pollicipes pollicipes.

BACKGROUND: The barnacles are a group of >2,000 species that have fascinated biologists, including Darwin, for centuries. Their lifestyles are extremely diverse, from free-swimming larvae to sessile adults, and even root-like endoparasites. Barnacles also cause hundreds of millions of dollars of losses annually due to biofouling. However, genomic resources for crustaceans, and barnacles in particular, are lacking. RESULTS: Using 62× Pacific Biosciences coverage, 189× Illumina whole-genome sequencing coverage, 203× HiC coverage, and 69× CHi-C coverage, we produced a chromosome-level genome assembly of the gooseneck barnacle Pollicipes pollicipes. The P. pollicipes genome is 770 Mb long and its assembly is one of the most contiguous and complete crustacean genomes available, with a scaffold N50 of 47 Mb and 90.5% of the BUSCO Arthropoda gene set. Using the genome annotation produced here along with transcriptomes of 13 other barnacle species, we completed phylogenomic analyses on a nearly 2 million amino acid alignment. Contrary to previous studies, our phylogenies suggest that the Pollicipedomorpha is monophyletic and sister to the Balanomorpha, which alters our understanding of barnacle larval evolution and suggests homoplasy in a number of naupliar characters. We also compared transcriptomes of P. pollicipes nauplius larvae and adults and found that nearly one-half of the genes in the genome are differentially expressed, highlighting the vastly different transcriptomes of larvae and adult gooseneck barnacles. Annotation of the genes with KEGG and GO terms reveals that these stages exhibit many differences including cuticle binding, chitin binding, microtubule motor activity, and membrane adhesion. CONCLUSION: This study provides high-quality genomic resources for a key group of crustaceans. This is especially valuable given the roles P. pollicipes plays in European fisheries, as a sentinel species for coastal ecosystems, and as a model for studying barnacle adhesion as well as its key position in the barnacle tree of life. A combination of genomic, phylogenetic, and transcriptomic analyses here provides valuable insights into the evolution and development of barnacles.

Expression of Hox, Cdx, and Six3/6 genes in the hoplonemertean Pantinonemertes californiensis offers insight into the evolution of maximally indirect development in the phylum Nemertea.

BACKGROUND: Maximally indirect development via a pilidium larva is unique to the pilidiophoran clade of phylum Nemertea. All other nemerteans have more or less direct development. The origin of pilidial development with disjunct invaginated juvenile rudiments and catastrophic metamorphosis remains poorly understood. While basal members of the phylum, the Palaeonemertea, do not appear to have ever had a pilidium, certain similarity exists in the development of the Pilidiophora and the sister clade, the Hoplonemertea. It is unclear whether this similarity represents the homology and whether pilidial development evolved before or after pilidiophorans diverged from hoplonemerteans. To gain insight into these questions, we examined the expression of Hox, Cdx, and Six3/6 genes in the development of the hoplonemertean Pantinonemertes californiensis and expression of Six3/6 in the pilidium of Micrura alaskensis. To further characterize the function of larval structures showing expression of these genes, we examined the serotonergic nervous system and cell proliferation in P. californiensis. RESULTS: We show that Hox and Cdx genes, which pattern the pilidial imaginal discs giving rise to the juvenile trunk, are expressed in paired posterior epidermal invaginations in P. californiensis larvae. We also show that Six3/6 patterns both the pilidial cephalic discs, which give rise to the juvenile head, and a pair of anterior epidermal invaginations in hoplonemertean development. We show that anterior invaginations in larval P. californiensis are associated with a pair of serotonergic neurons, and thus may have a role in the development of the juvenile nervous system. This is similar to the role of cephalic discs in pilidiophoran development. Finally, we show that four zones of high cell proliferation correspond to the paired invaginations in P. californiensis, suggesting that these invaginations may play a similar role in the development of the hoplonemertean juvenile to the role of imaginal discs in the pilidium, which also exhibit high rates of cell proliferation. CONCLUSIONS: Expression of Hox, Cdx, and Six3/6 genes supports the homology between the imaginal discs of the pilidium and the paired larval invaginations in hoplonemerteans. This suggests that invaginated juvenile rudiments (possible precursors to pilidial imaginal discs) may have been present in the most recent common ancestor of the Pilidiophora and Hoplonemertea.