Giants among Cnidaria: Large Nuclear Genomes and Rearranged Mitochondrial Genomes in Siphonophores.

Siphonophores (Cnidaria: Hydrozoa) are abundant predators found throughout the ocean and are important constituents of the global zooplankton community. They range in length from a few centimeters to tens of meters. They are gelatinous, fragile, and difficult to collect, so many aspects of the biology of these roughly 200 species remain poorly understood. To survey siphonophore genome diversity, we performed Illumina sequencing of 32 species sampled broadly across the phylogeny. Sequencing depth was sufficient to estimate nuclear genome size from k-mer spectra in six specimens, ranging from 0.7 to 2.3 Gb, with heterozygosity estimates between 0.69% and 2.32%. Incremental k-mer counting indicates k-mer peaks can be absent with nearly 20× read coverage, suggesting minimum genome sizes range from 1.4 to 5.6 Gb in the 25 samples without peaks in the k-mer spectra. This work confirms most siphonophore nuclear genomes are large relative to the genomes of other cnidarians, but also identifies several with reduced size that are tractable targets for future siphonophore nuclear genome assembly projects. We also assembled complete mitochondrial genomes for 33 specimens from these new data, indicating a conserved gene order shared among nonsiphonophore hydrozoans, Cystonectae, and some Physonectae, revealing the ancestral mitochondrial gene order of siphonophores. Our results also suggest extensive rearrangement of mitochondrial genomes within other Physonectae and in Calycophorae. Though siphonophores comprise a small fraction of cnidarian species, this survey greatly expands our understanding of cnidarian genome diversity. This study further illustrates both the importance of deep phylogenetic sampling and the utility of k-mer-based genome skimming in understanding the genomic diversity of a clade.

A chromosome-level reference genome for the common octopus, Octopus vulgaris (Cuvier, 1797).

Cephalopods are emerging animal models and include iconic species for studying the link between genomic innovations and physiological and behavioral complexities. Coleoid cephalopods possess the largest nervous system among invertebrates, both for cell counts and brain-to-body ratio. Octopus vulgaris has been at the center of a long-standing tradition of research into diverse aspects of cephalopod biology, including behavioral and neural plasticity, learning and memory recall, regeneration, and sophisticated cognition. However, no chromosome-scale genome assembly was available for O. vulgaris to aid in functional studies. To fill this gap, we sequenced and assembled a chromosome-scale genome of the common octopus, O. vulgaris. The final assembly spans 2.8 billion basepairs, 99.34% of which are in 30 chromosome-scale scaffolds. Hi-C heatmaps support a karyotype of 1n = 30 chromosomes. Comparisons with other octopus species' genomes show a conserved octopus karyotype and a pattern of local genome rearrangements between species. This new chromosome-scale genome of O. vulgaris will further facilitate research in all aspects of cephalopod biology, including various forms of plasticity and the neural machinery underlying sophisticated cognition, as well as an understanding of cephalopod evolution.

Cephalopod-omics: Emerging Fields and Technologies in Cephalopod Biology.

Few animal groups can claim the level of wonder that cephalopods instill in the minds of researchers and the general public. Much of cephalopod biology, however, remains unexplored: the largest invertebrate brain, difficult husbandry conditions, and complex (meta-)genomes, among many other things, have hindered progress in addressing key questions. However, recent technological advancements in sequencing, imaging, and genetic manipulation have opened new avenues for exploring the biology of these extraordinary animals. The cephalopod molecular biology community is thus experiencing a large influx of researchers, emerging from different fields, accelerating the pace of research in this clade. In the first post-pandemic event at the Cephalopod International Advisory Council (CIAC) conference in April 2022, over 40 participants from all over the world met and discussed key challenges and perspectives for current cephalopod molecular biology and evolution. Our particular focus was on the fields of comparative and regulatory genomics, gene manipulation, single-cell transcriptomics, metagenomics, and microbial interactions. This article is a result of this joint effort, summarizing the latest insights from these emerging fields, their bottlenecks, and potential solutions. The article highlights the interdisciplinary nature of the cephalopod-omics community and provides an emphasis on continuous consolidation of efforts and collaboration in this rapidly evolving field.

Ancient gene linkages support ctenophores as sister to other animals.

A central question in evolutionary biology is whether sponges or ctenophores (comb jellies) are the sister group to all other animals. These alternative phylogenetic hypotheses imply different scenarios for the evolution of complex neural systems and other animal-specific traits1-6. Conventional phylogenetic approaches based on morphological characters and increasingly extensive gene sequence collections have not been able to definitively answer this question7-11. Here we develop chromosome-scale gene linkage, also known as synteny, as a phylogenetic character for resolving this question12. We report new chromosome-scale genomes for a ctenophore and two marine sponges, and for three unicellular relatives of animals (a choanoflagellate, a filasterean amoeba and an ichthyosporean) that serve as outgroups for phylogenetic analysis. We find ancient syntenies that are conserved between animals and their close unicellular relatives. Ctenophores and unicellular eukaryotes share ancestral metazoan patterns, whereas sponges, bilaterians, and cnidarians share derived chromosomal rearrangements. Conserved syntenic characters unite sponges with bilaterians, cnidarians, and placozoans in a monophyletic clade to the exclusion of ctenophores, placing ctenophores as the sister group to all other animals. The patterns of synteny shared by sponges, bilaterians, and cnidarians are the result of rare and irreversible chromosome fusion-and-mixing events that provide robust and unambiguous phylogenetic support for the ctenophore-sister hypothesis. These findings provide a new framework for resolving deep, recalcitrant phylogenetic problems and have implications for our understanding of animal evolution.

Chromosome-level genome of the three-spot damselfish, Dascyllus trimaculatus.

Damselfishes (Family: Pomacentridae) are a group of ecologically important, primarily coral reef fishes that include over 400 species. Damselfishes have been used as model organisms to study recruitment (anemonefishes), the effects of ocean acidification (spiny damselfish), population structure, and speciation (Dascyllus). The genus Dascyllus includes a group of small-bodied species, and a complex of relatively larger bodied species, the Dascyllus trimaculatus species complex that is comprised of several species including D. trimaculatus itself. The three-spot damselfish, D. trimaculatus, is a widespread and common coral reef fish species found across the tropical Indo-Pacific. Here, we present the first-genome assembly of this species. This assembly contains 910 Mb, 90% of the bases are in 24 chromosome-scale scaffolds, and the Benchmarking Universal Single-Copy Orthologs score of the assembly is 97.9%. Our findings confirm previous reports of a karyotype of 2n = 47 in D. trimaculatus in which one parent contributes 24 chromosomes and the other 23. We find evidence that this karyotype is the result of a heterozygous Robertsonian fusion. We also find that the D. trimaculatus chromosomes are each homologous with single chromosomes of the closely related clownfish species, Amphiprion percula. This assembly will be a valuable resource in the population genomics and conservation of Damselfishes, and continued studies of the karyotypic diversity in this clade.

The complete mitochondrial genome of Dascyllus trimaculatus (Rüppell, 1829).

Damselfishes (family Pomacentridae) comprise approximately 400 species that play an important ecological role in temperate and coral reefs. Here, for the first time, we assemble and annotate the mitochondrial genome of Dascyllus trimaculatus, the three-spot dascyllus, a planktivorous damselfish that primarily recruits in anemones. The circular genome of D. trimaculatus is 16,967 bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. Gene arrangement and codon usage is similar to reported mitochondrial genomes of other damselfish genera, and a phylogenetic analysis of a set of damselfish representatives is consistent with known evolutionary analyses.

Speciation of pelagic zooplankton: Invisible boundaries can drive isolation of oceanic ctenophores.

The study of evolution and speciation in non-model systems provides us with an opportunity to expand our understanding of biodiversity in nature. Connectivity studies generally focus on species with obvious boundaries to gene flow, but in open-ocean environments, such boundaries are difficult to identify. Due to the lack of obvious boundaries, speciation and population subdivision in the pelagic environment remain largely unexplained. Comb jellies (Phylum Ctenophora) are mostly planktonic gelatinous invertebrates, many of which are considered to have freely interbreeding distributions worldwide. It is thought that the lobate ctenophore Bolinopsis infundibulum is distributed throughout cooler northern latitudes and B. vitrea warmer. Here, we examined the global population structure for species of Bolinopsis with genetic and morphological data. We found distinct evolutionary patterns within the genus, where B. infundibulum had a broad distribution from northern Pacific to Atlantic waters despite many physical barriers, while other species were geographically segregated despite few barriers. Divergent patterns of speciation within the genus suggest that oceanic currents, sea-level, and geological changes over time can act as either barriers or aids to dispersal in the pelagic environment. Further, we used population genomic data to examine evolution in the open ocean of a distinct lineage of Bolinopsis ctenophores from the North Eastern Pacific. Genetic information and morphological observations validated this as a separate species, Bolinopsis microptera, which was previously described but has recently been called B. infundibulum. We found that populations of B. microptera from California were in cytonuclear discordance, which indicates a secondary contact zone for previously isolated populations. Discordance at this scale is rare, especially in a continuous setting.

Deeply conserved synteny and the evolution of metazoan chromosomes.

Animal genomes show networks of deeply conserved gene linkages whose phylogenetic scope and chromosomal context remain unclear. Here, we report chromosome-scale conservation of synteny among bilaterians, cnidarians, and sponges and use comparative analysis to reconstruct ancestral chromosomes across major animal groups. Comparisons among diverse metazoans reveal the processes of chromosome evolution that produced contemporary karyotypes from their Precambrian progenitors. On the basis of these findings, we introduce a simple algebraic representation of chromosomal change and use it to establish a unified systematic framework for metazoan chromosome evolution. We find that fusion-with-mixing, a previously unappreciated mode of chromosome change, has played a central role. We find that relicts of several metazoan chromosomal units are preserved in unicellular eukaryotes. These conserved pre-metazoan linkages include the chromosomal unit that encodes the most diverse set of metazoan homeobox genes, suggesting a candidate genomic context for the early diversification of this key gene family.

Firefly genomes illuminate the evolution of beetle bioluminescent systems.

Fireflies are one of the best-known bioluminescent organisms, and the reaction mechanism and ecological utility of bioluminescence have been well-studied. Genome assemblies of six species of bioluminescent beetles have recently been published. These studies have focused on the evolution of novelties; luciferase, and the biosynthesis of luciferin and defensive chemicals. For example, clustering of the luciferase gene with acyl-CoA synthetase genes on a chromosome in luminous beetle genomes suggests the involvement of tandem gene duplications and neofunctionalization during the evolution of beetle bioluminescence. Several candidate genes for critical roles in beetle bioluminescence have been identified, but their functional analyses are still ongoing. The establishment of a long-term mass-rearing system and strain will be the key for the post-genome research on bioluminescent beetles. Lastly, the application of contemporary chromosome-scale genome assembly techniques to luminous beetles will help resolve outstanding evolutionary questions, such as how many times bioluminescence evolved in this clade.

Hidden diversity of Ctenophora revealed by new mitochondrial COI primers and sequences.

The mitochondrial gene cytochrome-c-oxidase subunit 1 (COI) is useful in many taxa for phylogenetics, population genetics, metabarcoding, and rapid species identifications. However, the phylum Ctenophora (comb jellies) has historically been difficult to study due to divergent mitochondrial sequences and the corresponding inability to amplify COI with degenerate and standard COI "barcoding" primers. As a result, there are very few COI sequences available for ctenophores, despite over 200 described species in the phylum. Here, we designed new primers and amplified the COI fragment from members of all major groups of ctenophores, including many undescribed species. Phylogenetic analyses of the resulting COI sequences revealed high diversity within many groups that was not evident from more conserved 18S rDNA sequences, in particular among the Lobata (Ctenophora; Tentaculata; Lobata). The COI phylogenetic results also revealed unexpected community structure within the genus Bolinopsis, suggested new species within the genus Bathocyroe, and supported the ecological and morphological differences of some species such as Lampocteis cruentiventer and similar undescribed lobates (Lampocteis sp. "V" stratified by depth, and "A" differentiated by colour). The newly designed primers reported herein provide important tools to enable researchers to illuminate the diversity of ctenophores worldwide via quick molecular identifications, improve the ability to analyse environmental DNA by improving reference libraries and amplifications, and enable a new breadth of population genetic studies.

A chromosome-scale genome assembly and karyotype of the ctenophore Hormiphora californensis.

Here, we present a karyotype, a chromosome-scale genome assembly, and a genome annotation from the ctenophore Hormiphora californensis (Ctenophora: Cydippida: Pleurobrachiidae). The assembly spans 110 Mb in 44 scaffolds and 99.47% of the bases are contained in 13 scaffolds. Chromosome micrographs and Hi-C heatmaps support a karyotype of 13 diploid chromosomes. Hi-C data reveal three large heterozygous inversions on chromosome 1, and one heterozygous inversion shares the same gene order found in the genome of the ctenophore Pleurobrachia bachei. We find evidence that H. californensis and P. bachei share thirteen homologous chromosomes, and the same karyotype of 1n = 13. The manually curated PacBio Iso-Seq-based genome annotation reveals complex gene structures, including nested genes and trans-spliced leader sequences. This chromosome-scale assembly is a useful resource for ctenophore biology and will aid future studies of metazoan evolution and phylogenetics.

Aequorea's secrets revealed: New fluorescent proteins with unique properties for bioimaging and biosensing.

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

Laboratory culture of the California Sea Firefly Vargula tsujii (Ostracoda: Cypridinidae): Developing a model system for the evolution of marine bioluminescence.

Bioluminescence, or the production of light by living organisms via chemical reaction, is widespread across Metazoa. Laboratory culture of bioluminescent organisms from diverse taxonomic groups is important for determining the biosynthetic pathways of bioluminescent substrates, which may lead to new tools for biotechnology and biomedicine. Some bioluminescent groups may be cultured, including some cnidarians, ctenophores, and brittle stars, but those use luminescent substrates (luciferins) obtained from their diets, and therefore are not informative for determination of the biosynthetic pathways of the luciferins. Other groups, including terrestrial fireflies, do synthesize their own luciferin, but culturing them is difficult and the biosynthetic pathway for firefly luciferin remains unclear. An additional independent origin of endogenous bioluminescence is found within ostracods from the family Cypridinidae, which use their luminescence for defense and, in Caribbean species, for courtship displays. Here, we report the first complete life cycle of a luminous ostracod (Vargula tsujii Kornicker & Baker, 1977, the California Sea Firefly) in the laboratory. We also describe the late-stage embryogenesis of Vargula tsujii and discuss the size classes of instar development. We find embryogenesis in V. tsujii ranges from 25-38 days, and this species appears to have five instar stages, consistent with ontogeny in other cypridinid lineages. We estimate a complete life cycle at 3-4 months. We also present the first complete mitochondrial genome for Vargula tsujii. Bringing a luminous ostracod into laboratory culture sets the stage for many potential avenues of study, including learning the biosynthetic pathway of cypridinid luciferin and genomic manipulation of an autogenic bioluminescent system.

Conserved novel ORFs in the mitochondrial genome of the ctenophore Beroe forskalii.

To date, five ctenophore species' mitochondrial genomes have been sequenced, and each contains open reading frames (ORFs) that if translated have no identifiable orthologs. ORFs with no identifiable orthologs are called unidentified reading frames (URFs). If truly protein-coding, ctenophore mitochondrial URFs represent a little understood path in early-diverging metazoan mitochondrial evolution and metabolism. We sequenced and annotated the mitochondrial genomes of three individuals of the beroid ctenophore Beroe forskalii and found that in addition to sharing the same canonical mitochondrial genes as other ctenophores, the B. forskalii mitochondrial genome contains two URFs. These URFs are conserved among the three individuals but not found in other sequenced species. We developed computational tools called pauvre and cuttlery to determine the likelihood that URFs are protein coding. There is evidence that the two URFs are under negative selection, and a novel Bayesian hypothesis test of trinucleotide frequency shows that the URFs are more similar to known coding genes than noncoding intergenic sequence. Protein structure and function prediction of all ctenophore URFs suggests that they all code for transmembrane transport proteins. These findings, along with the presence of URFs in other sequenced ctenophore mitochondrial genomes, suggest that ctenophores may have uncharacterized transmembrane proteins present in their mitochondria.

Luciferase of the Japanese syllid polychaete Odontosyllis undecimdonta.

Odontosyllis undecimdonta is a marine syllid polychaete that produces bright internal and exuded bioluminescence. Despite over fifty years of biochemical investigation into Odontosyllis bioluminescence, the light-emitting small molecule substrate and catalyzing luciferase protein have remained a mystery. Here we describe the discovery of a bioluminescent protein fraction from O. undecimdonta, the identification of the luciferase using peptide and RNA sequencing, and the in vitro reconstruction of the bioluminescence reaction using highly purified O. undecimdonta luciferin and recombinant luciferase. Lastly, we found no identifiably homologous proteins in publicly available datasets. This suggests that the syllid polychaetes contain an evolutionarily unique luciferase among all characterized luminous taxa.

NanoPack: visualizing and processing long-read sequencing data.

Summary: Here we describe NanoPack, a set of tools developed for visualization and processing of long-read sequencing data from Oxford Nanopore Technologies and Pacific Biosciences. Availability and implementation: The NanoPack tools are written in Python3 and released under the GNU GPL3.0 License. The source code can be found at https://github.com/wdecoster/nanopack, together with links to separate scripts and their documentation. The scripts are compatible with Linux, Mac OS and the MS Windows 10 subsystem for Linux and are available as a graphical user interface, a web service at http://nanoplot.bioinf.be and command line tools. Supplementary information: Supplementary data are available at Bioinformatics online.

Eco-evo bioluminescence on land and in the sea.

This review discusses the evolution of bioluminescence organisms that inhabit various environments based on the current understanding of their unique ecologies and biochemistries. As shown here, however, there are still many unanswered questions regarding the functions and mechanisms of bioluminescence, which should be investigated in further studies. To facilitate future research in this field, we introduce our recent attempt, the bioluminescent organism DNA barcode initiative. This genetic reference library will provide resources for other scientists to efficiently identify unstudied bioluminescent organisms, focus their biochemical and genetic research goals, and will generally promote bioluminescence as a field of scientific study.