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.

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.

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.

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.

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.

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.

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.

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.

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.

Comparative analysis of the multivariate genetic architecture of morphological traits in three species of Gomphocerine grasshoppers.

Evolutionary change is the change in trait values across generations, and usually occurs in multidimensional trait space rather than along isolated traits. Genetic covariation influences the magnitude and direction of evolutionary change and can be statistically summarized by the additive genetic (co)variance matrix, G. While G can affect the response to selection, it is exposed to evolutionary change by selection and genetic drift, but the magnitude and speed of these changes are poorly understood. We use comparative G matrix analyses to assess evolution of the shape and orientation of G over longer timescales in three species of Gomphocerine grasshoppers. We estimate 10 × 10 G matrices for five morphological traits expressed in both sexes. We find low-to-moderate heritabilities (average 0.36), mostly large cross-sex correlations (average 0.54) and moderate between-trait correlations (average 0.34). G matrices differ significantly among species with wing length contributing most to these differences. Wing length is the trait that is most divergent among species, suggesting it has been under selection during species divergence. The more distantly related species, Pseudochorthippus parallelus, was the most different in the shape of G. Projection of contemporary genetic variation into the divergence space D illustrates that the major axis of genetic variation in Gomphocerippus rufus is aligned with divergence from Chorthippus biguttulus, while the major axis of genetic variation in neither of the species is aligned with the divergence between Pseudochorthippus parallelus and the other two species. Our results demonstrate significant differences in G matrices with a phylogenetic signal in the differentiation.

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.

Transcriptome assembly for a colour-polymorphic grasshopper (Gomphocerus sibiricus) with a very large genome size.

BACKGROUND: The club-legged grasshopper Gomphocerus sibiricus is a Gomphocerinae grasshopper with a promising future as model species for studying the maintenance of colour-polymorphism, the genetics of sexual ornamentation and genome size evolution. However, limited molecular resources are available for this species. Here, we present a de novo transcriptome assembly as reference resource for gene expression studies. We used high-throughput Illumina sequencing to generate 5,070,036 paired-end reads after quality filtering. We then combined the best-assembled contigs from three different de novo transcriptome assemblers (Trinity, SOAPdenovo-trans and Oases/Velvet) into a single assembly. RESULTS: This resulted in 82,251 contigs with a N50 of 1357 and a TransRate assembly score of 0.325, which compares favourably with other orthopteran transcriptome assemblies. Around 87% of the transcripts could be annotated using InterProScan 5, BLASTx and the dammit! annotation pipeline. We identified a number of genes involved in pigmentation and green pigment metabolism pathways. Furthermore, we identified 76,221 putative single nucleotide polymorphisms residing in 8400 contigs. We also assembled the mitochondrial genome and investigated levels of sequence divergence with other species from the genus Gomphocerus. Finally, we detected and assembled Wolbachia sequences, which revealed close sequence similarity to the strain pel wPip. CONCLUSIONS: Our study has generated a significant resource for uncovering genotype-phenotype associations in a species with an extraordinarily large genome, while also providing mitochondrial and Wolbachia sequences that will be useful for comparative studies.

Genome-wide evidence supports mitochondrial relationships and pervasive parallel phenotypic evolution in open-habitat chats.

In wheatears and related species ('open-habitat chats'), molecular phylogenetics has led to a comprehensively revised understanding of species relationships and species diversity. Phylogenetic analyses have suggested that, in many cases, phenotypic similarities do not reflect species' relationships, revealing traditionally defined genera as non-monophyletic. This led to the suggestion of pervasive parallel evolution of open-habitat chats' plumage coloration and ecological phenotypes. However, to date, the molecular evidence for the phylogenetic relationships among open-habitat chats is mainly limited to mitochondrial DNA. Here, we assessed whether the mitochondrial relationships are supported by genome-wide data. To this end, we reconstructed the species tree among 14 open-habitat chat taxa using multi-species coalescent analyses based on ~1'300 SNPs. Our results confirm previous ones based chiefly on mitochondrial DNA; notably the paraphyly of the Oenanthe lugens complex and the clustering of individual species formerly placed in the genera Cercomela and Myrmecocichla within Oenanthe. Since several variable morphological and ecological characteristics occur in multiple places across the open-habitat chat phylogeny, our study consolidates the evidence for pervasive parallel evolution in the plumage coloration and ecology of open-habitat chats.

Direct and indirect genetic effects on reproductive investment in a grasshopper.

A fundamental part of the quantitative genetic theory deals with the partitioning of the phenotypic variance into additive genetic and environmental components. During interaction with conspecifics, the interaction partner becomes a part of the environment from the perspective of the focal individual. If the interaction effects have a genetic basis, they are called indirect genetic effects (IGEs) and can evolve along with direct genetic effects. Sexual reproduction is a classic context where potential conflict between males and females can arise from trade-offs between current and future investments. We studied five female fecundity traits, egg length and number, egg pod length and number and latency to first egg pod, and estimated the direct and IGEs using a half-sib breeding design in the grasshopper Chorthippus biguttulus. We found that the male IGEs were an order of magnitude lower than the direct genetic effects and were not significantly different from zero. However, there was some indication that IGEs were larger shortly after mating, consistent with the idea that IGEs fade with time after interaction. Female direct heritabilities were moderate to low. Simulation shows that the variance component estimates can appear larger with less data, calling for care when interpreting variance components estimated with low power. Our results illustrate that the contribution of male IGEs is overall low on the phenotypic variance of female fecundity traits. Thus, even in the relevant context of sexual conflict, the influence of male IGEs on the evolutionary trajectory of female reproductive traits is likely to be small.

Association mapping of morphological traits in wild and captive zebra finches: reliable within, but not between populations.

Identifying causal genetic variants underlying heritable phenotypic variation is a long-standing goal in evolutionary genetics. We previously identified several quantitative trait loci (QTL) for five morphological traits in a captive population of zebra finches (Taeniopygia guttata) by whole-genome linkage mapping. We here follow up on these studies with the aim to narrow down on the quantitative trait variants (QTN) in one wild and three captive populations. First, we performed an association study using 672 single nucleotide polymorphisms (SNPs) within candidate genes located in the previously identified QTL regions in a sample of 939 wild-caught zebra finches. Then, we validated the most promising SNP-phenotype associations (n = 25 SNPs) in 5228 birds from four populations. Genotype-phenotype associations were generally weak in the wild population, where linkage disequilibrium (LD) spans only short genomic distances. In contrast, in captive populations, where LD blocks are large, apparent SNP effects on morphological traits (i.e. associations) were highly repeatable with independent data from the same population. Most of those SNPs also showed significant associations with the same trait in other captive populations, but the direction and magnitude of these effects varied among populations. This suggests that the tested SNPs are not the causal QTN but rather physically linked to them, and that LD between SNPs and causal variants differs between populations due to founder effects. While the identification of QTN remains challenging in nonmodel organisms, we illustrate that it is indeed possible to confirm the location and magnitude of QTL in a population with stable linkage between markers and causal variants.

What triggers colour change? Effects of background colour and temperature on the development of an alpine grasshopper.

BACKGROUND: Colour polymorphisms are a fascinating facet of many natural populations of plants and animals, and the selective processes that maintain such variation are as relevant as the processes which promote their development. Orthoptera, the insect group that encompasses grasshoppers and bush crickets, includes a particularly large number of species that are colour polymorphic with a marked green-brown polymorphism being particularly widespread. Colour polymorphism has been associated with the need for crypsis and background matching and background-dependent homochromy has been described in a few species. However, when and how different environmental conditions influence variation in colour remains poorly understood. Here we test for effects of background colour and ambient temperature on the occurrence of colour morph switches (green to brown or brown to green) and developmental darkening in the alpine dwelling club-legged grasshopper Gomphocerus sibiricus. RESULTS: We monitored individually housed nymphae across three of their four developmental stages and into the first week after final ecdysis. Our data show an absence of colour morph switches in G. sibiricus, without a single switch observed in our sample. Furthermore, we test for an effect of temperature on colouration by manipulating radiant heat, a limiting factor in alpine habitats. Radiant heat had a significant effect on developmental darkening: individuals under low radiant heat tended to darken, while individuals under high radiant heat tended to lighten within nymphal stages. Young imagoes darkened under either condition. CONCLUSIONS: Our results indicate a plastic response to a variable temperature and indicate that melanin, a multipurpose pigment responsible for dark colouration and presumed to be costly, seems to be strategically allocated according to the current environmental conditions. Unlike other orthopterans, the species is apparently unable to switch colour morphs (green/brown) during development, suggesting that colour morphs are determined genetically (or very early during development) and that other processes have to contribute to crypsis and homochromy in this species.

A prezygotic transmission distorter acting equally in female and male zebra finches Taeniopygia guttata.

The two parental alleles at a specific locus are usually inherited with equal probability to the offspring. However, at least three processes can lead to an apparent departure from fair segregation: early viability selection, biased gene conversion and various kinds of segregation distortion. Here, we conduct a genome-wide scan for transmission distortion in a captive population of zebra finches (Taeniopygia guttata) using 1302 single-nucleotide polymorphisms (SNPs) followed by confirmatory analyses on independent samples from the same population. In the initial genome-wide scan, we found significant distortion at three linked loci on chromosome Tgu2 and we were able to replicate this finding in each of two follow-up data sets [overall transmission ratio = 0.567 (95% CI = 0.536-0.600), based on 1101 informative meioses]. Although the driving allele was preferentially transmitted by both heterozygous females [ratio = 0.560 (95% CI = 0.519-0.603)] and heterozygous males [ratio = 0.575 (95% CI = 0.531-0.623)], we could rule out postzygotic viability selection and biased gene conversion as possible mechanisms. Early postzygotic viability selection is unlikely, because it would result in eggs with no visible embryo and hence no opportunity for genotyping, and we confirmed that both females and males heterozygous for the driving allele did not produce a larger proportion of such eggs than homozygous birds. Biased gene conversion is expected to be rather localized, while we could trace transmission distortion in haplotypes of several megabases in a recombination desert. Thus, we here report the rare case of a prezygotically active transmission distorter operating equally effectively in female and male meioses.

Choosiness, a neglected aspect of preference functions: a review of methods, challenges and statistical approaches.

Animals are faced with many choices and a very important one is the choice of a mating partner. Inter-individual differences in mating preferences have been studied for some time, but most studies focus on the location of the peak preference rather than on other aspects of preference functions. In this review, we discuss the role of variation in choosiness in inter-sexual selection. We define individual-level choosiness as the change in mating propensity in response to different stimulus signals. We illustrate general issues in estimating aspects of preference functions and discuss experimental setups for quantifying variation in choosiness with a focus on choices based on acoustic signals in insects. One important consideration is whether preferences are measured sequentially one stimulus at a time or in competitive multiple-choice setups; the suitability of these alternatives depends on the ecology of the study species. Furthermore, we discuss the usefulness of behavioural proxies for determining preference functions, which can be misleading if the proxies are not linearly related to mating propensity. Finally, we address statistical approaches, including the use of function-valued trait analysis, for studying choosiness. Most of the conclusions can be generalized beyond acoustic signals in insects and to choices in non-sexual contexts.