Parasitic fish embryos do a "front-flip" on the yolk to resist expulsion from the host.

Embryonic development is often considered shielded from the effects of natural selection, being selected primarily for reliable development. However, embryos sometimes represent virulent parasites, triggering a coevolutionary "arms race" with their host. We have examined embryonic adaptations to a parasitic lifestyle in the bitterling fish. Bitterlings are brood parasites that lay their eggs in the gill chamber of host mussels. Bitterling eggs and embryos have adaptations to resist being flushed out by the mussel. These include a pair of projections from the yolk sac that act as an anchor. Furthermore, bitterling eggs all adopt a head-down position in the mussel gills which further increases their chances of survival. To examine these adaptations in detail, we have studied development in the rosy bitterling (Rhodeus ocellatus) using molecular markers, X-ray tomography, and time-lapse imaging. We describe a suite of developmental adaptations to brood parasitism in this species. We show that the mechanism underlying these adaptions is a modified pattern of blastokinesis-a process unique, among fish, to bitterlings. Tissue movements during blastokinesis cause the embryo to do an extraordinary "front-flip" on the yolk. We suggest that this movement determines the spatial orientation of the other developmental adaptations to parasitism, ensuring that they are optimally positioned to help resist the ejection of the embryo from the mussel. Our study supports the notion that natural selection can drive the evolution of a suite of adaptations, both embryonic and extra-embryonic, via modifications in early development.

Deep conservation and co-option of programmed cell death facilitates evolution of alternative phenotypes at multiple biological levels.

Alternative phenotypes, such as polyphenisms and sexual dimorphisms, are widespread in nature and appear at all levels of biological organization, from genes and cells to morphology and behavior. Yet, our understanding of the mechanisms through which alternative phenotypes develop and how they evolve remains understudied. In this review, we explore the association between alternative phenotypes and programmed cell death, a mechanism responsible for the elimination of superfluous cells during development. We discuss the ancient origins and deep conservation of programmed cell death (its function, forms and underlying core regulatory gene networks), and propose that it was co-opted repeatedly to generate alternative phenotypes at the level of cells, tissues, organs, external morphology, and even individuals. We review several examples from across the tree of life to explore the conditions under which programmed cell death is likely to facilitate the evolution of alternative phenotypes.

Onco-Breastomics: An Eco-Evo-Devo Holistic Approach.

Known as a diverse collection of neoplastic diseases, breast cancer (BC) can be hyperbolically characterized as a dynamic pseudo-organ, a living organism able to build a complex, open, hierarchically organized, self-sustainable, and self-renewable tumor system, a population, a species, a local community, a biocenosis, or an evolving dynamical ecosystem (i.e., immune or metabolic ecosystem) that emphasizes both developmental continuity and spatio-temporal change. Moreover, a cancer cell community, also known as an oncobiota, has been described as non-sexually reproducing species, as well as a migratory or invasive species that expresses intelligent behavior, or an endangered or parasite species that fights to survive, to optimize its features inside the host's ecosystem, or that is able to exploit or to disrupt its host circadian cycle for improving the own proliferation and spreading. BC tumorigenesis has also been compared with the early embryo and placenta development that may suggest new strategies for research and therapy. Furthermore, BC has also been characterized as an environmental disease or as an ecological disorder. Many mechanisms of cancer progression have been explained by principles of ecology, developmental biology, and evolutionary paradigms. Many authors have discussed ecological, developmental, and evolutionary strategies for more successful anti-cancer therapies, or for understanding the ecological, developmental, and evolutionary bases of BC exploitable vulnerabilities. Herein, we used the integrated framework of three well known ecological theories: the Bronfenbrenner's theory of human development, the Vannote's River Continuum Concept (RCC), and the Ecological Evolutionary Developmental Biology (Eco-Evo-Devo) theory, to explain and understand several eco-evo-devo-based principles that govern BC progression. Multi-omics fields, taken together as onco-breastomics, offer better opportunities to integrate, analyze, and interpret large amounts of complex heterogeneous data, such as various and big-omics data obtained by multiple investigative modalities, for understanding the eco-evo-devo-based principles that drive BC progression and treatment. These integrative eco-evo-devo theories can help clinicians better diagnose and treat BC, for example, by using non-invasive biomarkers in liquid-biopsies that have emerged from integrated omics-based data that accurately reflect the biomolecular landscape of the primary tumor in order to avoid mutilating preventive surgery, like bilateral mastectomy. From the perspective of preventive, personalized, and participatory medicine, these hypotheses may help patients to think about this disease as a process governed by natural rules, to understand the possible causes of the disease, and to gain control on their own health.

Bacteria- and temperature-regulated peptides modulate ß-catenin signaling in Hydra.

Animal development has traditionally been viewed as an autonomous process directed by the host genome. But, in many animals, biotic and abiotic cues, like temperature and bacterial colonizers, provide signals for multiple developmental steps. Hydra offers unique features to encode these complex interactions of developmental processes with biotic and abiotic factors, and we used it here to investigate the impact of bacterial colonizers and temperature on the pattern formation process. In Hydra, formation of the head organizer involves the canonical Wnt pathway. Treatment with alsterpaullone (ALP) results in acquiring characteristics of the head organizer in the body column. Intriguingly, germfree Hydra polyps are significantly more sensitive to ALP compared to control polyps. In addition to microbes, ß-catenin-dependent pattern formation is also affected by temperature. Gene expression analyses led to the identification of two small secreted peptides, named Eco1 and Eco2, being up-regulated in the response to both Curvibacter sp., the main bacterial colonizer of Hydra, and low temperatures. Loss-of-function experiments revealed that Eco peptides are involved in the regulation of pattern formation and have an antagonistic function to Wnt signaling in Hydra.

Nano-CT imaging of larvae in the ant Pheidole hyatti reveals coordinated growth of a rudimentary organ necessary for soldier development.

The growth of imaginal discs in holometabolous insects is coordinated with larval growth to ensure the symmetrical and proportional development of the adult appendages. In ants, the differential growth of these discs generates distinct castes-the winged male and queen castes and the wingless worker caste. In the hyperdiverse ant genus Pheidole, the worker caste is composed of two morphologically distinct subcastes: small-headed minor workers and larger, big-headed, soldiers. Although these worker subcastes are completely wingless, soldier larvae develop rudimentary forewing discs that function in generating the disproportionate head-to-body scaling and size of soldiers. It remains unclear, however, how rudimentary forewing discs in soldier larvae are coordinated with other imaginal discs. Here we show, using quantitative nano-CT imaging and three-dimensional analyses, that the increase in the volume of the soldier rudimentary forewing discs is coordinated with larval size as well as with the increase in the volume of the leg and eye-antennal (head) discs. However, relative to larval size, we found that when the rudimentary forewing discs appear during the last larval instar, they are relatively smaller but increase in volume faster than that of the head (eye-antennal) and leg discs. These findings show that the rudimentary wing disc in soldier larvae has evolved novel patterns of inter-organ coordination as compared with other insects to generate the big-headed soldier caste in Pheidole. More generally, our study raises the possibility that novel patterns of inter-organ coordination are a general feature of rudimentary organs that acquire novel regulatory functions during development and evolution.

Phenotypic plasticity rather than genotype drives reproductive choices in Hydra populations.

Facultative clonality is associated with complex life cycles where sexual and asexual forms can be exposed to contrasting selection pressures. Facultatively clonal animals often have distinct developmental capabilities that depend on reproductive mode (e.g., negligible senescence and exceptional regeneration ability in asexual individuals, which are lacking in sexual individuals). Understanding how these differences in life history strategies evolved is hampered by limited knowledge of the population structure underlying sexual and asexual forms in nature. Here we studied genetic differentiation of coexisting sexual and asexual Hydra oligactis polyps, a freshwater cnidarian where reproductive mode-dependent life history patterns are observed. We collected asexual and sexual polyps from 13 Central European water bodies and used restriction-site associated DNA sequencing to infer population structure. We detected high relatedness among populations and signs that hydras might spread with resting eggs through zoochory. We found no genetic structure with respect to mode of reproduction (asexual vs. sexual). On the other hand, clear evidence was found for phenotypic plasticity in mode of reproduction, as polyps inferred to be clones differed in reproductive mode. Moreover, we detected two cases of apparent sex change (males and females found within the same clonal lineages) in this species with supposedly stable sexes. Our study describes population genetic structure in Hydra for the first time, highlights the role of phenotypic plasticity in generating patterns of life history variation, and contributes to understanding the evolution of reproductive mode-dependent life history variation in coexisting asexual and sexual forms.

Age-dependent plasticity in reproductive investment, regeneration capacity and survival in a partially clonal animal (Hydra oligactis).

Asexual reproduction diversifies life-history priorities and is associated with unusual reproduction and somatic maintenance patterns, such as constant fertility with age, extensive regeneration ability and negligible senescence. While age-dependent plasticity in relative allocation to sexual versus asexual reproductive modes is relatively well studied, the modulation of somatic maintenance traits in parallel with age-dependent reproduction is much less well understood in clonal or partially clonal animals. Here, we asked how age-dependent investment into sexual and asexual reproduction co-varies with somatic maintenance such as regeneration in a partially clonal freshwater cnidarian Hydra oligactis, a species with remarkable regeneration abilities and experimentally inducible sex. We induced gametogenesis by lowering temperature at two ages, 1 or 4 weeks after detachment from an asexual parent, in animals of a male and a female clone. Then we measured phenotypically asexual and sexual reproductive traits (budding rate, start day and number of sexual organs) together with head regeneration rate, survival and the cellular background of these traits (number of reproductive and interstitial stem cells) for 2 or 5 months. Younger animals had higher asexual reproduction while individuals in the older group had more intensive gametogenesis and reproductive cell production. In parallel with these age-dependent reproductive differences, somatic maintenance of older individuals was also impacted: head regeneration, survival and interstitial stem cell numbers were reduced compared to younger polyps. Some of the traits investigated showed an ontogenetic effect, suggesting that age-dependent plasticity and a fixed ontogenetic response might both contribute to differences between age groups. We show that in H. oligactis asexual reproduction coupled with higher somatic maintenance is prioritized earlier in life, while sexual reproduction with higher maintenance costs occurs later if sex is induced. These findings confirm general life-history theory predictions on resource allocation between somatic maintenance and sexual reproduction applying in a partially clonal species. At the same time, our study also highlights the age-dependent integration of these resource allocation decisions with sexual/asexual strategies. Accounting for age-related differences might enhance repeatability of research done with clonal individuals derived from mass cultures.

Mechanisms related to sexual determination by temperature in reptiles.

A number of strategies have emerged that appear to relate to the evolution of mechanisms for sexual determination in vertebrates, among which are genetic sex determination caused by sex chromosomes and environmental sex determination, where environmental factors influence the phenotype of the sex of an individual. Within the reptile group, some orders such as: Chelonia, Crocodylia, Squamata and Rhynchocephalia, manifest one of the most intriguing and exciting environmental sexual determination mechanisms that exists, comprising temperature-dependent sex determination (TSD), where the temperature of incubation that the embryo experiences during its development is fundamental to establishing the sex of the individual. This makes them an excellent model for the study of sexual determination at the molecular, cellular and physiological level, as well as in terms of their implications at an evolutionary and ecological level. There are different hypotheses concerning how this process is triggered and this review aims to describe any new contributions to particular TSD hypotheses, analyzing them from the "eco-evo-devo" perspective.

Molecular Evolution of Insect Sociality: An Eco-Evo-Devo Perspective.

The evolution of eusociality is a perennial issue in evolutionary biology, and genomic advances have fueled steadily growing interest in the genetic changes underlying social evolution. Along with a recent flurry of research on comparative and evolutionary genomics in different eusocial insect groups (bees, ants, wasps, and termites), several mechanistic explanations have emerged to describe the molecular evolution of eusociality from solitary behavior. These include solitary physiological ground plans, genetic toolkits of deeply conserved genes, evolutionary changes in protein-coding genes, cis regulation, and the structure of gene networks, epigenetics, and novel genes. Despite this proliferation of ideas, there has been little synthesis, even though these ideas are not mutually exclusive and may in fact be complementary. We review available data on molecular evolution of insect sociality and highlight key biotic and abiotic factors influencing social insect genomes. We then suggest both phylogenetic and ecological evolutionary developmental biology (eco-evo-devo) perspectives for a more synthetic view of molecular evolution in insect societies.

Fast running restricts evolutionary change of the vertebral column in mammals.

The mammalian vertebral column is highly variable, reflecting adaptations to a wide range of lifestyles, from burrowing in moles to flying in bats. However, in many taxa, the number of trunk vertebrae is surprisingly constant. We argue that this constancy results from strong selection against initial changes of these numbers in fast running and agile mammals, whereas such selection is weak in slower-running, sturdier mammals. The rationale is that changes of the number of trunk vertebrae require homeotic transformations from trunk into sacral vertebrae, or vice versa, and mutations toward such transformations generally produce transitional lumbosacral vertebrae that are incompletely fused to the sacrum. We hypothesize that such incomplete homeotic transformations impair flexibility of the lumbosacral joint and thereby threaten survival in species that depend on axial mobility for speed and agility. Such transformations will only marginally affect performance in slow, sturdy species, so that sufficient individuals with transitional vertebrae survive to allow eventual evolutionary changes of trunk vertebral numbers. We present data on fast and slow carnivores and artiodactyls and on slow afrotherians and monotremes that strongly support this hypothesis. The conclusion is that the selective constraints on the count of trunk vertebrae stem from a combination of developmental and biomechanical constraints.

Major ontogenetic transitions during Volvox (Chlorophyta) evolution: when and where might they have occurred?

This paper represents an attempt to unify data from various lines of Volvox research: developmental biology, biogeography, and evolution. Several species (such as Volvox carteri and Volvox spermatosphaera) are characterized by rapid divisions of asexual reproductive cells, which may proceed in darkness. By contrast, several other species (such as Volvox aureus, Volvox globator, and Volvox tertius) exhibit slow and light/dependent divisions. The transition from the former pattern of asexual life cycle to the latter one has occurred in three lineages of the genus Volvox. Since V. aureus (unlike V. carteri) is able to complete the life cycle at a short photoperiod (8 h light/16 h dark regime), it is reasonable to suggest that the abovementioned evolutionary transitions might have occurred as adaptations to short winter days in high latitudes under warm climate conditions in the deep past. In the case of the lineage leading to V. tertius + Volvox dissipatrix, the crucial reorganizations of asexual life cycle might have occurred between about 45 and 60 million years ago in relatively high latitudes of Southern Hemisphere.

The role of vital dietary biomolecules in eco-evo-devo dynamics.

The physiological dependence of animals on dietary intake of vitamins, amino acids, and fatty acids is ubiquitous. Sharp differences in the availability of these vital dietary biomolecules among different resources mean that consumers must adopt a range of strategies to meet their physiological needs. We review the emerging work on omega-3 long-chain polyunsaturated fatty acids, focusing predominantly on predator-prey interactions, to illustrate that trade-off between capacities to consume resources rich in vital biomolecules and internal synthesis capacity drives differences in phenotype and fitness of consumers. This can then feedback to impact ecosystem functioning. We outline how focus on vital dietary biomolecules in eco-eco-devo dynamics can improve our understanding of anthropogenic changes across multiple levels of biological organization.

Transcriptomic thermal plasticity underlying gonadal development in a turtle with ZZ/ZW sex chromosomes despite canalized genotypic sex determination.

Understanding genome-wide responses to environmental conditions during embryogenesis is essential for discerning the evolution of developmental plasticity and canalization, two processes generating phenotypic variation targeted by natural selection. Here, we present the first comparative trajectory analysis of matched transcriptomic developmental time series from two reptiles incubated under identical conditions, a turtle with a ZZ/ZW system of genotypic sex determination (GSD), Apalone spinifera, and a turtle with temperature-dependent sex determination (TSD), Chrysemys picta. Results from our genome-wide, hypervariate gene expression analysis of sexed embryos across five developmental stages revealed that substantial transcriptional plasticity in the developing gonads can persist for >145 Myr, long after the canalization of sex determination via the evolution of sex chromosomes, while some gene-specific thermal sensitivity drifts or evolves anew. Such standing thermosensitivity represents an underappreciated evolutionary potential harbored by GSD species that may be co-opted during future adaptive shifts in developmental programing, such as a GSD to TSD reversal, if favored by ecological conditions. Additionally, we identified novel candidate regulators of vertebrate sexual development in GSD reptiles, including sex-determining candidate genes in a ZZ/ZW turtle.

Genome of the Rio Pearlfish (Nematolebias whitei), a bi-annual killifish model for Eco-Evo-Devo in extreme environments.

The Rio Pearlfish, Nematolebias whitei, is a bi-annual killifish species inhabiting seasonal pools in the Rio de Janeiro region of Brazil that dry twice per year. Embryos enter dormant diapause stages in the soil, waiting for the inundation of the habitat which triggers hatching and commencement of a new life cycle. Rio Pearlfish represents a convergent, independent origin of annualism from other emerging killifish model species. While some transcriptomic datasets are available for Rio Pearlfish, thus far, a sequenced genome has been unavailable. Here, we present a high quality, 1.2 Gb chromosome-level genome assembly, genome annotations, and a comparative genomic investigation of the Rio Pearlfish as representative of a vertebrate clade that evolved environmentally cued hatching. We show conservation of 3D genome structure across teleost fish evolution, developmental stages, tissues, and cell types. Our analysis of mobile DNA shows that Rio Pearlfish, like other annual killifishes, possesses an expanded transposable element profile with implications for rapid aging and adaptation to harsh conditions. We use the Rio Pearlfish genome to identify its hatching enzyme gene repertoire and the location of the hatching gland, a key first step in understanding the developmental genetic control of hatching. The Rio Pearlfish genome expands the comparative genomic toolkit available to study convergent origins of seasonal life histories, diapause, and rapid aging phenotypes. We present the first set of genomic resources for this emerging model organism, critical for future functional genetic, and multiomic explorations of "Eco-Evo-Devo" phenotypes of resilience and adaptation to extreme environments.

Non-pollinator selection for a floral homeotic mutant conferring loss of nectar reward in Aquilegia coerulea.

Here, we describe a polymorphic population of Aquilegia coerulea with a naturally occurring floral homeotic mutant, A. coerulea var. daileyae, where the characteristic petals with nectar spurs are replaced with a second set of sepals. Although it would be expected that this loss of pollinator reward would be disadvantageous to the mutant, we find that it has reached relatively high frequency (∼25%) and is under strong, positive selection across multiple seasons (s = 0.17-0.3) primarily due to reduced floral herbivory. We identify the underlying locus (APETALA3-3) and multiple causal loss-of-function mutations indicating an ongoing soft sweep. Elevated linkage disequilibrium around the two most common causal alleles indicates that positive selection has been occurring for many generations. Lastly, genotypic frequencies at AqAP3-3 indicate a degree of positive assortative mating by morphology. Together, these data provide both a compelling example that large-scale discontinuous morphological changes differentiating taxa can occur due to single mutations and a particularly clear example of linking genotype, phenotype, and fitness.

Slipper snail tales: How Crepidula fornicata and Crepidula atrasolea became model molluscs.

Despite the great abundance and diversity of molluscs, only a few have attained "model research organism" status. One of those species is the slipper snail Crepidula fornicata. Its embryos were first used for classical lineage tracing studies in the late 19th century, and over a 100 years later they were "re-discovered" by our labs and used for modern fate mapping, gene perturbation, in vivo imaging, transcriptomics, and the first application of CRISPR/Cas9-mediated genome editing among the Spiralia/Lophotrochozoa. Simultaneously, other labs made extensive examinations of taxonomy, phylogeny, ecology, life-history, mode of development, larval feeding behavior, and responses to the environment in members of the family Calyptraeidae, which includes the genus Crepidula. Recently, we developed tools, resources, and husbandry protocols for another, direct-developing species, Crepidula atrasolea. This species is an ideal "lab rat" among molluscs. Together these species will be valuable for probing the cellular and molecular mechanisms underlying molluscan biology and evolution.

My road to the ants: A model clade for eco-evo-devo.

This chapter is the story of how I pioneered ants as a system for studying eco-evo-devo, a field that integrates developmental biology with ecology and evolutionary biology. One aim of eco-evo-devo is to understand how the interactions between genes and their environments during development facilitates the origin and evolution of novel phenotypes. In a series of six parts, I review some of the key discoveries from my lab on how novel worker caste systems in ants--soldiers and supersoldiers--originated and evolved. I also discuss some of the ideas that emerged from these discoveries, including the role that polyphenisms, hidden developmental potentials, and rudimentary organs play in facilitating developmental and evolutionary change. As superorganisms, I argue that ants are uniquely positioned to reveal types of variation that are often difficult to observe in nature. In doing so, they have the potential to transform our view of biology and provide new perspectives in medicine, agriculture, and biodiversity conservation. With my story I hope to inspire the next generation of biologists to continue exploring the unknown regions of phenotypic space to solve some of our most pressing societal challenges.

The origin of wing polyphenism in ants: An eco-evo-devo perspective.

The evolution of eusociality, where solitary individuals integrate into a single colony, is a major transition in individuality. In ants, the origin of eusociality coincided with the origin of a wing polyphenism approximately 160 million years ago, giving rise to colonies with winged queens and wingless workers. As a consequence, both eusociality and wing polyphenism are nearly universal features of all ants. Here, we synthesize fossil, ecological, developmental, and evolutionary data in an attempt to understand the factors that contributed to the origin of wing polyphenism in ants. We propose multiple models and hypotheses to explain how wing polyphenism is orchestrated at multiple levels, from environmental cues to gene networks. Furthermore, we argue that the origin of wing polyphenism enabled the subsequent evolution of morphological diversity across the ants. We finally conclude by outlining several outstanding questions for future work.

Active Inferants: An Active Inference Framework for Ant Colony Behavior.

In this paper, we introduce an active inference model of ant colony foraging behavior, and implement the model in a series of in silico experiments. Active inference is a multiscale approach to behavioral modeling that is being applied across settings in theoretical biology and ethology. The ant colony is a classic case system in the function of distributed systems in terms of stigmergic decision-making and information sharing. Here we specify and simulate a Markov decision process (MDP) model for ant colony foraging. We investigate a well-known paradigm from laboratory ant colony behavioral experiments, the alternating T-maze paradigm, to illustrate the ability of the model to recover basic colony phenomena such as trail formation after food location discovery. We conclude by outlining how the active inference ant colony foraging behavioral model can be extended and situated within a nested multiscale framework and systems approaches to biology more generally.

Anemonefish, a model for Eco-Evo-Devo.

Anemonefish, are a group of about 30 species of damselfish (Pomacentridae) that have long aroused the interest of coral reef fish ecologists. Combining a series of original biological traits and practical features in their breeding that are described in this paper, anemonefish are now emerging as an experimental system of interest for developmental biology, ecology and evolutionary sciences. They are small sized and relatively easy to breed in specific husbandries, unlike the large-sized marine fish used for aquaculture. Because they live in highly structured social groups in sea anemones, anemonefish allow addressing a series of relevant scientific questions such as the social control of growth and sex change, the mechanisms controlling symbiosis, the establishment and variation of complex color patterns, and the regulation of aging. Combined with the use of behavioral experiments, that can be performed in the lab or directly in the wild, as well as functional genetics and genomics, anemonefish provide an attractive experimental system for Eco-Evo-Devo.