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.

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.

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.

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.

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.

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.

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.

"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 Quantitative Computational Framework for Allopolyploid Single-Cell Data Integration and Core Gene Ranking in Development.

Polyploidization drives regulatory and phenotypic innovation. How the merger of different genomes contributes to polyploid development is a fundamental issue in evolutionary developmental biology and breeding research. Clarifying this issue is challenging because of genome complexity and the difficulty in tracking stochastic subgenome divergence during development. Recent single-cell sequencing techniques enabled probing subgenome-divergent regulation in the context of cellular differentiation. However, analyzing single-cell data suffers from high error rates due to high dimensionality, noise, and sparsity, and the errors stack up in polyploid analysis due to the increased dimensionality of comparisons between subgenomes of each cell, hindering deeper mechanistic understandings. In this study, we develop a quantitative computational framework, called "pseudo-genome divergence quantification" (pgDQ), for quantifying and tracking subgenome divergence directly at the cellular level. Further comparing with cellular differentiation trajectories derived from single-cell RNA sequencing data allows for an examination of the relationship between subgenome divergence and the progression of development. pgDQ produces robust results and is insensitive to data dropout and noise, avoiding high error rates due to multiple comparisons of genes, cells, and subgenomes. A statistical diagnostic approach is proposed to identify genes that are central to subgenome divergence during development, which facilitates the integration of different data modalities, enabling the identification of factors and pathways that mediate subgenome-divergent activity during development. Case studies have demonstrated that applying pgDQ to single-cell and bulk tissue transcriptomic data promotes a systematic and deeper understanding of how dynamic subgenome divergence contributes to developmental trajectories in polyploid 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.

Incubation time, embryonic development and the vomeronasal organ of the Laysan albatross (Phoebastria immutabilis).

The effect of lengthened incubation periods on embryonic development, especially vestigial structures, is poorly understood. An example of which is the avesuchian vomeronasal organ (VNO), a nasal chemosensory organ found in many tetrapods but absent in adult avesuchians (crocodilians and birds) in whom it is presumed to be a transitory fetal structure. The Laysan Albatross (Phoebastria immutabilis) has an incubation period of their eggs of about 65 days. This incubation period is twice that of domestic fowl, wherein a putative VNO has been documented as an epithelial thickening. The purpose of this study is to document the development of a putative VNO in the albatross. Serial histological sections of nine albatross embryonic heads, across 6 stages (representing days 19 to 32: stages 31-39), were examined. A paired putative VNO was present as a short, tubular structure in the anterodorsal aspect on either side of the nasal septum from stage 32 onwards, getting steadily longer in later specimens. At the earliest stages, the epithelial walls of the tube resemble a neuroepithelium, but then becomes thinner and simpler in morphology. Based on our available age range, it is unclear whether it persists as a rudimentary structure (like that of the human) or if it is a transitory structure (like in chickens) in these mid embryonic stages. Though future studies must determine the fate of the Laysan albatross VNO (e.g., is it retained postnatally?), the role of incubation period length on embryonic development is a bigger question to be explored.

Comparative transcriptomics suggests a potential realizator gene for carapace expansion in longtail tadpole shrimp, Triops longicaudatus (Branchiopoda: Notostraca).

The origin of morphological innovation has been extensively studied within evolutionary developmental biology (evo-devo). Recent studies have demonstrated that the developmental module for double-layered epithelial outgrowths is conserved between the insect wings and branchiopod crustacean carapace, thereby introducing homology among these diverse structures. However, evo-devo studies on the branchiopod crustacean carapace have been primarily limited to a single species, the water flea Daphnia magna, leaving the gene regulatory network governing carapace development not comprehensively understood. Furthermore, realizator genes downstream of the character identity mechanism (ChIM) for bilayered epithelial development remain inadequately described. In this study, we analyzed tissue-specific transcriptional profiles in the developing longtail tadpole shrimp, Triops longicaudatus. We observed significant upregulation of papilin in the carapace-bearing head, along with its expression in both the carapace and the trunk limb lobes. Based on these results, we hypothesize that differential expression of papilin is involved in the disproportional growth of Triops carapace. Our findings will contribute to elucidating the diversification of double-layered epithelial outgrowths across distant arthropod lineages.

TF-High-Evolutionary: In Vivo Mutagenesis of Gene Regulatory Networks for the Study of the Genetics and Evolution of the Drosophila Regulatory Genome.

Understanding the evolutionary potential of mutations in gene regulatory networks is essential to furthering the study of evolution and development. However, in multicellular systems, genetic manipulation of regulatory networks in a targeted and high-throughput way remains challenging. In this study, we designed TF-High-Evolutionary (HighEvo), a transcription factor (TF) fused with a base editor (activation-induced deaminase), to continuously induce germline mutations at TF-binding sites across regulatory networks in Drosophila. Populations of flies expressing TF-HighEvo in their germlines accumulated mutations at rates an order of magnitude higher than natural populations. Importantly, these mutations accumulated around the targeted TF-binding sites across the genome, leading to distinct morphological phenotypes consistent with the developmental roles of the tagged TFs. As such, this TF-HighEvo method allows the interrogation of the mutational space of gene regulatory networks at scale and can serve as a powerful reagent for experimental evolution and genetic screens focused on the regulatory genome.

Auts2 enhances neurogenesis and promotes expansion of the cerebral cortex.

INTRODUCTION: The AUTS2 gene is associated with various neurodevelopmental and psychiatric disorders and has been suggested to play a role in acquiring human-specific traits. Functional analyses of Auts2 knockout mice have focused on postmitotic neurons, and the reported phenotypes do not faithfully recapitulate the whole spectrum of AUTS2-related human diseases. OBJECTIVE: The objective of the study is to assess the role of AUTS2 in the biology of neural progenitor cells, cortical neurogenesis and expansion; and understand how its deregulation leads to neurological disorders. METHODS: We screened the literature and conducted a time point analysis of AUTS2 expression during cortical development. We used in utero electroporation to acutely modulate the expression level of AUTS2 in the developing cerebral cortex in vivo, and thoroughly characterized cortical neurogenesis and morphogenesis using immunofluorescence, cell tracing and sorting, transcriptomic profiling, and gene ontology enrichment analyses. RESULTS: In addition to its expression in postmitotic neurons, we showed that AUTS2 is also expressed in neural progenitor cells at the peak of neurogenesis. Upregulation of AUTS2 dramatically altered the differentiation program and fate determination of cortical progenitors. Notably, it increased the number of basal progenitors and neurons and changed the expression of hundreds of genes, among which 444 have not been implicated in mouse brain development or function. CONCLUSION: The study provides evidence that AUTS2 is expressed in germinal zones and plays a key role in fate decision of neural progenitor cells with impact on corticogenesis. It also presents comprehensive lists of AUTS2 target genes thus advancing the molecular mechanisms underlying AUTS2-associated diseases and the evolutionary expansion of the cerebral cortex.