Emergence of novel genomic regulatory regions associated with light-organ development in the bobtail squid.

Light organs (LO) with symbiotic bioluminescent bacteria are hallmarks of many bobtail squid species. These organs possess structural and functional features to modulate light, analogous to those found in coleoid eyes. Previous studies identified four transcription factors and modulators (SIX, EYA, PAX6, DAC) associated with both eyes and light organ development, suggesting co-option of a highly conserved gene regulatory network. Using available topological, open chromatin, and transcriptomic data, we explore the regulatory landscape around the four transcription factors as well as genes associated with LO and shared LO/eye expression. This analysis revealed several closely associated and putatively co-regulated genes. Comparative genomic analyses identified distinct evolutionary origins of these putative regulatory associations, with the DAC locus showing a unique topological and evolutionarily recent organization. We discuss different scenarios of modifications to genome topology and how these changes may have contributed to the evolutionary emergence of the light organ.

Comparative Single-Cell Transcriptomics Reveals Novel Genes Involved in Bivalve Embryonic Shell Formation and Questions Ontogenetic Homology of Molluscan Shell Types.

Mollusks are known for their highly diverse repertoire of body plans that often includes external armor in form of mineralized hardparts. Representatives of the Conchifera, one of the two major lineages that comprises taxa which originated from a uni-shelled ancestor (Monoplacophora, Gastropoda, Cephalopoda, Scaphopoda, Bivalvia), are particularly relevant regarding the evolution of mollusk shells. Previous studies have found that the shell matrix of the adult shell (teleoconch) is rapidly evolving and that the gene set involved in shell formation is highly taxon-specific. However, detailed annotation of genes expressed in tissues involved in the formation of the embryonic shell (protoconch I) or the larval shell (protoconch II) are currently lacking. Here, we analyzed the genetic toolbox involved in embryonic and larval shell formation in the quagga mussel Dreissena rostriformis using single cell RNA sequencing. We found significant differences in genes expressed during embryonic and larval shell secretion, calling into question ontogenetic homology of these transitory bivalve shell types. Further ortholog comparisons throughout Metazoa indicates that a common genetic biomineralization toolbox, that was secondarily co-opted into molluscan shell formation, was already present in the last common metazoan ancestor. Genes included are engrailed, carbonic anhydrase, and tyrosinase homologs. However, we found that 25% of the genes expressed in the embryonic shell field of D. rostriformis lack an ortholog match with any other metazoan. This indicates that not only adult but also embryonic mollusk shells may be fast-evolving structures. We raise the question as to what degree, and on which taxonomic level, the gene complement involved in conchiferan protoconch formation may be lineage-specific or conserved across taxa.

Emergence of novel cephalopod gene regulation and expression through large-scale genome reorganization.

Coleoid cephalopods (squid, cuttlefish, octopus) have the largest nervous system among invertebrates that together with many lineage-specific morphological traits enables complex behaviors. The genomic basis underlying these innovations remains unknown. Using comparative and functional genomics in the model squid Euprymna scolopes, we reveal the unique genomic, topological, and regulatory organization of cephalopod genomes. We show that coleoid cephalopod genomes have been extensively restructured compared to other animals, leading to the emergence of hundreds of tightly linked and evolutionary unique gene clusters (microsyntenies). Such novel microsyntenies correspond to topological compartments with a distinct regulatory structure and contribute to complex expression patterns. In particular, we identify a set of microsyntenies associated with cephalopod innovations (MACIs) broadly enriched in cephalopod nervous system expression. We posit that the emergence of MACIs was instrumental to cephalopod nervous system evolution and propose that microsyntenic profiling will be central to understanding cephalopod innovations.

Adaptive venom evolution and toxicity in octopods is driven by extensive novel gene formation, expansion, and loss.

BACKGROUND: Cephalopods represent a rich system for investigating the genetic basis underlying organismal novelties. This diverse group of specialized predators has evolved many adaptations including proteinaceous venom. Of particular interest is the blue-ringed octopus genus (Hapalochlaena), which are the only octopods known to store large quantities of the potent neurotoxin, tetrodotoxin, within their tissues and venom gland. FINDINGS: To reveal genomic correlates of organismal novelties, we conducted a comparative study of 3 octopod genomes, including the Southern blue-ringed octopus (Hapalochlaena maculosa). We present the genome of this species and reveal highly dynamic evolutionary patterns at both non-coding and coding organizational levels. Gene family expansions previously reported in Octopus bimaculoides (e.g., zinc finger and cadherins, both associated with neural functions), as well as formation of novel gene families, dominate the genomic landscape in all octopods. Examination of tissue-specific genes in the posterior salivary gland revealed that expression was dominated by serine proteases in non-tetrodotoxin-bearing octopods, while this family was a minor component in H. maculosa. Moreover, voltage-gated sodium channels in H. maculosa contain a resistance mutation found in pufferfish and garter snakes, which is exclusive to the genus. Analysis of the posterior salivary gland microbiome revealed a diverse array of bacterial species, including genera that can produce tetrodotoxin, suggestive of a possible production source. CONCLUSIONS: We present the first tetrodotoxin-bearing octopod genome H. maculosa, which displays lineage-specific adaptations to tetrodotoxin acquisition. This genome, along with other recently published cephalopod genomes, represents a valuable resource from which future work could advance our understanding of the evolution of genomic novelty in this family.

Coupled Genomic Evolutionary Histories as Signatures of Organismal Innovations in Cephalopods: Co-evolutionary Signatures Across Levels of Genome Organization May Shed Light on Functional Linkage and Origin of Cephalopod Novelties.

How genomic innovation translates into organismal organization remains largely unanswered. Possessing the largest invertebrate nervous system, in conjunction with many species-specific organs, coleoid cephalopods (octopuses, squids, cuttlefishes) provide exciting model systems to investigate how organismal novelties evolve. However, dissecting these processes requires novel approaches that enable deeper interrogation of genome evolution. Here, the existence of specific sets of genomic co-evolutionary signatures between expanded gene families, genome reorganization, and novel genes is posited. It is reasoned that their co-evolution has contributed to the complex organization of cephalopod nervous systems and the emergence of ecologically unique organs. In the course of reviewing this field, how the first cephalopod genomic studies have begun to shed light on the molecular underpinnings of morphological novelty is illustrated and their impact on directing future research is described. It is argued that the application and evolutionary profiling of evolutionary signatures from these studies will help identify and dissect the organismal principles of cephalopod innovations. By providing specific examples, the implications of this approach both within and beyond cephalopod biology are discussed.

First molecular approach to the octopus fauna from the southern Caribbean.

The octopus fauna from the southern Caribbean is an understudied field. However, recent taxonomic work in the Colombian Caribbean has led to the discovery of several new species in the family Octopodidae. To provide molecular evidence for recent descriptions in the area (i.e., Octopus taganga, O. tayrona and Macrotritopus beatrixi) and contribute to the systematics of the family, we reconstructed the first molecular phylogenies of the family including Colombian Caribbean octopus species. Using cytochrome c oxidase subunit I and rhodopsin sequences from specimens collected in three sites (Santa Marta, Old Providence and San Andrés Islands) we inferred maximum-likelihood trees and delimited species with PTP. Our mitochondrial analysis supported the monophyly of species found in the area (i.e., O. taganga, O. hummelincki and O. briareus). The genetic distinction of the species O. tayrona and O. insularis was not resolved, as these were found in one clade together with Caribbean O. vulgaris and O. aff. tayrona species (O. spB) and delimited as a single species. Additionally, our results suggest a distant relationship of the Type I O. vulgaris group (Caribbean region) from the other forms of the species complex (Old World and Brazil). Lastly, the third newly described species M. beatrixi emerged as an independent lineage and was delimited as a single species. However, its relationship to other species of its genus remains unknown due to the lack of sequences in databases. Altogether, our molecular approach to the octopus fauna from the southern Caribbean adds on information to the relationship of Octopodidae species world-wide by providing sequences from recently described species from an understudied region. Further studies employing higher taxon sampling and more molecular information are needed to fill taxonomic gaps in the area and account for single-locus resolution on the systematics of this group.

Genomic signatures of G-protein-coupled receptor expansions reveal functional transitions in the evolution of cephalopod signal transduction.

Coleoid cephalopods show unique morphological and neural novelties, such as arms with tactile and chemosensory suckers and a large complex nervous system. The evolution of such cephalopod novelties has been attributed at a genomic level to independent gene family expansions, yet the exact association and the evolutionary timing remain unclear. In the octopus genome, one such expansion occurred in the G-protein-coupled receptors (GPCRs) repertoire, a superfamily of proteins that mediate signal transduction. Here, we assessed the evolutionary history of this expansion and its relationship with cephalopod novelties. Using phylogenetic analyses, at least two cephalopod- and two octopus-specific GPCR expansions were identified. Signatures of positive selection were analysed within the four groups, and the locations of these sequences in the Octopus bimaculoides genome were inspected. Additionally, the expression profiles of cephalopod GPCRs across various tissues were extracted from available transcriptomic data. Our results reveal the evolutionary history of cephalopod GPCRs. Unexpanded cephalopod GPCRs shared with other bilaterians were found to be mainly nervous tissue specific. By contrast, duplications that are shared between octopus and the bobtail squid or specific to the octopus' lineage generated copies with divergent expression patterns devoted to tissues outside of the brain. The acquisition of novel expression domains was accompanied by gene order rearrangement through either translocation or duplication and gene loss. Lastly, expansions showed signs of positive selection and some were found to form tandem clusters with shared conserved expression profiles in cephalopod innovations such as the axial nerve cord. Altogether, our results contribute to the understanding of the molecular and evolutionary history of signal transduction and provide insights into the role of this expansion during the emergence of cephalopod novelties and/or adaptations.

The survey and reference assisted assembly of the Octopus vulgaris genome.

The common octopus, Octopus vulgaris, is an active marine predator known for the richness and plasticity of its behavioral repertoire, and remarkable learning and memory capabilities. Octopus and other coleoid cephalopods, cuttlefish and squid, possess the largest nervous system among invertebrates, both for cell counts and body to brain size. O. vulgaris has been at the center of a long-tradition of research into diverse aspects of its biology. To leverage research in this iconic species, we generated 270 Gb of genomic sequencing data, complementing those available for the only other sequenced congeneric octopus, Octopus bimaculoides. We show that both genomes are similar in size, but display different levels of heterozygosity and repeats. Our data give a first quantitative glimpse into the rate of coding and non-coding regions and support the view that hundreds of novel genes may have arisen independently despite the close phylogenetic distance. We furthermore describe a reference-guided assembly and an open genomic resource (CephRes-gdatabase), opening new avenues in the study of genomic novelties in cephalopods and their biology.