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.

Genetic variability and transgenerational regulation of investment in sex in the monogonont rotifer Brachionus plicatilis.

In cyclical parthenogens such as aphids, cladocerans and rotifers, the coupling between sexual reproduction and the production of resting stages (diapausing eggs) imposes strong constraints on the timing of sex. Whereas induction of sex is generally triggered by environmental cues, the response to such cues may vary across individuals according to genetic and nongenetic factors. In this study, we explored genetic and epigenetic causes of variation for the propensity for sex using a collection of strains from a Spanish population of monogonont rotifers (Brachionus plicatilis) in which variation for the threshold population density at which sex is induced (mixis threshold) had been documented previously. Our results show significant variation for the mixis threshold among 20 clones maintained under controlled conditions for 15 asexual generations. The effect of the number of clonal generations since hatching of the diapausing egg on the mixis ratio (proportion of sexual offspring produced) was tested on 4 clones with contrasted mixis thresholds. The results show a negative correlation between the mixis threshold and mixis ratio, as well as a significant effect of the number of clonal generations since fertilization, sex being repressed during the first few generations after hatching of the diapausing egg.

Why outcross? The abandon-ship hypothesis in a facultative outcrossing/selfing fungal species.

Most species able to reproduce both sexually and asexually (facultative sexual species) invest more in sexual reproduction in stressful environment conditions. According to the abandon-ship hypothesis, plasticity for investment in sexual reproduction may have been selected in these species, allowing unfit genotypes to generate progeny carrying new advantageous allelic combinations. We tested this hypothesis in Aspergillus nidulans, a fungus able to reproduce asexually, or sexually, by outcrossing and/or haploid selfing (i.e. fusion of genetically identical haploid nuclei, causing immediate genome-wide homozygosity). We crossed various strains of A. nidulans in a non-stressful environment and a stressful environment containing a non-lethal dose of fungicide. Without stress, crosses preferentially generated haploselfed fruiting bodies, whereas stressful conditions significantly increased the outcrossing rate. Our results strongly support the abandon-ship hypothesis and suggest that, for parents with low fitness, the costs of investing in sexual reproduction may be compensated by the production of fitter progeny carrying beneficial allele combinations. Similarly, the progeny generated by outcrossing was less fit than that produced by haploid selfing in non-stressful environments, but fitter in stressful conditions, suggesting that outcrossing may have short-term advantages in stressful environments in A. nidulans.

ERK1/2 is an ancestral organising signal in spiral cleavage.

Animal development is classified as conditional or autonomous based on whether cell fates are specified through inductive signals or maternal determinants, respectively. Yet how these two major developmental modes evolved remains unclear. During spiral cleavage-a stereotypic embryogenesis ancestral to 15 invertebrate groups, including molluscs and annelids-most lineages specify cell fates conditionally, while some define the primary axial fates autonomously. To identify the mechanisms driving this change, we study Owenia fusiformis, an early-branching, conditional annelid. In Owenia, ERK1/2-mediated FGF receptor signalling specifies the endomesodermal progenitor. This cell likely acts as an organiser, inducing mesodermal and posterodorsal fates in neighbouring cells and repressing anteriorising signals. The organising role of ERK1/2 in Owenia is shared with molluscs, but not with autonomous annelids. Together, these findings suggest that conditional specification of an ERK1/2+ embryonic organiser is ancestral in spiral cleavage and was repeatedly lost in annelid lineages with autonomous development.

The Fox Gene Repertoire in the Annelid Owenia fusiformis Reveals Multiple Expansions of the foxQ2 Class in Spiralia.

Fox genes are a large and conserved family of transcription factors involved in many key biological processes, including embryogenesis and body patterning. Although the role of Fox genes has been studied in an array of model systems, comprehensive comparative studies in Spiralia-a large clade of invertebrate animals including molluscs and annelids-are scarce but much needed to better understand the evolutionary history of this gene family. Here, we reconstruct and functionally characterize the Fox gene complement in the annelid Owenia fusiformis, a slow evolving species and member of the sister group to all remaining annelids. The genome of O. fusiformis contains at least a single ortholog for 20 of the 22 Fox gene classes that are ancestral to Bilateria, including an ortholog of the recently discovered foxT class. Temporal and spatial expression dynamics reveal a conserved role of Fox genes in gut formation, mesoderm patterning, and apical organ and cilia formation in Annelida and Spiralia. Moreover, we uncover an ancestral expansion of foxQ2 genes in Spiralia, represented by 11 paralogs in O. fusiformis. Notably, although all foxQ2 copies have apical expression in O. fusiformis, they show variable spatial domains and staggered temporal activation, which suggest cooperation and sub-functionalization among foxQ2 genes for the development of apical fates in this annelid. Altogether, our study informs the evolution and developmental roles of Fox genes in Annelida and Spiralia generally, providing the basis to explore how regulatory changes in Fox gene expression might have contributed to developmental and morphological diversification in Spiralia.

Early embryogenesis and organogenesis in the annelid Owenia fusiformis.

BACKGROUND: Annelids are a diverse group of segmented worms within Spiralia, whose embryos exhibit spiral cleavage and a variety of larval forms. While most modern embryological studies focus on species with unequal spiral cleavage nested in Pleistoannelida (Sedentaria + Errantia), a few recent studies looked into Owenia fusiformis, a member of the sister group to all remaining annelids and thus a key lineage to understand annelid and spiralian evolution and development. However, the timing of early cleavage and detailed morphogenetic events leading to the formation of the idiosyncratic mitraria larva of O. fusiformis remain largely unexplored. RESULTS: Owenia fusiformis undergoes equal spiral cleavage where the first quartet of animal micromeres are slightly larger than the vegetal macromeres. Cleavage results in a coeloblastula approximately 5 h post-fertilization (hpf) at 19 °C. Gastrulation occurs via invagination and completes 4 h later, with putative mesodermal precursors and the chaetoblasts appearing 10 hpf at the dorso-posterior side. Soon after, at 11 hpf, the apical tuft emerges, followed by the first neurons (as revealed by the expression of elav1 and synaptotagmin-1) in the apical organ and the prototroch by 13 hpf. Muscles connecting the chaetal sac to various larval tissues develop around 18 hpf and by the time the mitraria is fully formed at 22 hpf, there are FMRFamide+ neurons in the apical organ and prototroch, the latter forming a prototrochal ring. As the mitraria feeds, it grows in size and the prototroch expands through active proliferation. The larva becomes competent after ~ 3 weeks post-fertilization at 15 °C, when a conspicuous juvenile rudiment has formed ventrally. CONCLUSIONS: Owenia fusiformis embryogenesis is similar to that of other equal spiral cleaving annelids, supporting that equal cleavage is associated with the formation of a coeloblastula, gastrulation via invagination, and a feeding trochophore-like larva in Annelida. The nervous system of the mitraria larva forms earlier and is more elaborated than previously recognized and develops from anterior to posterior, which is likely an ancestral condition to Annelida. Altogether, our study identifies the major developmental events during O. fusiformis ontogeny, defining a conceptual framework for future investigations.