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This is a pre-copyedited, author-produced PDF of an article accepted for publication in Molecular biology and evolution following peer review. The version of record Molecular biology and evolution 40.2 (2023): msac270 is available online at: https://academic.oup.com/search-results?page=1&q=10.1093%2Fmolbev%2Fmsac270&fl_SiteID=191&SearchSourceType=1&allJournals=1, Asymmetries are essential for proper organization and function of organ systems. Genetic studies in bilaterians have shown signaling through the Nodal/Smad2 pathway plays a key, conserved role in the establishment of body asymmetries. Although the main molecular players in the network for the establishment of left-right asymmetry (LRA) have been deeply described in deuterostomes, little is known about the regulation of Nodal signaling in spiralians. Here, we identified orthologs of the egf-cfc gene, a master regulator of the Nodal pathway in vertebrates, in several invertebrate species, which includes the first evidence of its presence in non-deuterostomes. Our functional experiments indicate that despite being present, egf-cfc does not play a role in the establishment of LRA in gastropods. However, experiments in zebrafish suggest that a single amino acid mutation in the egf-cfc gene in at least the common ancestor of chordates was the necessary step to induce a gain of function in LRA regulation. This study shows that the egf-cfc gene likely appeared in the ancestors of deuterostomes and "protostomes", before being adopted as a mechanism to regulate the Nodal pathway and the establishment of LRA in some lineages of deuterostomes

Asymmetries are essential for proper organization and function of organ systems. Genetic studies in deuterostomes have shown signaling through the Nodal/Smad2 pathway plays a key, conserved role in the establishment of body asymmetries. While Nodal signaling is required for the establishment of left-right asymmetry (LRA) across bilaterian species, little is known about the regulation of Nodal signaling in spiralians. Here, we identified orthologs of the egf-cfc gene, a master regulator of the Nodal pathway in vertebrates, in several invertebrate species, the first evidence of its presence in non-deuterostomes. Our functional experiments indicate that despite being present, egf-cfc does not play a role in the establishment LRA in gastropods. However, experiments in zebrafish suggest that a single amino acid mutation in the egf-cfc gene in the deuterostome common ancestor was the necessary step in inducing a gain-of-function in LRA regulation. This study shows that that the egf-cfc gene likely appeared in the bilaterian stem lineage, before being adopted as a master mechanism to regulate the Nodal pathway and the establishment of LRA in deuterostomes.

As complete genomes become easier to attain, even from previously difficult-to-sequence species, and as genomic resequencing becomes more routine, it is becoming obvious that genomic structural variation is more widespread than originally thought and plays an important role in maintaining genetic variation in populations. Structural variants (SVs) and associated gene presence-absence variation (PAV) can be important players in local adaptation, allowing the maintenance of genetic variation and taking part in other evolutionarily relevant phenomena. While recent studies have highlighted the importance of structural variation in Mollusca, the prevalence of this phenomenon in the broader context of marine organisms remains to be fully investigated.Here, we describe a straightforward and broadly applicable method for the identification of SVs in fully assembled diploid genomes, leveraging the same reads used for assembly. We also explain a gene PAV analysis protocol, which could be broadly applied to any species with a fully sequenced reference genome available. Although the strength of these approaches have been tested and proven in marine invertebrates, which tend to have high levels of heterozygosity, possibly due to their lifestyle traits, they are also applicable to other species across the tree of life, providing a ready means to begin investigations into this potentially widespread phenomena.

Spiders are a diverse order of chelicerates that diverged from other arthropods over 500 million years ago. Research on spider embryogenesis has made important contributions to understanding the evolution of animal development. In particular, studies of the common house spider Parasteatoda tepidariorum using developmental candidate gene approaches have provided key insights into the regulation and evolution of many processes including axis formation, segmentation and patterning. However, there remains a paucity of knowledge about the cells that build spider embryos, their gene expression profiles and fate. Single-cell transcriptomic analyses have been revolutionary in describing these complex landscapes of cellular genetics in a range of animals. Therefore, we carried out single-cell RNA sequencing of P. tepidariorum embryos at stages 7, 8 and 9, which encompass the establishment and patterning of the body plan, and initial differentiation of many tissues and organs. We identified 23 cell clusters marked by many developmental toolkit genes, as well as a plethora of non-candidate genes not previously investigated. We found many Hox genes were markers of cell clusters, and Hox gene paralogs often were present in different clusters. This provided further evidence of sub- and/or neo-functionalisation of these important developmental genes after the whole genome duplication in the arachnopulmonate ancestor. We also examined the spatial expression of marker genes for each cluster to generate a comprehensive cell atlas of these embryonic stages. This revealed new insights into the cellular basis and genetic regulation of head patterning, hematopoiesis, limb development, gut development and posterior segmentation. This atlas will serve as a platform for future analysis of spider cell specification and fate, and the evolution of these processes among animals at cellular resolution.

The endemic sponges of Lake Baikal are vital components of one of the most unique ecosystems on the planet. To date we have yet to satisfactorily resolve their systematic relationships. Their relat...

As complete genomes become easier to attain, even from previously difficult-to-sequence species, and as genomic resequencing becomes more routine, it is becoming obvious that genomic structural variation is more widespread than originally thought and plays an important role in maintaining genetic variation in populations. Structural variants (SVs) and associated gene presence-absence variation (PAV) can be important players in local adaptation, allowing the maintenance of genetic variation and taking part in other evolutionarily relevant phenomena. While recent studies have highlighted the importance of structural variation in Mollusca, the prevalence of this phenomenon in the broader context of marine organisms remains to be fully investigated.Here, we describe a straightforward and broadly applicable method for the identification of SVs in fully assembled diploid genomes, leveraging the same reads used for assembly. We also explain a gene PAV analysis protocol, which could be broadly applied to any species with a fully sequenced reference genome available. Although the strength of these approaches have been tested and proven in marine invertebrates, which tend to have high levels of heterozygosity, possibly due to their lifestyle traits, they are also applicable to other species across the tree of life, providing a ready means to begin investigations into this potentially widespread phenomena.

Freshwater sponges (Spongillida) are a unique lineage of demosponges that secondarily colonized lakes and rivers and are now found ubiquitously in these ecosystems. They developed specific adaptations to freshwater systems, including the ability to survive extreme thermal ranges, long-lasting dessication, anoxia, and resistance to a variety of pollutants. While spongillids have colonized all freshwater systems, the family Lubomirskiidae is endemic to Lake Baikal, and plays a range of key roles in this ecosystem. Our work compares the genomic content and microbiome of individuals of three species of the Lubomirskiidae, providing hypotheses for how molecular evolution has allowed them to adapt to their unique environments. We have sequenced deep (>92% of the metazoan ‘Benchmarking Universal Single-Copy Orthologs’ (BUSCO) set) transcriptomes from three species of Lubomirskiidae and a draft genome resource forLubomirskia baikalensis. We note Baikal sponges contain unicellular algal and bacterial symbionts, as well as the dinoflagellateGyrodinium. We investigated molecular evolution, gene duplication and novelty in freshwater sponges compared to marine lineages. Sixty one orthogroups have consilient evidence of positive selection. Transporters (e.g.zinc transporter-2),transcription factors (aristaless-related homeobox) and structural proteins (for exampleactin-3), alongside other genes, are under strong evolutionary pressure in freshwater, with duplication driving novelty across the Spongillida, but especially in the Lubomirskiidae. This addition to knowledge of freshwater sponge genetics provides a range of tools for understanding the molecular biology and, in the future, the ecology (for example, colonization and migration patterns) of these key species.

Dissolved silicon (DSi) is biologically processed to produce siliceous skeletons by a variety of organisms including radiolarians, silicoflagellates, choanoflagellates, plants, diatoms and some animals. In the photic ocean, diatoms are dominant consumers over competing other silicifiers. In Antarctica, where DSi is not particularly limiting, diatoms and sponges coexist in high abundances. Interestingly, diatom ingestion by sponges is a regular feeding strategy there. Although it was known that the diatom organic nutrients are readily metabolized by the sponges, what happened to the inorganic diatom silica skeleton remained unexplored. Here, we have conducted a multi-analytical approach to investigate the processing of diatom silica and whether it is reconverted into sponge silica. We have documented widespread diatom consumption by several demosponges, identifying storage vesicles for the diatom-derived silica by electron microscopy and microanalysis. Diatom-consuming sponges showed upregulation of silicatein and silicase genes, which in addition to the δ 30Si values of their silica, supports that the sponges are converting the ingested diatom silica into sponge silica without much further Si fractionation. Our multidisciplinary approach suggests that the reutilization of diatom silica by sponges is a common feature among Antarctic sponges, which should be further investigated in other latitudes and in other silicifiers.

Dorsoventral pattering relies on Toll and BMP signalling in all insects studied so far, with variations in the relative contributions of both pathways.Drosophilaand the beetleTriboliumshare extensive dependence on Toll, while representatives of more distantly related lineages like the waspNasoniaand bugOncopeltusrely more strongly on BMP signalling. Here, we show that in the cricketGryllus bimaculatus, an evolutionarily distant outgroup, Toll has, like inDrosophila, a direct patterning role for the ventral half of the embryo. In addition, Toll polarises BMP signalling, although this does not involve the conserved BMP inhibitor Sog/Chordin. Finally, Toll activation relies on ovarian patterning mechanisms with striking similarity toDrosophila. Our data suggest two surprising hypotheses: (1) that Toll's patterning function inGryllusandDrosophilais the result of convergent evolution or (2) aDrosophila-likesystem arose early in insect evolution and was extensively altered in multiple independent lineages.

Dorsoventral pattering relies on Toll and BMP signalling in all insects studied so far, with variations in the relative contributions of both pathways.Drosophilaand the beetleTriboliumshare extensive dependence on Toll, while representatives of more distantly related lineages like the waspNasoniaand bugOncopeltusrely more strongly on BMP signalling. Here, we show that in the cricketGryllus bimaculatus, an evolutionarily distant outgroup, Toll has, like inDrosophila, a direct patterning role for the ventral half of the embryo. In addition Toll polarizes BMP signalling, although this does not involve the conserved BMP inhibitor Sog/Chordin. Finally, Toll activation relies on ovarian patterning mechanisms with striking similarity toDrosophila.Our data suggest two surprising hypotheses: 1) that Toll’s patterning function inGryllusandDrosophilais the result of convergent evolution or 2) aDrosophila-likesystem arose early in insect evolution, and was extensively altered in multiple independent lineages.

The advent of complete genomic sequencing has opened a window into genomic phenomena obscured by fragmented assemblies. A good example of these is the existence of hemizygous regions of autosomal chromosomes, which can result in marked differences in gene content between individuals within species. While these hemizygous regions, and presence/absence variation of genes that can result, are well known in plants, firm evidence has only recently emerged for their existence in metazoans. Here, we use recently published, complete genomes from wild-caught molluscs to investigate the prevalence of hemizygosity across a well-known and ecologically important clade. We show that hemizygous regions are widespread in mollusc genomes, not clustered in individual chromosomes, and often contain genes linked to transposition, DNA repair and stress response. With targeted investigations of HSP70-12 and C1qDC , we also show how individual gene families are distributed within pan-genomes. This work suggests that extensive pan-genomes are widespread across the conchiferan Mollusca, and represent useful tools for genomic evolution, allowing the maintenance of additional genetic diversity within the population. As genomic sequencing and re-sequencing becomes more routine, the prevalence of hemizygosity, and its impact on selection and adaptation, are key targets for research across the tree of life. This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.

Single-cell sequencing technologies are revolutionizing biology, but they are limited by the need to dissociate live samples. Here, we present ACME (ACetic-MEthanol), a dissociation approach for single-cell transcriptomics that simultaneously fixes cells. ACME-dissociated cells have high RNA integrity, can be cryopreserved multiple times, and are sortable and permeable. As a proof of principle, we provide single-cell transcriptomic data of different species, using both droplet-based and combinatorial barcoding single-cell methods. ACME uses affordable reagents, can be done in most laboratories and even in the field, and thus will accelerate our knowledge of cell types across the tree of life. This work was supported by an MRC grant (MR/S007849/1) and a Royal Society Grant (RGS\R1\191278) to JS. HG-C was supported by a Nigel Groome studentship from Oxford Brookes University. PA-C was supported by an EMBO Long Term Fellowship (ALTF-217-2018). JN was supported by funding from a BBSRC grant (BB/M011224/1) and the Osk. Huttunen Foundation (Doctoral grant)

Single-cell sequencing technologies are revolutionizing biology, but are limited by the need to dissociate fresh samples that can only be fixed at later stages. We present ACME (ACetic-MEthanol) dissociation, a cell dissociation approach that fixes cells as they are being dissociated. ACME-dissociated cells have high RNA integrity, can be cryopreserved multiple times, can be sorted by Fluorescence-Activated Cell Sorting (FACS) and are permeable, enabling combinatorial single-cell transcriptomic approaches. As a proof of principle, we have performed SPLiT-seq with ACME cells to obtain around ∼34K single cell transcriptomes from two planarian species and identified all previously described cell types in similar proportions. ACME is based on affordable reagents, can be done in most laboratories and even in the field, and thus will accelerate our knowledge of cell types across the tree of life.

BackgroundThe King Scallop, Pecten maximus, is distributed in shallow waters along the Atlantic coast of Europe. It forms the basis of a valuable commercial fishery and its ubiquity means that it plays a key role in coastal ecosystems and food webs. Like other filter feeding bivalves it can accumulate potent phytotoxins, to which it has evolved some immunity. The molecular origins of this immunity are of interest to evolutionary biologists, pharmaceutical companies and fisheries management.FindingsHere we report the genome sequencing of this species, conducted as part of the Wellcome Sanger 25 Genomes Project. This genome was assembled from PacBio reads and scaffolded with 10x Chromium and Hi-C data, and its 3,983 scaffolds have an N50 of 44.8 Mb (longest scaffold 60.1 Mb), with 92% of the assembly sequence contained in 19 scaffolds, corresponding to the 19 chromosomes found in this species. The total assembly spans 918.3 Mb, and is the best-scaffolded marine bivalve genome published to date, exhibiting 95.5% recovery of the metazoan BUSCO set. Gene annotation resulted in 67,741 gene models. Analysis of gene content revealed large numbers of gene duplicates, as previously seen in bivalves, with little gene loss, in comparison with the sequenced genomes of other marine bivalve species.ConclusionsThe genome assembly of Pecten maximus and its annotated gene set provide a high-quality platform for a wide range of investigations, including studies on such disparate topics as shell biomineralization, pigmentation, vision and resistance to algal toxins. As a result of our findings we highlight the sodium channel gene Nav1, known as a gene conferring resistance to saxitoxin and tetrodotoxin, as a candidate for further studies investigating immunity to domoic acid.

The genomes of non-bilaterian metazoans are key to understanding the molecular basis of early animal evolution. However, a full comprehension of how animal-specific traits, such as nervous systems, arose is hindered by the scarcity and fragmented nature of genomes from key taxa, such as Porifera. Ephydatia muelleri is a freshwater sponge found across the northern hemisphere. Here, we present its 326 Mb genome, assembled to high contiguity (N50: 9.88 Mb) with 23 chromosomes on 24 scaffolds. Our analyses reveal a metazoan-typical genome architecture, with highly shared synteny across Metazoa, and suggest that adaptation to the extreme temperatures and conditions found in freshwater often involves gene duplication. The pancontinental distribution and ready laboratory culture of E. muelleri make this a highly practical model system which, with RNAseq, DNA methylation and bacterial amplicon data spanning its development and range, allows exploration of genomic changes both within sponges and in early animal evolution., Reconstructing the early molecular evolution of animals requires genomic resources for non-bilaterian animals. Here, the authors present the chromosome-level genome of a freshwater sponge together with analyses of its genome architecture, methylation, developmental gene expression, and microbiome.

Molluscan eyes exhibit an enormous range of morphological variation, ranging from tiny pigment-cup eyes in limpets, compound eyes in ark clams and pinhole eyes in Nautilus, through to concave mirror eyes in scallops and the large camera-type eyes of the more derived cephalopods. Here we assess the potential of non-destructive micro-computed tomography (µ-CT) for investigating the anatomy of molluscan eyes in three species of the family Solariellidae, a group of small, deep-sea gastropods. We compare our results directly with those from traditional histological methods applied to the same specimens, and show not only that eye microstructure can be visualised in sufficient detail for meaningful comparison even in very small animals, but also that μ-CT can provide additional insight into gross neuroanatomy without damaging rare and precious specimens. Data from μ-CT scans also show that neurological innervation of eyes is reduced in dark-adapted snails when compared with the innervation of cephalic tentacles, which are involved in mechanoreception and possibly chemoreception. Molecular tests also show that the use of µ-CT and phosphotungstic acid stain do not prevent successful downstream DNA extraction, PCR amplification or sequencing. The use of µ-CT methods is therefore highly recommended for the investigation of difficult-to-collect or unique specimens.

For their apparent morphological simplicity, the Platyhelminthes or “flatworms” are a diverse clade found in a broad range of habitats. Their body plans have however made them difficult to robustly classify. Molecular evidence is only beginning to uncover the true evolutionary history of this clade. Here we present nine novel mitochondrial genomes from the still undersampled orders Polycladida and Rhabdocoela, assembled from short Illumina reads. In particular we present for the first time in the literature the mitochondrial sequence of a Rhabdocoel, Bothromesostoma personatum (Typhloplanidae, Mesostominae). The novel mitochondrial genomes examined generally contained the 36 genes expected in the Platyhelminthes, with all possessing 12 of the 13 protein-coding genes normally found in metazoan mitochondrial genomes (ATP8 being absent from all Platyhelminth mtDNA sequenced to date), along with two ribosomal RNA genes. The majority presented possess 22 transfer RNA genes, and a single tRNA gene was absent from two of the nine assembled genomes. By comparison of mitochondrial gene order and phylogenetic analysis of the protein coding and ribosomal RNA genes contained within these sequences with those of previously sequenced species we are able to gain a firm molecular phylogeny for the inter-relationships within this clade. Our phylogenetic reconstructions, using both nucleotide and amino acid sequences under several models and both Bayesian and Maximum Likelihood methods, strongly support the monophyly of Polycladida, and the monophyly of Acotylea and Cotylea within that clade. They also allow us to speculate on the early emergence of Macrostomida, the monophyly of a “Turbellarian-like” clade, the placement of Rhabditophora, and that of Platyhelminthes relative to the Lophotrochozoa (=Spiralia). The data presented here therefore represent a significant advance in our understanding of platyhelminth phylogeny, and will form the basis of a range of future research in the still-disputed classifications within this taxon., CG was for a portion of the time spent on this project a “Ramon y Cajal” postdoctoral fellow supported by the Spanish MEC (Ministerio de Economia y Competividad) and the UAM and funded by project no CGL2011-29916. NJK is currently supported by a H2020 MSCA grant [IF750937].

The sponge class Demospongiae is the most speciose and morphologically diverse in the phylum Porifera, and the species within it are vital components of a range of ecosystems worldwide. Despite their ubiquity, a number of recalcitrant problems still remain to be solved regarding their phylogenetic inter-relationships, the timing of their appearance, and their mitochondrial biology, the latter of which is only beginning to be investigated., Here we generated 14 new demosponge mitochondrial genomes which, alongside previously published mitochondrial resources, were used to address these issues. In addition to phylogenomic analysis, we have used syntenic data and analysis of coding regions to forge a framework for understanding the inter–relationships between Demospongiae sub-classes and orders. We have also leveraged our new resources to study the mitochondrial biology of these clades in terms of codon usage, optimisation and gene expression, to understand how these vital cellular components may have contributed to the success of the Porifera., Our results strongly support a sister relationship between Keratosa and (Verongimorpha + Heteroscleromorpha), contradicting previous studies using nuclear markers. Our study includes one species of Clionaida, and show for the first time support for a grouping of Suberitida+(Clionaida+(Tethyida + Poecilosclerida). The findings of our phylogenetic analyses are supported by in-depth examination of structural and coding-level evidence from our mitochondrial data. A time-calibrated phylogeny estimated the origin of Demospongiae in the Cambrian (~529 Mya), and suggests that most demosponge order crown-groups emerged in the Mesozoic. This work therefore provides a robust basis for considering demosponge phylogenetic relationships, as well as essential mitochondrial data for understanding the biological basis for their success and diversity., xpeditions to the North Atlantic, and BP, VK, AS, PC and AR were supported by the EU SponGES project, which received funding from the European Union’s Horizon 2020 research and innovation programme [under grant agreement No 679849]. AR and Szon 2020 research and innovation program [under the Marie Sklodowska-Curie grant agreement No 750937]. AR and AS received funding from VILLUM FONDEN [Grant No. 16518], and AS also from Strategic Funding through national funds provided by the T received funding from the Spanish government, Ministry of Science and Innovation [CTM2010-17415/ANT and CTM2013-42667/ANT]. NJK received funding from the European Union's HoriFoundation for Science and Technology (FCT) [UID/Multi/04423/2019] and the European Regional Development Fund (ERDF) [in the framework of the programme PT2020]. Additional funding was provided by an internal departmental fund of the NHM to AR [SDR17012]. ST received funding from the Juan de la Cierva Incorporación postdoctoral program [IJCI-2017-33116].

Antarctic shallow-water invertebrates are exceptional candidates to study population genetics and evolution, because of their peculiar evolutionary history and adaptation to extreme habitats that expand and retreat with the ice sheets. Among them, sponges are one of the major components, yet population connectivity of none of their many Antarctic species has been studied. To investigate gene flow, local adaptation and resilience to near-future changes caused by global warming, we sequenced 62 individuals of the sponge Dendrilla antarctica along the Western Antarctic Peninsula (WAP) and the South Shetlands (spanning ~900 km). We obtained information from 577 double digest restriction site-associated DNA sequencing (ddRADseq)-derived single nucleotide polymorphism (SNP), using RADseq techniques for the first time with shallow-water sponges. In contrast to other studies in sponges, our 389 neutral SNPs data set showed high levels of gene flow, with a subtle substructure driven by the circulation system of the studied area. However, the 140 outlier SNPs under positive selection showed signals of population differentiation, separating the central-southern WAP from the Bransfield Strait area, indicating a divergent selection process in the study area despite panmixia. Fourteen of these outliers were annotated, being mostly involved in immune and stress responses. We suggest that the main selective pressure on D. antarctica might be the difference in the planktonic communities present in the central-southern WAP compared to the Bransfield Strait area, ultimately depending on sea-ice control of phytoplankton blooms. Our study unveils an unexpectedly long-distance larval dispersal exceptional in Porifera, broadening the use of genome-wide markers within nonmodel Antarctic organisms.

Stolonization in syllid annelids is a unique mode of reproduction among animals. During the breeding season, a structure resembling the adult but containing only gametes, called stolon, is formed generally at the posterior end of the animal. When stolons mature, they detach from the adult and gametes are released into the water column. The process is synchronized within each species, and it has been reported to be under environmental and endogenous control, probably via endocrine regulation. To further understand reproduction in syllids and to elucidate the molecular toolkit underlying stolonization, we generated Illumina RNA-seq data from different tissues of reproductive and nonreproductive individuals of Syllis magdalena and characterized gene expression during the stolonization process. Several genes involved in gametogenesis (ovochymase, vitellogenin, testis-specific serine/threonine-kinase), immune response (complement receptor 2), neuronal development (tyrosine-protein kinase Src42A), cell proliferation (alpha-1D adrenergic receptor), and steroid metabolism (hydroxysteroid dehydrogenase 2) were found differentially expressed in the different tissues and conditions analyzed. In addition, our findings suggest that several neurohormones, such as methyl farnesoate, dopamine, and serotonin, might trigger stolon formation, the correct maturation of gametes and the detachment of stolons when gametogenesis ends. The process seems to be under circadian control, as indicated by the expression patterns of r-opsins. Overall, our results shed light into the genes that orchestrate the onset of gamete formation and improve our understanding of how some hormones, previously reported to be involved in reproduction and metamorphosis processes in other invertebrates, seem to also regulate reproduction via stolonization. This research received funding from the European Union’s (European Atomic Energy Community’s) Seventh Framework Program (FP7/2007–2013; FP7/2007–2011) under grant agreement 227799 to P.A.-C. Sequencing and analyses were conducted with internal MCZ funds to G.G. and with the support of the Center for Systems Biology and the Research Computing group, both from the Faculty of Arts and Sciences (Harvard University), and with internal funds from the Department of Life Sciences (Natural History Museum of London) to AR. One anonymous reviewer and Associate Editor Mandë Holford provided constructive criticism which helped to improve this study.

Stolonization in syllid annelids is a unique mode of reproduction among animals. During the breeding season, a structure resembling the adult but containing only gametes, called stolon, is formed at the posterior end of the animal. When the stolons mature, they detach from the adult and the gametes are released into the water column. The process is synchronized within each species, and it has been reported to be under environmental and endogenous control, probably via endocrine regulation. To further understand the reproduction in syllids and to elucidate the molecular toolkit underlying stolonization, we generated Illumina RNA-seq data from different tissues of reproductive and non-reproductive individuals ofSyllis magdalena, and characterized gene expression during the stolonization process. Several genes involved in gametogenesis (ovochymase,vitellogenin,testis-specific serine/threonine-kinase), immune response (complement receptor 2), neuronal development (tyrosine-protein kinase Src42A), cell proliferation (alpha-1D adrenergic receptor), and steroid metabolism (hydroxysteroid dehydrogenase2) were found differentially expressed in the different tissues and conditions analyzed. In addition, our findings suggest that several neurohormones, such as methyl farnesoate, dopamine and serotonin, might trigger the stolon formation, the correct maturation of gametes and the detachment of stolons when gametogenesis is complete. The process seems to be under circadian control, as indicated by the expression patterns ofr-opsins. Overall, our results shed light into the genes that orchestrate the onset of gamete formation, and improve our understanding of how some hormones, previously reported to be involved in reproduction and metamorphosis processes in other invertebrates, seem to also regulate reproduction via stolonization.

The phylum Arthropoda contains the largest number of described living animal species, with insects and crustaceans dominating the terrestrial and aquatic environments, respectively. Their successful radiations have long been linked to their rigid exoskeleton in conjunction with their specialized endocrine systems. In order to understand how hormones can contribute to the evolution of these animals, here, we have categorized the sesquiterpenoid and ecdysteroid pathway genes in the noninsect arthropod genomes, which are known to play important roles in the regulation of molting and metamorphosis in insects. In our analyses, the majority of gene homologs involved in the biosynthetic, degradative, and signaling pathways of sesquiterpenoids and ecdysteroids can be identified, implying these two hormonal systems were present in the last common ancestor of arthropods. Moreover, we found that the “Broad-Complex” was specifically gained in the Pancrustacea, and the innovation of juvenile hormone (JH) in the insect linage correlates with the gain of the JH epoxidase (CYP15A1/C1) and the key residue changes in the binding domain of JH receptor (“Methoprene-tolerant”). Furthermore, the gain of “Phantom” differentiates chelicerates from the other arthropods in using ponasterone A rather than 20-hydroxyecdysone as molting hormone. This study establishes a comprehensive framework for interpreting the evolution of these vital hormonal pathways in these most successful animals, the arthropods, for the first time.

Although the sesquiterpenoid juvenile hormone (JH) and the steroidal ecdysteroids are of vital importance to the development and reproduction of insects, our understanding of the evolution of these crucial hormonal regulators in other arthropods is limited. To better understand arthropod hormone evolution and regulation, here we describe the hormonal pathway genes (e.g. those involved in hormone biosynthesis, degradation, regulation and signal transduction) of a new decapod model, the shrimp Neocaridina denticulata. The majority of known insect sesquiterpenoid and ecdysteroid pathway genes and their regulators are contained in the N. denticulata genome. In the sesquiterpenoid pathway, these include biosynthetic pathway components: juvenile hormone acid methyltransferase (JHAMT); hormone binding protein: juvenile hormone binding protein (JHBP); and degradation pathway components: juvenile hormone esterase (JHE), juvenile hormone esterase binding protein (JHEBP) and juvenile hormone epoxide hydrolase (JHEH), with the JHBP, JHEBP and JHEH genes being discovered in a crustacean for the first time here. Ecdysteroid biosynthetic pathway genes identified include spook, phantom, disembodied, shadow and CYP18. Potential hormonal regulators and signal transducers such as allatostatins (ASTs), Methoprene-tolerant (Met), Retinoid X receptor (RXR), Ecdysone receptor (EcR), calponin-like protein Chd64, FK509-binding protein (FKBP39), Broad-complex (Br-c), and crustacean hyperglycemic hormone/molt-inhibiting hormone/gonad-inhibiting hormone (CHH/MIH/GIH) genes are all present in the shrimp N. denticulata. To our knowledge, this is the first report of these hormonal pathways and their regulatory genes together in a single decapod, providing a vital resource for further research into development, reproduction, endocrinology and evolution of crustaceans, and arthropods in general.

The increasing availability of genomic information from the Arthropoda continues to revolutionize our understanding of the biology of this most diverse animal phylum. However, our sampling of arthropod diversity remains uneven, and key clade such as the Myriapoda are severely underrepresented. Here we present the genome of the cosmopolitanly distributed Rusty Millipede Trigoniulus corallinus, which represents the first diplopod genome to be published, and the second example from the Myriapoda as a whole. This genomic resource contains the majority of core eukaryotic genes (94.3%), and key transcription factor classes that were thought to be lost in the Ecdysozoa. Mitochondrial genome and gene family (transcription factor, Dscam, circadian clock-driving protein, odorant receptor cassette, bioactive compound, and cuticular protein) analyses were also carried out to shed light on their states in the Diplopoda and Myriapoda. The ready availability of T. corallinus recommends it as a new model for evolutionary developmental biology, and the data set described here will be of widespread utility in investigating myriapod and arthropod genomics and evolution.

Kenny, Nathan J. et al., Whole-genome duplication (WGD) results in new genomic resources that can be exploited by evolution for rewiring genetic regulatory networks in organisms. In metazoans, WGD occurred before the last common ancestor of vertebrates, and has been postulated as a major evolutionary force that contributed to their speciation and diversification of morphological structures. Here, we have sequenced genomes from three of the four extant species of horseshoe crabs - Carcinoscorpius rotundicauda, Limulus polyphemus and Tachypleus tridentatus. Phylogenetic and sequence analyses of their Hox and other homeobox genes, which encode crucial transcription factors and have been used as indicators of WGD in animals, strongly suggests that WGD happened before the last common ancestor of these marine chelicerates >135 million years ago. Signatures of subfunctionalisation of paralogues of Hox genes are revealed in the appendages of two species of horseshoe crabs. Further, residual homeobox pseudogenes are observed in the three lineages. The existence of WGD in the horseshoe crabs, noted for relative morphological stasis over geological time, suggests that genomic diversity need not always be reflected phenotypically, in contrast to the suggested situation in vertebrates. This study provides evidence of ancient WGD in the ecdysozoan lineage, and reveals new opportunities for studying genomic and regulatory evolution after WGD in the Metazoa., This study was supported by a Direct Grant (4053034) of The Chinese University of Hong Kong (JHLH), and Collaborative Research Fund of the Research Grants Council, HKSAR Government (C4042-14G) (TFC, HSK, KHC, JHLH). PWHH was supported by the ERC.

Regeneration is an essential process for all multicellular organisms, allowing them to recover effectively from internal and external injury. This process has been studied extensively in a medical context in vertebrates, with pathways often investigated mechanistically, both to derive increased understanding and as potential drug targets for therapy. Several species from other parts of the metazoan tree of life, noted for their regenerative prowess, have previously been targeted for study. This allows us to understand their regenerative mechanisms and see how they could be adapted for use in medicine. Species in clades such asHydra,planarians and echinoderms can regenerate large portions of their body, the former two clades being able to completely regenerate from even a small portion of their somatic tissue. Less well-documented for their regenerative abilities are sponges. This is surprising, as they are both one of the earliest-branching extant metazoan phyla on Earth, and are rapidly able to respond to injury. Their sessile lifestyle, lack of an external protective layer, inability to respond to predation and filter-feeding strategy all mean that regeneration is often required. In particular the demosponge genusHalisarcahas been noted for its fast cell turnover and ability to quickly adjust its cell kinetic properties to repair damage through regeneration. However, while the rate and structure of regeneration in sponges has begun to be investigated, the molecular mechanisms behind this ability are yet to be catalogued.Here we describe the assembly of a reference transcriptome forHalisarca caerulea,along with additional transcriptomes noting response to injury before, shortly following (2 hrs post-), and 12 hrs after trauma. RNAseq reads were assembled using Trinity, annotated, and samples compared, to allow initial insight into the transcriptomic basis of sponge regenerative processes. These resources are deep, with our reference assembly containing more than 92.6% of the BUSCO Metazoa set of genes, and well-assembled (N50s of 836, 957, 1,688 and 2,032 for untreated, 2h, 12h and reference transcriptomes respectively), and therefore represent excellent initial resources as a bedrock for future study. The generation of transcriptomic resources from sponges before and following deliberate damage has allowed us to study particular pathways within this species responsible for repairing damage. We note particularly the involvement of the Wnt cascades in this process in this species, and detail the contents of this cascade, along with cell cycle, extracellular matrix and apoptosis-linked genes in this work.This resource represents an excellent starting point for the continued development of this knowledge, givenH. caerulea’s ability to regenerate and position as an outgroup for comparing the process of regeneration across metazoan lineages. With this resource in place, we can begin to infer the regenerative capacity of the common ancestor of all extant animal life, and unravel the elements of regeneration in an often-overlooked clade.

Organizers play important roles during the embryonic development of many animals. The most famous example is the Spemann organizer that sets up embryonic axes in amphibian embryos. In spiders, a group of BMP secreting mesenchymal cells (the cumulus) functions as an organizer of the dorsoventral axis. Similar to experiments performed with the Spemann organizer, transplantation of the cumulus is able to induce a secondary axis in spiders. Despite the importance of this structure, it is unknown which factors are needed to activate cumulus specific gene expression. To address this question, we performed a transcriptomic analysis of early embryonic development in the spider Parasteatoda tepidariorum. Through this work, we found that the transcription factor Pt-Ets4 is needed for cumulus integrity, dorsoventral patterning and for the activation of Pt-hunchback and Pt-twist expression. Furthermore, ectopic expression of Pt-Ets4 is sufficient to induce cell delamination and migration by inducing a mesoderm-like cell fate. DOI: http://dx.doi.org/10.7554/eLife.27590.001, eLife digest At the earliest stages of animal development, embryos consisting of only a handful of cells must figure out where each part of their body will come from. The first step in this process is to determine what will be their head versus their tail, and what will be their front versus their back. Many animals use specialized groups of cells, called “organizers”, to make this decision. This occurs in backboned animals – including humans – and also in distantly related animals such as spiders. In spiders, the developing embryo must form an organizer called the “cumulus” or the spiderling will not develop correctly. In order to form and maintain the cumulus, various genes must be turned on in a carefully controlled order in exactly the right cells. Pechmann et al. have now discovered the role of a previously unknown gene (called Pt-Ets4) that marks the spot where the cumulus forms. This gene is required for cumulus maintenance and it also helps to activate a number of other cumulus-specific genes. When this gene is disrupted, the spider embryo does not properly differentiate its front from its back. The findings presented by Pechmann et al. add to a growing foundation of studies aiming to understand how genes ‘talk’ to one another and organize embryos as they develop. In years to come, the unraveling of these gene pathways, where genes sequentially turn other genes on and off, will allow us to more fully understand how a single cell can grow into a complete adult animal. DOI: http://dx.doi.org/10.7554/eLife.27590.002

The abyssal demosponge Plenaster craigi is endemic to the Clarion - Clipperton Zone (CCZ) in the NE Pacific, a region with abundant seafloor polymetallic nodules and of potential interest for mining. Plenaster craigi encrusts on these nodules and is an abundant component of the ecosystem. To assess the impact of mining operations, it is crucial to understand the genetics of this species, because its genetic diversity and connectivity across the area may be representative of other nodule-encrusting invertebrate epifauna. Here we describe and characterize 14 polymorphic microsatellite markers from this keystone species using Illumina MiSeq, tested for 75 individuals from three different areas across the CCZ, including an Area of Particular Environmental Interest (APEI-6) and two areas within the adjacent UK1 mining exploration area. The number of alleles per locus ranged from 3 to 30 (13.33 average alleles for all loci across areas). Observed and expected heterozygosity ranged from 0.909–0.048 and from 0.954–0.255, respectively. Several loci displayed significant deviation from the Hardy-Weinberg equilibrium, which appears to be common in other sponge studies. The microsatellite loci described here will be used to assess the genetic structure and connectivity on populations of the sponge across the CCZ, which will be invaluable for monitoring the impact of mining operations on its habitat. Also, we provide the annotated mitochondrial genome of P. craigi, compare its arrangement with other closely related species, and discuss the phylogenetic framework for the sponge after Maximum Likelihood and Bayesian Inference analyses using nucleotide and amino acid sequences data sets separately. Electronic supplementary material The online version of this article (10.1007/s12526-017-0786-0) contains supplementary material, which is available to authorized users.

The speciose Crustacea is the largest subphylum of arthropods on the planet after the Insecta. To date, however, the only publically available sequenced crustacean genome is that of the water flea, Daphnia pulex, a member of the Branchiopoda. While Daphnia is a well-established ecotoxicological model, previous study showed that one-third of genes contained in its genome are lineage-specific and could not be identified in any other metazoan genomes. To better understand the genomic evolution of crustaceans and arthropods, we have sequenced the genome of a novel shrimp model, Neocaridina denticulata, and tested its experimental malleability. A library of 170-bp nominal fragment size was constructed from DNA of a starved single adult and sequenced using the Illumina HiSeq2000 platform. Core eukaryotic genes, the mitochondrial genome, developmental patterning genes (such as Hox) and microRNA processing pathway genes are all present in this animal, suggesting it has not undergone massive genomic loss. Comparison with the published genome of Daphnia pulex has allowed us to reveal 3750 genes that are indeed specific to the lineage containing malacostracans and branchiopods, rather than Daphnia-specific (E-value: 10−6). We also show the experimental tractability of N. denticulata, which, together with the genomic resources presented here, make it an ideal model for a wide range of further aquacultural, developmental, ecotoxicological, food safety, genetic, hormonal, physiological and reproductive research, allowing better understanding of the evolution of crustaceans and other arthropods.

Directional left/right (LR) asymmetries, in which there are consistent, heritable differences in morphology between the left and right sides of bilaterally symmetrical organisms, are found in animals across the Bilateria. For many years, we have lacked evidence for shared mechanisms underlying their development. This led to the supposition that the mechanisms driving establishment of LR asymmetries, and consequently the asymmetries themselves, had evolved separately in the three major Superphyla that constitute the Bilateria. The recent discovery that the transforming growth factor-beta (TGF-B) ligand Nodal plays a role in the regulation of LR asymmetry in both Deuterostomia and Lophotrochozoa has reignited debate in this field, as it suggests that at least this aspect of the development of the LR axis is conserved. In this review, we discuss evidence for shared mechanisms of LR asymmetry establishment across the bilaterian tree of life and consider how these mechanisms might have diverged across the Metazoa over the last 500 million years or so of evolution. As well as the likelihood that Nodal is an ancestral mechanism for regulating LR asymmetry, we reemphasize cytoskeletal architecture as a potential shared mechanism underlying symmetry breaking. However, convergent evolution remains a distinct possibility and study of a wider diversity of species will be needed to distinguish between conserved and lineage-specific mechanisms.

Since the discovery that the TGF-β signalling molecule Nodal and its downstream effector Pitx have a parallel role in establishing asymmetry between molluscs and deuterostomes the debate over the degree to which this signalling pathway is conserved across the Bilateria as a whole has been ongoing. Further taxon sampling is critical to understand the evolution and divergence of this signalling pathway in animals. Using genome and transcriptome mining we confirmed the presence of nodal and Pitx in a range of additional animal taxa for which their presence has not yet been described. In situ hybridization was used to show the embryonic expression of these genes in brachiopods and planarians. We show that both nodal and Pitx genes are broadly conserved across the Spiralia, and nodal likely appeared in the Bilaterian stem lineage after the divergence of the Acoelomorpha. Furthermore, both nodal and Pitx mRNA appears to be expressed in an asymmetric fashion in the brachiopod Terebratalia transversa. No evidence for the presence of a Lefty ortholog could be found in the non-deuterostome genomic resources examined. Nodal expression is asymmetric in a number of spiralian lineages, indicating a possible ancestral role of the Nodal/Pitx cascade in the establishment of asymmetries across the Bilateria.

As the last few decades of work has shown, precise regulation of biosynthesis and release of arthropod hormones is essential to cope with environmental stresses and challenges. In crustaceans and insects, the sesquiterpenoids methyl farnesoate (MF), farnesoic acid (FA) and juvenile hormone (JH) regulate many developmental, physiological, and reproductive processes. In this review, we discuss how comparative genomics has and will impact our views on arthropod endocrinology. We will also highlight the current knowledge of regulation of genes involved in arthropod hormone biosynthesis by microRNAs, and describe the potential insights into arthropod endocrinology, evolution, and adaptation that are likely to come from the study of microRNAs.

Despite recent efforts to sample broadly across metazoan and insect diversity, current sequence resources in the Coleoptera do not adequately describe the diversity of the clade. Here we present deep, staged transcriptomic data for two coleopteran species, Atrachya menetriesi (Faldermann 1835) and Callosobruchus maculatus (Fabricius 1775). Our sampling covered key stages in ovary and early embryonic development in each species. We utilized this data to build combined assemblies for each species which were then analysed in detail. The combined A. menetriesi assembly consists of 228,096 contigs with an N50 of 1,598 bp, while the combined C. maculatus assembly consists of 128,837 contigs with an N50 of 2,263 bp. For these assemblies, 34.6% and 32.4% of contigs were identified using Blast2GO, and 97% and 98.3% of the BUSCO set of metazoan orthologs were present, respectively. We also carried out manual annotation of developmental signalling pathways and found that nearly all expected genes were present in each transcriptome. Our analyses show that both transcriptomes are of high quality. Lastly, we performed read mapping utilising our timed, stage specific RNA samples to identify differentially expressed contigs. The resources presented here will provide a firm basis for a variety of experimentation, both in developmental biology and in comparative genomic studies.

Background: The gastropod mollusc Biomphalaria glabrata is well known as a vector for the tropical disease schistosomiasis, which affects nearly 200 million people worldwide. Despite intensive study, our understanding of the genetic basis of B. glabrata development, growth and disease resistance is constrained by limited genetic resources, constraints for which next-generation sequencing methods provide a ready solution. Methods: Illumina sequencing and de novo assembly using the Trinity program was used to generate a high-quality transcriptomic dataset spanning the entirety of in ovo development in schistosomiasis-free B. glabrata. This was subjected to automated (KEGG, BLAST2GO) and manual annotation efforts, allowing insight into the gene complements of this species in a number of contexts. Results: Excellent dataset recovery was observed, with 133,084 contigs produced of mean size 2219.48 bp. 80,952 (60.8 %) returned a BLASTx hit with an E value of less than 10, and 74,492 (55.97 %) were either mapped or assigned a GO identity using the BLAST2GO program. The CEGMA set of core eukaryotic genes was found to be 99.6 % present, indicating exceptional transcriptome completeness. We were able to identify a wealth of disease-pathway related genes within our dataset, including the Wnt, apoptosis and Notch pathways. This provides an invaluable reference point for further work into molluscan development and evolution, for studying the impact of schistosomiasis in this species, and perhaps providing targets for the treatment of this widespread disease. Conclusions: Here we present a deep transcriptome of an embryonic sample of schistosomiasis-free B. glabrata, presenting a comprehensive dataset for comparison to disease-affected specimens and from which conclusions can be drawn about the genetics of this widespread medical model. Furthermore, the dataset provided by this sequencing provides a useful reference point for comparison to other mollusc species, which can be used to better understand the evolution of this commercially, ecologically and medically important phylum., Clarendon Fund studentship

TGF-β signalling plays a key role in the patterning of metazoan body plans and growth. It is widely regarded as a 'module' capable of co-option into novel functions. The TGF-β pathway arose in the Metazoan lineage, and while it is generally regarded as well conserved across evolutionary time, its components have been largely studied in the Ecdysozoa and Deuterostomia. The recent discovery of the Nodal molecule in molluscs has underlined the necessity of untangling this signalling network in lophotrochozoans in order to truly comprehend the evolution, conservation and diversification of this key pathway. Three novel genome resources, the mollusc Patella vulgata, annelid Pomatoceros lamarcki and rotifer Brachionus plicatilis, along with other publicly available data, were searched for the presence of TGF-β pathway genes. Bayesian and Maximum Likelihood analyses, along with some consideration of conserved domain structure, was used to confirm gene identity. Analysis revealed conservation of key components within the canonical pathway, allied with extensive diversification of TGF-β ligands and partial loss of genes encoding pathway inhibitors in some lophotrochozoan lineages. We fully describe the TGF-β signalling cassette of a range of lophotrochozoans, allowing firm inference to be drawn as to the ancestral state of this pathway in this Superphylum. The TGF-β signalling cascade's reputation as being highly conserved across the Metazoa is reinforced. Diversification within the activin-like complement, as well as potential wide loss of regulatory steps in some Phyla, hint at specific evolutionary implications for aspects of this cascade's functionality in this Superphylum.

Our study reports on the occurrence of a new species of Leucetta (Calcinea, Calcarea) from the Southern Ocean, Leucetta giribeti, sp. nov., collected in the shallow waters (15m depth) of Deception Island, South Shetland Islands. This new taxon is described based on a combination of morphological and molecular data, including the description of oocytes, embryos, larvae and sperm found in the choanosome. While female reproductive elements showed great similarities with those of other calcineans, sperm is reported here for the first time in the whole Calcinea subclass. Sperm cells are flagellated and possess a typical spermatic mid-piece, which is usually observed in cnidarians. In our phylogenetic analyses, we recovered Leucetta giribeti, sp. nov. as sister species of a clade formed by species of the genera Leucetta, Pericharax and Leucettusa. Although the clade in which Leucetta giribeti, sp. nov. is placed is supported by molecular and morphological features, we cannot propose a new genus due to uncertainties regarding the type species of the genus, Leucetta primigenia Haeckel, 1872. Our study reinforces the relevance of integrative approaches in the description of new taxa and contributes to resolving the poorly known reproductive patterns of Antarctic sponge species.

Directional left/right (LR) asymmetries, in which there are consistent, heritable differences in morphology between the left and right sides of bilaterally symmetrical organisms, are found in animals across the Bilateria. For many years, we have lacked evidence for shared mechanisms underlying their development. This led to the supposition that the mechanisms driving establishment of LR asymmetries, and consequently the asymmetries themselves, had evolved separately in the three major Superphyla that constitute the Bilateria. The recent discovery that the transforming growth factor-beta (TGF-B) ligand Nodal plays a role in the regulation of LR asymmetry in both Deuterostomia and Lophotrochozoa has reignited debate in this field, as it suggests that at least this aspect of the development of the LR axis is conserved. In this review, we discuss evidence for shared mechanisms of LR asymmetry establishment across the bilaterian tree of life and consider how these mechanisms might have diverged across the Metazoa over the last 500 million years or so of evolution. As well as the likelihood that Nodal is an ancestral mechanism for regulating LR asymmetry, we reemphasize cytoskeletal architecture as a potential shared mechanism underlying symmetry breaking. However, convergent evolution remains a distinct possibility and study of a wider diversity of species will be needed to distinguish between conserved and lineage-specific mechanisms.

The C termini of N-methyl-d-aspartate (NMDA) receptor NR2 subunits are thought to play a major role in the molecular establishment of memory across the Bilateria, via the phenomenon known as long-term potentiation (LTP). Despite their long history of use as models in the study of memory, the expression and structure of the NR2 subunit in the Lophotrochozoa has remained uncategorized. Here, we report the phylogenic relationships of NR subunits across the Bilateria, and the cloning and in situ analysis of expression of NMDA NR1 and NR2 subunits in the monogont rotifer Brachionus plicatilis. RNA in situ hybridization suggests expression of NMDA receptor subunits in B. plicatilis is neural, consistent with expression observed in other species, and ours is the first report confirming NR2 expression in the lophotrochozoan clade. However, the single NR2 subunit identified in B. plicatilis was found to lack the long C terminal domain found in vertebrates, which is believed to modulate LTP. Further investigation revealed that mollusc and annelid NR2 subunits possess long intracellular C terminal domains. As data from molluscs (and particularly Aplysia californica) are the basis for much of our understanding of LTP, understanding how these diverse lophotrochozoan C termini function in vivo will have many implications for how we consider the evolution of the molecular control of learning and memory across the Metazoa as a whole and interpret the results of experiments into this vital component of cognition.