Stepwise emergence of the neuronal gene expression program in early animal evolution.

The assembly of the neuronal and other major cell type programs occurred early in animal evolution. We can reconstruct this process by studying non-bilaterians like placozoans. These small disc-shaped animals not only have nine morphologically described cell types and no neurons but also show coordinated behaviors triggered by peptide-secreting cells. We investigated possible neuronal affinities of these peptidergic cells using phylogenetics, chromatin profiling, and comparative single-cell genomics in four placozoans. We found conserved cell type expression programs across placozoans, including populations of transdifferentiating and cycling cells, suggestive of active cell type homeostasis. We also uncovered fourteen peptidergic cell types expressing neuronal-associated components like the pre-synaptic scaffold that derive from progenitor cells with neurogenesis signatures. In contrast, earlier-branching animals like sponges and ctenophores lacked this conserved expression. Our findings indicate that key neuronal developmental and effector gene modules evolved before the advent of cnidarian/bilaterian neurons in the context of paracrine cell signaling.

Left-Right Asymmetry in Invertebrates: From Molecules to Organisms.

Although most animals appear symmetric externally, they exhibit chirality within their body cavity, i.e., in terms of asymmetric organ position, directional organ looping, and lateralized organ function. Left-right (LR) asymmetry is determined genetically by intricate molecular interactions that occur during development. Key genes have been elucidated in several species. There are common mechanisms in vertebrates and invertebrates, but some appear to exhibit unique mechanisms. This review focuses on LR asymmetry formation in invertebrates, particularly Drosophila, ascidians, and mollusks. It aims to understand the role of the genes that are key to creating LR asymmetry and how chirality information is converted/transmitted across the hierarchies from molecules to cells and from cells to tissues.

The Genetics of Aging: A Vertebrate Perspective.

Aging negatively impacts vitality and health. Many genetic pathways that regulate aging were discovered in invertebrates. However, the genetics of aging is more complex in vertebrates because of their specialized systems. This Review discusses advances in the genetic regulation of aging in vertebrates from work in mice, humans, and organisms with exceptional lifespans. We highlight challenges for the future, including sex-dependent differences in lifespan and the interplay between genes and environment. We also discuss how the identification of reliable biomarkers of age and development of new vertebrate models can be leveraged for personalized interventions to counter aging and age-related diseases.

Structural Basis of Egg Coat-Sperm Recognition at Fertilization.

Recognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1-3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion.

Molecular evolution of the Thrombospondin superfamily.

Thrombospondins (TSPs) are multidomain, calcium-binding glycoproteins that have wide-ranging roles in vertebrates in cell interactions, extracellular matrix (ECM) organisation, angiogenesis, tissue remodelling, synaptogenesis, and also in musculoskeletal and cardiovascular functions. Land animals encode five TSPs, which assembly co-translationally either as trimers (subgroup A) or pentamers (subgroup B). The vast majority of research has focused on this canonical TSP family, which evolved through the whole-genome duplications that took place early in the vertebrate lineage. With benefit of the growth in genome- and transcriptome-predicted proteomes of a much wider range of animal species, examination of TSPs throughout metazoan phyla has revealed extensive conservation of subgroup B-type TSPs in invertebrates. In addition, these searches established that canonical TSPs are, in fact, one branch within a TSP superfamily that includes other clades designated mega-TSPs, sushi-TSPs and poriferan-TSPs. Despite the apparent simplicity of poriferans and cnidarians as organisms, these phyla encode a greater diversity of TSP superfamily members than vertebrates. We discuss here the molecular characteristics of the TSP superfamily members, current knowledge of their expression profiles and functions in invertebrates, and models for the evolution of this complex ECM superfamily.

Bacterial origin of a key innovation in the evolution of the vertebrate eye.

The vertebrate eye was described by Charles Darwin as one of the greatest potential challenges to a theory of natural selection by stepwise evolutionary processes. While numerous evolutionary transitions that led to the vertebrate eye have been explained, some aspects appear to be vertebrate specific with no obvious metazoan precursor. One critical difference between vertebrate and invertebrate vision hinges on interphotoreceptor retinoid-binding protein (IRBP, also known as retinol-binding protein, RBP3), which enables the physical separation and specialization of cells in the vertebrate visual cycle by promoting retinoid shuttling between cell types. While IRBP has been functionally described, its evolutionary origin has remained elusive. Here, we show that IRBP arose via acquisition of novel genetic material from bacteria by interdomain horizontal gene transfer (iHGT). We demonstrate that a gene encoding a bacterial peptidase was acquired prior to the radiation of extant vertebrates >500 Mya and underwent subsequent domain duplication and neofunctionalization to give rise to vertebrate IRBP. Our phylogenomic analyses on >900 high-quality genomes across the tree of life provided the resolution to distinguish contamination in genome assemblies from true instances of horizontal acquisition of IRBP and led us to discover additional independent transfers of the same bacterial peptidase gene family into distinct eukaryotic lineages. Importantly, this work illustrates the evolutionary basis of a key transition that led to the vertebrate visual cycle and highlights the striking impact that acquisition of bacterial genes has had on vertebrate evolution.

Evidence for an ancient aquatic origin of the RNA viral order Articulavirales.

The emergence of previously unknown disease-causing viruses in mammals is in part the result of a long-term evolutionary process. Reconstructing the deep phylogenetic histories of viruses helps identify major evolutionary transitions and contextualizes the emergence of viruses in new hosts. We used a combination of total RNA sequencing and transcriptome data mining to extend the diversity and evolutionary history of the RNA virus order Articulavirales, which includes the influenza viruses. We identified instances of Articulavirales in the invertebrate phylum Cnidaria (including corals), constituting a novel and divergent family that we provisionally named the "Cnidenomoviridae." We further extended the evolutionary history of the influenza virus lineage by identifying four divergent, fish-associated influenza-like viruses, thereby supporting the hypothesis that fish were among the first hosts of influenza viruses. In addition, we substantially expanded the phylogenetic diversity of quaranjaviruses and proposed that this genus be reclassified as a family-the "Quaranjaviridae." Within this putative family, we identified a novel arachnid-infecting genus, provisionally named "Cheliceravirus." Notably, we observed a close phylogenetic relationship between the Crustacea- and Chelicerata-infecting "Quaranjaviridae" that is inconsistent with virus-host codivergence. Together, these data suggest that the Articulavirales has evolved over at least 600 million years, first emerging in aquatic animals. Importantly, the evolution of the Articulavirales was likely shaped by multiple aquatic-terrestrial transitions and substantial host jumps, some of which are still observable today.

Progressive modifications during evolution involving epigenetic changes have determined loss of regeneration mainly in terrestrial animals: A hypothesis.

In order to address a biological explanation for the different regenerative abilities present among animals, a new evolutionary speculation is presented. It is hypothesized that epigenetic mechanisms have lowered or erased regeneration during the evolution of terrestrial invertebrates and vertebrates. The hypothesis indicates that a broad regeneration can only occur in marine or freshwater conditions, and that life on land does not allow for high regeneration. This is due to the physical, chemical and microbial conditions present in the terrestrial environment with respect to those of the aquatic environment. The present speculation provides examples of hypothetic evolutionary animal lineages that colonized the land, such as parasitic annelids, terrestrial mollusks, arthropods and amniotes. These are the animals where regeneration is limited or absent and their injuries are only repaired through limited healing or scarring. It is submitted that this loss derived from changes in the developmental gene pathways sustaining regeneration in the aquatic environment but that cannot be expressed on land. Once regeneration was erased in terrestrial species, re-adaptation to freshwater niches could not reactivate the previously altered gene pathways that determined regeneration. Therefore a broad regeneration was no longer possible or became limited and heteromorphic in the derived, extant animals. Only in few cases extensive healing abilities or regengrow, a healing process where regeneration overlaps with somatic growth, have evolved among arthropods and amniotes. The present paper is an extension of previous speculations trying to explain in biological terms the different regenerative abilities present among metazoans.

Combinatorial Wnt signaling landscape during brachiopod anteroposterior patterning.

BACKGROUND: Wnt signaling pathways play crucial roles in animal development. They establish embryonic axes, specify cell fates, and regulate tissue morphogenesis from the early embryo to organogenesis. It is becoming increasingly recognized that these distinct developmental outcomes depend upon dynamic interactions between multiple ligands, receptors, antagonists, and other pathway modulators, consolidating the view that a combinatorial "code" controls the output of Wnt signaling. However, due to the lack of comprehensive analyses of Wnt components in several animal groups, it remains unclear if specific combinations always give rise to specific outcomes, and if these combinatorial patterns are conserved throughout evolution. RESULTS: In this work, we investigate the combinatorial expression of Wnt signaling components during the axial patterning of the brachiopod Terebratalia transversa. We find that T. transversa has a conserved repertoire of ligands, receptors, and antagonists. These genes are expressed throughout embryogenesis but undergo significant upregulation during axial elongation. At this stage, Frizzled domains occupy broad regions across the body while Wnt domains are narrower and distributed in partially overlapping patches; antagonists are mostly restricted to the anterior end. Based on their combinatorial expression, we identify a series of unique transcriptional subregions along the anteroposterior axis that coincide with the different morphological subdivisions of the brachiopod larval body. When comparing these data across the animal phylogeny, we find that the expression of Frizzled genes is relatively conserved, whereas the expression of Wnt genes is more variable. CONCLUSIONS: Our results suggest that the differential activation of Wnt signaling pathways may play a role in regionalizing the anteroposterior axis of brachiopod larvae. More generally, our analyses suggest that changes in the receptor context of Wnt ligands may act as a mechanism for the evolution and diversification of the metazoan body axis.

Global level of methylation in the sea lamprey (jawless vertebrate) genome is intermediate between invertebrate and jawed vertebrate genomes.

In eukaryotes, cytosine methylation is a primary heritable epigenetic modification of the genome that regulates many cellular processes. In invertebrate, methylated cytosine generally located on specific genomic elements (e.g., gene bodies and silenced repetitive elements) to show a "mosaic" pattern. While in jawed vertebrate (teleost and tetrapod), highly methylated cytosine located genome-wide but only absence at regulatory regions (e.g., promoter and enhancer). Many studies imply that the evolution of DNA methylation reprogramming may have helped the transition from invertebrates to jawed vertebrates, but the detail remains largely elusive. In this study, we used the whole-genome bisulfite-sequencing technology to investigate the genome-wide methylation in three tissues (heart, muscle, and sperm) from the sea lamprey, an extant agnathan (jawless) vertebrate. Strikingly, we found that the methylation level of the sea lamprey is very similar to that in sea urchin (a deuterostome) and sea squirt (a chordate) invertebrates. In sum, the global pattern in sea lamprey is intermediate methylation level (around 30%), that is higher than methylation level in the genomes of pre-bilaterians and protostomes (1%-10%), but lower than methylation level appeared in jawed vertebrates (around 70%, teleost and tetrapod). We anticipate that, in addition to genetic dynamics such as genome duplications, epigenetic dynamics such as global methylation reprograming was also orchestrated toward the emergence and evolution of vertebrates.

Anciently duplicated genes continuously recruited to heart expression in vertebrate evolution are associated with heart chamber increase.

Although gene/genome duplications in the early stage of vertebrates have been thought to provide major resources of raw genetic materials for evolutionary innovations, it is unclear whether they continuously contribute to the evolution of morphological complexity during the course of vertebrate evolution, such as the evolution from two heart chambers (fishes) to four heart chambers (mammals and birds). We addressed this issue by our heart RNA-Seq experiments combined with published data, using 13 vertebrates and one invertebrate (sea squirt, as an outgroup). Our evolutionary transcriptome analysis showed that number of ancient paralogous genes expressed in heart tends to increase with the increase of heart chamber number along the vertebrate phylogeny, in spite that most of them were duplicated at the time near to the origin of vertebrates or even more ancient. Moreover, those paralogs expressed in heart exert considerably different functions from heart-expressed singletons: the former are functionally enriched in cardiac muscle and muscle contraction-related categories, whereas the latter play more basic functions of energy generation like aerobic respiration. These findings together support the notion that recruiting anciently paralogous genes that are expressed in heart is associated with the increase of chamber number in vertebrate evolution.

RNA-seq analysis of parasitism by Intoshia linei (Orthonectida) reveals protein effectors of defence, communication, feeding and growth.

Orthonectida is a group of multicellular endoparasites of a wide range of marine invertebrates. Their parasitic stage is a multinuclear shapeless plasmodium infiltrating host tissues. The development of the following worm-like sexual generation takes place within the cytoplasm of the plasmodium. The existence of the plasmodial stage and the development of a sexual stage within the plasmodium are unique features to Bilateria. However, the molecular mechanisms that maintain this peculiar organism, and hence enable parasitism in orthonectids, are unknown. Here, we present the first-ever RNA-seq analysis of the plasmodium, aimed at the identification and characterization of the plasmodium-specific protein-coding genes and corresponding hypothetical proteins that distinguish the parasitic plasmodium stage from the sexual stage of the orthonectid Intoshia linei Giard, 1877, parasite of nemertean Lineus ruber Müller, 1774. We discovered 119 plasmodium-specific proteins, 82 of which have inferred functions based on known domains. Thirty-five of the detected proteins are orphans, at least part of which may reflect the unique evolutionary adaptations of orthonectids to parasitism. Some of the identified proteins are known effector molecules of other endoparasites suggesting convergence. Our data indicate that the plasmodium-specific proteins might be involved in the plasmodium defense against the host, host-parasite communication, feeding and nutrient uptake, growth within the host, and support of the sexual stage development. These molecular processes in orthonectids have not been described before, and the particular protein effectors remained unknown until now.

Genomic evidence for the widespread presence of GH45 cellulases among soil invertebrates.

Lignocellulose is a major component of vascular plant biomass. Its decomposition is crucial for the terrestrial carbon cycle. Microorganisms are considered primary decomposers, but evidence increases that some invertebrates may also decompose lignocellulose. We investigated the taxonomic distribution and evolutionary origins of GH45 hydrolases, important enzymes for the decomposition of cellulose and hemicellulose, in a collection of soil invertebrate genomes. We found that these genes are common in springtails and oribatid mites. Phylogenetic analysis revealed that cellulase genes were acquired early in the evolutionary history of these groups. Domain architectures and predicted 3D enzyme structures indicate that these cellulases are functional. Patterns of presence and absence of these genes across different lineages prompt further investigation into their evolutionary and ecological benefits. The ubiquity of cellulase genes suggests that soil invertebrates may play a role in lignocellulose decomposition, independently or in synergy with microorganisms. Understanding the ecological and evolutionary implications might be crucial for understanding soil food webs and the carbon cycle.

An ultraconserved element probe set for velvet worms (Onychophora).

Onychophora are cryptic, soil-dwelling invertebrates known for their biogeographic affinities, diversity of reproductive modes, close phylogenetic relationship to arthropods, and peculiar prey capture mechanism. The 216 valid species of Onychophora are grouped into two families - Peripatopsidae and Peripatidae - and apart from a few relationships among major lineages within these two families, a stable phylogenetic backbone for the phylum has yet to be resolved. This has hindered our understanding of onychophoran biogeographic patterns, evolutionary history, and systematics. Neopatida, the Neotropical clade of peripatids, has proved particularly difficult, with recalcitrant nodes and low resolution, potentially due to rapid radiation of the group during the Cretaceous. Previous studies have had to compromise between number of loci and number of taxa due to limitations of Sanger sequencing and phylotranscriptomics, respectively. Additionally, aspects of their genome size and structure have made molecular phylogenetics difficult and data matrices have been affected by missing data. To address these issues, we leveraged recent, published transcriptomes and the first high quality genome for the phylum and designed a high affinity ultraconserved element (UCE) probe set for Onychophora. This new probe set, consisting of âˆ¼ 20,000 probes that target 1,465 loci across both families, has high locus recovery and phylogenetic utility. Phylogenetic analyses recovered the monophyly of major clades of Onychophora and revealed a novel lineage from the Neotropics that challenges our current understanding of onychophoran biogeographic endemicity. This new resource could drastically increase the power of molecular datasets and potentially allow access to genomic scale data from archival museum specimens to further tackle the issues exasperating onychophoran systematics.

Functional Characterization of Gonadotropin-Releasing Hormone and Corazonin Signaling Systems in Pacific Abalone: Toward Reclassification of Invertebrate Neuropeptides.

INTRODUCTION: The proposed evolutionary origins and corresponding nomenclature of bilaterian gonadotropin-releasing hormone (GnRH)-related neuropeptides have changed tremendously with the aid of receptor deorphanization. However, the reclassification of the GnRH and corazonin (CRZ) signaling systems in Lophotrochozoa remains unclear. METHODS: We characterized GnRH and CRZ receptors in the mollusk Pacific abalone, Haliotis discus hannai (Hdh), by phylogenetic and gene expression analyses, bioluminescence-based reporter, Western blotting, substitution of peptide amino acids, in vivo neuropeptide injection, and RNA interference assays. RESULTS: Two Hdh CRZ-like receptors (Hdh-CRZR-A and Hdh-CRZR-B) and three Hdh GnRH-like receptors (Hdh-GnRHR1-A, Hdh-GnRHR1-B, and Hdh-GnRHR2) were identified. In phylogenetic analysis, Hdh-CRZR-A and -B grouped within the CRZ-type receptors, whereas Hdh-GnRHR1-A/-B and Hdh-GnRHR2 clustered within the GnRH/adipokinetic hormone (AKH)/CRZ-related peptide-type receptors. Hdh-CRZR-A/-B and Hdh-GnRHR1-A were activated by Hdh-CRZ (pQNYHFSNGWHA-NH2) and Hdh-GnRH (pQISFSPNWGT-NH2), respectively. Hdh-CRZR-A/-B dually coupled with the Gαq and Gαs signaling pathways, whereas Hdh-GnRHR1-A was linked only with Gαq signaling. Analysis of substituted peptides, [I2S3]Hdh-CRZ and [N2Y3H4]Hdh-GnRH, and in silico docking models revealed that the N-terminal amino acids of the peptides are critical for the selectivity of Hdh-CRZR and Hdh-GnRHR. Two precursor transcripts for Hdh-CRZ and Hdh-GnRH peptides and their receptors were mainly expressed in the neural ganglia, and their levels increased in starved abalones. Injection of Hdh-CRZ peptide into abalones decreased food consumption, whereas Hdh-CRZR knockdown increased food consumption. Moreover, Hdh-CRZ induced germinal vesicle breakdown in mature oocytes. CONCLUSION: Characterization of Hdh-CRZRs and Hdh-GnRHRs and their cognate peptides provides new insight into the evolutionary route of GnRH-related signaling systems in bilaterians.

Immunological Diversity Is a Cornerstone of Organismal Defense and Allorecognition across Metazoa.

The ongoing arms race between hosts and microbes has fueled the evolution of novel strategies for diversifying the molecules involved in immune responses. Characterization of immune systems from an ever-broadening phylogenetic range of organisms reveals that there are many mechanisms by which this diversity can be generated and maintained. Diversification strategies operate at the level of populations, genomes, genes, and even individual transcripts. Lineage-specific innovations have been cataloged within the immune systems of both invertebrates and vertebrates. Furthermore, somatic diversification of immune receptor genes has now been described in jawless vertebrates and some invertebrate species. In addition to pathogen detection, immunological diversity plays important roles in several distinct allorecognition systems. In this Brief Review, we highlight some of the evolutionary innovations employed by a variety of metazoan species to generate the molecular diversity required to detect a vast array of molecules in the context of both immune response and self/nonself-recognition.

Comparative mitogenomics of Brachiopods reveals conservatism in articulate species and unusualness in inarticulate species.

BACKGROUND: Brachiopods are a phylum of marine invertebrates with over 10,000 fossil species. Today, there are fewer than 500 extant species assigned to the class Articulata or Inarticulata and for which knowledge of evolutionary genetics and genomics is still poor. Until now, complete mitogenome sequences of two inarticulate species and four articulate species were available. METHODS AND RESULTS: The complete mitogenome of the inarticulate brachiopod species Lingula reevii (20,778 bp) was obtained by using next generation sequencing. It contains 12 protein-coding genes (the annotation of atp8 is unsure), two ribosomal RNA genes, 26 transfer RNA genes, and one supernumerary ORF that is also conserved in the inarticulate species Lingula anatina. It is hypothesized that this ORF could represent a Lingula-specific mtORFan gene (without obvious homology to other genes). Comparative mitogenomics indicate the mitochondrial gene order of L. reevii is unique among brachiopods, and that compared to articulate species, inarticulate species exhibit massive mitogenome rearrangements, deviant ATP8 protein sequences and supernumerary ORFs, possibly representing species- or lineage-specific mtORFan genes. CONCLUSION: The results of this study enrich genetics knowledge of extant brachiopods, which may eventually help to test hypotheses about their decline.

Cost-efficient PCR based DNA barcoding of marine invertebrate specimens with NovaSeq amplicon sequencing.

BACKGROUND: The marine environment harbors high biodiversity; however, it is poorly understood. Nucleotide sequence data of all marine organisms should be accumulated before natural and/or anthropogenic environmental changes jeopardize the marine environment. In this study, we report a cost-effective and easy DNA barcoding method. This method can be readily adopted without using library preparation kits. It includes multiplex PCR of short targets, indexing PCR, and outsourcing to a sequencing service using the NovaSeq system. METHODS AND RESULTS: We targeted four mitochondrial genes [cytochrome c oxidase subunit I (COI), COIII, 16S rRNA (16S), and 12S rRNA (12S)] and three nuclear genes [18S rRNA (18S), 28S rRNA (28S), internal transcribed spacer 2 (ITS2)] in 95 marine invertebrate specimens, which were primarily annelids. The primers, including adapters and indices for NovaSeq sequencing, were newly designed. Two PCR runs were conducted. The 1st PCR amplified specific loci with universal primers and the 2nd added sequencing adapters and indices to the 1st PCR products. The gene sequences obtained from the FASTQ files were subjected to BLAST search and phylogenetic analyses. One run using 95 specimens yielded sequences averaging 2816 bp per specimen for a total length of six loci. Nuclear genes were more successfully assembled compared with mitochondrial genes. A weak but significantly negative correlation was observed between the average length of each locus and success rate of the assembly. Some of the sequences were almost identical to the sequences obtained from specimens collected far from Japan, indicating the presence of potentially invasive species identified for the first time. CONCLUSIONS: We obtained gene sequences efficiently using next-generation sequencing rather than Sanger sequencing. Although this method requires further optimization to increase the success rate for some loci, it is used as a first step to select specimens for further analyses by determining the specific loci of the targets.

CompoDynamics: a comprehensive database for characterizing sequence composition dynamics.

Sequence compositions of nucleic acids and proteins have significant impact on gene expression, RNA stability, translation efficiency, RNA/protein structure and molecular function, and are associated with genome evolution and adaptation across all kingdoms of life. Therefore, a devoted resource of sequence compositions and associated features is fundamentally crucial for a wide range of biological research. Here, we present CompoDynamics (https://ngdc.cncb.ac.cn/compodynamics/), a comprehensive database of sequence compositions of coding sequences (CDSs) and genomes for all kinds of species. Taking advantage of the exponential growth of RefSeq data, CompoDynamics presents a wealth of sequence compositions (nucleotide content, codon usage, amino acid usage) and derived features (coding potential, physicochemical property and phase separation) for 118 689 747 high-quality CDSs and 34 562 genomes across 24 995 species. Additionally, interactive analytical tools are provided to enable comparative analyses of sequence compositions and molecular features across different species and gene groups. Collectively, CompoDynamics bears the great potential to better understand the underlying roles of sequence composition dynamics across genes and genomes, providing a fundamental resource in support of a broad spectrum of biological studies.