Ocean to Tree: Leveraging Single-Molecule RNA-Seq to Repair Genome Gene Models and Improve Phylogenomic Analysis of Gene and Species Evolution.

Understanding gene evolution across genomes and organisms, including ctenophores, can provide unexpected biological insights. It enables powerful integrative approaches that leverage sequence diversity to advance biomedicine. Sequencing and bioinformatic tools can be inexpensive and user-friendly, but numerous options and coding can intimidate new users. Distinct challenges exist in working with data from diverse species but may go unrecognized by researchers accustomed to gold-standard genomes. Here, we provide a high-level workflow and detailed pipeline to enable animal collection, single-molecule sequencing, and phylogenomic analysis of gene and species evolution. As a demonstration, we focus on (1) PacBio RNA-seq of the genome-sequenced ctenophore Mnemiopsis leidyi, (2) diversity and evolution of the mechanosensitive ion channel Piezo in genetic models and basal-branching animals, and (3) associated challenges and solutions to working with diverse species and genomes, including gene model updating and repair using single-molecule RNA-seq. We provide a Python Jupyter Notebook version of our pipeline (GitHub Repository: Ctenophore-Ocean-To-Tree-2023 https://github.com/000generic/Ctenophore-Ocean-To-Tree-2023 ) that can be run for free in the Google Colab cloud to replicate our findings or modified for specific or greater use. Our protocol enables users to design new sequencing projects in ctenophores, marine invertebrates, or other novel organisms. It provides a simple, comprehensive platform that can ease new user entry into running their evolutionary sequence analyses.

Ctenophora: Illustrated Guide and Taxonomy.

Ctenophores or comb jellies represent the first diverging lineage of extant animals - sister to all other Metazoa. As a result, they occupy a unique place in the biological sciences. Despite their importance, this diverse group of marine predators has remained relatively poorly known, with both the species and higher-level taxonomy of the phylum in need of attention. We present a checklist of the phylum based on a review of the current taxonomic literature and illustrate their diversity with images. The current classification presented remains substantially in conflict with recent phylogenetic results, and many of the taxa are not monophyletic or untested. This chapter summarizes the existing classification focusing on recognized families and genera with 185 currently accepted, extant species listed. We provide illustrative examples of ctenophore diversity covering all but one of the 33 families and 47 of the 48 genera, as well as about 25-30 undescribed species. We also list the 14 recognized ctenophore fossil species and note others that have been controversially attributed to the phylum. Analyses of unique ctenophore adaptations are critical to understanding early animal evolution and adaptive radiation of this clade of basal metazoans.

Evolutionary origin of the nervous system from Ctenophora prospective.

Nervous system is one of the key adaptations underlying the evolutionary success of the majority of animal groups. Ctenophores (or comb jellies) are gelatinous marine invertebrates that were probably the first lineage to diverge from the rest of animals. Due to the key phylogenetic position and multiple unique adaptations, the noncentralized nervous system of comb jellies has been in the center of the debate around the origin of the nervous system in the animal kingdom and whether it happened only once or twice. Here, we discuss the latest findings in ctenophore neuroscience and multiple challenges on the way to build a clear evolutionary picture of the origin of the nervous system.

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.

Evidence for sponges as sister to all other animals from partitioned phylogenomics with mixture models and recoding.

Resolving the relationships between the major lineages in the animal tree of life is necessary to understand the origin and evolution of key animal traits. Sponges, characterized by their simple body plan, were traditionally considered the sister group of all other animal lineages, implying a gradual increase in animal complexity from unicellularity to complex multicellularity. However, the availability of genomic data has sparked tremendous controversy as some phylogenomic studies support comb jellies taking this position, requiring secondary loss or independent origins of complex traits. Here we show that incorporating site-heterogeneous mixture models and recoding into partitioned phylogenomics alleviates systematic errors that hamper commonly-applied phylogenetic models. Testing on real datasets, we show a great improvement in model-fit that attenuates branching artefacts induced by systematic error. We reanalyse key datasets and show that partitioned phylogenomics does not support comb jellies as sister to other animals at either the supermatrix or partition-specific level.

Animal Phylogeny: Resolving the Slugfest of Ctenophores, Sponges and Acoels?

Animal phylogeny has always been controversial, but a new study brings some much-needed order for two infamous wandering groups, the ctenophores and the Xenacoelomorphs. The study introduces an innovative approach to dissect systematic errors in the underlying methodology of molecular phylogenies.

Molecular Insights Into the Ctenophore Genus Beroe in Europe: New Species, Spreading Invaders.

The genus Beroe Browne, 1756 (Ctenophora, Beroidae) occurs worldwide, with 25 currently-described species. Because the genus is poorly studied, the definitive number of species is uncertain. Recently, a possible new Beroe species was suggested based on internal transcribed spacer 1 (ITS1) sequences from samples collected in Svalbard, Norway. Another species, Beroe ovata, was introduced to Europe from North America, initially in the Black Sea and subsequently (and possibly secondarily) into the Mediterranean and Baltic Seas. In areas where ctenophores have been introduced, they have often had significant detrimental ecological effects. The potential for other cryptic and/or undescribed Beroe species and history of spread of some species in the genus give reason for additional study. When alive, morphological hallmarks may be challenging to spot and photograph owing to the animals' transparency and near-constant motion. We sampled and analyzed 109 putative Beroe specimens from Europe, using morphological and molecular approaches. DNA analyses were conducted using cytochrome oxidase 1 and internal transcribed spacer sequences and, together with published sequences from GenBank, phylogenetic relationships of the genus were explored. Our study suggests the presence of at least 5 genetic lineages of Beroe in Europe, of which 3 could be assigned to known species: Beroe gracilis Künne 1939; Beroe cucumis Fabricius, 1780; and Beroe ovata sensu Mayer, 1912. The other 2 lineages (here provisionally named Beroe "norvegica" and Beroe "anatoliensis") did not clearly coincide with any known species and might therefore reflect new species, but confirmation of this requires further study.

The Diversification of Early Emerging Metazoans: A Window into the Evolution of Animal Multicellularity.

The biannual international workshop entitled "The diversification of early emerging metazoans: A window into animal evolution?" took place at the Evangelische Akademie Tutzing, Germany, 11-14. September 2017. It was organized by Thomas Bosch (Kiel), Thomas Holstein (Heidelberg), and Ulrich Technau (Vienna), and it was sponsored by the Deutsche Forschungsgemeinschaft (DFG). The meeting gathered over 140 researchers to discuss the contribution of non-bilaterian metazoan models (Porifera, Ctenophora, Placozoa, and Cnidaria) to our understanding of: a. The evolution of metazoan developmental processes; b. Fundamental molecular mechanisms underlying metazoan features; and c. The complex interactions that animals establish with their environment.

Ctenophore relationships and their placement as the sister group to all other animals.

Ctenophora, comprising approximately 200 described species, is an important lineage for understanding metazoan evolution and is of great ecological and economic importance. Ctenophore diversity includes species with unique colloblasts used for prey capture, smooth and striated muscles, benthic and pelagic lifestyles, and locomotion with ciliated paddles or muscular propulsion. However, the ancestral states of traits are debated and relationships among many lineages are unresolved. Here, using 27 newly sequenced ctenophore transcriptomes, publicly available data and methods to control systematic error, we establish the placement of Ctenophora as the sister group to all other animals and refine the phylogenetic relationships within ctenophores. Molecular clock analyses suggest modern ctenophore diversity originated approximately 350 million years ago ± 88 million years, conflicting with previous hypotheses, which suggest it originated approximately 65 million years ago. We recover Euplokamis dunlapae-a species with striated muscles-as the sister lineage to other sampled ctenophores. Ancestral state reconstruction shows that the most recent common ancestor of extant ctenophores was pelagic, possessed tentacles, was bioluminescent and did not have separate sexes. Our results imply at least two transitions from a pelagic to benthic lifestyle within Ctenophora, suggesting that such transitions were more common in animal diversification than previously thought.

Molecular diversity of benthic ctenophores (Coeloplanidae).

Coeloplanidae, the largest family of benthic ctenophores, comprises 33 species, all described based on traditional morphological characteristics, such as coloration, length, and number of aboral papillae, which are highly variable and can be affected by fixation methods and environmental conditions. Thus, there is a need for reliable genetic markers to complement the morphological identifications at the species level. Here, we analyzed 95 specimens from 11 morphologically distinct species of benthic ctenophores from the Red Sea and Sulu Sea, and tested selected regions of four genetic markers (ITS1, 18S rRNA, 28S rRNA and COI) for their ability to differentiate between species. We show that the barcoding region of the mitochondrial gene, cytochrome oxidase subunit I (COI), is highly variable among species of Coeloplanidae, and effectively discriminates between species in this family. The average Kimura-2-parameter (K2P) distance between species-level clades was 10%, while intraspecific variation was ~30 times lower (0.36%). COI-based phylogeny supported the delineation of four recently described new species from the Red Sea. The other nuclear markers tested were found to be too conserved in order to separate between species. We conclude that COI is a potential molecular barcode for the family Coeloplanidae and suggest to test it in pelagic ctenophores.

Census of Cnidaria (Medusozoa) and Ctenophora from South American marine waters.

We have compiled available records in the literature for medusozoan cnidarians and ctenophores of South America. New records of species are also included. Each entry (i.e., identified species or still as yet not determined species referred to as "sp." in the literature) includes a synonymy list for South America, taxonomical remarks, notes on habit, and information on geographical occurrence. We have listed 800 unique determined species, in 958 morphotype entries: 5 cubozoans, 905 hydrozoans, 25 scyphozoans, 3 staurozoans, and 20 ctenophores. Concerning nomenclatural and taxonomical decisions, two authors of this census (Miranda, T.P. & Marques, A.C.) propose Podocoryna quitus as a nomen novum for the junior homonym Hydractinia reticulata (Fraser, 1938a); Euphysa monotentaculata Zamponi, 1983b as a new junior synonym of Euphysa aurata Forbes, 1848; and Plumularia spiralis Milstein, 1976 as a new junior synonym of Plumularia setacea (Linnaeus, 1758). Finally, we also reassign Plumularia oligopyxis Kirchenpauer, 1876 as Kirchenpaueria oligopyxis (Kirchenpauer, 1876) and Sertularella margaritacea Allman, 1885 as Symplectoscyphus margaritaceus (Allman, 1885).

Non-excitable fluorescent protein orthologs found in ctenophores.

BACKGROUND: Fluorescent proteins are optically active proteins found across many clades in metazoans. A fluorescent protein was recently identified in a ctenophore, but this has been suggested to derive from a cnidarian, raising again the question of origins of this group of proteins. RESULTS: Through analysis of transcriptome data from 30 ctenophores, we identified a member of an orthologous group of proteins similar to fluorescent proteins in each of them, as well as in the genome of Mnemiopsis leidyi. These orthologs lack canonical residues involved in chromophore formation, suggesting another function. CONCLUSIONS: The phylogenetic position of the ctenophore protein family among fluorescent proteins suggests that this gene was present in the common ancestor of all ctenophores and that the fluorescent protein previously found in a ctenophore actually derives from a siphonophore.

Genomic data do not support comb jellies as the sister group to all other animals.

Understanding how complex traits, such as epithelia, nervous systems, muscles, or guts, originated depends on a well-supported hypothesis about the phylogenetic relationships among major animal lineages. Traditionally, sponges (Porifera) have been interpreted as the sister group to the remaining animals, a hypothesis consistent with the conventional view that the last common animal ancestor was relatively simple and more complex body plans arose later in evolution. However, this premise has recently been challenged by analyses of the genomes of comb jellies (Ctenophora), which, instead, found ctenophores as the sister group to the remaining animals (the "Ctenophora-sister" hypothesis). Because ctenophores are morphologically complex predators with true epithelia, nervous systems, muscles, and guts, this scenario implies these traits were either present in the last common ancestor of all animals and were lost secondarily in sponges and placozoans (Trichoplax) or, alternatively, evolved convergently in comb jellies. Here, we analyze representative datasets from recent studies supporting Ctenophora-sister, including genome-scale alignments of concatenated protein sequences, as well as a genomic gene content dataset. We found no support for Ctenophora-sister and conclude it is an artifact resulting from inadequate methodology, especially the use of simplistic evolutionary models and inappropriate choice of species to root the metazoan tree. Our results reinforce a traditional scenario for the evolution of complexity in animals, and indicate that inferences about the evolution of Metazoa based on the Ctenophora-sister hypothesis are not supported by the currently available data.

Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes.

Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.

Occurrence of Isopenicillin-N-Synthase Homologs in Bioluminescent Ctenophores and Implications for Coelenterazine Biosynthesis.

The biosynthesis of the luciferin coelenterazine has remained a mystery for decades. While not all organisms that use coelenterazine appear to make it themselves, it is thought that ctenophores are a likely producer. Here we analyze the transcriptome data of 24 species of ctenophores, two of which have published genomes. The natural precursors of coelenterazine have been shown to be the amino acids L-tyrosine and L-phenylalanine, with the most likely biosynthetic pathway involving cyclization and further modification of the tripeptide Phe-Tyr-Tyr ("FYY"). Therefore, we searched the ctenophore transcriptome data for genes with the short peptide "FYY" as part of their coding sequence. We recovered a group of candidate genes for coelenterazine biosynthesis in the luminous species which encode a set of highly conserved non-heme iron oxidases similar to isopenicillin-N-synthase. These genes were absent in the transcriptomes and genome of the two non-luminous species. Pairwise identities and substitution rates reveal an unusually high degree of identity even between the most unrelated species. Additionally, two related groups of non-heme iron oxidases were found across all ctenophores, including those which are non-luminous, arguing against the involvement of these two gene groups in luminescence. Important residues for iron-binding are conserved across all proteins in the three groups, suggesting this function is still present. Given the known functions of other members of this protein superfamily are involved in heterocycle formation, we consider these genes to be top candidates for laboratory characterization or gene knockouts in the investigation of coelenterazine biosynthesis.

Ctenophores from the Oaxaca coast, including a checklist of species from the Pacific coast of Mexico.

Ctenophores are poorly known in the tropical eastern Pacific, including the southern coast of Mexico. Previous records of ctenophores along the Pacific coast have been provided mainly from northern waters. For the coast of Oaxaca state, their occurrence has only been mentioned before at phylum level. In this paper, we provide the first three records of ctenophores for the Oaxacan coast, which represent new records of Beroe forskalii and Bolinopsis vitrea as well as the first record of Ocyropsis maculata in the tropical eastern Pacific. Descriptions of these three species, as well as a checklist of the ctenophores from the west coast of Mexico are provided.

Employing Phylogenomics to Resolve the Relationships among Cnidarians, Ctenophores, Sponges, Placozoans, and Bilaterians.

Despite an explosion in the amount of sequence data, phylogenomics has failed to settle controversy regarding some critical nodes on the animal tree of life. Understanding relationships among Bilateria, Ctenophora, Cnidaria, Placozoa, and Porifera is essential for studying how complex traits such as neurons, muscles, and gastrulation have evolved. Recent studies have cast doubt on the historical viewpoint that sponges are sister to all other animal lineages with recent studies recovering ctenophores as sister. However, the ctenophore-sister hypothesis has been criticized as unrealistic and caused by systematic error. We review past phylogenomic studies and potential causes of systematic error in an effort to identify areas that can be improved in future studies. Increased sampling of taxa, less missing data, and a priori removal of sequences and taxa that may cause systematic error in phylogenomic inference will likely be the most fruitful areas of focus when assembling future datasets. Ultimately, we foresee metazoan relationships being resolved with higher support in the near future, and we caution against dismissing novel hypotheses merely because they conflict with historical viewpoints of animal evolution.