Evolution and development of complex floral displays.

Flowering plants - angiosperms - display an astounding diversity of floral features, which have evolved in response to animal pollination and have resulted in the most species-rich plant clade. Combinations of macroscale (e.g. colour, symmetry, organ number) and microscale (e.g. cell type, tissue patterning) features often lead to highly elaborate floral displays. Most studies have focused on model species with simple floral displays to uncover the genetic and evolutionary mechanisms involved in flower evolution, yet few studies have focused on complex floral displays. Here, we review current knowledge on the development and evolution of complex floral displays. We review gene regulatory networks involved in four developmental pathways contributing to overall floral display (inflorescence architecture, organ identity, flower symmetry and flower colour) in classical plant models. We then discuss how evolutionary modification of one or more of these pathways has resulted in the production of a range of complex floral displays. Finally, we explore modular systems in which multiple pathways have been modified simultaneously, generating the most elaborate floral displays.

Transcriptomic analyses in the gametophytes of the apomictic fern Dryopteris affinis.

MAIN CONCLUSION: A novel genomic map of the apogamous gametophyte of the fern Dryopteris affinis unlocks oldest hindrance with this complex plant group, to gain insight into evo-devo approaches. The gametophyte of the fern Dryopteris affinis ssp. affinis represents a good model to explore the molecular basis of vegetative and reproductive development, as well as stress responses. Specifically, this fern reproduces asexually by apogamy, a peculiar case of apomixis whereby a sporophyte forms directly from a gametophytic cell without fertilization. Using RNA-sequencing approach, we have previously annotated more than 6000 transcripts. Here, we selected 100 of the inferred proteins homolog to those of Arabidopsis thaliana, which were particularly interesting for a detailed study of their potential functions, protein-protein interactions, and distance trees. As expected, a plethora of proteins associated with gametogenesis and embryogenesis in angiosperms, such as FERONIA (FER) and CHROMATING REMODELING 11 (CHR11) were identified, and more than a dozen candidates potentially involved in apomixis, such as ARGONAUTE family (AGO4, AGO9, and AGO 10), BABY BOOM (BBM), FASCIATED STEM4 (FAS4), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), and MATERNAL EFFECT EMBRYO ARREST29 (MEE29). In addition, proteins involved in the response to biotic and abiotic stresses were widely represented, as shown by the enrichment of heat-shock proteins. Using the String platform, the interactome revealed that most of the protein-protein interactions were predicted based on experimental, database, and text mining datasets, with MULTICOPY SUPPRESSOR OF IRA4 (MSI4) showing the highest number of interactions: 16. Lastly, some proteins were studied through distance trees by comparing alignments with respect to more distantly or closely related plant groups. This analysis identified DCL4 as the most distant protein to the predicted common ancestor. New genomic information in relation to gametophyte development, including apomictic reproduction, could expand our current vision of evo-devo approaches.

Fin elaboration via anterior-posterior constraint by hhip on Hedgehog signaling in teleosts.

Pectoral fins, the anterior paired fins in fish, have enhanced maneuvering abilities due to morphological changes. Teleosts have fewer radial bones in their pectoral fins than basal species, resulting in more elaborate fins. The mechanism behind this radial constraint change in teleosts is unclear. Here, we found that mutations in hhip, an antagonist of Hedgehog signaling, lead to an increase in radial bones in a localized region. The shh genes, ligands of Hedgehog signaling, were expressed coinciding with notable hhip expression specifically during early development. We suggest that a negative feedback effect of Hedgehog signaling by hhip regulates the constraint of the pectoral fin in zebrafish. Additionally, the expression reanalysis of Hhip-related genes implied that the notable hhip expression during early development was a characteristic of zebrafish, not observed in basal species. Region-specific expression of Hox13 genes indicated that hhip-/- zebrafish expanded the median region of the pectoral fin, analogous to the region with abundant radials in basal species. The data underscore potential morphological evolution through constrained diversity.

Co-option of the trichome-forming network initiated the evolution of a morphological novelty in Drosophila eugracilis.

Identifying the molecular origins by which new morphological structures evolve is one of the long-standing problems in evolutionary biology. To date, vanishingly few examples provide a compelling account of how new morphologies were initially formed, thereby limiting our understanding of how diverse forms of life derived their complex features. Here, we provide evidence that the large projections on the Drosophila eugracilis phallus that are implicated in sexual conflict have evolved through the partial co-option of the trichome genetic network. These unicellular apical projections on the phallus postgonal sheath are reminiscent of trichomes that cover the Drosophila body but are up to 20-fold larger in size. During their development, they express the transcription factor Shavenbaby, the master regulator of the trichome network. Consistent with the co-option of the Shavenbaby network during the evolution of the D. eugracilis projections, somatic mosaic CRISPR-Cas9 mutagenesis shows that shavenbaby is necessary for their proper length. Moreover, misexpression of Shavenbaby in the sheath of D. melanogaster, a naive species that lacks these projections, is sufficient to induce small trichomes. These induced projections rely on a genetic network that is shared to a large extent with the D. eugracilis projections, indicating its partial co-option but also some genetic rewiring. Thus, by leveraging a genetically tractable evolutionary novelty, our work shows that the trichome-forming network is flexible enough that it can be partially co-opted in a new context and subsequently refined to produce unique apical projections that are barely recognizable compared with their simpler ancestral beginnings.

The legacy and evolvability of Pere Alberch's ideas.

Pere Alberch played a pivotal role in shaping the field of evolutionary developmental biology during the 1980s and 1990s. Whereas initially his contributions were sidelined by the empirical advancements of the molecular revolution in developmental and evolutionary biology, his theoretical insights have left a lasting impact on the discipline. This article provides a comprehensive review of the legacy and evolvability of Alberch's ideas in contemporary evo-devo, which included the study of morphogenesis as the proper level of developmental causation, the interplay between developmental constraints and natural selection, the epistemic role of teratologies, the origin of evolutionary novelties and the concept of evolvability.

The logic of monsters: development and morphological diversity in stem-cell-based embryo models.

Organoids and stem-cell-based embryo models (SEMs) are imperfect organ or embryo representations that explore a much larger space of possible forms, or morphospace, compared to their in vivo counterparts. Here, we discuss SEM biology in light of seminal work by Pere Alberch, a leading figure in early evo-devo, interpreting SEMs as developmental 'monstrosities' in the Alberchian sense. Alberch suggested that ordered patterns in aberrant development-i.e. 'the logic of monsters'-reveal developmental constraints on possible morphologies. In the same vein, we detail how SEMs have begun to shed light on structural features of normal development, such as developmental variability, the relative importance of internal versus external constraints, boundary conditions and design principles governing robustness and canalization. We argue that SEMs represent a powerful experimental tool to explore and expand developmental morphospace and propose that the 'monstrosity' of SEMs can be leveraged to uncover the 'hidden' rules and developmental constraints that robustly shape and pattern the embryo.

The ivory lncRNA regulates seasonal color patterns in buckeye butterflies.

Long noncoding RNAs (lncRNAs) are transcribed elements increasingly recognized for their roles in regulating gene expression. Thus far, however, we have little understanding of how lncRNAs contribute to evolution and adaptation. Here, we show that a conserved lncRNA, ivory, is an important color patterning gene in the buckeye butterfly Junonia coenia. ivory overlaps with cortex, a locus linked to multiple cases of crypsis and mimicry in Lepidoptera. Along with a companion paper by Livraghi et al., we argue that ivory, not cortex, is the color pattern gene of interest at this locus. In J. coenia, a cluster of cis-regulatory elements (CREs) in the first intron of ivory are genetically associated with natural variation in seasonal color pattern plasticity, and targeted deletions of these CREs phenocopy seasonal phenotypes. Deletions of different ivory CREs produce other distinct phenotypes as well, including loss of melanic eyespot rings, and positive and negative changes in overall wing pigmentation. We show that the color pattern transcription factors Spineless, Bric-a-brac, and Ftz-f1 bind to the ivory promoter during wing pattern development, suggesting that they directly regulate ivory. This case study demonstrates how cis-regulation of a single noncoding RNA can exert diverse and nuanced effects on the evolution and development of color patterns, including modulating seasonally plastic color patterns.

Deep-time origin of tympanic hearing in crown reptiles.

The invasion of terrestrial ecosystems by tetrapods (c. 375 million years [Ma]) represents one of the major evolutionary transitions in the history of life on Earth. The success of tetrapods on land is linked to evolutionary novelties. Among these, the evolution of a tympanic ear contributed to mitigating the problem of an impedance mismatch between the air and the fluid embedding sound-detecting hair cells in the inner ear.1,2,3 Pioneering studies advocated that similarities in the tympanic ear of tetrapods could only result from a single origin of this structure in the group,4,5 an idea later challenged by paleontological and developmental data.4,6,7,8 Current evidence suggests that this sensory structure evolved independently in amphibians, mammals, and reptiles,1,6 but it remains uncertain how many times tympanic hearing originated in crown reptiles.9,10 We combine developmental information with paleontological data to evaluate the evolution of the tympanic ear in reptiles from two complementary perspectives. Phylogenetically informed ancestral reconstruction analyses of a taxonomically broad sample of early reptiles point to the presence of a tympanic membrane as the ancestral condition of the crown group. Consistently, comparative analyses using embryos of lizards and crocodylians reveal similarities, including the formation of the tympanic membrane within the second pharyngeal arch, which has been previously reported for birds. Therefore, both our developmental and paleontological data suggest a single origin for the tympanic middle ear in the group, challenging the current paradigm of multiple acquisitions of tympanic hearing in living reptiles.

Understanding developmental system drift.

Developmental system drift (DSD) occurs when the genetic basis for homologous traits diverges over time despite conservation of the phenotype. In this Review, we examine the key ideas, evidence and open problems arising from studies of DSD. Recent work suggests that DSD may be pervasive, having been detected across a range of different organisms and developmental processes. Although developmental research remains heavily reliant on model organisms, extrapolation of findings to non-model organisms can be error-prone if the lineages have undergone DSD. We suggest how existing data and modelling approaches may be used to detect DSD and estimate its frequency. More direct study of DSD, we propose, can inform null hypotheses for how much genetic divergence to expect on the basis of phylogenetic distance, while also contributing to principles of gene regulatory evolution.

Ancient Secretory Pathways Contributed to the Evolutionary Origin of an Ecologically Impactful Bioluminescence System.

Evolutionary innovations in chemical secretion-such as the production of secondary metabolites, pheromones, and toxins-profoundly impact ecological interactions across a broad diversity of life. These secretory innovations may involve a "legacy-plus-innovation" mode of evolution, whereby new biochemical pathways are integrated with conserved secretory processes to create novel products. Among secretory innovations, bioluminescence is important because it evolved convergently many times to influence predator-prey interactions, while often producing courtship signals linked to increased rates of speciation. However, whether or not deeply conserved secretory genes are used in secretory bioluminescence remains unexplored. Here, we show that in the ostracod Vargula tsujii, the evolutionary novel c-luciferase gene is co-expressed with many conserved genes, including those related to toxin production and high-output protein secretion. Our results demonstrate that the legacy-plus-innovation mode of secretory evolution, previously applied to sensory modalities of olfaction, gustation, and nociception, also encompasses light-producing signals generated by bioluminescent secretions. This extension broadens the paradigm of secretory diversification to include not only chemical signals but also bioluminescent light as an important medium of ecological interaction and evolutionary innovation.

Developmental bias as a cause and consequence of adaptive radiation and divergence.

Efforts to reconcile development and evolution have demonstrated that development is biased, with phenotypic variation being more readily produced in certain directions. However, how this "developmental bias" can influence micro- and macroevolution is poorly understood. In this review, we demonstrate that defining features of adaptive radiations suggest a role for developmental bias in driving adaptive divergence. These features are i) common ancestry of developmental systems; ii) rapid evolution along evolutionary "lines of least resistance;" iii) the subsequent repeated and parallel evolution of ecotypes; and iv) evolutionary change "led" by biased phenotypic plasticity upon exposure to novel environments. Drawing on empirical and theoretical data, we highlight the reciprocal relationship between development and selection as a key driver of evolutionary change, with development biasing what variation is exposed to selection, and selection acting to mold these biases to align with the adaptive landscape. Our central thesis is that developmental biases are both the causes and consequences of adaptive radiation and divergence. We argue throughout that incorporating development and developmental bias into our thinking can help to explain the exaggerated rate and scale of evolutionary processes that characterize adaptive radiations, and that this can be best achieved by using an eco-evo-devo framework incorporating evolutionary biology, development, and ecology. Such a research program would demonstrate that development is not merely a force that imposes constraints on evolution, but rather directs and is directed by evolutionary forces. We round out this review by highlighting key gaps in our understanding and suggest further research programs that can help to resolve these issues.

The case against simplistic genetic explanations of evolution.

Humans are curious to understand the causes of traits that distinguish us from other animals and that distinguish vastly different species from one another. We also have a proclivity for simple stories and sometimes tend toward seeking and accepting simple genetic explanations for large evolutionary shifts, even to a single gene. Here, I reveal how a biased expectation of mechanistic simplicity threads through the long history of evolutionary and developmental genetics. I argue, however, that expecting a simple mechanism threatens a deeper understanding of evolution, and I define the limitations for interpreting experimental evidence in evolutionary developmental genetics.

Delphinium as a model for development and evolution of complex zygomorphic flowers.

The complex zygomorphic flowers of the early-diverging eudicot Delphinium provide an opportunity to explore intriguing evolutionary, developmental, and genetic questions. The dorsal perianth organs, consisting of a spurred sepal and the nectar-bearing spurred petal(s) in Delphinium, contribute to the dorso-ventralization and zygomorphic flower morphology. The seamless integration of the two or three dorsal petaloid spurred organs is considered a synorganization, and the resulting organ complex is referred to as a hyperorgan. The hyperorgan shows variability within the tribe due to variation in the number, size, and shape of the spurs. Research in recent decades within this tribe has enhanced our understanding of morphological evolution of flowers. More recently, functional studies using the RNAi approach of Virus-Induced Gene Silencing (VIGS) have unraveled interesting results highlighting the role of gene duplication in the functional diversification of organ identity and symmetry genes. Research in this early-diverging eudicot genus bridges the gaps in understanding the morphological innovations that are mostly studied in model grass and core eudicot clades. This first comprehensive review synthesizes eco-evo-devo research on Delphinium, developing a holistic understanding of recent advancements and establishing the genus as an exceptional model for addressing fundamental questions in developmental genetics, particularly in the evolution of complex flowers. This progress highlights Delphinium's significant potential for future studies in this field.

Analysis of auxin responses in the fern Ceratopteris richardii identifies the developmental phase as a major determinant for response properties.

The auxin signaling molecule regulates a range of plant growth and developmental processes. The core transcriptional machinery responsible for auxin-mediated responses is conserved across all land plants. Genetic, physiological and molecular exploration in bryophyte and angiosperm model species have shown both qualitative and quantitative differences in auxin responses. Given the highly divergent ontogeny of the dominant gametophyte (bryophytes) and sporophyte (angiosperms) generations, however, it is unclear whether such differences derive from distinct phylogeny or ontogeny. Here, we address this question by comparing a range of physiological, developmental and molecular responses to auxin in both generations of the model fern Ceratopteris richardii. We find that auxin response in Ceratopteris gametophytes closely resembles that of a thalloid bryophyte, whereas the sporophyte mimics auxin response in flowering plants. This resemblance manifests both at the phenotypic and transcriptional levels. Furthermore, we show that disrupting auxin transport can lead to ectopic sporophyte induction on the gametophyte, suggesting a role for auxin in the alternation of generations. Our study thus identifies developmental phase, rather than phylogeny, as a major determinant of auxin response properties in land plants.

Deciphering the evolutionary development of the "Chinese lantern" within Solanaceae.

MAIN CONCLUSION: The key genetic variation underlying the evo-devo of ICS in Solanaceae may be further pinpointed using an integrated strategy of forward and reverse genetics studies under the framework of phylogeny. The calyx of Physalis remains persistent throughout fruit development. Post-flowering, the fruiting calyx is inflated rapidly to encapsulate the berry, giving rise to a "Chinese lantern" structure called inflated calyx syndrome (ICS). It is unclear how this novelty arises. Over the past 2 decades, the role of MADS-box genes in the evolutionary development (evo-devo) of ICS has mainly been investigated within Solanaceae. In this review, we analyze the main achievements, challenges, and new progress. ICS acts as a source for fruit development, provides a microenvironment to protect fruit development, and assists in long-distance fruit dispersal. ICS is a typical post-floral trait, and the onset of its development is triggered by specific developmental signals that coincide with fertilization. These signals can be replaced by exogenous gibberellin and cytokinin application. MPF2-like heterotopic expression and MBP21-like loss have been proposed to be two essential evolutionary events for ICS origin, and manipulating the related MADS-box genes has been shown to affect the ICS size, sepal organ identity, and/or male fertility, but not completely disrupt ICS. Therefore, the core genes or key links in the ICS biosynthesis pathways may have undergone secondary mutations during evolution, or they have not yet been pinpointed. Recently, we have made some encouraging progress in acquiring lantern mutants in Physalis floridana. In addition to technological innovation, we propose an integrated strategy to further analyze the evo-devo mechanisms of ICS in Solanaceae using forward and reverse genetics studies under the framework of phylogeny.

A Rhodnius prolixus catalytically inactive Calpain protease patterns the insect embryonic dorsal-ventral axis.

The calcium dependent Calpain proteases are modulatory enzymes with important roles in cell cycle control, development and immunity. In the fly model Drosophila melanogaster Calpain A cleaves Cactus/IkappaB and consequently modifies Toll signals during embryonic dorsal-ventral (DV) patterning. Here we explore the role of Calpains in the hemiptera Rhodnius prolixus, an intermediate germband insect where the Bone Morphogenetic Protein (BMP) instead of the Toll pathway plays a major role in DV patterning. Phylogenetic analysis of Calpains in species ranging from Isoptera to Diptera indicates an increase of Calpain sequences in the R. prolixus genome and other hemimetabolous species. One locus encoding each of the CalpC, CalpD and Calp7 families, and seven Calpain A/B loci are present in the R. prolixus genome. Several predicted R. prolixus Calpains display a unique architecture, such as loss of Calcium-binding EF-hand domains and loss of catalytic residues in the active site CysPc domain, yielding catalytically dead Calpains A/B. Knockdown for one of these inactive Calpains results in embryonic DV patterning defects, with expansion of ventral and lateral gene expression domains and consequent failure of germ band elongation. In conclusion, our results reveal that Calpains may exert a conserved function in insect DV patterning, despite the changing role of the Toll and BMP pathways in defining gene expression territories along the insect DV axis.

"The Logic of Monsters:" Pere Alberch and the Evolutionary Significance of Experimental Teratology.

This paper offers an historical introduction to Pere Alberch's evolutionary thought and his contributions to Evo-Devo, based on his unique approach to experimental teratology. We will take as our point of reference the teratogenic experiments developed by Alberch and Emily A. Gale during the 1980s, aimed at producing monstrous variants of frogs and salamanders. We will analyze his interpretation of the results of these experiments within the framework of the emergence of evolutionary developmental biology (or "Evo-Devo"). The aim is understand how Alberch interpreted teratological anomalies as highly revealing objects of study for understanding the development of organic form, not only in an ontogenetic sense-throughout embryonic development-but also phylogenetically-throughout the evolution of species. Alberch's interpretation of monsters reflects the influence of a long tradition of non-Darwinian evolutionary thought, which began in the nineteenth century and was continued in the twentieth century by people such as Richard Goldschmidt, Conrad H. Waddington, and Stephen Jay Gould. They all proposed various non-gradualist models of evolution, in which embryonic development played a central role. Following this tradition, Alberch argued that, in order to attain a correct understanding of the role of embryological development in evolution, it was necessary to renounce the gradualist paradigm associated with the Darwinian interpretation of evolution, which understood nature as a continuum. According to Alberch, the study of monstrous abnormalities was of great value in understanding how certain epigenetic restrictions in development could give rise to discontinuities and directionality in morphological transformations throughout evolution.

A long noncoding RNA at the cortex locus controls adaptive coloration in butterflies.

Evolutionary variation in the wing pigmentation of butterflies and moths offers striking examples of adaptation by crypsis and mimicry. The cortex locus has been independently mapped as the locus controlling color polymorphisms in 15 lepidopteran species, suggesting that it acts as a genomic hotspot for the diversification of wing patterns, but functional validation through protein-coding knockouts has proven difficult to obtain. Our study unveils the role of a long noncoding RNA (lncRNA) which we name ivory, transcribed from the cortex locus, in modulating color patterning in butterflies. Strikingly, ivory expression prefigures most melanic patterns during pupal development, suggesting an early developmental role in specifying scale identity. To test this, we generated CRISPR mosaic knock-outs in five nymphalid butterfly species and show that ivory mutagenesis yields transformations of dark pigmented scales into white or light-colored scales. Genotyping of Vanessa cardui germline mutants associates these phenotypes to small on-target deletions at the conserved first exon of ivory. In contrast, cortex germline mutant butterflies with confirmed null alleles lack any wing phenotype and exclude a color patterning role for this adjacent gene. Overall, these results show that a lncRNA gene acts as a master switch of color pattern specification and played key roles in the adaptive diversification of wing patterns in butterflies.

Age-associated growth control modifies leaf proximodistal symmetry and enabled leaf shape diversification.

Biological shape diversity is often manifested in modulation of organ symmetry and modification of the patterned elaboration of repeated shape elements.1,2,3,4,5 Whether and how these two aspects of shape determination are coordinately regulated is unclear.5,6,7 Plant leaves provide an attractive system to investigate this problem, because they often show asymmetries along the proximodistal (PD) axis of their blades, along which they can also produce repeated marginal outgrowths such as serrations or leaflets.1 One aspect of leaf shape diversity is heteroblasty, where the leaf form in a single genotype is modified with progressive plant age.8,9,10,11 In Arabidopsis thaliana, a plant with simple leaves, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) controls heteroblasty by activating CyclinD3 expression, thereby sustaining proliferative growth and retarding differentiation in adult leaves.12,13 However, the precise significance of SPL9 action for leaf symmetry and marginal patterning is unknown. By combining genetics, quantitative shape analyses, and time-lapse imaging, we show that PD symmetry of the leaf blade in A. thaliana decreases in response to an age-dependent SPL9 expression gradient, and that SPL9 action coordinately regulates the distribution and shape of marginal serrations and overall leaf form. Using comparative analyses, we demonstrate that heteroblastic growth reprogramming in Cardamine hirsuta, a complex-leafed relative of A. thaliana, also involves prolonging the duration of cell proliferation and delaying differentiation. We further provide evidence that SPL9 enables species-specific action of homeobox genes that promote leaf complexity. In conclusion, we identified an age-dependent layer of organ PD growth regulation that modulates leaf symmetry and has enabled leaf shape diversification.