Comparative Analysis of Maternal Gene Expression Patterns Unravels Evolutionary Signatures Across Reproductive Modes.

Maternal genes have a pivotal role in regulating metazoan early development. As such their functions have been extensively studied since the dawn of developmental biology. The temporal and spatial dynamics of their transcripts have been thoroughly described in model organisms and their functions have been undergoing heavy investigations. Yet, less is known about the evolutionary changes shaping their presence within diverse oocytes. Due to their unique maternal inheritance pattern, a high degree is predicted to be present when it comes to their expression. Insofar only limited and conflicting results have emerged around it. Here, we set out to elucidate which evolutionary changes could be detected in the maternal gene expression patterns using phylogenetic comparative methods on RNAseq data from 43 species. Using normalized gene expression values and fold change information throughout early development we set out to find the best-fitting evolutionary model. Through modeling, we find evidence supporting both the high degree of divergence and constraint on gene expression values, together with their temporal dynamics. Furthermore, we find that maternal gene expression alone can be used to explain the reproductive modes of different species. Together, these results suggest a highly dynamic evolutionary landscape of maternal gene expression. We also propose a possible functional dichotomy of maternal genes which is influenced by the reproductive strategy undertaken by examined species.

A sea star is only a head.

Where are the front and back ends in a sea star? Formery et al. recently tackled this long-standing mystery using state-of-the-art molecular tools, leading them to suggest that a sea star may be constructed from components that, in other animals, would constitute only the head.

Marine animal evolutionary developmental biology-Advances through technology development.

Evolutionary developmental biology, the interdisciplinary effort of illuminating the conserved similarities and differences during animal development across all phylogenetic clades, has gained renewed interest in the past decades. As technology (immunohistochemistry, next-generation sequencing, advanced imaging, and computational resources) has advanced, so has our ability of resolving fundamental hypotheses and overcoming the genotype-phenotype gap. This rapid progress, however, has also exposed gaps in the collective knowledge around the choice and representation of model organisms. It has become clear that evo-devo requires a comparative, large-scale approach including marine invertebrates to resolve some of the most urgent questions about the phylogenetic positioning and character traits of the last common ancestors. Many invertebrates at the base of the tree of life inhabit marine environments and have been used for some years due to their accessibility, husbandry, and morphology. Here, we briefly review the major concepts of evolutionary developmental biology and discuss the suitability of established model organisms to address current research questions, before focussing on the importance, application, and state-of-the-art of marine evo-devo. We highlight novel technical advances that progress evo-devo as a whole.

Peripheral and central employment of acid-sensing ion channels during early bilaterian evolution.

Nervous systems are endowed with rapid chemosensation and intercellular signaling by ligand-gated ion channels (LGICs). While a complex, bilaterally symmetrical nervous system is a major innovation of bilaterian animals, the employment of specific LGICs during early bilaterian evolution is poorly understood. We therefore questioned bilaterian animals' employment of acid-sensing ion channels (ASICs), LGICs that mediate fast excitatory responses to decreases in extracellular pH in vertebrate neurons. Our phylogenetic analysis identified an earlier emergence of ASICs from the overarching DEG/ENaC (degenerin/epithelial sodium channel) superfamily than previously thought and suggests that ASICs were a bilaterian innovation. Our broad examination of ASIC gene expression and biophysical function in each major bilaterian lineage of Xenacoelomorpha, Protostomia, and Deuterostomia suggests that the earliest bilaterian ASICs were probably expressed in the periphery, before being incorporated into the brain as it emerged independently in certain deuterostomes and xenacoelomorphs. The loss of certain peripheral cells from Ecdysozoa after they separated from other protostomes likely explains their loss of ASICs, and thus the absence of ASICs from model organisms Drosophila and Caenorhabditis elegans. Thus, our use of diverse bilaterians in the investigation of LGIC expression and function offers a unique hypothesis on the employment of LGICs in early bilaterian evolution.

Most animals on Earth, from worms to chimpanzees, belong to a group known as the bilaterians. Despite their rich variety of shapes and lifestyles, all these creatures share similarities ­ in particular, a complex nervous system where neurons can quickly relay electric signals. This is made possible by a class of proteins, known as ligand-gated ion channels, which are studded through the membrane of cells. There, they help neurons efficiently communicate with each other by converting external chemical information into internal electrical signals. Yet despite their importance, how and when these proteins have evolved remains poorly understood. Marti-Solans et al. decided to explore this question by focusing on acid-sensing ion channels, a family which often forms the linchpin of bilaterian neural networks. They examined when these proteins first evolved (that is, in which putative ancestral animals) and where in the body. To do so, they combed through genetic data from all major bilaterian lineages as well as from non-biletarian groups; this included previously unexplored datasets that give insight into the type of cells in which a particular gene is active. The analyses revealed that the channels are specific to bilaterians, but that they appeared earlier than previously thought, being present in the very first members of this group. However, at this stage, the proteins were mainly located in cells at the periphery of the body rather than in those from emerging neural circuits. This suggests that the channels were co-opted by nerve cells later on, when the nervous systems became more complex. The proteins being initially located in cells at the outer edge of the body could also explain why they are absent in bilaterian creatures such as fruit flies and nematode worms; these animals all belong to a lineage where growth takes place by shedding their external layers. Acid-sensing ion channels are an important group of potential drug targets, often being implicated in pain and diseases of the nervous system. The work of Marti-Solans et al. offers an insight into the diversity of roles these proteins can play in the body, demonstrating once again how evolution can repurpose the same biophysical functions to serve a range of needs inside an organism.

Annelid functional genomics reveal the origins of bilaterian life cycles.

Indirect development with an intermediate larva exists in all major animal lineages1, which makes larvae central to most scenarios of animal evolution2-11. Yet how larvae evolved remains disputed. Here we show that temporal shifts (that is, heterochronies) in trunk formation underpin the diversification of larvae and bilaterian life cycles. We performed chromosome-scale genome sequencing in the annelid Owenia fusiformis with transcriptomic and epigenomic profiling during the life cycles of this and two other annelids. We found that trunk development is deferred to pre-metamorphic stages in the feeding larva of O. fusiformis but starts after gastrulation in the non-feeding larva with gradual metamorphosis of Capitella teleta and the direct developing embryo of Dimorphilus gyrociliatus. Accordingly, the embryos of O. fusiformis develop first into an enlarged anterior domain that forms larval tissues and the adult head12. Notably, this also occurs in the so-called 'head larvae' of other bilaterians13-17, with which the O. fusiformis larva shows extensive transcriptomic similarities. Together, our findings suggest that the temporal decoupling of head and trunk formation, as maximally observed in head larvae, facilitated larval evolution in Bilateria. This diverges from prevailing scenarios that propose either co-option9,10 or innovation11 of gene regulatory programmes to explain larva and adult origins.

The localization of Toll and Imd pathway and complement system components and their response to Vibrio infection in the nemertean Lineus ruber.

BACKGROUND: Innate immunity is the first line of defense against pathogens. In animals, the Toll pathway, the Imd pathway, the complement system, and lectins are well-known mechanisms involved in innate immunity. Although these pathways and systems are well understood in vertebrates and arthropods, they are understudied in other invertebrates. RESULTS: To shed light on immunity in the nemertean Lineus ruber, we performed a transcriptomic survey and identified the main components of the Toll pathway (e.g., myD88, dorsal/dif/NFκB-p65), the Imd pathway (e.g., imd, relish/NFκB-p105/100), the complement system (e.g., C3, cfb), and some lectins (FreD-Cs and C-lectins). In situ hybridization showed that TLRß1, TLRß2, and imd are expressed in the nervous system; the complement gene C3-1 is expressed in the gut; and the lectins are expressed in the nervous system, the blood, and the gut. To reveal their potential role in defense mechanisms, we performed immune challenge experiments, in which Lineus ruber specimens were exposed to the gram-negative bacteria Vibrio diazotrophicus. Our results show the upregulation of specific components of the Toll pathway (TLRα3, TLRß1, and TLRß2), the complement system (C3-1), and lectins (c-lectin2 and fred-c5). CONCLUSIONS: Therefore, similarly to what occurs in other invertebrates, our study shows that components of the Toll pathway, the complement system, and lectins are involved in the immune response in the nemertean Lineus ruber. The presence of these pathways and systems in Lineus ruber, but also in other spiralians; in ecdysozoans; and in deuterostomes suggests that these pathways and systems were involved in the immune response in the stem species of Bilateria.

Brachiopod and mollusc biomineralisation is a conserved process that was lost in the phoronid-bryozoan stem lineage.

BACKGROUND: Brachiopods and molluscs are lophotrochozoans with hard external shells which are often believed to have evolved convergently. While palaeontological data indicate that both groups are descended from biomineralising Cambrian ancestors, the closest relatives of brachiopods, phoronids and bryozoans, are mineralised to a much lower extent and are comparatively poorly represented in the Palaeozoic fossil record. Although brachiopod and mollusc shells are structurally analogous, genomic and proteomic evidence indicates that their formation involves a complement of conserved, orthologous genes. Here, we study a set of genes comprised of 3 homeodomain transcription factors, one signalling molecule and 6 structural proteins which are implicated in mollusc and brachiopod shell formation, search for their orthologs in transcriptomes or genomes of brachiopods, phoronids and bryozoans, and present expression patterns of 8 of the genes in postmetamorphic juveniles of the rhynchonelliform brachiopod T. transversa. RESULTS: Transcriptome and genome searches for the 10 target genes in the brachiopods Terebratalia transversa, Lingula anatina, Novocrania anomala, the bryozoans Bugula neritina and Membranipora membranacea, and the phoronids Phoronis australis and Phoronopsis harmeri resulted in the recovery of orthologs of the majority of the genes in all taxa. While the full complement of genes was present in all brachiopods with a single exception in L. anatina, a bloc of four genes could consistently not be retrieved from bryozoans and phoronids. The genes engrailed, distal-less, ferritin, perlucin, sp1 and sp2 were shown to be expressed in the biomineralising mantle margin of T. transversa juveniles. CONCLUSIONS: The gene expression patterns we recovered indicate that while mineralised shells in brachiopods and molluscs are structurally analogous, their formation builds on a homologous process that involves a conserved complement of orthologous genes. Losses of some of the genes related to biomineralisation in bryozoans and phoronids indicate that loss of the capacity to form mineralised structures occurred already in the phoronid-bryozoan stem group and supports the idea that mineralised skeletons evolved secondarily in some of the bryozoan subclades.

The evolution of the metazoan Toll receptor family and its expression during protostome development.

BACKGROUND: Toll-like receptors (TLRs) play a crucial role in immunity and development. They contain leucine-rich repeat domains, one transmembrane domain, and one Toll/IL-1 receptor domain. TLRs have been classified into V-type/scc and P-type/mcc TLRs, based on differences in the leucine-rich repeat domain region. Although TLRs are widespread in animals, detailed phylogenetic studies of this gene family are lacking. Here we aim to uncover TLR evolution by conducting a survey and a phylogenetic analysis in species across Bilateria. To discriminate between their role in development and immunity we furthermore analyzed stage-specific transcriptomes of the ecdysozoans Priapulus caudatus and Hypsibius exemplaris, and the spiralians Crassostrea gigas and Terebratalia transversa. RESULTS: We detected a low number of TLRs in ecdysozoan species, and multiple independent radiations within the Spiralia. V-type/scc and P-type/mcc type-receptors are present in cnidarians, protostomes and deuterostomes, and therefore they emerged early in TLR evolution, followed by a loss in xenacoelomorphs. Our phylogenetic analysis shows that TLRs cluster into three major clades: clade α is present in cnidarians, ecdysozoans, and spiralians; clade ß in deuterostomes, ecdysozoans, and spiralians; and clade γ is only found in spiralians. Our stage-specific transcriptome and in situ hybridization analyses show that TLRs are expressed during development in all species analyzed, which indicates a broad role of TLRs during animal development. CONCLUSIONS: Our findings suggest that a clade α TLR gene (TLR-Ca) and a clade ß/γ TLR gene (TLR-Cß/γ) were already present in the cnidarian-bilaterian common ancestor. However, although TLR-Ca was conserved in cnidarians, TLR-Cß/γ was lost during the early evolution of these taxa. Moreover, TLR-Cß/γ duplicated to generate TLR-Cß and TLR-Cγ in the lineage to the last common protostome-deuterostome ancestor. TLR-Ca, TLR-Cß and TLR-Cγ further expanded generating the three major TLR clades. While all three clades radiated in several spiralian lineages, specific TLRs clades have been presumably lost in other lineages. Furthermore, the expression of the majority of these genes during protostome ontogeny suggests a likely role in development.

Molecular and morphological analysis of the developing nemertean brain indicates convergent evolution of complex brains in Spiralia.

BACKGROUND: The brain anatomy in the clade Spiralia can vary from simple, commissural brains (e.g., gastrotrichs, rotifers) to rather complex, partitioned structures (e.g., in cephalopods and annelids). How often and in which lineages complex brains evolved still remains unclear. Nemerteans are a clade of worm-like spiralians, which possess a complex central nervous system (CNS) with a prominent brain, and elaborated chemosensory and neuroglandular cerebral organs, which have been previously suggested as homologs to the annelid mushroom bodies. To understand the developmental and evolutionary origins of the complex brain in nemerteans and spiralians in general, we investigated details of the neuroanatomy and gene expression in the brain and cerebral organs of the juveniles of nemertean Lineus ruber. RESULTS: In the juveniles, the CNS is already composed of all major elements present in the adults, including the brain, paired longitudinal lateral nerve cords, and an unpaired dorsal nerve cord, which suggests that further neural development is mostly related with increase in the size but not in complexity. The ultrastructure of the juvenile cerebral organ revealed that it is composed of several distinct cell types present also in the adults. The 12 transcription factors commonly used as brain cell type markers in bilaterians show region-specific expression in the nemertean brain and divide the entire organ into several molecularly distinct areas, partially overlapping with the morphological compartments. Additionally, several of the mushroom body-specific genes are expressed in the developing cerebral organs. CONCLUSIONS: The dissimilar expression of molecular brain markers between L. ruber and the annelid Platynereis dumerilii indicates that the complex brains present in those two species evolved convergently by independent expansions of non-homologous regions of a simpler brain present in their last common ancestor. Although the same genes are expressed in mushroom bodies and cerebral organs, their spatial expression within organs shows apparent differences between annelids and nemerteans, indicating convergent recruitment of the same genes into patterning of non-homologous organs or hint toward a more complicated evolutionary process, in which conserved and novel cell types contribute to the non-homologous structures.

Nemertean, Brachiopod, and Phoronid Neuropeptidomics Reveals Ancestral Spiralian Signaling Systems.

Neuropeptides are diverse signaling molecules in animals commonly acting through G-protein coupled receptors (GPCRs). Neuropeptides and their receptors underwent extensive diversification in bilaterians and the relationships of many peptide-receptor systems have been clarified. However, we lack a detailed picture of neuropeptide evolution in lophotrochozoans as in-depth studies only exist for mollusks and annelids. Here, we analyze peptidergic systems in Nemertea, Brachiopoda, and Phoronida. We screened transcriptomes from 13 nemertean, 6 brachiopod, and 4 phoronid species for proneuropeptides and neuropeptide GPCRs. With mass spectrometry from the nemertean Lineus longissimus, we validated several predicted peptides and identified novel ones. Molecular phylogeny combined with peptide-sequence and gene-structure comparisons allowed us to comprehensively map spiralian neuropeptide evolution. We found most mollusk and annelid peptidergic systems also in nemerteans, brachiopods, and phoronids. We uncovered previously hidden relationships including the orthologies of spiralian CCWamides to arthropod agatoxin-like peptides and of mollusk APGWamides to RGWamides from annelids, with ortholog systems in nemerteans, brachiopods, and phoronids. We found that pleurin neuropeptides previously only found in mollusks are also present in nemerteans and brachiopods. We also identified cases of gene family duplications and losses. These include a protostome-specific expansion of RFamide/Wamide signaling, a spiralian expansion of GnRH-related peptides, and duplications of vasopressin/oxytocin before the divergence of brachiopods, phoronids, and nemerteans. This analysis expands our knowledge of peptidergic signaling in spiralians and other protostomes. Our annotated data set of nearly 1,300 proneuropeptide sequences and 600 GPCRs presents a useful resource for further studies of neuropeptide signaling.

Molecular evidence for a single origin of ultrafiltration-based excretory organs.

Excretion is an essential physiological process, carried out by all living organisms, regardless of their size or complexity.1-3 Both protostomes (e.g., flies and flatworms) and deuterostomes (e.g., humans and sea urchins) possess specialized excretory organs serving that purpose. Those organs exhibit an astonishing diversity, ranging from units composed of just few distinct cells (e.g., protonephridia) to complex structures, built by millions of cells of multiple types with divergent morphology and function (e.g., vertebrate kidneys).4,5 Although some molecular similarities between the development of kidneys of vertebrates and the regeneration of the protonephridia of flatworms have been reported,6,7 the molecular underpinnings of the development of excretory organs have never been systematically studied in a comparative context.4 Here, we show that a set of transcription factors (eya, six1/2, pou3, sall, lhx1/5, and osr) and structural proteins (nephrin, kirre, and zo1) is expressed in the excretory organs of a phoronid, brachiopod, annelid, onychophoran, priapulid, and hemichordate that represent major protostome lineages and non-vertebrate deuterostomes. We demonstrate that the molecular similarity observed in the vertebrate kidney and flatworm protonephridia6,7 is also seen in the developing excretory organs of those animals. Our results show that all types of ultrafiltration-based excretory organs are patterned by a conserved set of developmental genes, an observation that supports their homology. We propose that the last common ancestor of protostomes and deuterostomes already possessed an ultrafiltration-based organ that later gave rise to the vast diversity of extant excretory organs, including both proto- and metanephridia.

FGF signaling acts on different levels of mesoderm development within Spiralia.

FGF signaling is involved in mesoderm induction in members of deuterostomes (e.g. tunicates, hemichordates), but not in flies and nematodes, in which it has a role in mesoderm patterning and migration. However, we need comparable studies in other protostome taxa in order to decipher whether this mesoderm-inducing function of FGF extends beyond the lineage of deuterostomes. Here, we investigated the role of FGF signaling in mesoderm development in three species of lophophorates, a clade within the protostome group Spiralia. Our gene expression analyses show that the mesodermal molecular patterning is conserved between brachiopods and phoronids, but the spatial and temporal recruitment of transcription factors differs significantly. Moreover, the use of the inhibitor SU5402 demonstrates that FGF signaling is involved in different steps of mesoderm development, as well as in morphogenetic movements of gastrulation and axial elongation. Our findings suggest that the mesoderm-inducing role of FGF extends beyond the group of deuterostomes.

Conservative route to genome compaction in a miniature annelid.

The causes and consequences of genome reduction in animals are unclear because our understanding of this process mostly relies on lineages with often exceptionally high rates of evolution. Here, we decode the compact 73.8-megabase genome of Dimorphilus gyrociliatus, a meiobenthic segmented worm. The D. gyrociliatus genome retains traits classically associated with larger and slower-evolving genomes, such as an ordered, intact Hox cluster, a generally conserved developmental toolkit and traces of ancestral bilaterian linkage. Unlike some other animals with small genomes, the analysis of the D. gyrociliatus epigenome revealed canonical features of genome regulation, excluding the presence of operons and trans-splicing. Instead, the gene-dense D. gyrociliatus genome presents a divergent Myc pathway, a key physiological regulator of growth, proliferation and genome stability in animals. Altogether, our results uncover a conservative route to genome compaction in annelids, reminiscent of that observed in the vertebrate Takifugu rubripes.

A developmental perspective on the evolution of the nervous system.

The evolution of nervous systems in animals has always fascinated biologists, and thus multiple evolutionary scenarios have been proposed to explain the appearance of neurons and complex neuronal centers. However, the absence of a robust phylogenetic framework for animal interrelationships, the lack of a mechanistic understanding of development, and a recapitulative view of animal ontogeny have traditionally limited these scenarios. Only recently, the integration of advanced molecular and morphological studies in a broad range of animals has allowed to trace the evolution of developmental and neuronal characters on a better-resolved animal phylogeny. This has falsified most traditional scenarios for nervous system evolution, paving the way for the emergence of new testable hypotheses. Here we summarize recent progress in studies of nervous system development in major animal lineages and formulate some of the arising questions. In particular, we focus on how lineage analyses of nervous system development and a comparative study of the expression of neural-related genes has influenced our understanding of the evolution of an elaborated central nervous system in Bilateria. We argue that a phylogeny-guided study of neural development combining thorough descriptive and functional analyses is key to establish more robust scenarios for the origin and evolution of animal nervous systems.

Hox gene expression during development of the phoronid Phoronopsis harmeri.

BACKGROUND: Phoronida is a small group of marine worm-like suspension feeders, which together with brachiopods and bryozoans form the clade Lophophorata. Although their development is well studied on the morphological level, data regarding gene expression during this process are scarce and restricted to the analysis of relatively few transcription factors. Here, we present a description of the expression patterns of Hox genes during the embryonic and larval development of the phoronid Phoronopsis harmeri. RESULTS: We identified sequences of eight Hox genes in the transcriptome of Ph. harmeri and determined their expression pattern during embryonic and larval development using whole mount in situ hybridization. We found that none of the Hox genes is expressed during embryonic development. Instead their expression is initiated in the later developmental stages, when the larval body is already formed. In the investigated initial larval stages the Hox genes are expressed in the non-collinear manner in the posterior body of the larvae: in the telotroch and the structures that represent rudiments of the adult worm. Additionally, we found that certain head-specific transcription factors are expressed in the oral hood, apical organ, preoral coelom, digestive system and developing larval tentacles, anterior to the Hox-expressing territories. CONCLUSIONS: The lack of Hox gene expression during early development of Ph. harmeri indicates that the larval body develops without positional information from the Hox patterning system. Such phenomenon might be a consequence of the evolutionary intercalation of the larval form into an ancestral life cycle of phoronids. The observed Hox gene expression can also be a consequence of the actinotrocha representing a "head larva", which is composed of the most anterior body region that is devoid of Hox gene expression. Such interpretation is further supported by the expression of head-specific transcription factors. This implies that the Hox patterning system is used for the positional information of the trunk rudiments and is, therefore, delayed to the later larval stages. We propose that a new body form was intercalated to the phoronid life cycle by precocious development of the anterior structures or by delayed development of the trunk rudiment in the ancestral phoronid larva.

Molecular patterning during the development of Phoronopsis harmeri reveals similarities to rhynchonelliform brachiopods.

BACKGROUND: Phoronids, rhynchonelliform and linguliform brachiopods show striking similarities in their embryonic fate maps, in particular in their axis specification and regionalization. However, although brachiopod development has been studied in detail and demonstrated embryonic patterning as a causal factor of the gastrulation mode (protostomy vs deuterostomy), molecular descriptions are still missing in phoronids. To understand whether phoronids display underlying embryonic molecular mechanisms similar to those of brachiopods, here we report the expression patterns of anterior (otx, gsc, six3/6, nk2.1), posterior (cdx, bra) and endomesodermal (foxA, gata4/5/6, twist) markers during the development of the protostomic phoronid Phoronopsis harmeri. RESULTS: The transcription factors foxA, gata4/5/6 and cdx show conserved expression in patterning the development and regionalization of the phoronid embryonic gut, with foxA expressed in the presumptive foregut, gata4/5/6 demarcating the midgut and cdx confined to the hindgut. Furthermore, six3/6, usually a well-conserved anterior marker, shows a remarkably dynamic expression, demarcating not only the apical organ and the oral ectoderm, but also clusters of cells of the developing midgut and the anterior mesoderm, similar to what has been reported for brachiopods, bryozoans and some deuterostome Bilateria. Surprisingly, brachyury, a transcription factor often associated with gastrulation movements and mouth and hindgut development, seems not to be involved with these patterning events in phoronids. CONCLUSIONS: Our description and comparison of gene expression patterns with other studied Bilateria reveals that the timing of axis determination and cell fate distribution of the phoronid shows highest similarity to that of rhynchonelliform brachiopods, which is likely related to their shared protostomic mode of development. Despite these similarities, the phoronid Ph. harmeri also shows particularities in its development, which hint to divergences in the arrangement of gene regulatory networks responsible for germ layer formation and axis specification.

Convergent evolution of a vertebrate-like methylome in a marine sponge.

Vertebrates have highly methylated genomes at CpG positions, whereas invertebrates have sparsely methylated genomes. This increase in methylation content is considered a major regulatory innovation of vertebrate genomes. However, here we report that a sponge, proposed as the potential sister group to the rest of animals, has a highly methylated genome. Despite major differences in genome size and architecture, we find similarities between the independent acquisitions of the hypermethylated state. Both lineages show genome-wide CpG depletion, conserved strong transcription factor methyl-sensitivity and developmental methylation dynamics at 5-hydroxymethylcytosine enriched regions. Together, our findings trace back patterns associated with DNA methylation in vertebrates to the early steps of animal evolution. Thus, the sponge methylome challenges previous hypotheses concerning the uniqueness of vertebrate genome hypermethylation and its implications for regulatory complexity.

Morphology of the nervous system of monogonont rotifer Epiphanes senta with a focus on sexual dimorphism between feeding females and dwarf males.

BACKGROUND: Monogononta is a large clade of rotifers comprised of diverse morphological forms found in a wide range of ecological habitats. Most monogonont species display cyclical parthenogenesis, where generations of asexually reproducing females are interspaced by mixis events when sexual reproduction occurs between mictic females and dwarf, haploid males. The morphology of monogonont feeding females is relatively well described, however data on male anatomy are very limited. Thus far, male musculature of only two species has been described with confocal laser scanning microscopy (CLSM) and it remains unknown how dwarfism influences the neuroanatomy of males on detailed level. RESULTS: Here, we provide a CLSM-based description of the nervous system of both sexes of Epiphanes senta, a freshwater monogonont rotifer. The general nervous system architecture is similar between males and females and shows a similar level of complexity. However, the nervous system in males is more compact and lacks a stomatogastric part. CONCLUSION: Comparison of the neuroanatomy between male and normal-sized feeding females provides a better understanding of the nature of male dwarfism in Monogononta. We propose that dwarfism of monogonont non-feeding males is the result of a specific case of heterochrony, called "proportional dwarfism" as they, due to their inability to feed, retain a juvenile body size, but still develop a complex neural architecture comparable to adult females. Reduction of the stomatogastric nervous system in the males correlates with the loss of the entire digestive tract and associated morphological structures.

Active mode of excretion across digestive tissues predates the origin of excretory organs.

Most bilaterian animals excrete toxic metabolites through specialized organs, such as nephridia and kidneys, which share morphological and functional correspondences. In contrast, excretion in non-nephrozoans is largely unknown, and therefore the reconstruction of ancestral excretory mechanisms is problematic. Here, we investigated the excretory mode of members of the Xenacoelomorpha, the sister group to Nephrozoa, and Cnidaria, the sister group to Bilateria. By combining gene expression, inhibitor experiments, and exposure to varying environmental ammonia conditions, we show that both Xenacoelomorpha and Cnidaria are able to excrete across digestive-associated tissues. However, although the cnidarian Nematostella vectensis seems to use diffusion as its main excretory mode, the two xenacoelomorphs use both active transport and diffusion mechanisms. Based on these results, we propose that digestive-associated tissues functioned as excretory sites before the evolution of specialized organs in nephrozoans. We conclude that the emergence of a compact, multiple-layered bilaterian body plan necessitated the evolution of active transport mechanisms, which were later recruited into the specialized excretory organs.