BackgroundWhat 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.ResultsWe 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1468-3) contains supplementary material, which is available to authorized users.

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.

In vertebrates, DNA methylation is an epigenetic mechanism that modulates gene transcription, and plays crucial roles during development, cell fate maintenance, germ cell pluripotency and inheritable genome imprinting. DNA methylation might also play a role as a genome defense mechanism against the mutational activity derived from transposon mobility. In contrast to the heavily methylated genomes in vertebrates, most genomes in invertebrates are poorly or just moderately methylated, and the function of DNA methylation remains unclear. Here, we review the DNA methylation system in the cephalochordate amphioxus, which belongs to the most basally divergent group of our own phylum, the chordates. First, surveys of the amphioxus genome database reveal the presence of the DNA methylation machinery, DNA methyltransferases and methyl-CpG-binding domain proteins. Second, comparative genomics and analyses of conserved synteny between amphioxus and vertebrates provide robust evidence that the DNA methylation machinery of amphioxus represents the ancestral toolkit of chordates, and that its expansion in vertebrates was originated by the two rounds of whole-genome duplication that occurred in stem vertebrates. Third, in silico analysis of CpGo/e ratios throughout the amphioxus genome suggests a bimodal distribution of DNA methylation, consistent with a mosaic pattern comprising domains of methylated DNA interspersed with domains of unmethylated DNA, similar to the situation described in ascidians, but radically different to the globally methylated vertebrate genomes. Finally, we discuss potential roles of the DNA methylation system in amphioxus in the context of chordate genome evolution and the origin of vertebrates.

Summary: The genome sequencing and the development of RNAi knockdown technologies in the urochordate Oikopleura dioica are making this organism an attractive emergent model in the field of EvoDevo. To succeed as a new animal model, however, an organism needs to be easily and affordably cultured in the laboratory. Nowadays, there are only two facilities in the world capable to indefinitely maintain Oikopleura dioica, one in the SARS institute (Bergen, Norway) and the other in the Osaka University (Japan). Here, we describe the setup of a new facility in the University of Barcelona (Spain) in which we have modified previously published husbandry protocols to optimize the weekly production of thousands of embryos and hundreds of mature animals using the minimum amount of space, human resources, and technical equipment. This optimization includes novel protocols of cryopreservation and solid cultures for long-term maintenance of microalgal stocks-Chaetoceros calcitrans, Isochrysis sp., Rhinomonas reticulate, and Synechococcus sp.-needed for Oikopleura dioica feeding. Our culture system maintains partially inbred lines healthy with similar characteristics to wild animals, and it is easily expandable to satisfy on demand the needs of any laboratory that may wish to use Oikopleura dioica as a model organism. genesis 53: 183-193,

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.

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.

Acid-sensing ion channels (ASICs) are membrane proteins that endow vertebrate neurons with fast excitatory responses to decreases in extracellular pH. Although previous studies suggest that ASICs are found in certain invertebrates, the lineage in which ASICs emerged and the functional role of ASICs beyond the vertebrates is unknown. We reconstructed ASIC evolution by surveying metazoan ASICs and performing a phylogenetic analysis, which suggests that ASICs evolved in an early bilaterian. This was supported by electrophysiological measurements of proton-gated currents at heterologous channels from diverse bilaterians. This also revealed substantial variation in biophysical properties of broadly related ASICs, with selective sodium/potassium ion permeability ranging from 3- to 36-fold and half-maximal activating pH from 4.2 to 8.1. Furthermore, we studied the expression of ASICs in species outside the vertebrates and found expression in the central nervous system of certain bilaterian lineages but in the periphery, including nervous system, digestive system, and motile ciliated epithelia, of most lineages. The loss of such epithelial cells in Ecdysozoa might explain the loss of ASICs from this major animal lineage. Our results suggest that ASICs emerged in an early bilaterian, and expression in the central nervous system was accompanied or even pre-dated by expression in the periphery.

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.