The 3D-Evo Space: Evolution of Gene Expression and Alternative Splicing Regulation.

Animal species present relatively high levels of gene conservation, and yet they display a great variety of cell type and tissue phenotypes. These diverse phenotypes are mainly specified through differential gene usage, which relies on several mechanisms. Two of the most relevant mechanisms are regulated gene transcription, usually referred to as gene expression (rGE), and regulated alternative splicing (rAS). Several works have addressed how either rGE or rAS contributes to phenotypic diversity throughout evolution, but a back-to-back comparison between the two molecular mechanisms, specifically highlighting both their common regulatory principles and unique properties, is still missing. In this review, we propose an innovative framework for the unified comparison between rGE and rAS from different perspectives: the three-dimensional (3D)-evo space. We use the 3D-evo space to comprehensively (a) review the molecular basis of rGE and rAS (i.e., the molecular axis), (b) depict the tissue-specific phenotypes they contribute to (i.e., the tissue axis), and (c) describe the determinants that drive the evolution of rGE and rAS programs (i.e., the evolution axis). Finally, we unify the perspectives emerging from the three axes by discussing general trends and specific examples of rGE and rAS tissue program evolution.

Annelid Comparative Genomics and the Evolution of Massive Lineage-Specific Genome Rearrangement in Bilaterians.

The organization of genomes into chromosomes is critical for processes such as genetic recombination, environmental adaptation, and speciation. All animals with bilateral symmetry inherited a genome structure from their last common ancestor that has been highly conserved in some taxa but seemingly unconstrained in others. However, the evolutionary forces driving these differences and the processes by which they emerge have remained largely uncharacterized. Here, we analyze genome organization across the phylum Annelida using 23 chromosome-level annelid genomes. We find that while many annelid lineages have maintained the conserved bilaterian genome structure, the Clitellata, a group containing leeches and earthworms, possesses completely scrambled genomes. We develop a rearrangement index to quantify the extent of genome structure evolution and show that, compared to the last common ancestor of bilaterians, leeches and earthworms have among the most highly rearranged genomes of any currently sampled species. We further show that bilaterian genomes can be classified into two distinct categories-high and low rearrangement-largely influenced by the presence or absence, respectively, of chromosome fission events. Our findings demonstrate that animal genome structure can be highly variable within a phylum and reveal that genome rearrangement can occur both in a gradual, stepwise fashion, or rapid, all-encompassing changes over short evolutionary timescales.

Highly conserved and extremely evolvable: BMP signalling in secondary axis patterning of Cnidaria and Bilateria.

Bilateria encompass the vast majority of the animal phyla. As the name states, they are bilaterally symmetric, that is with a morphologically clear main body axis connecting their anterior and posterior ends, a second axis running between their dorsal and ventral surfaces, and with a left side being roughly a mirror image of their right side. Bone morphogenetic protein (BMP) signalling has widely conserved functions in the formation and patterning of the second, dorso-ventral (DV) body axis, albeit to different extents in different bilaterian species. Whilst initial findings in the fruit fly Drosophila and the frog Xenopus highlighted similarities amongst these evolutionarily very distant species, more recent analyses featuring other models revealed considerable diversity in the mechanisms underlying dorsoventral patterning. In fact, as phylogenetic sampling becomes broader, we find that this axis patterning system is so evolvable that even its core components can be deployed differently or lost in different model organisms. In this review, we will try to highlight the diversity of ways by which BMP signalling controls bilaterality in different animals, some of which do not belong to Bilateria. Future research combining functional analyses and modelling is bound to give us some understanding as to where the limits to the extent of the evolvability of BMP-dependent axial patterning may lie.

Loss of Timeless Underlies an Evolutionary Transition within the Circadian Clock.

Most organisms possess time-keeping devices called circadian clocks. At the molecular level, circadian clocks consist of transcription-translation feedback loops (TTFLs). Although some components of the negative TTFL are conserved across the animals, important differences exist between typical models, such as mouse and the fruit fly. In Drosophila, the key components are PERIOD (PER) and TIMELESS (TIM-d) proteins, whereas the mammalian clock relies on PER and CRYPTOCHROME (CRY-m). Importantly, how the clock has maintained functionality during evolutionary transitions between different states remains elusive. Therefore, we systematically described the circadian clock gene setup in major bilaterian lineages and identified marked lineage-specific differences in their clock constitution. Then we performed a thorough functional analysis of the linden bug Pyrrhocoris apterus, an insect species comprising features characteristic of both the Drosophila and the mammalian clocks. Unexpectedly, the knockout of timeless-d, a gene essential for the clock ticking in Drosophila, did not compromise rhythmicity in P. apterus, it only accelerated its pace. Furthermore, silencing timeless-m, the ancestral timeless type ubiquitously present across animals, resulted in a mild gradual loss of rhythmicity, supporting its possible participation in the linden bug clock, which is consistent with timeless-m role suggested by research on mammalian models. The dispensability of timeless-d in P. apterus allows drawing a scenario in which the clock has remained functional at each step of transition from an ancestral state to the TIM-d-independent PER + CRY-m system operating in extant vertebrates, including humans.

Left-Right Reversal Recurrently Evolved Regardless of Diaphanous-Related Formin Gene Duplication or Loss in Snails.

Bilateria exhibit whole-body handedness in internal structure. This left-right polarity is evolutionarily conserved with virtually no reversed extant lineage, except in molluscan Gastropoda. Phylogenetically independent snail groups contain both clockwise-coiled (dextral) and counterclockwise-coiled (sinistral) taxa that are reversed from each other in bilateral handedness as well as in coiling direction. Within freshwater Hygrophila, Lymnaea with derived dextrality have diaphanous related formin (diaph) gene duplicates, while basal sinistral groups possess one diaph gene. In terrestrial Stylommatophora, dextral Bradybaena also have diaph duplicates. Defective maternal expression of one of those duplicates gives rise to sinistral hatchlings in Lymnaea and handedness-mixed broods in Bradybaena, through polarity change in spiral cleavage of embryos. These findings led to the hypothesis that diaph duplication was crucial for the evolution of dextrality by reversal. The present study discovered that diaph duplication independently occurred four times and its duplicate became lost twice in gastropods. The dextrality of Bradybaena represents the ancestral handedness conserved across gastropods, unlike the derived dextrality of Lymnaea. Sinistral lineages recurrently evolved by reversal regardless of whether diaph had been duplicated. Amongst the seven formin gene subfamilies, diaph has most thoroughly been conserved across eukaryotes of the 14 metazoan phyla and choanoflagellate. Severe embryonic mortalities resulting from insufficient expression of the duplicate in both of Bradybaena and Lymnaea also support that diaph duplicates bare general roles for cytoskeletal dynamics other than controlling spiralian handedness. Our study rules out the possibility that diaph duplication or loss played a primary role for reversal evolution.

Chemical cognition: chemoconnectomics and convergent evolution of integrative systems in animals.

Neurons underpin cognition in animals. However, the roots of animal cognition are elusive from both mechanistic and evolutionary standpoints. Two conceptual frameworks both highlight and promise to address these challenges. First, we discuss evidence that animal neural and other integrative systems evolved more than once (convergent evolution) within basal metazoan lineages, giving us unique experiments by Nature for future studies. The most remarkable examples are neural systems in ctenophores and neuroid-like systems in placozoans and sponges. Second, in addition to classical synaptic wiring, a chemical connectome mediated by hundreds of signal molecules operates in tandem with neurons and is the most information-rich source of emerging properties and adaptability. The major gap-dynamic, multifunctional chemical micro-environments in nervous systems-is not understood well. Thus, novel tools and information are needed to establish mechanistic links between orchestrated, yet cell-specific, volume transmission and behaviors. Uniting what we call chemoconnectomics and analyses of the cellular bases of behavior in basal metazoan lineages arguably would form the foundation for deciphering the origins and early evolution of elementary cognition and intelligence.

TRF2 and the evolution of the bilateria.

The development of a complex body plan requires a diversity of regulatory networks. Here we consider the concept of TATA-box-binding protein (TBP) family proteins as "system factors" that each supports a distinct set of transcriptional programs. For instance, TBP activates TATA-box-dependent core promoters, whereas TBP-related factor 2 (TRF2) activates TATA-less core promoters that are dependent on a TCT or downstream core promoter element (DPE) motif. These findings led us to investigate the evolution of TRF2. TBP occurs in Archaea and eukaryotes, but TRF2 evolved prior to the emergence of the bilateria and subsequent to the evolutionary split between bilaterians and nonbilaterian animals. Unlike TBP, TRF2 does not bind to the TATA box and could thus function as a new system factor that is largely independent of TBP. We postulate that this TRF2-based system served as the foundation for new transcriptional programs, such as those involved in triploblasty and body plan development, that facilitated the evolution of bilateria.

On the evolution of bilaterality.

Bilaterality - the possession of two orthogonal body axes - is the name-giving trait of all bilaterian animals. These body axes are established during early embryogenesis and serve as a three-dimensional coordinate system that provides crucial spatial cues for developing cells, tissues, organs and appendages. The emergence of bilaterality was a major evolutionary transition, as it allowed animals to evolve more complex body plans. Therefore, how bilaterality evolved and whether it evolved once or several times independently is a fundamental issue in evolutionary developmental biology. Recent findings from non-bilaterian animals, in particular from Cnidaria, the sister group to Bilateria, have shed new light into the evolutionary origin of bilaterality. Here, we compare the molecular control of body axes in radially and bilaterally symmetric cnidarians and bilaterians, identify the minimal set of traits common for Bilateria, and evaluate whether bilaterality arose once or more than once during evolution.

Lateral neural borders as precursors of peripheral nervous systems: A comparative view across bilaterians.

The nervous systems in most bilaterians are centralized, composed of central nervous systems (CNS) and peripheral nervous systems (PNS). Common molecular and cellular patterns of medial nerve cords have been observed in various distantly related bilaterians, suggesting deep homology of CNS. The development patterns of PNS, however, are more diverse than CNS across different phylogenetic lineages and the evolution of PNS so far has been thought to be polygenic. The molecular and cellular programs during the development of PNS among different bilaterian branches are drastically different. For example, vertebrate PNS is essentially derived from neural crest cells and placodes, which are largely vertebrate innovations and do not exist in invertebrates. On the other hand, the lack of common precursor cell types does not necessarily lead to the conclusion of different evolutionary origins. Homology needs to be examined with a deeper and broader scope. In this review, we examined the molecular, cellular and developmental characteristics of PNS in a broad range of bilaterians to summarize our current understanding of variation and potentially conserved themes. These comparisons demonstrate that there exist both migratory and non-migratory neuroblasts in the lateral border of CNS precursors in most model bilaterian animals. These lateral border neuroblasts are specified by conserved gene regulatory network and give rise to sensory neurons, suggesting that lateral border neuroblasts represent the progenitor of PNS and share deep homology among different branches of Bilateria. Future studies are needed to elucidate the evo-devo mechanisms of the lateral neural borders as PNS progenitors.

Possible rules for the ancestral origin of Hox gene collinearity.

The Hox gene cluster is believed to have formed from a single ProtoHox gene by repeated cycles of the following events: tandem gene duplication, mutation to generate a new expression boundary along the embryonic axis, and acquisition of a new Hox patterning function. The Hox cluster in Bilateria evolved in compliance with the so-called collinearity rule. That is, the order of the genes along the chromosome corresponds with the order of their embryonic expression domains along the head-tail axis. Gaunt (2015) suggested that collinearity may have arisen as a mechanism to minimise the incidence of boundaries between active and inactive genes within the Hox cluster. We now attempt to clarify the model by presenting it in the form of three rules: 1) no two Hox genes may persist in the same cluster with the same anterior boundary of activity in the same tissue; 2) an inactive Hox gene must not be flanked by two active Hox genes; 3) an active Hox gene must not be flanked by two inactive genes. We provide evidence and illustrative computer simulations to show that these rules, which can apply only to partially overlapping patterns of Hox activity, may account for the ancestral origin of Hox gene collinearity.

Hidden diversity of Acoelomorpha revealed through metabarcoding.

Animals with bilateral symmetry comprise the majority of the described species within Metazoa. However, the nature of the first bilaterian animal remains unknown. As most recent molecular phylogenies point to Xenacoelomorpha as the sister group to the rest of Bilateria, understanding their biology, ecology and diversity is key to reconstructing the nature of the last common bilaterian ancestor (Urbilateria). To date, sampling efforts have focused mainly on coastal areas, leaving potential gaps in our understanding of the full diversity of xenacoelomorphs. We therefore analysed 18S rDNA metabarcoding data from three marine projects covering benthic and pelagic habitats worldwide. Our results show that acoels have a greater richness in planktonic environments than previously described. Interestingly, we also identified a putative novel clade of acoels in the deep benthos that branches as sister group to the rest of Acoela, thus representing the earliest-branching acoel clade. Our data highlight deep-sea environments as an ideal habitat to sample acoels with key phylogenetic positions, which might be useful for reconstructing the early evolution of Bilateria.

Experimental taphonomy of Artemia reveals the role of endogenous microbes in mediating decay and fossilization.

Exceptionally preserved fossils provide major insights into the evolutionary history of life. Microbial activity is thought to play a pivotal role in both the decay of organisms and the preservation of soft tissue in the fossil record, though this has been the subject of very little experimental investigation. To remedy this, we undertook an experimental study of the decay of the brine shrimp Artemia, examining the roles of autolysis, microbial activity, oxygen diffusion and reducing conditions. Our findings indicate that endogenous gut bacteria are the main factor controlling decay. Following gut wall rupture, but prior to cuticle failure, gut-derived microbes spread into the body cavity, consuming tissues and forming biofilms capable of mediating authigenic mineralization, that pseudomorph tissues and structures such as limbs and the haemocoel. These observations explain patterns observed in exceptionally preserved fossil arthropods. For example, guts are preserved relatively frequently, while preservation of other internal anatomy is rare. They also suggest that gut-derived microbes play a key role in the preservation of internal anatomy and that differential preservation between exceptional deposits might be because of factors that control autolysis and microbial activity. The findings also suggest that the evolution of a through gut and its bacterial microflora increased the potential for exceptional fossil preservation in bilaterians, providing one explanation for the extreme rarity of internal preservation in those animals that lack a through gut.

Right across the tree of life: the evolution of left-right asymmetry in the Bilateria.

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.

Decline and fall of the Ediacarans: late-Neoproterozoic extinctions and the rise of the modern biosphere.

The end-Neoproterozoic transition marked a gradual but permanent shift between distinct configurations of Earth's biosphere. This interval witnessed the demise of the enigmatic Ediacaran Biota, ushering in the structured trophic webs and disparate animal body plans of Phanerozoic ecosystems. However, little consensus exists on the reality, drivers, and macroevolutionary implications of end-Neoproterozoic extinctions. Here we evaluate potential drivers of late-Neoproterozoic turnover by addressing recent findings on Ediacaran geochronology, the persistence of classical Ediacaran macrobionts into the Cambrian, and the existence of Ediacaran crown-group eumetazoans. Despite renewed interest in the possibility of Phanerozoic-style 'mass extinctions' in the latest Neoproterozoic, our synthesis of the available evidence does not support extinction models based on episodic geochemical triggers, nor does it validate simple ecological interpretations centred on direct competitive displacement. Instead, we argue that the protracted and indirect effects of early bilaterian innovations, including escalations in sediment engineering, predation, and the largely understudied impacts of reef-building, may best account for the temporal structure and possible selectivity of late-Neoproterozoic extinctions. We integrate these processes into a generalised model of early eumetazoan-dominated ecologies, charting the disruption of spatial and temporal isotropy on the Ediacaran benthos as a consequence of diversifying macrofaunal interactions. Given the nature of resource distribution in Ediacaran ecologies, the continuities among Ediacaran and Cambrian faunas, and the convergent origins of ecologically disruptive innovations among bilaterians we suggest that the rise of Phanerozoic-type biotas may have been unstoppable.

Evolutionary conserved peptide and glycoprotein hormone-like neuroendocrine systems in C. elegans.

Peptides and protein hormones form the largest group of secreted signals that mediate intercellular communication and are central regulators of physiology and behavior in all animals. Phylogenetic analyses and biochemical identifications of peptide-receptor systems reveal a broad evolutionary conservation of these signaling systems at the molecular level. Substantial progress has been made in recent years on characterizing the physiological and putative ancestral roles of many peptide systems through comparative studies in invertebrate models. Several peptides and protein hormones are not only molecularly conserved but also have conserved roles across animal phyla. Here, we focus on functional insights gained in the nematode Caenorhabditis elegans that, with its compact and well-described nervous system, provides a powerful model to dissect neuroendocrine signaling networks involved in the control of physiology and behavior. We summarize recent discoveries on the evolutionary conservation and knowledge on the functions of peptide and protein hormone systems in C. elegans.

Analysis of SMAD1/5 target genes in a sea anemone reveals ZSWIM4-6 as a novel BMP signaling modulator.

BMP signaling has a conserved function in patterning the dorsal-ventral body axis in Bilateria and the directive axis in anthozoan cnidarians. So far, cnidarian studies have focused on the role of different BMP signaling network components in regulating pSMAD1/5 gradient formation. Much less is known about the target genes downstream of BMP signaling. To address this, we generated a genome-wide list of direct pSMAD1/5 target genes in the anthozoan Nematostella vectensis, several of which were conserved in Drosophila and Xenopus. Our ChIP-seq analysis revealed that many of the regulatory molecules with documented bilaterally symmetric expression in Nematostella are directly controlled by BMP signaling. We identified several so far uncharacterized BMP-dependent transcription factors and signaling molecules, whose bilaterally symmetric expression may be indicative of their involvement in secondary axis patterning. One of these molecules is zswim4-6, which encodes a novel nuclear protein that can modulate the pSMAD1/5 gradient and potentially promote BMP-dependent gene repression.

Seeking Sense in the Hox Gene Cluster.

The Hox gene cluster, responsible for patterning of the head-tail axis, is an ancestral feature of all bilaterally symmetrical animals (the Bilateria) that remains intact in a wide range of species. We can say that the Hox cluster evolved successfully only once since it is commonly the same in all groups, with labial-like genes at one end of the cluster expressed in the anterior embryo, and Abd-B-like genes at the other end of the cluster expressed posteriorly. This review attempts to make sense of the Hox gene cluster and to address the following questions. How did the Hox cluster form in the protostome-deuterostome last common ancestor, and why was this with a particular head-tail polarity? Why is gene clustering usually maintained? Why is there collinearity between the order of genes along the cluster and the positions of their expressions along the embryo? Why do the Hox gene expression domains overlap along the embryo? Why have vertebrates duplicated the Hox cluster? Why do Hox gene knockouts typically result in anterior homeotic transformations? How do animals adapt their Hox clusters to evolve new structural patterns along the head-tail axis?

Filtering artifactual signal increases support for Xenacoelomorpha and Ambulacraria sister relationship in the animal tree of life.

Conflicting studies place a group of bilaterian invertebrates containing xenoturbellids and acoelomorphs, the Xenacoelomorpha, as either the primary emerging bilaterian phylum1,2,3,4,5,6 or within Deuterostomia, sister to Ambulacraria.7,8,9,10,11 Although their placement as sister to the rest of Bilateria supports relatively simple morphology in the ancestral bilaterian, their alternative placement within Deuterostomia suggests a morphologically complex ancestral bilaterian along with extensive loss of major phenotypic traits in the Xenacoelomorpha. Recent studies have questioned whether Deuterostomia should be considered monophyletic at all.10,12,13 Hidden paralogy and poor phylogenetic signal present a major challenge for reconstructing species phylogenies.14,15,16,17,18 Here, we assess whether these issues have contributed to the conflict over the placement of Xenacoelomorpha. We reanalyzed published datasets, enriching for orthogroups whose gene trees support well-resolved clans elsewhere in the animal tree.16 We find that most genes in previously published datasets violate incontestable clans, suggesting that hidden paralogy and low phylogenetic signal affect the ability to reconstruct branching patterns at deep nodes in the animal tree. We demonstrate that removing orthogroups that cannot recapitulate incontestable relationships alters the final topology that is inferred, while simultaneously improving the fit of the model to the data. We discover increased, but ultimately not conclusive, support for the existence of Xenambulacraria in our set of filtered orthogroups. At a time when we are progressing toward sequencing all life on the planet, we argue that long-standing contentious issues in the tree of life will be resolved using smaller amounts of better quality data that can be modeled adequately.19.

HERMES: An improved method to test mitochondrial genome molecular synapomorphies among clades.

Mitochondrial chromosomes have diversified among eukaryotes and many different architectures and features are now acknowledged for this genome. Here we present the improved HERMES index, which can measure and quantify the amount of molecular change experienced by mitochondrial genomes. We test the improved approach with ten molecular phylogenetic studies based on complete mitochondrial genomes, representing six bilaterian Phyla. In most cases, HERMES analysis spotted out clades or single species with peculiar molecular synapomorphies, allowing to identify phylogenetic and ecological patterns. The software presented herein handles linear, circular, and multi-chromosome genomes, thus widening the HERMES scope to the complete eukaryotic domain.

Conserved Patterns in Developmental Processes and Phases, Rather than Genes, Unite the Highly Divergent Bilateria.

Bilateria are the predominant clade of animals on Earth. Despite having evolved a wide variety of body plans and developmental modes, they are characterized by common morphological traits. By default, researchers have tried to link clade-specific genes to these traits, thus distinguishing bilaterians from non-bilaterians, by their gene content. Here we argue that it is rather biological processes that unite Bilateria and set them apart from their non-bilaterian sisters, with a less complex body morphology. To test this hypothesis, we compared proteomes of bilaterian and non-bilaterian species in an elaborate computational pipeline, aiming to search for a set of bilaterian-specific genes. Despite the limited confidence in their bilaterian specificity, we nevertheless detected Bilateria-specific functional and developmental patterns in the sub-set of genes conserved in distantly related Bilateria. Using a novel multi-species GO-enrichment method, we determined the functional repertoire of genes that are widely conserved among Bilateria. Analyzing expression profiles in three very distantly related model species-D. melanogaster, D. rerio and C. elegans-we find characteristic peaks at comparable stages of development and a delayed onset of expression in embryos. In particular, the expression of the conserved genes appears to peak at the phylotypic stage of different bilaterian phyla. In summary, our study illustrate how development connects distantly related Bilateria after millions of years of divergence, pointing to processes potentially separating them from non-bilaterians. We argue that evolutionary biologists should return from a purely gene-centric view of evolution and place more focus on analyzing and defining conserved developmental processes and periods.