Ontogeny of color development in two green-brown polymorphic grasshopper species.

Many insects, including several orthopterans, undergo dramatic changes in body coloration during ontogeny. This variation is particularly intriguing in gomphocerine grasshoppers, where the green and brown morphs appear to be genetically determined (Schielzeth & Dieker, 2020, BMC Evolutionary Biology, 20, 63; Winter et al., 2021, Heredity, 127, 66). A better understanding of how these color morphs develop during ontogeny can provide valuable insights into the evolution and ecology of such a widespread color polymorphism. Here, we focus on the color development of two green-brown polymorphic species, the club-legged grasshopper Gomphocerus sibiricus and the steppe grasshopper Chorthippus dorsatus. By following the color development of individuals from hatching to adulthood, we found that color morph differences begin to develop during the second nymphal stage, are clearly defined by the third nymphal stage, and remain stable throughout the life of an individual. Interestingly, we also observed that shed skins of late nymphal stages are identifiable by color morphs based on their yellowish coloration, rather than the green that marks green body parts. Furthermore, by assessing how these colors are perceived by different visual systems, we found that certain potential predators can chromatically discriminate between morphs, while others may not. These results suggest that the putative genes controlling color morph are active during the early stages of ontogeny, and that green color is likely composed of two components, one present in the cuticle and one not. In addition, the effectiveness of camouflage appears to vary depending on the specific predator involved.

Condition dependence of (un)predictability in escape behavior of a grasshopper species.

(Un)predictability has only recently been recognized as an important dimension of animal behavior. Currently, we neither know if (un)predictability encompasses one or multiple traits nor how (un)predictability is dependent on individual conditions. Knowledge about condition dependence, in particular, could inform us about whether predictability or unpredictability is costly in a specific context. Here, we study the condition dependence of (un)predictability in the escape behavior of the steppe grasshopper Chorthippus dorsatus. Predator-prey interactions represent a behavioral context in which we expect unpredictability to be particularly beneficial. By exposing grasshoppers to an immune challenge, we explore if individuals in poor condition become more or less predictable. We quantified three aspects of escape behavior (flight initiation distance, jump distance, and jump angle) in a standardized setup and analyzed the data using a multivariate double-hierarchical generalized linear model. The immune challenge did not affect (un)predictability in flight initiation distance and jump angle, but decreased unpredictability in jump distances, suggesting that unpredictability can be costly. Variance decomposition shows that 3-7% of the total phenotypic variance was explained by individual differences in (un)predictability. Covariation between traits was found both among averages and among unpredictabilities for one of the three trait pairs. The latter might suggest an (un)predictability syndrome, but the lack of (un)predictability correlation in the third trait suggests modularity. Our results indicated condition dependence of (un)predictability in grasshopper escape behavior in one of the traits, and illustrate the value of mean and residual variance decomposition for analyzing animal behavior.

Conditional on the social environment? Roots of repeatability in hormone concentrations of male guinea pigs.

Individual differences in behavioral and physiological traits among members of the same species are increasingly being recognized as important in animal research. On the group level, shaping of behavioral and hormonal phenotypes by environmental factors has been reported in different taxa. The extent to which the environment impacts behavior and hormones on the individual level, however, is rather unexplored. Hormonal phenotypes of guinea pigs can be shaped by the social environment on the group level: pair-housed and colony-housed males differ systematically in average testosterone and stressor-induced cortisol levels (i.e. cortisol responsiveness). The aim of the present study was to evaluate whether repeatability and individual variance components (i.e. between- and within-individual variation) of hormonal phenotypes also differ in different social environments. To test this, we determined baseline testosterone, baseline cortisol, and cortisol responsiveness after challenge in same-aged pair-housed and colony-housed guinea pig males over a period of four months. We found comparable repeatability for baseline cortisol and cortisol responsiveness in males from both social conditions. In contrast, baseline testosterone was repeatable only in males from colonies. Interestingly, this result was brought about by significantly larger between-individual variation of testosterone, that was not explained by differences in dominance rank. Individualized social niches differentiated under complex colony, but not pair housing, could be an explanation for this finding.

A Phylogenomic Assessment of Processes Underpinning Convergent Evolution in Open-Habitat Chats.

Insights into the processes underpinning convergent evolution advance our understanding of the contributions of ancestral, introgressed, and novel genetic variation to phenotypic evolution. Phylogenomic analyses characterizing genome-wide gene tree heterogeneity can provide first clues about the extent of ILS and of introgression and thereby into the potential of these processes or (in their absence) the need to invoke novel mutations to underpin convergent evolution. Here, we were interested in understanding the processes involved in convergent evolution in open-habitat chats (wheatears of the genus Oenanthe and their relatives). To this end, based on whole-genome resequencing data from 50 taxa of 44 species, we established the species tree, characterized gene tree heterogeneity, and investigated the footprints of ILS and introgression within the latter. The species tree corroborates the pattern of abundant convergent evolution, especially in wheatears. The high levels of gene tree heterogeneity in wheatears are explained by ILS alone only for 30% of internal branches. For multiple branches with high gene tree heterogeneity, D-statistics and phylogenetic networks identified footprints of introgression. Finally, long branches without extensive ILS between clades sporting similar phenotypes provide suggestive evidence for the role of novel mutations in the evolution of these phenotypes. Together, our results suggest that convergent evolution in open-habitat chats involved diverse processes and highlight that phenotypic diversification is often complex and best depicted as a network of interacting lineages.

Once an optimist, always an optimist? Studying cognitive judgment bias in mice.

Individuals differ in the way they judge ambiguous information: some individuals interpret ambiguous information in a more optimistic, and others in a more pessimistic way. Over the past two decades, such "optimistic" and "pessimistic" cognitive judgment biases (CJBs) have been utilized in animal welfare science as indicators of animals' emotional states. However, empirical studies on their ecological and evolutionary relevance are still lacking. We, therefore, aimed at transferring the concept of "optimism" and "pessimism" to behavioral ecology and investigated the role of genetic and environmental factors in modulating CJB in mice. In addition, we assessed the temporal stability of individual differences in CJB. We show that the chosen genotypes (C57BL/6J and B6D2F1N) and environments ("scarce" and "complex") did not have a statistically significant influence on the responses in the CJB test. By contrast, they influenced anxiety-like behavior with C57BL/6J mice and mice from the "complex" environment displaying less anxiety-like behavior than B6D2F1N mice and mice from the "scarce" environment. As the selected genotypes and environments did not explain the existing differences in CJB, future studies might investigate the impact of other genotypes and environmental conditions on CJB, and additionally, elucidate the role of other potential causes like endocrine profiles and epigenetic modifications. Furthermore, we show that individual differences in CJB were repeatable over a period of seven weeks, suggesting that CJB represents a temporally stable trait in laboratory mice. Therefore, we encourage the further study of CJB within an animal personality framework.

How Individualized Niches Arise: Defining Mechanisms of Niche Construction, Niche Choice, and Niche Conformance.

Organisms interact with their environments in various ways. We present a conceptual framework that distinguishes three mechanisms of organism-environment interaction. We call these NC3 mechanisms: niche construction, in which individuals make changes to the environment; niche choice, in which individuals select an environment; and niche conformance, in which individuals adjust their phenotypes in response to the environment. Each of these individual-level mechanisms affects an individual's phenotype-environment match, its fitness, and its individualized niche, defined in terms of the environmental conditions under which the individual can survive and reproduce. Our framework identifies how individuals alter the selective regimes that they and other organisms experience. It also places clear emphasis on individual differences and construes niche construction and other processes as evolved mechanisms. The NC3 mechanism framework therefore helps to integrate population-level and individual-level research.

Evidence for morph-specific substrate choice in a green-brown polymorphic grasshopper.

Orthopteran insects are characterized by high variability in body coloration, in particular featuring a widespread green-brown color polymorphism. The mechanisms that contribute to the maintenance of this apparently balanced polymorphism are not yet understood. To investigate whether morph-dependent microhabitat choice might contribute to the continued coexistence of multiple morphs, we studied substrate choice in the meadow grasshopper Pseudochorthippus parallelus. The meadow grasshopper occurs in multiple discrete, genetically determined color morphs that range from uniform brown to uniform green. We tested whether three common morphs preferentially choose differently colored backgrounds in an experimental arena. We found that a preference for green backgrounds was most pronounced in uniform green morphs. If differential choices improve morph-specific performance in natural habitats via crypsis and/or thermoregulatory benefits, they could help to equalize fitness differences among color morphs and potentially produce frequency-dependent microhabitat competition, though difference appear too small to serve as the only explanation. We also measured the reflectance of the grasshoppers and backgrounds and used visual modeling to quantify the detectability of the different morphs to a range of potential predators. Multiple potential predators, including birds and spiders, are predicted to distinguish between morphs chromatically, while other species, possibly including grasshoppers themselves, will perceive only differences in brightness. Our study provides the first evidence that morph-specific microhabitat choice might be relevant to the maintenance of the green-brown polymorphisms in grasshoppers and shows that visual distinctness of color morphs varies between perceivers.

partR2: partitioning R2 in generalized linear mixed models.

The coefficient of determination R 2 quantifies the amount of variance explained by regression coefficients in a linear model. It can be seen as the fixed-effects complement to the repeatability R (intra-class correlation) for the variance explained by random effects and thus as a tool for variance decomposition. The R 2 of a model can be further partitioned into the variance explained by a particular predictor or a combination of predictors using semi-partial (part) R 2 and structure coefficients, but this is rarely done due to a lack of software implementing these statistics. Here, we introduce partR2, an R package that quantifies part R 2 for fixed effect predictors based on (generalized) linear mixed-effect model fits. The package iteratively removes predictors of interest from the model and monitors the change in the variance of the linear predictor. The difference to the full model gives a measure of the amount of variance explained uniquely by a particular predictor or a set of predictors. partR2 also estimates structure coefficients as the correlation between a predictor and fitted values, which provide an estimate of the total contribution of a fixed effect to the overall prediction, independent of other predictors. Structure coefficients can be converted to the total variance explained by a predictor, here called 'inclusive' R 2, as the square of the structure coefficients times total R 2. Furthermore, the package reports beta weights (standardized regression coefficients). Finally, partR2 implements parametric bootstrapping to quantify confidence intervals for each estimate. We illustrate the use of partR2 with real example datasets for Gaussian and binomial GLMMs and discuss interactions, which pose a specific challenge for partitioning the explained variance among predictors.

Simple inheritance of color and pattern polymorphism in the steppe grasshopper Chorthippus dorsatus.

The green-brown polymorphism of grasshoppers and bush-crickets represents one of the most penetrant polymorphisms in any group of organisms. This poses the question of why the polymorphism is shared across species and how it is maintained. There is mixed evidence for whether and in which species it is environmentally or genetically determined in Orthoptera. We report breeding experiments with the steppe grasshopper Chorthippus dorsatus, a polymorphic species for the presence and distribution of green body parts. Morph ratios did not differ between sexes, and we find no evidence that the rearing environment (crowding and habitat complexity) affected the polymorphism. However, we find strong evidence for genetic determination for the presence/absence of green and its distribution. Results are most parsimoniously explained by three autosomal loci with two alleles each and simple dominance effects: one locus influencing the ability to show green color, with a dominant allele for green; a locus with a recessive allele suppressing green on the dorsal side; and a locus with a recessive allele suppressing green on the lateral side. Our results contribute to the emerging contrast between the simple genetic inheritance of green-brown polymorphisms in the subfamily Gomphocerinae and environmental determination in other subfamilies of grasshoppers. In three out of four species of Gomphocerinae studied so far, the results suggest one or a few loci with a dominance of alleles allowing the occurrence of green. This supports the idea that brown individuals differ from green individuals by homozygosity for loss-of-function alleles preventing green pigment production or deposition.

Individuality meets plasticity: Endocrine phenotypes across male dominance rank acquisition in guinea pigs living in a complex social environment.

The time of dominance rank acquisition is a crucial phase in male life history that often affects reproductive success and hence fitness. Hormones such as testosterone and glucocorticoids can influence as well as be affected by this process. At the same time, hormone concentrations can show large individual variation. The extent to which such variation is repeatable, particularly in dynamic social settings, is a question of current interest. The aim of the present study was therefore to investigate how dominance rank and individual differences contribute to variance in hormone concentrations during male rank acquisition in a complex social environment. For this purpose, dominance rank as well as baseline testosterone, baseline cortisol, and cortisol responsiveness after exposure to a novel environment were determined in colony-housed guinea pig males from late adolescence through adulthood. Hormone-dominance relationships and repeatability of hormone measures beyond their relation to rank were assessed. There was a significant positive relationship between baseline testosterone and rank, but this link became weaker with increasing age. Baseline cortisol or cortisol responsiveness, in contrast, were not significantly related to dominance. Notably, all three endocrine parameters were significantly repeatable independent of dominance rank from late adolescence through adulthood. Baseline testosterone and cortisol responsiveness showed a significantly higher repeatability than baseline cortisol. This suggests that testosterone titres and cortisol responsiveness represent stable individual attributes even under complex social conditions.

Poor nutritional condition promotes high-risk behaviours: a systematic review and meta-analysis.

Animal behaviour can lead to varying levels of risk, and an individual's physical condition can alter the potential costs and benefits of undertaking risky behaviours. How risk-taking behaviour depends on condition is subject to contrasting hypotheses. The asset protection principle proposes that individuals in better condition should be more risk averse, as they have higher future reproductive potential (i.e. more to lose). The state-dependent safety hypothesis proposes that high-condition individuals that are more likely to survive and maximise the benefits of risky situations may make apparently riskier choices, as their individual risk is in fact lower. We systematically searched for studies that experimentally manipulated animals' nutritional or energetic condition through diet treatments, and subsequently measured risk-taking behaviour in contexts relating to predation, novelty and exploration. Our meta-analysis quantified condition effects on risk-taking behaviour at both the mean and variance level. We preregistered our methods and hypotheses prior to conducting the study. Phylogenetic multilevel meta-analysis revealed that the lower-nutritional-condition individuals showed on average ca. 26% greater tendency towards risk than high-condition individuals (95% confidence interval: 15-38%; N = 126 studies, 1297 effect sizes). Meta-regressions revealed several factors influencing the overall effect, such as the experimental context used to measure risk-taking behaviour, and the life stage when condition was manipulated. Meta-analysis of variance revealed no clear overall effect of condition on behavioural variance (on average ca. 3% decrease in variance in low- versus high-condition groups; 95% confidence interval: -8 to 3%; N = 119 studies, 1235 effect sizes), however, the experimental context was an important factor influencing the strength and direction of the variance effect. Our comprehensive systematic review and meta-analysis provide insights into the roles of state dependency and plasticity in intraspecific behavioural variation. While heterogeneity among effect sizes was high, our results show that poor nutritional state on average increases risk taking in ecological contexts involving predation, novelty and exploration.

Collision between biological process and statistical analysis revealed by mean centring.

Animal ecologists often collect hierarchically structured data and analyse these with linear mixed-effects models. Specific complications arise when the effect sizes of covariates vary on multiple levels (e.g. within vs. among subjects). Mean centring of covariates within subjects offers a useful approach in such situations, but is not without problems. A statistical model represents a hypothesis about the underlying biological process. Mean centring within clusters assumes that the lower level responses (e.g. within subjects) depend on the deviation from the subject mean (relative) rather than on the absolute scale of the covariate. This may or may not be biologically realistic. We show that mismatch between the nature of the generating (i.e. biological) process and the form of the statistical analysis produce major conceptual and operational challenges for empiricists. We explored the consequences of mismatches by simulating data with three response-generating processes differing in the source of correlation between a covariate and the response. These data were then analysed by three different analysis equations. We asked how robustly different analysis equations estimate key parameters of interest and under which circumstances biases arise. Mismatches between generating and analytical equations created several intractable problems for estimating key parameters. The most widely misestimated parameter was the among-subject variance in response. We found that no single analysis equation was robust in estimating all parameters generated by all equations. Importantly, even when response-generating and analysis equations matched mathematically, bias in some parameters arose when sampling across the range of the covariate was limited. Our results have general implications for how we collect and analyse data. They also remind us more generally that conclusions from statistical analysis of data are conditional on a hypothesis, sometimes implicit, for the process(es) that generated the attributes we measure. We discuss strategies for real data analysis in face of uncertainty about the underlying biological process.

Sperm velocity in a promiscuous bird across experimental media of different viscosities.

In species with internal fertilization, the female genital tract appears challenging to sperm, possibly resulting from selection on for example ovarian fluid to control sperm behaviour and, ultimately, fertilization. Few studies, however, have examined the effects of swimming media viscosities on sperm performance. We quantified effects of media viscosities on sperm velocity in promiscuous willow warblers Phylloscopus trochilus. We used both a reaction norm and a character-state approach to model phenotypic plasticity of sperm behaviour across three experimental media of different viscosities. Compared with a standard medium (Dulbecco's Modified Eagle Medium, DMEM), media enriched with 1% or 2% w/v methyl cellulose decreased sperm velocity by up to about 50%. Spermatozoa from experimental ejaculates of different males responded similarly to different viscosities, and a lack of covariance between elevations and slopes of individual velocity-by-viscosity reaction norms indicated that spermatozoa from high- and low-velocity ejaculates were slowed down by a similar degree when confronted with high-viscosity environments. Positive cross-environment (1% versus 2% cellulose) covariances of sperm velocity under the character-state approach suggested that sperm performance represents a transitive trait, with rank order of individual ejaculates maintained when expressed against different environmental backgrounds. Importantly, however, a lack of significant covariances in sperm velocity involving a cellulose concentration of 0% indicated that pure DMEM represented a qualitatively different environment, questioning the validity of this widely used standard medium for assaying sperm performance. Enriching sperm environments along ecologically relevant gradients prior to assessing sperm performance will strengthen explanatory power of in vitro studies of sperm behaviour.

Author Correction: Reproducibility of animal research in light of biological variation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Comparative Analysis of Genomic Repeat Content in Gomphocerine Grasshoppers Reveals Expansion of Satellite DNA and Helitrons in Species with Unusually Large Genomes.

Eukaryotic organisms vary widely in genome size and much of this variation can be explained by differences in the abundance of repetitive elements. However, the phylogenetic distributions and turnover rates of repetitive elements are largely unknown, particularly for species with large genomes. We therefore used de novo repeat identification based on low coverage whole-genome sequencing to characterize the repeatomes of six species of gomphocerine grasshoppers, an insect clade characterized by unusually large and variable genome sizes. Genome sizes of the six species ranged from 8.4 to 14.0 pg DNA per haploid genome and thus include the second largest insect genome documented so far (with the largest being another acridid grasshopper). Estimated repeat content ranged from 79% to 96% and was strongly correlated with genome size. Averaged over species, these grasshopper repeatomes comprised significant amounts of DNA transposons (24%), LINE elements (21%), helitrons (13%), LTR retrotransposons (12%), and satellite DNA (8.5%). The contribution of satellite DNA was particularly variable (ranging from <1% to 33%) as was the contribution of helitrons (ranging from 7% to 20%). The age distribution of divergence within clusters was unimodal with peaks ∼4-6%. The phylogenetic distribution of repetitive elements was suggestive of an expansion of satellite DNA in the lineages leading to the two species with the largest genomes. Although speculative at this stage, we suggest that the expansion of satellite DNA could be secondary and might possibly have been favored by selection as a means of stabilizing greatly expanded genomes.

The green-brown polymorphism of the club-legged grasshopper Gomphocerus sibiricus is heritable and appears genetically simple.

BACKGROUND: Local coexistence of distinct, genetically determined color morphs can be unstable and transitional. Stable, long-term coexistence requires some form of balancing selection to protect morphs from getting lost by directional selection or genetic drift. However, not all phenotypic polymorphism need to have a genetic basis. We here report on the genetic basis of two color polymorphisms in the club-legged grasshopper Gomphocerus sibiricus: a green-brown polymorphism that is phylogenetically and geographically widespread among orthopteran insects and a pied-brown pattern polymorphism that is shared among many gomphocerine grasshoppers. RESULTS: We found a remarkably clear outcome of matings within and between morph that suggest not only that the green-brown polymorphism is heritable in this species, but that results can be most parsimoniously explained by a single autosomal locus with two alleles in which the green allele is dominant over the brown allele. A few individuals did not match this pattern and suggest the existence of genetic modifiers and/or developmental phenocopies. We also show that the pied-brown polymorphism is highly heritable, although the evidence for the involvement of one or more loci is less clear-cut. CONCLUSIONS: Overall, our data demonstrate that the two polymorphisms are heritable in the club-legged grasshopper and appear genetically simple, at least with respect to green morphs. The results are consistent with the idea that the synthesis or transport of a pigment involved in the production of green coloration (likely biliverdin) is lost by homozygosity for loss-of-function alleles in brown individuals. The apparently simple genetic architecture of the green-brown polymorphism offer potential for studying balancing selection in the field and for genetic mapping in this species.

Reproducibility of animal research in light of biological variation.

Context-dependent biological variation presents a unique challenge to the reproducibility of results in experimental animal research, because organisms' responses to experimental treatments can vary with both genotype and environmental conditions. In March 2019, experts in animal biology, experimental design and statistics convened in Blonay, Switzerland, to discuss strategies addressing this challenge. In contrast to the current gold standard of rigorous standardization in experimental animal research, we recommend the use of systematic heterogenization of study samples and conditions by actively incorporating biological variation into study design through diversifying study samples and conditions. Here we provide the scientific rationale for this approach in the hope that researchers, regulators, funders and editors can embrace this paradigm shift. We also present a road map towards better practices in view of improving the reproducibility of animal research.

Linked-read sequencing enables haplotype-resolved resequencing at population scale.

The feasibility to sequence entire genomes of virtually any organism provides unprecedented insights into the evolutionary history of populations and species. Nevertheless, many population genomic inferences - including the quantification and dating of admixture, introgression and demographic events, and inference of selective sweeps - are still limited by the lack of high-quality haplotype information. The newest generation of sequencing technology now promises significant progress. To establish the feasibility of haplotype-resolved genome resequencing at population scale, we investigated properties of linked-read sequencing data of songbirds of the genus Oenanthe across a range of sequencing depths. Our results based on the comparison of downsampled (25×, 20×, 15×, 10×, 7×, and 5×) with high-coverage data (46-68×) of seven bird genomes mapped to a reference suggest that phasing contiguities and accuracies adequate for most population genomic analyses can be reached already with moderate sequencing effort. At 15× coverage, phased haplotypes span about 90% of the genome assembly, with 50% and 90% of phased sequences located in phase blocks longer than 1.25-4.6 Mb (N50) and 0.27-0.72 Mb (N90). Phasing accuracy reaches beyond 99% starting from 15× coverage. Higher coverages yielded higher contiguities (up to about 7 Mb/1 Mb [N50/N90] at 25× coverage), but only marginally improved phasing accuracy. Phase block contiguity improved with input DNA molecule length; thus, higher-quality DNA may help keeping sequencing costs at bay. In conclusion, even for organisms with gigabase-sized genomes like birds, linked-read sequencing at moderate depth opens an affordable avenue towards haplotype-resolved genome resequencing at population scale.