Brief Communication: The Predictable Network Topology of Evolutionary Genomic Constraint.

Large-scale comparative genomics studies offer valuable resources for understanding both functional and evolutionary rate constraints. It is suggested that constraint aligns with the topology of genomic networks, increasing toward the center, with intermediate nodes combining relaxed constraint with higher contributions to the phenotype due to pleiotropy. However, this pattern has yet to be demonstrated in vertebrates. This study shows that constraint intensifies toward the network's center in placental mammals. Genes with rate changes associated with emergence of hibernation cluster mostly toward intermediate positions, with higher constraint in faster-evolving genes, which is indicative of a "sweet spot" for adaptation. If this trend holds universally, network node metrics could predict high-constraint regions even in clades lacking empirical constraint data.

Heat induces multiomic and phenotypic stress propagation in zebrafish embryos.

Heat alters biology from molecular to ecological levels, but may also have unknown indirect effects. This includes the concept that animals exposed to abiotic stress can induce stress in naive receivers. Here, we provide a comprehensive picture of the molecular signatures of this process, by integrating multiomic and phenotypic data. In individual zebrafish embryos, repeated heat peaks elicited both a molecular response and a burst of accelerated growth followed by a growth slowdown in concert with reduced responses to novel stimuli. Metabolomes of the media of heat treated vs. untreated embryos revealed candidate stress metabolites including sulfur-containing compounds and lipids. These stress metabolites elicited transcriptomic changes in naive receivers related to immune response, extracellular signaling, glycosaminoglycan/keratan sulfate, and lipid metabolism. Consequently, non-heat-exposed receivers (exposed to stress metabolites only) experienced accelerated catch-up growth in concert with reduced swimming performance. The combination of heat and stress metabolites accelerated development the most, mediated by apelin signaling. Our results prove the concept of indirect heat-induced stress propagation toward naive receivers, inducing phenotypes comparable with those resulting from direct heat exposure, but utilizing distinct molecular pathways. Group-exposing a nonlaboratory zebrafish line, we independently confirm that the glycosaminoglycan biosynthesis-related gene chs1 and the mucus glycoprotein gene prg4a, functionally connected to the candidate stress metabolite classes sugars and phosphocholine, are differentially expressed in receivers. This hints at the production of Schreckstoff-like cues in receivers, leading to further stress propagation within groups, which may have ecological and animal welfare implications for aquatic populations in a changing climate.

Investigating the effects of arginine methylation inhibitors on microdissected brain tumour biopsies maintained in a miniaturised perfusion system.

Arginine methylation is a post-translational modification that consists of the transfer of one or two methyl (CH3) groups to arginine residues in proteins. Several types of arginine methylation occur, namely monomethylation, symmetric dimethylation and asymmetric dimethylation, which are catalysed by different protein arginine methyltransferases (PRMTs). Inhibitors of PRMTs have recently entered clinical trials to target several types of cancer, including gliomas (NCT04089449). People with glioblastoma (GBM), the most aggressive form of brain tumour, are among those with the poorest quality of life and likelihood of survival of anyone diagnosed with cancer. There is currently a lack of (pre)clinical research on the possible application of PRMT inhibitors to target brain tumours. Here, we set out to investigate the effects of clinically-relevant PRMT inhibitors on GBM biopsies. We present a new, low-cost, easy to fabricate perfusion device that can maintain GBM tissue in a viable condition for at least eight days post-surgical resection. The miniaturised perfusion device enables the treatment of GBM tissue with PRMT inhibitors ex vivo, and we observed a two-fold increase in apoptosis in treated samples compared to parallel control experiments. Mechanistically, we show thousands of differentially expressed genes after treatment, and changes in the type of arginine methylation of the RNA binding protein FUS that are consistent with hundreds of differential gene splicing events. This is the first time that cross-talk between different types of arginine methylation has been observed in clinical samples after treatment with PRMT inhibitors.

Sex and gametogenesis stage are strong drivers of gene expression in Mytilus edulis exposed to environmentally relevant plasticiser levels and pH 7.7.

Plastic pollution and changes in oceanic pH are both pressing environmental issues. Little emphasis, however, has been placed on the influence of sex and gametogenesis stage when investigating the effects of such stressors. Here, we examined histology and molecular biomarkers of blue mussels Mytilus edulis exposed for 7 days to a pH 7.7 scenario (- 0.4 units) in combination with environmentally relevant concentrations (0, 0.5 and 50 µg/L) of the endocrine disrupting plasticiser di-2-ethylhexyl phthalate (DEHP). Through a factorial design, we investigated the gametogenesis cycle and sex-related expression of genes involved in pH homeostasis, stress response and oestrogen receptor-like pathways after the exposure to the two environmental stressors. As expected, we found sex-related differences in the proportion of developing, mature and spawning gonads in histological sections. Male gonads also showed higher levels of the acid-base regulator CA2, but females had a higher expression of stress response-related genes (i.e. sod, cat, hsp70). We found a significant effect of DEHP on stress response-related gene expression that was dependent on the gametogenesis stage, but there was only a trend towards downregulation of CA2 in response to pH 7.7. In addition, differences in gene expression between males and females were most pronounced in experimental conditions containing DEHP and/or acidified pH but never the control, indicating that it is important to consider sex and gametogenesis stage when studying the response of mussels to diverse stressors.

Functional genomics of abiotic environmental adaptation in lacertid lizards and other vertebrates.

Understanding the genomic basis of adaptation to different abiotic environments is important in the context of climate change and resulting short-term environmental fluctuations. Using functional and comparative genomics approaches, we here investigated whether signatures of genomic adaptation to a set of environmental parameters are concentrated in specific subsets of genes and functions in lacertid lizards and other vertebrates. We first identify 200 genes with signatures of positive diversifying selection from transcriptomes of 24 species of lacertid lizards and demonstrate their involvement in physiological and morphological adaptations to climate. To understand how functionally similar these genes are to previously predicted candidate functions for climate adaptation and to compare them with other vertebrate species, we then performed a meta-analysis of 1,100 genes under selection obtained from -omics studies in vertebrate species adapted to different abiotic factors. We found that the vertebrate gene set formed a tightly connected interactome, which was to 23% enriched in previously predicted functions of adaptation to climate, and to a large part (18%) involved in organismal stress response. We found a much higher degree of identical genes being repeatedly selected among different animal groups (43.6%), and of functional similarity and post-translational modifications than expected by chance, and no clear functional division between genes used for ectotherm and endotherm physiological strategies. In total, 171 out of 200 genes of Lacertidae were part of this network. These results highlight an important role of a comparatively small set of genes and their functions in environmental adaptation and narrow the set of candidate pathways and markers to be used in future research on adaptation and stress response related to climate change.

Thermal stress induces a positive phenotypic and molecular feedback loop in zebrafish embryos.

Aquatic organisms must cope with both rising and rapidly changing temperatures. These thermal changes can affect numerous traits, from molecular to ecological scales. Biotic stressors are already known to induce the release of chemical cues which trigger behavioural responses in other individuals. In this study, we infer whether fluctuating temperature, as an abiotic stressor, may similarly induce stress-like responses in individuals not directly exposed to the stressor. To test this hypothesis, zebrafish (Danio rerio) embryos were exposed for 24 h to fluctuating thermal stress, to medium in which another embryo was thermally stressed before ("stress medium"), and to a combination of these. Growth, behaviour, expression of molecular markers, and of whole-embryo cortisol were used to characterise the thermal stress response and its propagation between embryos. Both fluctuating high temperature and stress medium significantly accelerated development, by shifting stressed embryos from segmentation to pharyngula stages, and altered embryonic activity. Importantly, we found that the expression of sulfide:quinone oxidoreductase (SQOR), the antioxidant gene SOD1, and of interleukin-1ß (IL-1ß) were significantly altered by stress medium. This study illustrates the existence of positive thermal stress feedback loops in zebrafish embryos where heat stress can induce stress-like responses in conspecifics, but which might operate via different molecular pathways. If similar effects also occur under less severe heat stress regimes, this mechanism may be relevant in natural settings as well.

Consequences of combined exposure to thermal stress and the plasticiser DEHP in Mytilus spp. differ by sex.

Little is known about the combined effect of environmental factors and contaminants on commercially important marine species, and whether this effect differs by sex. In this study, blue mussels were exposed for seven days to both single and combined stressors (i.e., +3 °C elevated temperature and two environmentally relevant concentrations of the plastic softener DEHP, 0.5 and 50 µg/l) in a factorial design. Males were observed to be more sensitive to high temperature, demonstrated by the significant increase in out-of-season spawning gonads and higher gene expression of the antioxidant catalase and the estrogen receptor genes. On the other hand, while the gametogenesis cycle in females was more resilient than in males, DEHP exposure altered the estrogen-related receptor gene expression. We show that the combined stressors DEHP and increased temperature, in environmentally relevant magnitudes, have different consequences in male and female mussels, with the potential to impact the timing and breeding season success in Mytilus spp.

Aligning functional network constraint to evolutionary outcomes.

BACKGROUND: Functional constraint through genomic architecture is suggested to be an important dimension of genome evolution, but quantitative evidence for this idea is rare. In this contribution, existing evidence and discussions on genomic architecture as constraint for convergent evolution, rapid adaptation, and genic adaptation are summarized into alternative, testable hypotheses. Network architecture statistics from protein-protein interaction networks are then used to calculate differences in evolutionary outcomes on the example of genomic evolution in yeast, and the results are used to evaluate statistical support for these longstanding hypotheses. RESULTS: A discriminant function analysis lent statistical support to classifying the yeast interactome into hub, intermediate and peripheral nodes based on network neighborhood connectivity, betweenness centrality, and average shortest path length. Quantitative support for the existence of genomic architecture as a mechanistic basis for evolutionary constraint is then revealed through utilizing these statistical parameters of the protein-protein interaction network in combination with estimators of protein evolution. CONCLUSIONS: As functional genetic networks are becoming increasingly available, it will now be possible to evaluate functional genetic network constraint against variables describing complex phenotypes and environments, for better understanding of commonly observed deterministic patterns of evolution in non-model organisms. The hypothesis framework and methodological approach outlined herein may help to quantify the extrinsic versus intrinsic dimensions of evolutionary constraint, and result in a better understanding of how fast, effectively, or deterministically organisms adapt.

Transcriptomic Signatures of Experimental Alkaloid Consumption in a Poison Frog.

In the anuran family Dendrobatidae, aposematic species obtain their toxic or unpalatable alkaloids from dietary sources, a process known as sequestering. To understand how toxicity evolved in this family, it is paramount to elucidate the pathways of alkaloid processing (absorption, metabolism, and sequestering). Here, we used an exploratory skin gene expression experiment in which captive-bred dendrobatids were fed alkaloids. Most of these experiments were performed with Dendrobates tinctorius, but some trials were performed with D. auratus, D. leucomelas and Allobates femoralis to explore whether other dendrobatids would show similar patterns of gene expression. We found a consistent pattern of up-regulation of genes related to muscle and mitochondrial processes, probably due to the lack of mutations related to alkaloid resistance in these species. Considering conserved pathways of drug metabolism in vertebrates, we hypothesize alkaloid degradation is a physiological mechanism of resistance, which was evidenced by a strong upregulation of the immune system in D. tinctorius, and of complement C2 across the four species sampled. Probably related to this strong immune response, we found several skin keratins downregulated, which might be linked to a reduction of the cornified layer of the epidermis. Although not conclusive, our results offer candidate genes and testable hypotheses to elucidate alkaloid processing in poison frogs.

Environmental temperatures shape thermal physiology as well as diversification and genome-wide substitution rates in lizards.

Climatic conditions changing over time and space shape the evolution of organisms at multiple levels, including temperate lizards in the family Lacertidae. Here we reconstruct a dated phylogenetic tree of 262 lacertid species based on a supermatrix relying on novel phylogenomic datasets and fossil calibrations. Diversification of lacertids was accompanied by an increasing disparity among occupied bioclimatic niches, especially in the last 10 Ma, during a period of progressive global cooling. Temperate species also underwent a genome-wide slowdown in molecular substitution rates compared to tropical and desert-adapted lacertids. Evaporative water loss and preferred temperature are correlated with bioclimatic parameters, indicating physiological adaptations to climate. Tropical, but also some populations of cool-adapted species experience maximum temperatures close to their preferred temperatures. We hypothesize these species-specific physiological preferences may constitute a handicap to prevail under rapid global warming, and contribute to explaining local lizard extinctions in cool and humid climates.

Patterns, Mechanisms and Genetics of Speciation in Reptiles and Amphibians.

In this contribution, the aspects of reptile and amphibian speciation that emerged from research performed over the past decade are reviewed. First, this study assesses how patterns and processes of speciation depend on knowing the taxonomy of the group in question, and discuss how integrative taxonomy has contributed to speciation research in these groups. This study then reviews the research on different aspects of speciation in reptiles and amphibians, including biogeography and climatic niches, ecological speciation, the relationship between speciation rates and phenotypic traits, and genetics and genomics. Further, several case studies of speciation in reptiles and amphibians that exemplify many of these themes are discussed. These include studies of integrative taxonomy and biogeography in South American lizards, ecological speciation in European salamanders, speciation and phenotypic evolution in frogs and lizards. The final case study combines genomics and biogeography in tortoises. The field of amphibian and reptile speciation research has steadily moved forward from the assessment of geographic and ecological aspects, to incorporating other dimensions of speciation, such as genetic mechanisms and evolutionary forces. A higher degree of integration among all these dimensions emerges as a goal for future research.

Plant Phenology Supports the Multi-emergence Hypothesis for Ebola Spillover Events.

Ebola virus disease outbreaks in animals (including humans and great apes) start with sporadic host switches from unknown reservoir species. The factors leading to such spillover events are little explored. Filoviridae viruses have a wide range of natural hosts and are unstable once outside hosts. Spillover events, which involve the physical transfer of viral particles across species, could therefore be directly promoted by conditions of host ecology and environment. In this report, we outline a proof of concept that temporal fluctuations of a set of ecological and environmental variables describing the dynamics of the host ecosystem are able to predict such events of Ebola virus spillover to humans and animals. We compiled a data set of climate and plant phenology variables and Ebola virus disease spillovers in humans and animals. We identified critical biotic and abiotic conditions for spillovers via multiple regression and neural network-based time series regression. Phenology variables proved to be overall better predictors than climate variables. African phenology variables are not yet available as a comprehensive online resource. Given the likely importance of phenology for forecasting the likelihood of future Ebola spillover events, our results highlight the need for cost-effective transect surveys to supply phenology data for predictive modelling efforts.

Genomic and phenotypic signatures of climate adaptation in an Anolis lizard.

Integrated knowledge on phenotype, physiology, and genomic adaptations is required to understand the effects of climate on evolution. The functional genomic basis of organismal adaptation to changes in the abiotic environment, its phenotypic consequences, and its possible convergence across vertebrates are still understudied. In this study, we use a comparative approach to verify predicted gene functions for vertebrate thermal adaptation with observed functions underlying repeated genomic adaptations in response to elevation in the lizard Anolis cybotes. We establish a direct link between recurrently evolved phenotypes and functional genomics of altitude-related climate adaptation in three highland and lowland populations in the Dominican Republic. We show that across vertebrates, genes contained in this interactome are expressed within the brain, the endocrine system, and during development. These results are relevant to elucidate the effect of global climate change across vertebrates and might aid in furthering insight into gene-environment relationships under disturbances to homeostasis.

Transcriptomic and macroevolutionary evidence for phenotypic uncoupling between frog life history phases.

Anuran amphibians undergo major morphological transitions during development, but the contribution of their markedly different life-history phases to macroevolution has rarely been analysed. Here we generate testable predictions for coupling versus uncoupling of phenotypic evolution of tadpole and adult life-history phases, and for the underlying expression of genes related to morphological feature formation. We test these predictions by combining evidence from gene expression in two distantly related frogs, Xenopus laevis and Mantidactylus betsileanus, with patterns of morphological evolution in the entire radiation of Madagascan mantellid frogs. Genes linked to morphological structure formation are expressed in a highly phase-specific pattern, suggesting uncoupling of phenotypic evolution across life-history phases. This gene expression pattern agrees with uncoupled rates of trait evolution among life-history phases in the mantellids, which we show to have undergone an adaptive radiation. Our results validate a prevalence of uncoupling in the evolution of tadpole and adult phenotypes of frogs.

Opposing Patterns of Seasonal Change in Functional and Phylogenetic Diversity of Tadpole Assemblages.

Assemblages that are exposed to recurring temporal environmental changes can show changes in their ecological properties. These can be expressed by differences in diversity and assembly rules. Both can be identified using two measures of diversity: functional (FD) and phylogenetic diversity (PD). Frog communities are understudied in this regard, especially during the tadpole life stage. We utilised tadpole assemblages from Madagascan rainforest streams to test predictions of seasonal changes on diversity and assemblage composition and on diversity measures. From the warm-wet to the cool-dry season, species richness (SR) of tadpole assemblages decreased. Also FD and PD decreased, but FD less and PD more than expected by chance. During the dry season, tadpole assemblages were characterised by functional redundancy (among assemblages-with increasing SR), high FD (compared to a null model), and low PD (phylogenetic clustering; compared to a null model). Although mutually contradictory at first glance, these results indicate competition as tadpole community assembly driving force. This is true during the limiting cool-dry season but not during the more suitable warm-wet season. We thereby show that assembly rules can strongly depend on season, that comparing FD and PD can reveal such forces, that FD and PD are not interchangeable, and that conclusions on assembly rules based on FD alone are critical.

Secondary Data Analytics of Aquaporin Expression Levels in Glioblastoma Stem-Like Cells.

Glioblastoma is the most common brain tumor in adults in which recurrence has been attributed to the presence of cancer stem cells in a hypoxic microenvironment. On the basis of tumor formation in vivo and growth type in vitro, two published microarray gene expression profiling studies grouped nine glioblastoma stem-like (GS) cell lines into one of two groups: full (GSf) or restricted (GSr) stem-like phenotypes. Aquaporin-1 (AQP1) and aquaporin-4 (AQP4) are water transport proteins that are highly expressed in primary glial-derived tumors. However, the expression levels of AQP1 and AQP4 have not been previously described in a panel of 92 glioma samples. Therefore, we designed secondary data analytics methods to determine the expression levels of AQP1 and AQP4 in GS cell lines and glioblastoma neurospheres. Our investigation also included a total of 2,566 expression levels from 28 Affymetrix microarray probe sets encoding 13 human aquaporins (AQP0-AQP12); CXCR4 (the receptor for stromal cell derived factor-1 [SDF-1], a potential glioma stem cell therapeutic target]); and PROM1 (gene encoding CD133, the widely used glioma stem cell marker). Interactive visual representation designs for integrating phenotypic features and expression levels revealed that inverse expression levels of AQP1 and AQP4 correlate with distinct phenotypes in a set of cell lines grouped into full and restricted stem-like phenotypes. Discriminant function analysis further revealed that AQP1 and AQP4 expression are better predictors for tumor formation and growth types in glioblastoma stem-like cells than are CXCR4 and PROM1. Future investigations are needed to characterize the molecular mechanisms for inverse expression levels of AQP1 and AQP4 in the glioblastoma stem-like neurospheres.

Evidence for an intrinsic factor promoting landscape genetic divergence in Madagascan leaf-litter frogs.

The endemic Malagasy frog radiations are an ideal model system to study patterns and processes of speciation in amphibians. Large-scale diversity patterns of these frogs, together with other endemic animal radiations, led to the postulation of new and the application of known hypotheses of species diversification causing diversity patterns in this biodiversity hotspot. Both extrinsic and intrinsic factors have been studied in a comparative framework, with extrinsic factors usually being related to the physical environment (landscape, climate, river catchments, mountain chains), and intrinsic factors being clade-specific traits or constraints (reproduction, ecology, morphology, physiology). Despite some general patterns emerging from such large-scale comparative analyses, it became clear that the mechanism of diversification in Madagascar may vary among clades, and may be a multifactorial process. In this contribution, I test for intrinsic factors promoting population-level divergence within a clade of terrestrial, diurnal leaf-litter frogs (genus Gephyromantis) that has previously been shown to diversify according to extrinsic factors. Landscape genetic analyses of the microendemic species Gephyromantis enki and its widely distributed, larger sister species Gephyromantis boulengeri over a rugged landscape in the Ranomafana area shows that genetic variance of the smaller species cannot be explained by landscape resistance alone. Both topographic and riverine barriers are found to be important in generating this divergence. This case study yields additional evidence for the probable importance of body size in lineage diversification.

A candidate multimodal functional genetic network for thermal adaptation.

Vertebrate ectotherms such as reptiles provide ideal organisms for the study of adaptation to environmental thermal change. Comparative genomic and exomic studies can recover markers that diverge between warm and cold adapted lineages, but the genes that are functionally related to thermal adaptation may be difficult to identify. We here used a bioinformatics genome-mining approach to predict and identify functions for suitable candidate markers for thermal adaptation in the chicken. We first established a framework of candidate functions for such markers, and then compiled the literature on genes known to adapt to the thermal environment in different lineages of vertebrates. We then identified them in the genomes of human, chicken, and the lizard Anolis carolinensis, and established a functional genetic interaction network in the chicken. Surprisingly, markers initially identified from diverse lineages of vertebrates such as human and fish were all in close functional relationship with each other and more associated than expected by chance. This indicates that the general genetic functional network for thermoregulation and/or thermal adaptation to the environment might be regulated via similar evolutionarily conserved pathways in different vertebrate lineages. We were able to identify seven functions that were statistically overrepresented in this network, corresponding to four of our originally predicted functions plus three unpredicted functions. We describe this network as multimodal: central regulator genes with the function of relaying thermal signal (1), affect genes with different cellular functions, namely (2) lipoprotein metabolism, (3) membrane channels, (4) stress response, (5) response to oxidative stress, (6) muscle contraction and relaxation, and (7) vasodilation, vasoconstriction and regulation of blood pressure. This network constitutes a novel resource for the study of thermal adaptation in the closely related nonavian reptiles and other vertebrate ectotherms.