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The vertebrate skull is a complex structure, and studies of skull shape have yielded considerable insight into the evolutionary forces shaping specialized phenotypes in organisms as diverse as bats, frogs, and fossorial animals. Here, we used phylogenetic comparative analyses of CT scans of male skulls from 57 species of Sceloporus lizards to explore patterns of skull evolution in a group of generalist taxa. We found that most interspecific variation is in terms of skull elongation such that some species have long, narrow skulls, whereas others exhibit more compact and robust skulls. We also found strong links to overall body size, with evolutionary shifts to larger bodies being associated with more compact skulls and slower evolutionary rates. This is the opposite of the pattern in most mammals in which larger bodied species have longer snouts, and more like the pattern in frogs in which function has played a more important evolutionary role. Also, unlike other vertebrates, the jaw, anterior, and posterior parts of the Sceloporus skull are largely integrated, having evolved independently of each other only to a limited, albeit significant, degree. Our results emphasize the importance of body size in the evolutionary shaping of the skull and suggest that additional studies of behavioral function in a generalist group are warranted., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). Ecology and Evolution published by John Wiley & Sons Ltd.)

Certain individuals have a disproportionate effect on group responses. Characteristics may include susceptibility to pollutants, such as cadmium (Cd), a potent trace metal. Here, we show how a pair of Cd-exposed individuals can impact the behavior of unexposed groups. We used behavioral assessments to characterize the extent of the effects of the Cd-exposed individuals on group boldness, cohesion, foraging, activity, and responses to plants. We found that groups with a pair of Cd-exposed fish remained closer to novel stimuli and plants than did groups with untreated (control) fish. The presence of plants reduced Cd-induced differences in shoal cohesion and delays feeding in male shoals. Shoals with Cd- and water-treated fish were equally active. The results suggest that fish acutely exposed to environmentally relevant Cd concentrations can have profound effects on the un-exposed majority. However, the presence of plants may mitigate the effects of contaminants on some aspects of social behavior., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Delia Shelton reports financial support was provided by National Institutes of Health. Delia Shelton reports financial support was provided by National Institute of Environmental Health Sciences. Delia Shelton reports a relationship with National Institutes of Health that includes: funding grants. Delia Shelton reports a relationship with National Science Foundation that includes: funding grants. Emilia Martins reports a relationship with National Science Foundation that includes: funding grants. Piyumika Suriyampola reports a relationship with National Science Foundation that includes: funding grants. Joseph Shaw reports a relationship with National Science Foundation that includes: funding grants. Joseph Shaw reports a relationship with National Institutes of Health that includes: funding grants. Stephen P. Glaholt reports a relationship with National Science Foundation that includes: funding grants. Stephen P. Glaholt reports a relationship with National Institutes of Health that includes: funding grants. Zoe Dinges reports a relationship with National Science Foundation that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Weak, but environmentally relevant concentrations of contaminants can have subtle, yet important, impacts on organisms, which are often overlooked due to the lack of acute impacts and the timing of exposure. Thus, recognizing simple, non-invasive markers of contamination events is essential for early detection and addressing the effects of exposure to weak environmental contaminants. Here, we tested whether exposure to an environmentally relevant concentration of Bisphenol-A (BPA), a common and persistent contaminant in aquatic systems, affects the lateralization of adult zebrafish (Danio rerio), a widely used model organism in ecotoxicology. We found that 73.5% of adult zebrafish displayed a left-side bias when they approached a visual cue, but that those exposed to weak BPA (0.02 mg/L) for 7 days did not exhibit laterality. Only 47.1% displayed a left-side bias. We found no differences in activity level and visual sensitivity, motor and sensory mechanisms, that regulate lateralized responses and that were unaffected by weak BPA exposure. These findings indicate the reliability of laterality as a simple measure of contaminant exposure and for future studies of the detailed mechanisms underlying subtle and complex behavioral effects to pollutants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Many organisms communicate using signals in different sensory modalities (multicomponent or multimodal). When one signal or component is lost over evolutionary time, it may be indicative of changes in other characteristics of the signalling system, including the sensory organs used to perceive and process signals. Sceloporus lizards predominantly use chemical and visual signals to communicate, yet some species have lost the ancestral ventral colour patch used in male-male agonistic interactions and exhibit increased chemosensory behaviour. Here, we asked whether evolutionary loss of this sexual signal is associated with larger vomeronasal organ (VNO) volumes (an organ that detects chemical scents) compared with species that have retained the colour patch. We measured VNO coronal section areas of 7-8 adult males from each of 11 Sceloporus species (4 that lost and 7 that retained the colour patch), estimated sensory and total epithelium volume, and compared volumes using phylogenetic analysis of covariance, controlling for body size. Contrary to expectations, we found that species retaining the ventral patch had similar relative VNO volumes as did species that had lost the ancestral patch, and that body size explains VNO epithelium volume. Visual signal loss may be sufficiently compensated for by increased chemosensory behaviour, and the allometric pattern may indicate sensory system trade-offs for large-bodied species., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Evolutionary Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The long non-coding RNA HOX transcript antisense intergenic RNA ( HOTAIR ) is associated with oncogenic features in bladder cancer and is predictive of poor clinical outcomes in patients diagnosed with this disease. In this study, we evaluated the impact of the HOTAIR single nucleotide polymorphisms rs920778 and rs12826786 on bladder cancer risk and survival. This case-control study included 106 bladder cancer patients and 199 cancer-free controls. Polymorphisms were evaluated through PCR-restriction fragment length polymorphism. The odds ratio and 95% confidence intervals were tested using univariable and multivariable logistic regressions. The effects on patient survival were evaluated using the log-rank test and Cox regression models. Our data showed that the HOTAIR rs920778 and rs12826786 genetic variants are not associated with the risk of developing bladder cancer. Nevertheless, survival analyses suggested that the HOTAIR rs920778 TT genotype and rs12826786 CC genotype are associated with increased survival in male bladder cancer patients and in patients, both male and female, who have primary tumors with a pathological stage of pT2. Together, these results suggest that, despite not being associated with bladder cancer risk, HOTAIR rs920778 and rs12826786 polymorphisms might represent new prognostic factors in this type of cancer. This is particularly important as these polymorphisms might be easily evaluated in bladder cancer patients in a minimally invasive manner to better predict their clinical outcomes.

Phenotypes that allow animals to detect, weather, and predict changes efficiently are essential for survival in fluctuating environments. Some phenotypes may remain specialized to suit an environment perfectly, while others become more plastic or generalized, shifting flexibly to match current context or adopting a form that can utilize a wide range of contexts. Here, we tested the differences in behavior, morphology, sensory and metabolic physiology between wild zebrafish (Danio rerio) in highly variable fast-flowing rivers and still-water sites. We found that river zebrafish moved at higher velocities than did still-water fish, had lower oxygen demands, and responded less vigorously to small changes in flow rate, as we might expect for fish that are well-suited to high-flow environments. River zebrafish also had less streamlined bodies and were more behaviorally plastic than were still-water zebrafish, both features that may make them better-suited to a transitional lifestyle. Our results suggest that zebrafish use distinct sensory mechanisms and metabolic physiology to reduce energetic costs of living in fast-flowing water while relying on morphology and behavior to create flexible solutions to a challenging habitat. Insights on animals' reliance on traits with different outcomes provide a framework to better understand their survival in future environmental fluctuations., (© 2023. Springer Nature Limited.)

The development of new drugs through studies of candidate molecules is a complex undertaking; however, computational or in silico approaches aimed at optimizing molecules with greater development potential are being utilized for predictions of pharmacokinetic properties such as absorption, distribution, metabolism and excretion (ADME) as well as toxicological parameters. The objective of this study was to examine in silico and in vivo pharmacokinetic and toxicological properties of the chemical constituents present in the essential oil of Croton heliotropiifolius Kunth leaves. The following Pubchem platform as well as Software SwissADME and PreADMET software were employed for in silico studies while micronucleus (MN) testing for in vivo determination of mutagenicity, using Swiss adult male Mus musculus mice. In silico findings demonstrated that all chemical constituents presented (1) high oral absorption (2) medium cellular permeability and (3) high blood brain permeability. As for toxicity, these chemical constituents exhibited low to medium risk of occurrence of cytotoxicity. Regarding in vivo evaluation, peripheral blood samples obtained from animals tested with the oil showed no significant differences in number of MN compared to negative controls. Data indicate that further investigations are necessary to corroborate the findings of this study. Our data suggest that essential oil extracted from Croton heliotropiifolius Kunth leaves may serve as a candidate for new drug development.

To account for global contamination events, we must identify direct and indirect pollutant effects. Although pollutants can have direct effects on individuals, it is unknown how a few contaminated individuals affect groups, a widespread social organization. We show environmentally relevant levels of cadmium (Cd) can have indirect social effects revealed in the social context of a larger group. Cd-contaminated individuals had poor vision and more aggressive responses, but no other behavioral effects. The presence of experienced Cd-exposed pairs in the groups had an indirect effect on the un-exposed individual's social interactions leading to the shoal becoming bolder and moving closer to a novel object than control groups. Because a few directly affected individuals could indirectly affect social behavior of the un-exposed majority, we believe that such acute but potentially important heavy metal toxicity could inform reliable predictions about the consequences of their use in a changing world., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Clear cell renal cell carcinoma (ccRCC) is highly prone to metastasize and displays an extremely low 5-year survival rate. Not only miRNAs (miRs) are key gene expression regulators but can also be epigenetically modified. Abnormal miR expression has been linked with epithelial-mesenchymal transition (EMT)-driven ccRCC progression. MiR-30a/c-5p were found downregulated in ccRCC and associated with aggressiveness. Herein, we sought to unravel miR-30a/c-5p mechanistic role in ccRCC. RNA sequencing and genome-wide methylome data of ccRCC and normal tissue samples from The Cancer Genome Atlas database were integrated to identify candidate miRs cytosine-phosphate-guanine (CpG) loci deregulated in ccRCC. TargetScan was searched to identify miR putative targets. MiR-30a/c-5p expression and promoter methylation was evaluated in vitro, by PCR. Western blot, functional and luciferase assays were performed after cell transfection with either pre-miR, antimiR, or siRNA against twinfilin-1 (TWF1). Immunohistochemistry (IHC) was performed in ccRCC tissues. We found miR-30c-5p downregulation and aberrant promoter methylation in ccRCC tissues. In vitro studies revealed concomitant miR-30a/c-5p downregulation and increased promoter methylation, as well as a significant re-expression following decitabine treatment. Functional assays demonstrated that both miRs significantly decreased cell aggressiveness and the protein levels of EMT-promoting players, while upregulating epithelial markers, namely Claudin-1 and ZO-1. Importantly, we confirmed TWF1 as a direct target of both miRs, and its potential involvement in epithelial-mesenchymal transition/mesenchymal-epithelial transition regulation. IHC analysis revealed higher TWF1 expression in primary tissues from patients that developed metastases, after surgical treatment. Our results implicate miR-30a/c-5p in ccRCC cells' aggressiveness attenuation by directly targeting TWF1 and hampering EMT., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. The prognosis of patients is very poor, with a median overall survival of ~ 15 months after diagnosis. Cadherin-3 (also known as P-cadherin), a cell-cell adhesion molecule encoded by the CDH3 gene, is deregulated in several cancer types, but its relevance in GBM is unknown. In this study, we investigated the functional roles, the associated molecular signatures, and the prognostic value of CDH3/P-cadherin in this highly malignant brain tumor. CDH3/P-cadherin mRNA and protein levels were evaluated in human glioma samples. Knockdown and overexpression models of P-cadherin in GBM were used to evaluate its functional role in vitro and in vivo. CDH3-associated gene signatures were identified by enrichment analyses and correlations. The impact of CDH3 in the survival of GBM patients was assessed in independent cohorts using both univariable and multivariable models. We found that P-cadherin protein is expressed in a subset of gliomas, with an increased percentage of positive samples in grade IV tumors. Concordantly, CDH3 mRNA levels in glioma samples from The Cancer Genome Atlas (TCGA) database are increased in high-grade gliomas. P-cadherin displays oncogenic functions in multiple knockdown and overexpression GBM cell models by affecting cell viability, cell cycle, cell invasion, migration, and neurosphere formation capacity. Genes that were positively correlated with CDH3 are enriched for oncogenic pathways commonly activated in GBM. In vivo, GBM cells expressing high levels of P-cadherin generate larger subcutaneous tumors and cause shorter survival of mice in an orthotopic intracranial model. Concomitantly, high CDH3 expression is predictive of shorter overall survival of GBM patients in independent cohorts. Together, our results show that CDH3/P-cadherin expression is associated with aggressiveness features of GBM and poor patient prognosis, suggesting that it may be a novel therapeutic target for this deadly brain tumor., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Social context may influence the perception of sensory cues and the ability to display refined behavioral responses. Previous work suggests that effective responses to environmental cues can be contingent on having a sufficient number of individuals in a group. Thus, the changes in group size may have profound impacts, particularly on the behavior of small social groups. Using zebrafish ( Danio rerio ), here we examined how changes in group size influence the ability to respond to changes in water flow. We found that fish in relatively larger groups displayed stronger rheotaxis even when comparing pairs of fish with groups of four fish, indicating that a small increase in group size can enhance the responsiveness to environmental change. Individual fish in relatively larger groups also spent less time in the energetically costly leading position compared to individuals in pairs, indicating that even a small increase in group size may provide energetic benefits. We also found that the shoal cohesion was dependent on the size of the group but within a given group size, shoal cohesion did not vary with flow rate. Our study highlights that even a small change in group size could significantly affect the way social fish respond to the changes in water flow, which could be an important attribute that shapes the resilience of social animals in changing environments., Competing Interests: Disclosure Statement: No conflicting areas of interests are involved with this experiment.

The existence of a clear association between stress and cancer is still a matter of debate. Recent studies suggest that chronic stress is associated with some cancer types and may influence tumor initiation and patient prognosis, but its role in brain tumors is not known. Glioblastoma (GBM) is a highly malignant primary brain cancer, for which effective treatments do not exist. Understanding how chronic stress, or its effector hormones glucocorticoids (GCs), may modulate GBM aggressiveness is of great importance. To address this, we used both syngeneic and xenograft in vivo orthotopic mouse models of GBM, in immunocompetent C57BL/6J or immunodeficient NSG mice, respectively, to evaluate how different paradigms of stress exposure could influence GBM aggressiveness and animals' overall survival (OS). Our results demonstrated that a previous exposure to exogenous corticosterone administration, chronic restraint stress, or chronic unpredictable stress do not impact the OS of these mice models of GBM. Concordantly, ex vivo analyses of various GBM-relevant genes showed similar intra-tumor expression levels across all experimental groups. These findings suggest that corticosterone and chronic stress do not significantly affect GBM aggressiveness in murine models., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lopes, Vieira de Castro, Pojo, Gonçalves, Martins, Coimbra, Sotiropoulos, Sousa, Rodrigues and Costa.)

Colorful ornaments are important visual signals for animal communication that can provide critical information about the quality of the signaler. In this study, we focused on different color characteristics of the abdominal patches of males of six lizard species from the genus Sceloporus. We addressed three main objectives. First, we examined if size, brightness, saturation, and conspicuousness of these ornaments are indicative of body size, condition, immune function, or levels of testosterone and corticosterone. Second, we evaluated if the distinct components of these abdominal patches (blue or green patches and black stripes) transmit similar information about the signaler, which would support the redundant signal hypothesis, or if these components are related to different phenotypic traits, which would support the multiple message hypothesis. Third, we compared the phenotypic correlates of these ornaments among our six species to understand the degree of conservatism in the signaling patterns or to find species-specific signals. Using data collected from males in natural conditions and a multi-model inference framework, we found that in most species the area of the patches and the brightness of the blue component are positively related to body size. Thus, these color characteristics are presumably indicative of the physical strength and competitive ability of males and these shared signals were likely inherited from a common ancestor. In half of the species, males in good body condition also exhibit relatively larger blue and black areas, suggesting that the expression of these ornaments is condition-dependent. Abdominal patches also provide information about immunocompetence of the males as indicated by different correlations between certain color characteristics and ectoparasite load, counts of heterophils, and the heterophil:lymphocyte ratio. Our findings reveal that area and brightness of the abdominal patches signal the size and body condition of males, whereas blue saturation and conspicuousness with respect to the surrounding substrate are indicative of immune condition, thus supporting the multiple message hypothesis. However, some of these correlations were not shared by all species and, hence, point to intriguing species-specific signals., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Anthropogenic activities often lead to alterations in the natural environment via multiple routes. Simultaneous occurrence of interacting environmental perturbations may influence animals via more complex pathways than when being exposed to environmental stressors discretely. In our study, we investigated the interactive effects of poor visual environment and exposure to an environmentally realistic concentration of a common contaminant on the behavior of larval zebrafish, Danio rerio. Specifically, we tested the sensory-motor behavior of zebrafish larvae by exposing them to low-light conditions and a low concentration of bisphenol-A (BPA) for 7 days postfertilization. We found that zebrafish exposed to both BPA and low-light conditions had significantly weaker response to a moving-visual cue. However, those exposed to only one of these treatments did not have altered response to visual cues. Since the response to a moving, visual cue involves locomotion, we also examined the distance they traveled as a proxy for activity level of individuals across treatments. However, the distance traveled by individuals did not significantly differ across treatments, suggesting that the differences in response are linked to visual sensory pathways. Here, we emphasize that the adverse effects of environmental stressors, particularly of those that occur at environmentally relevant concentrations, may emerge only when they co-occur with another environmental stressor. These findings highlight the need to incorporate multiple environmental stressors to comprehensively assess impacts that human activities have on behavioral strategies of animals., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Species with multimodal communication integrate information from social cues in different modalities into behavioral responses that are mediated by changes in gene expression in the brain. Differences in patterns of gene expression between signal modalities may shed light on the neuromolecular mechanisms underlying multisensory processing. Here, we use RNA-Seq to analyze brain transcriptome responses to either chemical or visual social signals in a territorial lizard with multimodal communication. Using an intruder challenge paradigm, we exposed 18 wild-caught, adult, male Sceloporus jarrovii to either male conspecific scents (femoral gland secretions placed on a small pebble), the species-specific push-up display (a programmed robotic model), or a control (an unscented pebble). We conducted differential expression analysis with both a de novo S. jarrovii transcriptome assembly and the reference genome of a closely related species, Sceloporus undulatus. Despite some inter-individual variation, we found significant differences in gene expression in the brain across signal modalities and the control in both analyses. The most notable differences occurred between chemical and visual stimulus treatments, closely followed by visual stimulus versus the control. Altered expression profiles could explain documented aggression differences in the immediate behavioral response to conspecific signals from different sensory modalities. Shared differentially expressed genes between visually- or chemically-stimulated males are involved in neural activity and neurodevelopment and several other differentially expressed genes in stimulus-challenged males are involved in conserved signal-transduction pathways associated with the social stress response, aggression and the response to territory intruders across vertebrates., (© 2021 International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.)

Environmental change poses a devastating risk to human and environmental health. Rapid assessment of water conditions is necessary for monitoring, evaluating, and addressing this global health danger. Sentinels or biological monitors can be deployed in the field using minimal resources to detect water quality changes in real time, quickly and cheaply. Zebrafish ( Danio rerio ) are ideal sentinels for detecting environmental changes due to their biomedical tool kit, widespread geographic distribution, and well-characterized phenotypic responses to environmental disturbances. Here, we demonstrate the utility of zebrafish sentinels by characterizing phenotypic differences in wild zebrafish between two field sites in India. Site 1 was a rural environment with flowing water, low-hypoxic conditions, minimal human-made debris, and high iron and lead concentrations. Site 2 was an urban environment with still water, hypoxic conditions, plastic pollution, and high arsenic, iron, and chromium concentrations. We found that zebrafish from Site 2 were smaller, more cohesive, and less active than Site 1 fish. We also found sexually dimorphic body shapes within the Site 2, but not the Site 1, population. Advancing zebrafish sentinel research and development will enable rapid detection, evaluation, and response to emerging global health threats.

The work herein presented reports the development of fucoidan/chitosan nanoparticles (NPs) loaded with gemcitabine and functionalized with ErbB-2 antibody at their surface (NPs + Gem + Ab). The maximum immobilization of ErbB-2 on NPs' surface was set at 10 μg mL -1 and resulted in NPs with a size around 160 nm, a polydispersity index of 0.18, and a zeta potential of 21 mV. ErbB-2 is overexpressed in some subtypes of breast cancers, and the targeting capability of the NPs + Gem + Ab system was confirmed by an increased cellular uptake of SKBR3 cells (ErbB-2 positive) when compared to MDA-MB-231 (ErbB-2 negative). To validate the targeting efficacy of NPs + Gem + Ab, a co-culture system with human endothelial and SKBR3 cells was established. Cytotoxic effects over endothelial cells were similar for all the tested conditions (between 25 and 30%). However, the NPs + Gem + Ab system presented increased toxicity over breast cancer cells, above 80% after 24 h, when compared to free Gem and NPs + Gem (around 15% and 20%, respectively). In vivo studies demonstrated that the developed targeting system significantly reduced tumor growth and the appearance of lung metastasis compared to untreated controls. In summary, the efficacy of the NPs + Gem + Ab system to target cancer cells was established and validated both in vitro and in vivo, being a compelling alternative strategy to current chemotherapeutic approaches., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Behavioural responses to communicative signals combine input from multiple sensory modalities and signal compensation theory predicts that evolutionary shifts in one sensory modality could impact the response to signals in other sensory modalities. Here, we conducted two types of field experiments with 11 species spread across the lizard genus Sceloporus to test the hypothesis that the loss of visual signal elements affects behavioural responses to a chemical signal (conspecific scents) or to a predominantly visual signal (a conspecific lizard), both of which are used in intraspecific communication. We found that three species that have independently lost a visual signal trait, a colourful belly patch, responded to conspecific scents with increased chemosensory behaviour compared to a chemical control, while species with the belly patch did not. However, most species, with and without the belly patch, responded to live conspecifics with increased visual displays of similar magnitude. While aggressive responses to visual stimuli are taxonomically widespread in Sceloporus , our results suggest that increased chemosensory response behaviour is linked to colour patch loss. Thus, interactions across sensory modalities could constrain the evolution of complex signalling phenotypes, thereby influencing signal diversity.

Network science has long been recognized as a well-established discipline across many biological domains. In the particular case of cancer genomics, network discovery is challenged by the multitude of available high-dimensional heterogeneous views of data. Glioblastoma (GBM) is an example of such a complex and heterogeneous disease that can be tackled by network science. Identifying the architecture of molecular GBM networks is essential to understanding the information flow and better informing drug development and pre-clinical studies. Here, we review network-based strategies that have been used in the study of GBM, along with the available software implementations for reproducibility and further testing on newly coming datasets. Promising results have been obtained from both bulk and single-cell GBM data, placing network discovery at the forefront of developing a molecularly-informed-based personalized medicine.

As shown from several long-term and time-intensive studies, closely related, sympatric species can impose strong selection on one another, leading to dramatic examples of phenotypic evolution. Here, we use occurrence data to identify clusters of sympatric Sceloporus lizard species and to test whether Sceloporus species tend to coexist with other species that differ in body size, as we would expect when there is competition between sympatric congeners. We found that Sceloporus species can be grouped into 16 unique bioregions. Bioregions that are located at higher latitudes tend to be larger and have fewer species, following Rapoport's rule and the latitudinal diversity gradient. Species richness was positively correlated with the number of biomes and elevation heterogeneity of each bioregion. Additionally, most bioregions show signs of phylogenetic underdispersion, meaning closely related species tend to occur in close geographic proximity. Finally, we found that although Sceloporus species that are similar in body size tend to cluster geographically, small-bodied Sceloporus species are more often in sympatry with larger-bodied Sceloporus species than expected by chance alone, whereas large-bodied species cluster with each other geographically and phylogenetically. These results suggest that community composition in extant Sceloporus species is the result of allopatric evolution, as closely related species move into different biomes, and interspecies interactions, with sympatry between species of different body sizes. Our phyloinformatic approach offers unique and detailed insights into how a clade composed of ecologically and morphologically disparate species are distributed over large geographic space and evolutionary time., Competing Interests: None declared., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

This study aimed to estimate the impact of an abandoned copper (Cu) mine on ecosystem environmental quality, using integrated ecological and biogeochemical analyses. Through a controlled experiment, the amount of Cu released by waste rocks, Cu adsorbed in soils, Cu geochemical behaviour and its leached amount were measured. Furthermore, to investigate the impacts of mine drainage on the adjacent ecosystem, samples of sediments, water and aquatic macroinvertebrates were analysed. We found that waste rocks still have high Cu concentration even after 30 years under weathering, ranging from 7782 to 8717 mg kg -1 , associated mainly with carbonates, amorphous oxides and sulphides. It was estimated that 7.2 tonnes of Cu were released by waste rocks into the environment over last 30 years. The concentration of Cu observed in Ubari stream water was (

-1 ), in sediments (28.0-1185 mg kg -1 ) and in macroinvertebrates (1.3-28.9 mg kg -1 d/w). The ecological indexes showed that near mine discharge a significance decrease in the density of aquatic macroinvertebrates and a significance increase of Cu in biological tissues occurs, causing disturbances in biodiversity. The results showed that, even after long periods, the waste rocks from abandoned mines still contain high levels of metal, that are gradually released into the environment through weathering and erosion, representing a potential source of environmental pollution and a clear threat to the environmental quality of adjacent ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Anthropogenic change is expected to alter environments at alarming rates. To predict the impact of modified environments on social behavior, we must study the relationship between environmental features and collective behavior in a genetically tractable model, zebrafish ( Danio rerio ). Here, we conducted a field study to examine the relationship between salient environmental features and collective behavior in four populations of zebrafish. We found zebrafish in flowing water formed volatile groups, whereas those in still water had more consistent membership and leadership. Groups in fast-flowing water were large (up to 2000 fish) and tightly knit with short nearest neighbor distances, whereas group sizes were smaller (11 fish/group) with more space between individual fish in still and slow-flowing water. These observations point to a possible profound role of water flow in influencing collective behavior in wild zebrafish.

Animals can shift their reliance on different sensory modalities in response to environmental conditions, and knowing the degree to which traits are reversible may help us to predict their chances of survival in a changing environment. Here, using adult zebrafish (Danio rerio), we found that 6 weeks in different light environments alone were sufficient to shift whether fish approached visual or chemical cues first, and that a subsequent reversal of lighting conditions also reversed their sensory preferences. In addition, we measured simple behavioral responses to sensory stimuli presented alone, and found that zebrafish housed in dim light for 6 weeks responded weakly to an optomotor assay, but strongly to an olfactory cue, whereas fish experiencing bright light for 6 weeks responded strongly to the visual optomotor stimulus and weakly in an olfactory assay. Visual and olfactory responses were equally reversible, and shifted to the opposite pattern when we reversed lighting conditions for 6 weeks. In contrast, we did not find a change in activity level, suggesting that changes in multiple sensory modalities can buffer animals from changes in more complex forms of behavior. This reversal of sensory response provides insight into how animals may use sensory shifts to keep up with environmental change., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Animal signals evolve by striking a balance between the need to convey information through particular habitats and the limitations of what types of signals can most easily be produced and perceived. Here, we present new results from field measures of undisturbed behavior and biochemical analyses of scent marks from 12 species of Sceloporus lizards to explore whether evolutionary changes in chemical composition are better predicted by measures of species behavior, particularly those associated with visual displays, chemoreception, and locomotion, or by measures of habitat climate (precipitation and temperature). We found that more active lizard species used fewer compounds in their volatile scent marks, perhaps conveying less specific information about individual and species identity. Scent marks from more active lizard species also had higher proportions of saturated fatty acids, and the evolution of these compounds has been tracking the phylogeny closely as we would expect for a metabolic byproduct. In contrast, the proportions of unsaturated fatty acids were better explained by evolutionary shifts in habitat temperature (and not precipitation), with species in warmer climates using almost no volatile unsaturated fatty acids. The proportion of aldehydes was explained by both behavior and environment, decreasing with behavioral activity and increasing with habitat temperature. Our results highlight the evolutionary flexibility of complex chemical signals, with different chemical compounds responding to different elements of the selective landscape over evolutionary time., (© The Author(s) 2020. Published by Oxford University Press on behalf of the International Society for Behavioral Ecology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Despite much effort to improve treatments, patients with malignant glioma still present a very poor prognosis that has not changed significantly in the last decades. In this context, it is crucial to better understand glioma pathogenesis to identify new molecular prognostic subgroups and therapeutic targets. WNT6 was recently identified as a new oncogenic molecule in glioblastoma (GBM), with prognostic value in patients, but the mechanisms underlying WNT6 aberrant expression in glioma are still unknown. WNT6 was overexpressed in a subset of gliomas independently of IDH mutations, 1p/19q codeletion status, and WNT6 gene copy number. Interestingly, WNT6 expression is associated with the DNA methylation levels of particular CpG regions at both the WNT6 promoter and the gene body in glioma patient samples. HOXA9, a transcription factor previously associated with poorer clinical outcome in GBM, was identified as a novel transcriptional regulator of WNT6, activating the WNT/β-catenin pathway in vitro and in vivo. In various cohorts of glioma patients, mRNA levels of WNT6 and HOXA9 were significantly correlated, extending our in vitro and in vivo findings into the clinical setting. Interestingly, this novel molecular link between WNT6 and HOXA9 was not limited to glioma, as they were co-expressed also in patients with other tumor types. Clinically, WNT6 was a prognostic biomarker of shorter survival in GBM, independently of HOXA9 expression. Concomitant high expression of both WNT6 and HOXA9 identified a subgroup of patients with particularly dismal survival. These findings describe novel WNT6 regulatory mechanisms in GBM, establishing particular DNA methylation patterns and HOXA9 as critical regulators of WNT6 expression in glioma. This HOXA9-WNT6 molecular link supports WNT signaling in GBM cells and is a powerful prognostic biomarker, highlighting the clinical relevance of this axis in patients. Novel therapies targeting WNT6-HOXA9 signaling may thus be useful for this deadly disease., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Single substances within complex vertebrate chemical signals could be physiologically or behaviourally active. However, the vast diversity in chemical structure, physical properties and molecular size of semiochemicals makes identifying pheromonally active compounds no easy task. Here, we identified two volatile cyclic dipeptides, cyclo(L-Leu-L-Pro) and cyclo(L-Pro-L-Pro), from the complex mixture of a chemical signal in terrestrial vertebrates (lizard genus Sceloporus), synthesised one of them and investigated their biological activity in male intra-specific communication. In a series of behavioural trials, lizards performed more chemosensory behaviour (tongue flicks, lip smacks and substrate lickings) when presented with the synthesised cyclo(L-Pro-L-Pro) chemical blend, compared to the controls, the cyclo(L-Leu-L-Pro) blend, or a combined blend with both cyclic dipeptides. The results suggest a potential semiochemical role of cyclo(L-Pro-L-Pro) and a modulating effect of cyclo(L-Leu-L-Pro) that may depend on the relative concentration of both compounds in the chemical signal. In addition, our results stress how minor compounds in complex mixtures can produce a meaningful behavioural response, how small differences in structural design are crucial for biological activity, and highlight the need for more studies to determine the complete functional landscape of biologically relevant compounds.

Phylogenetic comparative methods use tree topology, branch lengths, and models of phenotypic change to take into account nonindependence in statistical analysis. However, these methods normally assume that trees and models are known without error. Approaches relying on evolutionary regimes also assume specific distributions of character states across a tree, which often result from ancestral state reconstructions that are subject to uncertainty. Several methods have been proposed to deal with some of these sources of uncertainty, but approaches accounting for all of them are less common. Here, we show how Bayesian statistics facilitates this task while relaxing the homogeneous rate assumption of the well-known phylogenetic generalized least squares (PGLS) framework. This Bayesian formulation allows uncertainty about phylogeny, evolutionary regimes, or other statistical parameters to be taken into account for studies as simple as testing for coevolution in two traits or as complex as testing whether bursts of phenotypic change are associated with evolutionary shifts in intertrait correlations. A mixture of validation approaches indicates that the approach has good inferential properties and predictive performance. We provide suggestions for implementation and show its usefulness by exploring the coevolution of ankle posture and forefoot proportions in Carnivora., (© 2019 The Authors. Evolution © 2019 The Society for the Study of Evolution.)

Animals may exhibit preference for colors that match their environment or the resources in the environment. These preferences may impact ability to learn associations with these colors and revert the associations when the reward contingency is modified. We used zebrafish Danio rerio from four populations to test if color preferences impact associative and reversal learning ability. First, we tested if preference for blue or green impact associative ability. We subjected individual fish through eight trials to associate a social stimulus with blue or green. Next, we tested if preference for red or green impact associative reversal learning ability. We trained fish in groups of three to associate a social stimulus with red or green over three trials, and reversed the reward contingency during the following session. Results showed that zebrafish preferred green over blue and domesticated fish chose green more than blue when there was a reward attached. Zebrafish also preferred red over green. Fish from one wild population learned with both colors and reversed learning only from green to red and not vice-versa. Fish from another population showed an overwhelming preference for red irrespective of what was rewarded. Domesticated fish did not show reversal learning ability.

Colour signalling traits are often lost over evolutionary time, perhaps because they increase vulnerability to visual predators or lose relevance in terms of sexual selection. Here, we used spectrometric and phylogenetic comparative analyses to ask whether four independent losses of a sexually selected blue patch are spectrally similar, and whether these losses equate to a decrease in conspicuousness or to loss of a signal. We found that patches were lost in two distinct ways: either increasing reflectance primarily at very long or at very short wavelengths, and that species with additional colour elements (UV, green and pink) may be evolutionary intermediates. In addition, we found that patch spectral profiles of all species were closely aligned with visual receptors in the receiver's retina. We found that loss of the blue patch makes males less conspicuous in terms of chromatic conspicuousness, but more conspicuous in terms of achromatic contrast, and that sexual dimorphism often persists regardless of patch loss. Dorsal surfaces were considerably more cryptic than were ventral surfaces, and species in which male bellies were the most similar in conspicuousness to their dorsal surfaces were also the most sexually dimorphic. These results emphasize the consistent importance of sexual selection and its flexible impact on different signal components through evolutionary time., (© 2019 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2019 European Society For Evolutionary Biology.)

Many lizards are olfactory foragers and prey upon herbivorous arthropods, yet their responses to common herbivore-associated plant volatiles remain unknown. As such, their role in mediating plant indirect defenses also remains largely obscured. In this paper, we use a cotton-swab odor presentation assay to ask whether lizards respond to two arthropod-associated plant-derived volatile compounds: 2-( E )-hexenal and hexanoic acid. We studied the response of two lizard species, Sceloporus virgatus and Aspidoscelis exsanguis , because they differ substantially in their foraging behavior. We found that the actively foraging A. exsanguis responded strongly to hexanoic acid, whereas the ambush foraging S. virgatus responded to 2-( E )-hexenal-an herbivore-associated plant volatile involved in indirect defense against herbivores. These findings indicate that S. virgatus may contribute to plant indirect defense and that a species' response to specific odorants is linked with foraging mode. Future studies can elucidate how lizards use various compounds to locate prey and how these responses impact plant-herbivore interactions., Competing Interests: None declared.

Many animals exhibit behavioral plasticity as they move between habitats seasonally, reside in fluctuating environments, or respond to human-induced environmental change. We know that physical environment during early development can have a lasting impact on behavior, and on the neural mechanisms that shape behavior. In adults, social context can have similar persistent effects on behavior and the brain. Here, we asked whether physical context impacts adult social behavior in a novel environment. We placed groups of adult zebrafish (Danio rerio) in two different physical contexts. After two weeks, we measured group behavior in a novel context, and found that zebrafish with recent experience in a more-complex physical environment charged each other more often and tended to form tighter shoals than did fish that had been housed in less-complex environments. These differences were present regardless of the novel context in which we assayed behavior, and were not easily explained by differences in activity level. Our results demonstrate the impact of recent experiences on adult behavior, and highlight the importance of physical as well as social history in predicting animal behavior in novel situations., Competing Interests: The authors have declared that no competing interests exist.

Glioblastoma (GBM) is a universally fatal brain cancer, for which novel therapies targeting specific underlying oncogenic events are urgently needed. While the WNT pathway has been shown to be frequently activated in GBM, constituting a potential therapeutic target, the relevance of WNT6, an activator of this pathway, remains unknown. Methods: WNT6 protein and mRNA levels were evaluated in GBM. WNT6 levels were silenced or overexpressed in GBM cells to assess functional effects in vitro and in vivo . Phospho-kinase arrays and TCF/LEF reporter assays were used to identify WNT6-signaling pathways, and significant associations with stem cell features and cancer-related pathways were validated in patients. Survival analyses were performed with Cox regression and Log-rank tests. Meta-analyses were used to calculate the estimated pooled effect. Results: We show that WNT6 is significantly overexpressed in GBMs, as compared to lower-grade gliomas and normal brain, at mRNA and protein levels. Functionally, WNT6 increases typical oncogenic activities in GBM cells, including viability, proliferation, glioma stem cell capacity, invasion, migration, and resistance to temozolomide chemotherapy. Concordantly, in in vivo orthotopic GBM mice models, using both overexpressing and silencing models, WNT6 expression was associated with shorter overall survival, and increased features of tumor aggressiveness. Mechanistically, WNT6 contributes to activate typical oncogenic pathways, including Src and STAT, which intertwined with the WNT pathway may be critical effectors of WNT6-associated aggressiveness in GBM. Clinically, we establish WNT6 as an independent prognostic biomarker of shorter survival in GBM patients from several independent cohorts. Conclusion: Our findings establish WNT6 as a novel oncogene in GBM, opening opportunities to develop more rational therapies to treat this highly aggressive tumor., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Territorial animals lay scent marks around their territories to broadcast their presence, but these olfactory signals can both attract and repel con-specifics. Attraction or aversion can have a profound impact in terms of space use and thereby influence an individual's access to resources and mates. Here, we test the impact of chemical signals on the long-term space use and activity of receivers, comparing the response of males and females, territory holders, and temporary visitors in Sceloporus undulatus lizards in the field. We placed either male femoral gland secretions (chemical) or blank (control) cues on resident male landmarks, repeatedly over 5 d, while monitoring the activity and location of all lizards in the vicinity. We found that resident males and females, but not non-resident males, were active on more days near landmarks treated with chemical cues than landmarks treated with control cues. Non-resident males remained closer to chemical than control cues. These results suggest that territorial scent marks are attractive to conspecifics and impact space use, but that the specific effects depend on receiver sex and residency status. Such subtle or gradual changes in behavior may frequently be overlooked by short-term choice experiments. Future studies investigating the behavioral significance of a communicative signal should consider these finer details of behavior for a more comprehensive assessment.

We present a new phylogenetic comparative method-phylogenetic analysis of covariance (PANCOVA)-that uses interspecific data and a phylogeny to estimate the effects of major events on both the rate of phenotypic evolution and the association between traits. It could be used, for example, to model the impact of a key innovation, colonization of a new habitat, or environmental change. The approach is optimized with maximum likelihood and is formulated under the familiar phylogenetic generalized least squares framework, which is flexible and easily extended to incorporate other factors and parameters. As an example, we explore the relationship between parental investment and relative telencephalon size in birds and contrast the results of PANCOVA with those from other phylogenetic comparative methods.

Many evolutionary forces can shape the evolution of communicative signals, and the long-term impact of each force may depend on relative timing and magnitude. We use a phylogenetic analysis to infer the history of blue belly patches of Sceloporus lizards, and a detailed spectrophotometric analysis of four species to explore the specific forces shaping evolutionary change. We find that the ancestor of Sceloporus had blue patches. We then focus on four species; the first evolutionary shift (captured by comparison of S. merriami and S. siniferus ) represents an ancient loss of the belly patch by S. siniferus , and the second evolutionary shift, bounded by S. undulatus and S. virgatus , represents a more recent loss of blue belly patch by S. virgatus . Conspicuousness measurements suggest that the species with the recent loss ( S. virgatus ) is the least conspicuous. Results for two other species ( S. siniferus and S. merriami ) suggest that over longer periods of evolutionary time, new signal colours have arisen which minimize absolute contrast with the habitat while maximizing conspicuousness to a lizard receiver. Specifically, males of the species representing an ancient loss of blue patch ( S. siniferus ) are more conspicuous than are females in the UV, whereas S. merriami males have evolved a green element that makes their belly patches highly sexually dimorphic but no more conspicuous than the white bellies of S. merriami females. Thus, our results suggest that natural selection may act more immediately to reduce conspicuousness, whereas sexual selection may have a more complex impact on communicative signals through the introduction of new colours.

In vertebrates, viviparity has evolved independently multiple times, apparently increasing morphological diversification and speciation rates as a consequence. We tested whether the evolution of viviparity has also increased diversification of life-history traits by estimating evolutionary rates of lizards from the North American family Phrynosomatidae. Using modern phylogenetic comparative methods, we compared these rates between oviparous and viviparous species, and found no support for this hypothesis. Instead, we found higher evolutionary rates for oviparous species in some life-history traits. Our results suggest that the evolution of viviparity may have constrained rather than facilitated evolution of life histories., (© 2016 The Author(s).)

Animals rely on multimodal signals to obtain information from conspecifics through alternative sensory systems, and the evolutionary loss of a signal in one modality may lead to compensation through increased use of signals in an alternative modality. We investigated associations between chemical signaling and evolutionary loss of abdominal color patches in males of four species (two plain-bellied and two colorful-bellied) of Sceloporus lizards. We conducted field trials to compare behavioral responses of male lizards to swabs with femoral gland (FG) secretions from conspecific males and control swabs (clean paper). We also analyzed the volatile organic compound (VOC) composition of male FG secretions by stir bar extraction and gas chromatography-mass spectrometry (GC-MS) to test the hypothesis that loss of the visual signal is associated with elaboration of the chemical signal. Males of plain-bellied, but not colorful-bellied species exhibited different rates of visual displays when exposed to swabs of conspecific FG secretions relative to control swabs. The VOC composition of male Sceloporus FG secretions was similar across all four species, and no clear association between relative abundances of VOCs and evolutionary loss of abdominal color patches was observed. The emerging pattern is that behavioral responses to conspecific chemical signals are species- and context-specific in male Sceloporus, and compensatory changes in receivers, but not signalers may be involved in mediating increased responsiveness to chemical signals in males of plain-bellied species.

Fossils and other paleontological information can improve phylogenetic comparative method estimates of phenotypic evolution and generate hypotheses related to species diversification. Here, we use fossil information to calibrate ancestral reconstructions of suitable climate for Sceloporus lizards in North America. Integrating data from the fossil record, general circulation models of paleoclimate during the Miocene, climate envelope modeling, and phylogenetic comparative methods provides a geographically and temporally explicit species distribution model of Sceloporus-suitable habitat through time. We provide evidence to support the historic biogeographic hypothesis of Sceloporus diversification in warm North American deserts and suggest a relatively recent Sceloporus invasion into Mexico around 6 Ma. We use a physiological model to map extinction risk. We suggest that the number of hours of restriction to a thermal refuge limited Sceloporus from inhabiting Mexico until the climate cooled enough to provide suitable habitat at approximately 6 Ma. If the future climate returns to the hotter climates of the past, Mexico, the place of highest modern Sceloporus richness, will no longer provide suitable habitats for Sceloporus to survive and reproduce.

Wild zebrafish exhibit a wide range of behavior. We found abundant wild zebrafish in flowing rivers and still water, in large, tightly-knit groups of hundreds of individuals, as well as in small, loose shoals. In two still-water populations, zebrafish were quite small in body size, common, and in tight groups of up to 22 fish. As in earlier laboratory studies, these zebrafish exhibited very low levels of aggression. In slowly flowing water in central India, zebrafish were relatively rare and gathered in small shoals (4-12 fish), often with other small fish, such as Rasbora daniconius. These stream zebrafish were larger in body size (27 mm TL) and much more aggressive than those in still water. In a second river population with much faster flowing water, zebrafish were abundant and again relatively large (21 mm TL). These zebrafish occurred in very large (up to 300 individuals) and tightly-knit (nearest-neighbor distances up to 21 mm) groups that exhibited collective rheotaxis and almost no aggression. This remarkable variation in social behavior of wild zebrafish offers an opportunity for future studies of behavioral genetics, development, and neuroscience.

Plastic responses can have adaptive significance for organisms occurring in unpredictable environments, migratory species and organisms occupying novel environments. Zebrafish (Danio rerio) occur in a wide range of habitats and environments that fluctuate frequently across seasons and habitats. We expect wild populations of fish to be behaviorally more flexible than fish reared in conventional laboratory and hatchery environments. We measured three behavioral traits among 2 wild (U and PN) and 1 laboratory bred (SH) zebrafish populations in four environments differing in water flow and vegetation regimes. We found that the degree of plasticity varied with the type of behavior and also among populations. In general, vegetation increased aggression and water flow decreased latency to feed after a disturbance, but the patterns were population dependent. For example, while wild U fish fed more readily after a disturbance in vegetated and/or flowing habitats, fish from the wild PN population and lab-reared SH strain showed little variation in foraging across different environmental conditions. Zebrafish from all the three populations were more aggressive when tested in an arena with vegetation. In contrast, while there was an inter-population difference in shoaling distances, variation in shoaling distance across environmental conditions within populations was not significant. These results suggest that both foraging and aggression in zebrafish are more plastic and influenced by immediate context than is shoaling distance, which may have a stronger genetic basis. Our findings point to different underlying mechanisms influencing the expression of these traits and warrants further investigations.

The neural and molecular mechanisms underlying social behavior - including their functional significance and evolution - can only be fully understood using data obtained under multiple social, environmental, and physiological conditions. Understanding the complexity of social behavior requires integration across levels of analysis in both laboratory and field settings. However, there is currently a disconnect between the systems studied in the laboratory versus the field. We argue that recent conceptual and technical advances provide exciting new opportunities to close this gap by making non-model organisms accessible to modern approaches in both laboratory and nature., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Signals that convey related information may impose selection on each other, creating evolutionary links between different components of the communicative repertoire. Here, we ask about the consequences of the evolutionary loss of one signal (a colour patch) on another (a motion display) in Sceloporus lizards. We present data on male lizards of four species: two pairs of sister taxa representing two independent evolutionary losses of the static colour patch ( Sceloporus cozumelae and Sceloporus parvus ; Sceloporus siniferus and Sceloporus merriami ). Males of the two species that have undergone an evolutionary loss of blue-belly patches ( S. cozumelae, S. siniferus ) were less active than their blue-bellied sister taxa ( S. parvus, S. merriami ), consistent with the suggestion that the belly patches were lost to reduce conspicuousness of species with high predation pressure. In contrast, the headbob display appears to have become more, rather than less, conspicuous over evolutionary time. The colour patch is exhibited primarily during aggressive encounters, whereas headbob displays are multifunction signals used in several different contexts, including aggressive encounters. Males of species that have lost the colour patch produced more motion displays, and the structure of those motion displays were more similar to those produced during combat. In both evolutionary episodes, a static colour signal appears to have been replaced by dynamic motion displays that can be turned off in the presence of predators and other unwanted receivers. The predominant pattern is one of evolutionary compensation and interactions between multiple signals that convey related information.