Extreme genome scrambling in marine planktonic Oikopleura dioica cryptic species.

Genome structural variations within species are rare. How selective constraints preserve gene order and chromosome structure is a central question in evolutionary biology that remains unsolved. Our sequencing of several genomes of the appendicularian tunicate Oikopleura dioica around the globe reveals extreme genome scrambling caused by thousands of chromosomal rearrangements, although showing no obvious morphological differences between these animals. The breakpoint accumulation rate is an order of magnitude higher than in ascidian tunicates, nematodes, Drosophila, or mammals. Chromosome arms and sex-specific regions appear to be the primary unit of macrosynteny conservation. At the microsyntenic level, scrambling did not preserve operon structures, suggesting an absence of selective pressure to maintain them. The uncoupling of the genome scrambling with morphological conservation in O. dioica suggests the presence of previously unnoticed cryptic species and provides a new biological system that challenges our previous vision of speciation in which similar animals always share similar genome structures.

Developmental toxicity of pre-production plastic pellets affects a large swathe of invertebrate taxa.

Microplastics pose risks to marine organisms through ingestion, entanglement, and as carriers of toxic additives and environmental pollutants. Plastic pre-production pellet leachates have been shown to affect the development of sea urchins and, to some extent, mussels. The extent of those developmental effects on other animal phyla remains unknown. Here, we test the toxicity of environmental mixed nurdle samples and new PVC pellets for the embryonic development or asexual reproduction by regeneration of animals from all the major animal superphyla (Lophotrochozoa, Ecdysozoa, Deuterostomia and Cnidaria). Our results show diverse, concentration-dependent impacts in all the species sampled for new pellets, and for molluscs and deuterostomes for environmental samples. Embryo axial formation, cell specification and, specially, morphogenesis seem to be the main processes affected by plastic leachate exposure. Our study serves as a proof of principle for the potentially catastrophic effects that increasing plastic concentrations in the oceans and other ecosystems can have across animal populations from all major animal superphyla.

Cardiopharyngeal deconstruction and ancestral tunicate sessility.

A central question in chordate evolution is the origin of sessility in adult ascidians, and whether the appendicularian complete free-living style represents a primitive or derived condition among tunicates1. According to the 'a new heart for a new head' hypothesis, the evolution of the cardiopharyngeal gene regulatory network appears as a pivotal aspect to understand the evolution of the lifestyles of chordates2-4. Here we show that appendicularians experienced massive ancestral losses of cardiopharyngeal genes and subfunctions, leading to the 'deconstruction' of two ancestral modules of the tunicate cardiopharyngeal gene regulatory network. In ascidians, these modules are related to early and late multipotency, which is involved in lineage cell-fate determination towards the first and second heart fields and siphon muscles. Our work shows that the deconstruction of the cardiopharyngeal gene regulatory network involved the regressive loss of the siphon muscle, supporting an evolutionary scenario in which ancestral tunicates had a sessile ascidian-like adult lifestyle. In agreement with this scenario, our findings also suggest that this deconstruction contributed to the acceleration of cardiogenesis and the redesign of the heart into an open-wide laminar structure in appendicularians as evolutionary adaptations during their transition to a complete pelagic free-living style upon the innovation of the food-filtering house5.

Modular Evolution and Population Variability of Oikopleura dioica Metallothioneins.

Chordate Oikopleura dioica probably is the fastest evolving metazoan reported so far, and thereby, a suitable system in which to explore the limits of evolutionary processes. For this reason, and in order to gain new insights on the evolution of protein modularity, we have investigated the organization, function and evolution of multi-modular metallothionein (MT) proteins in O. dioica. MTs are a heterogeneous group of modular proteins defined by their cysteine (C)-rich domains, which confer the capacity of coordinating different transition metal ions. O. dioica has two MTs, a bi-modular OdiMT1 consisting of two domains (t-12C and 12C), and a multi-modular OdiMT2 with six t-12C/12C repeats. By means of mass spectrometry and spectroscopy of metal-protein complexes, we have shown that the 12C domain is able to autonomously bind four divalent metal ions, although the t-12C/12C pair -as it is found in OdiMT1- is the optimized unit for divalent metal binding. We have also shown a direct relationship between the number of the t-12C/12C repeats and the metal-binding capacity of the MTs, which means a stepwise mode of functional and structural evolution for OdiMT2. Finally, after analyzing four different O. dioica populations worldwide distributed, we have detected several OdiMT2 variants with changes in their number of t-12C/12C domain repeats. This finding reveals that the number of repeats fluctuates between current O. dioica populations, which provides a new perspective on the evolution of domain repeat proteins.

Tunicates Illuminate the Enigmatic Evolution of Chordate Metallothioneins by Gene Gains and Losses, Independent Modular Expansions, and Functional Convergences.

To investigate novel patterns and processes of protein evolution, we have focused in the metallothioneins (MTs), a singular group of metal-binding, cysteine-rich proteins that, due to their high degree of sequence diversity, still represents a "black hole" in Evolutionary Biology. We have identified and analyzed more than 160 new MTs in nonvertebrate chordates (especially in 37 species of ascidians, 4 thaliaceans, and 3 appendicularians) showing that prototypic tunicate MTs are mono-modular proteins with a pervasive preference for cadmium ions, whereas vertebrate and cephalochordate MTs are bimodular proteins with diverse metal preferences. These structural and functional differences imply a complex evolutionary history of chordate MTs-including de novo emergence of genes and domains, processes of convergent evolution, events of gene gains and losses, and recurrent amplifications of functional domains-that would stand for an unprecedented case in the field of protein evolution.

Massive Gene Loss and Function Shuffling in Appendicularians Stretch the Boundaries of Chordate Wnt Family Evolution.

Gene loss is a pervasive source of genetic variation that influences species evolvability, biodiversity and the innovation of evolutionary adaptations. To better understand the evolutionary patterns and impact of gene loss, here we investigate as a case study the evolution of the wingless (Wnt) family in the appendicularian tunicate Oikopleura dioica, an emergent EvoDevo model characterized by its proneness to lose genes among chordates. Genome survey and phylogenetic analyses reveal that only four of the thirteen Wnt subfamilies have survived in O. dioica-Wnt5, Wnt10, Wnt11, and Wnt16,-representing the minimal Wnt repertoire described in chordates. While the loss of Wnt4 and Wnt8 likely occurred in the last common ancestor of tunicates, representing therefore a synapomorphy of this subphylum, the rest of losses occurred during the evolution of appendicularians. This work provides the first complete Wnt developmental expression atlas in a tunicate and the first insights into the evolution of Wnt developmental functions in appendicularians. Our work highlights three main evolutionary patterns of gene loss: (1) conservation of ancestral Wnt expression domains not affected by gene losses; (2) function shuffling among Wnt paralogs accompanied by gene losses; and (3) extinction of Wnt expression in certain embryonic directly correlated with gene losses. Overall our work reveals that in contrast to "conservative" pattern of evolution of cephalochordates and vertebrates, O. dioica shows an even more radical "liberal" evolutionary pattern than that described ascidian tunicates, stretching the boundaries of the malleability of Wnt family evolution in chordates.

Oikopleura dioica: An Emergent Chordate Model to Study the Impact of Gene Loss on the Evolution of the Mechanisms of Development.

The urochordate Oikopleura dioica is emerging as a nonclassical animal model in the field of evolutionary developmental biology (a.k.a. evo-devo) especially attractive for investigating the impact of gene loss on the evolution of mechanisms of development. This is because this organism fulfills the requirements of an animal model (i.e., has a simple and accessible morphology, a short generation time and life span, and affordable culture in the laboratory and amenable experimental manipulation), but also because O. dioica occupies a key phylogenetic position to understand the diversification and origin of our own phylum, the chordates. During its evolution, O. dioica genome has suffered a drastic process of compaction, becoming the smallest known chordate genome, a process that has been accompanied by exacerbating amount of gene losses. Interestingly, however, despite the extensive gene losses, including entire regulatory pathways essential for the embryonic development of other chordates, O. dioica retains the typical chordate body plan. This unexpected situation led to the formulation of the so-called inverse paradox of evo-devo, that is, when a genetic diversity is able to maintain a phenotypic unity. This chapter reviews the biological features of O. dioica as a model animal, along with the current data on the evolution of its genes and genome. We pay special attention to the numerous examples of gene losses that have taken place during the evolution of this unique animal model, which is helping us to understand to which the limits of evo-devo can be pushed off.

Developmental atlas of appendicularian Oikopleura dioica actins provides new insights into the evolution of the notochord and the cardio-paraxial muscle in chordates.

Locomotion by tail beating powered by a system of bilateral paraxial muscle and notochord is likely one of the key evolutionary innovations that facilitated the origin and radiation of chordates. The innovation of paraxial muscle was accompanied by gene duplications in stem chordates that gave rise to muscular actins from cytoplasmic ancestral forms, which acquired contractile capability thanks to the recruitment of the myosin motor-machinery. To better understand the role of actin diversification during the evolution of chordates, in this work we have characterized the complete actin catalogue of the appendicularian Oikopleura dioica, an urochordate that maintains a chordate body plan throughout its life, including the notochord in a muscled tail that confers an active free-living pelagic style. Our genomic survey, phylogenetic analyses and Diagnostic-Actin-Values (DAVs) reveal that O. dioica has four muscular actins (ActnM1-4) and three cytoplasmic actins (ActnC1-3), most of which originated by independent gene duplications during the evolution of the appendicularian lineage. Detailed developmental expression atlas of the complete actin catalogue of O. dioica reveals differences in the temporal-regulation and tissue-specificity of different actin paralogs, suggesting complex processes of subfunctionalization during the evolution of urochordates. Our results suggest the presence of a "cardio-paraxial" muscular actin at least in the last common ancestor of Olfactores (i.e. vertebrates+urochordates). Our results reveal highly dynamic tissue-specific expression patterns for some cytoplasmic actins, including the notochord, ciliated cells and neurons with axonal projections, which challenge the classic housekeeping notion ascribed to these genes. Considering that previous work had demonstrated the existence of notochord-specific actins in cephalochordates, the tissue-specific expression of two cytoplasmic actins in the notochord of O. dioica suggests that this pattern plausibly reflects the ancestral condition of chordates, and provides new insights to better understand the evolutionary origin of the notochord.

Amphioxus functional genomics and the origins of vertebrate gene regulation.

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that-in vertebrates-over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.

Metallothioneins of the urochordate Oikopleura dioica have Cys-rich tandem repeats, large size and cadmium-binding preference.

The increasing levels of heavy metals derived from human activity are poisoning marine environments, threating zooplankton and ocean food webs. To protect themselves from the harmful effects of heavy metals, living beings have different physiological mechanisms, one of which is based on metallothioneins (MTs), a group of small cysteine-rich proteins that can bind heavy metals counteracting their toxicity. The MT system of urochordate appendicularians, an ecologically relevant component of the zooplankton, remained, however, unknown. In this work, we have characterized the MTs of the appendicularian species Oikopleura dioica, revealing that O. dioica has two MT genes, named OdMT1 and OdMT2, which encode for Cys-rich proteins, the former with 72 amino acids comparable with the small size MTs of other organisms, but the second with 399 amino acids representing the longest MT reported to date for any living being. Sequence analysis revealed that OdMT2 gene arose from a duplication of an ancestral OdMT1 gene followed by up to six tandem duplications of an ancestral repeat unit (RU) in the current OdMT2 gene. Interestingly, each RU contained, in turn, an internal repeat of a 7-Cys subunit (X3CX3CX2CX2CX3-6CX2CXCX), which is repeated up to 12 times in OdMT2. Finally, ICP-AES analyses of heterologously expressed OdMT proteins showed that both MTs were capable to form metal-complexes, with preference for cadmium ions. Collectively, our results provide the first characterization of the MT system in an appendicularian species as an initial step to understand the zooplankton response to metal toxicity and other environmental stress situations.

Diatom bloom-derived biotoxins cause aberrant development and gene expression in the appendicularian chordate Oikopleura dioica.

Investigating environmental hazards than could affect appendicularians is of prime ecological interest because they are among the most abundant components of the mesozooplankton. This work shows that embryo development of the appendicularian Oikopleura dioica is compromised by diatom bloom-derived biotoxins, even at concentrations in the same range as those measured after blooms. Developmental gene expression analysis of biotoxin-treated embryos uncovers an aberrant golf ball-like phenotype affecting morphogenesis, midline convergence, and tail elongation. Biotoxins induce a rapid upregulation of defensome genes, and considerable delay and silencing of zygotic transcription of developmental genes. Upon a possible future intensification of blooms associated with ocean warming and acidification, our work puts an alert on the potential impact that an increase of biotoxins may have on marine food webs, and points to defensome genes as molecular biosensors that marine ecologists could use to monitor the genetic stress of natural populations exposed to microalgal blooms.

Gene losses did not stop the evolution of big brains.

Elephants and fruit bats have evolved large brains even though they have lost a gene that is fundamental to the supply of energy to the brain when glucose is not available.

Wnt evolution and function shuffling in liberal and conservative chordate genomes.

BACKGROUND: What impact gene loss has on the evolution of developmental processes, and how function shuffling has affected retained genes driving essential biological processes, remain open questions in the fields of genome evolution and EvoDevo. To investigate these problems, we have analyzed the evolution of the Wnt ligand repertoire in the chordate phylum as a case study. RESULTS: We conduct an exhaustive survey of Wnt genes in genomic databases, identifying 156 Wnt genes in 13 non-vertebrate chordates. This represents the most complete Wnt gene catalog of the chordate subphyla and has allowed us to resolve previous ambiguities about the orthology of many Wnt genes, including the identification of WntA for the first time in chordates. Moreover, we create the first complete expression atlas for the Wnt family during amphioxus development, providing a useful resource to investigate the evolution of Wnt expression throughout the radiation of chordates. CONCLUSIONS: Our data underscore extraordinary genomic stasis in cephalochordates, which contrasts with the liberal and dynamic evolutionary patterns of gene loss and duplication in urochordate genomes. Our analysis has allowed us to infer ancestral Wnt functions shared among all chordates, several cases of function shuffling among Wnt paralogs, as well as unique expression domains for Wnt genes that likely reflect functional innovations in each chordate lineage. Finally, we propose a potential relationship between the evolution of WntA and the evolution of the mouth in chordates.

Reporter Analyses Reveal Redundant Enhancers that Confer Robustness on Cis-Regulatory Mechanisms.

Reporter analyses of Hox1 and Brachyury (Bra) genes have revealed examples of redundant enhancers that provide regulatory robustness. Retinoic acid (RA) activates through an RA-response element the transcription of Hox1 in the nerve cord of the ascidian Ciona intestinalis. We also found a weak RA-independent neural enhancer within the second intron of Hox1. The Hox1 gene in the larvacean Oikopleura dioica is also expressed in the nerve cord. The O. dioica genome, however, does not contain the RA receptor-encoding gene, and the expression of Hox1 has become independent of RA. We have found that the upstream sequence of the O. dioica Hox1 was able to activate reporter gene expression in the nerve cord of the C. intestinalis embryo, suggesting that an RA-independent regulatory system in the nerve cord might be common in larvaceans and ascidians. This RA-independent redundant regulatory system may have facilitated the Oikopleura ancestor losing RA signaling without an apparent impact on Hox1 expression domains. On the other hand, vertebrate Bra is expressed in the ventral mesoderm and notochord, whereas its ascidian ortholog is exclusively expressed in the notochord. Fibroblast growth factor (FGF) induces Bra in the ventral mesoderm in vertebrates, whereas it induces Bra in the notochord in ascidians. Disruption of the FGF signal does not completely silence Bra expression in ascidians, suggesting that FGF-dependent and independent enhancers might comprise a redundant regulatory system in ascidians. The existence of redundant enhancers, therefore, provides regulatory robustness that may facilitate the acquisition of new expression domains.

PTH Reloaded: A New Evolutionary Perspective.

The parathyroid hormone (PTH) family is a group of structurally-related secreted peptides involved in bone mineral homeostasis and multitude of developmental processes in vertebrates. These peptides mediate actions through PTH receptors (PTHRs), which belong to the transmembrane G protein-coupled receptor group. To date, genes encoding for PTH and PTHR have only been identified in chordates, suggesting that this signaling pathway may be an evolutionary innovation of our phylum. In vertebrates, we found up to six PTH and three PTHR different paralogs, varying in number between mammals and teleost fishes due to the different rounds of whole-genome duplication and specific gene losses suffered between the two groups of animals. The diversification of the PTH gene family has been accompanied by both functional divergence and convergence, making sometimes difficult the comparison between PTH peptides of teleosts and mammals. Here, we review the roles of all Pth peptides in fishes, and based on the evolutionary history of PTH paralogs, we propose a new and simple nomenclature from PTH1 to PTH4. Moreover, the recent characterization of the Pth4 in zebrafish allows us to consider the prominent role of the brain-to-bone signaling pathway in the regulation of bone development and homeostasis. Finally, comparison between PTH peptides of fish and mammals allows us to discuss an evolutionary model for PTH functions related to bone mineral balance during the vertebrate transition from an aquatic to a terrestrial environment.

Pth4, an ancient parathyroid hormone lost in eutherian mammals, reveals a new brain-to-bone signaling pathway.

Regulation of bone development, growth, and remodeling traditionally has been thought to depend on endocrine and autocrine/paracrine modulators. Recently, however, brain-derived signals have emerged as key regulators of bone metabolism, although their mechanisms of action have been poorly understood. We reveal the existence of an ancient parathyroid hormone (Pth)4 in zebrafish that was secondarily lost in the eutherian mammals' lineage, including humans, and that is specifically expressed in neurons of the hypothalamus and appears to be a central neural regulator of bone development and mineral homeostasis. Transgenic fish lines enabled mapping of axonal projections leading from the hypothalamus to the brainstem and spinal cord. Targeted laser ablation demonstrated an essential role for of pth4-expressing neurons in larval bone mineralization. Moreover, we show that Runx2 is a direct regulator of pth4 expression and that Pth4 can activate cAMP signaling mediated by Pth receptors. Finally, gain-of-function experiments show that Pth4 can alter calcium/phosphorus levels and affect expression of genes involved in phosphate homeostasis. Based on our discovery and characterization of Pth4, we propose a model for evolution of bone homeostasis in the context of the vertebrate transition from an aquatic to a terrestrial lifestyle.-Suarez-Bregua, P., Torres-Nuñez, E., Saxena, A., Guerreiro, P., Braasch, I., Prober, D. A., Moran, P., Cerda-Reverter, J. M., Du, S. J., Adrio, F., Power, D. M., Canario, A. V. M., Postlethwait, J. H., Bronner, M E., Cañestro, C., Rotllant, J. Pth4, an ancient parathyroid hormone lost in eutherian mammals, reveals a new brain-to-bone signaling pathway.

Coelimination and Survival in Gene Network Evolution: Dismantling the RA-Signaling in a Chordate.

The bloom of genomics is revealing gene loss as a pervasive evolutionary force generating genetic diversity that shapes the evolution of species. Outside bacteria and yeast, however, the understanding of the process of gene loss remains elusive, especially in the evolution of animal species. Here, using the dismantling of the retinoic acid metabolic gene network (RA-MGN) in the chordate Oikopleura dioica as a case study, we combine approaches of comparative genomics, phylogenetics, biochemistry, and developmental biology to investigate the mutational robustness associated to biased patterns of gene loss. We demonstrate the absence of alternative pathways for RA-synthesis in O. dioica, which suggests that gene losses of RA-MGN were not compensated by mutational robustness, but occurred in a scenario of regressive evolution. In addition, the lack of drastic phenotypic changes associated to the loss of RA-signaling provides an example of the inverse paradox of Evo-Devo. This work illustrates how the identification of patterns of gene coelimination-in our case five losses (Rdh10, Rdh16, Bco1, Aldh1a, and Cyp26)-is a useful strategy to recognize gene network modules associated to distinct functions. Our work also illustrates how the identification of survival genes helps to recognize neofunctionalization events and ancestral functions. Thus, the survival and extensive duplication of Cco and RdhE2 in O. dioica correlated with the acquisition of complex compartmentalization of expression domains in the digestive system and a process of enzymatic neofunctionalization of the Cco, while the surviving Aldh8 could be related to its ancestral housekeeping role against toxic aldehydes.

Evolution by gene loss.

The recent increase in genomic data is revealing an unexpected perspective of gene loss as a pervasive source of genetic variation that can cause adaptive phenotypic diversity. This novel perspective of gene loss is raising new fundamental questions. How relevant has gene loss been in the divergence of phyla? How do genes change from being essential to dispensable and finally to being lost? Is gene loss mostly neutral, or can it be an effective way of adaptation? These questions are addressed, and insights are discussed from genomic studies of gene loss in populations and their relevance in evolutionary biology and biomedicine.

The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons.

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences.