Domestic dog lineages reveal genetic drivers of behavioral diversification.

Selective breeding of domestic dogs has generated diverse breeds often optimized for performing specialized tasks. Despite the heritability of breed-typical behavioral traits, identification of causal loci has proven challenging due to the complexity of canine population structure. We overcome longstanding difficulties in identifying genetic drivers of canine behavior by developing a framework for understanding relationships between breeds and the behaviors that define them, utilizing genetic data for over 4,000 domestic, semi-feral, and wild canids and behavioral survey data for over 46,000 dogs. We identify ten major canine genetic lineages and their behavioral correlates and show that breed diversification is predominantly driven by non-coding regulatory variation. We determine that lineage-associated genes converge in neurodevelopmental co-expression networks, identifying a sheepdog-associated enrichment for interrelated axon guidance functions. This work presents a scaffold for canine diversification that positions the domestic dog as an unparalleled system for revealing the genetic origins of behavioral diversity.

VEGAS as a Platform for Facile Directed Evolution in Mammalian Cells.

Directed evolution, artificial selection toward designed objectives, is routinely used to develop new molecular tools and therapeutics. Successful directed molecular evolution campaigns repeatedly test diverse sequences with a designed selective pressure. Unicellular organisms and their viral pathogens are exceptional for this purpose and have been used for decades. However, many desirable targets of directed evolution perform poorly or unnaturally in unicellular backgrounds. Here, we present a system for facile directed evolution in mammalian cells. Using the RNA alphavirus Sindbis as a vector for heredity and diversity, we achieved 24-h selection cycles surpassing 10-3 mutations per base. Selection is achieved through genetically actuated sequences internal to the host cell, thus the system's name: viral evolution of genetically actuating sequences, or "VEGAS." Using VEGAS, we evolve transcription factors, GPCRs, and allosteric nanobodies toward functional signaling endpoints each in less than 1 weeks' time.

Genetic Mapping and Biochemical Basis of Yellow Feather Pigmentation in Budgerigars.

Parrot feathers contain red, orange, and yellow polyene pigments called psittacofulvins. Budgerigars are parrots that have been extensively bred for plumage traits during the last century, but the underlying genes are unknown. Here we use genome-wide association mapping and gene-expression analysis to map the Mendelian blue locus, which abolishes yellow pigmentation in the budgerigar. We find that the blue trait maps to a single amino acid substitution (R644W) in an uncharacterized polyketide synthase (MuPKS). When we expressed MuPKS heterologously in yeast, yellow pigments accumulated. Mass spectrometry confirmed that these yellow pigments match those found in feathers. The R644W substitution abolished MuPKS activity. Furthermore, gene-expression data from feathers of different bird species suggest that parrots acquired their colors through regulatory changes that drive high expression of MuPKS in feather epithelia. Our data also help formulate biochemical models that may explain natural color variation in parrots. VIDEO ABSTRACT.

Synthetic gene drives as an anthropogenic evolutionary force.

Genetic drive represents a fundamental evolutionary force that can exact profound change to the genetic composition of populations by biasing allele transmission. Herein I propose that the use of synthetic homing gene drives, the human-mediated analog of endogenous genetic drives, warrants the designation of 'genetic welding' as an anthropogenic evolutionary force. Conceptually, this distinction parallels that of artificial and natural selection. Genetic welding is capable of imposing complex and rapid heritable phenotypic change on entire populations, whether motivated by biodiversity conservation or public health. Unanticipated possible long-term evolutionary outcomes, however, demand further investigation and bioethical consideration. The emerging importance of genetic welding also compels our explicit recognition of genetic drive as an addition to the other four fundamental forces of evolution.

Promotion of apoplastic oxidative burst by artificially selected GhCBSX3A enhances Verticillium dahliae resistance in upland cotton.

Verticillium wilt (VW) is a devasting disease affecting various plants, including upland cotton, a crucial fiber crop. Despite its impact, the genetic basis underlying cotton's susceptibility or defense against VW remains unclear. Here, we conducted a genome-wide association study on VW phenotyping in upland cotton and identified a locus on A13 that is significantly associated with VW resistance. We then identified a cystathionine ß-synthase domain gene at A13 locus, GhCBSX3A, which was induced by Verticillium dahliae. Functional analysis, including expression silencing in cotton and overexpression in Arabidopsis thaliana, confirmed that GhCBSX3A is a causal gene at the A13 locus, enhancing SAR-RBOHs-mediated apoplastic oxidative burst. We found allelic variation on the TATA-box of GhCBSX3A promoter attenuated its expression in upland cotton, thereby weakening VW resistance. Interestingly, we discovered that altered artificial selection of GhCBSX3A_R (an elite allele for VW) under different VW pressures during domestication and other improved processes allows specific human needs to be met. Our findings underscore the importance of GhCBSX3A in response to VW, and we propose a model for defense-associated genes being selected depending on the pathogen's pressure. The identified locus and gene serve as promising targets for VW resistance enhancement in cotton through genetic engineering.

Historic dog Furs Unravel the Origin and Artificial Selection of Modern Nordic Lapphund and Elkhound dog Breeds.

The origins and extreme morphological evolution of the modern dog breeds are poorly studied because the founder populations are extinct. Here, we analyse eight 100 to 200 years old dog fur samples obtained from traditional North Swedish clothing, to explore the origin and artificial selection of the modern Nordic Lapphund and Elkhound dog breeds. Population genomic analysis confirmed the Lapphund and Elkhound breeds to originate from the local dog population, and showed a distinct decrease in genetic diversity in agreement with intense breeding. We identified eleven genes under positive selection during the breed development. In particular, the MSRB3 gene, associated with breed-related ear morphology, was selected in all Lapphund and Elkhound breeds, and functional assays showed that a SNP mutation in the 3'UTR region suppresses its expression through miRNA regulation. Our findings demonstrate analysis of near-modern dog artifacts as an effective tool for interpreting the origin and artificial selection of the modern dog breeds.

Multiple Origins and Genomic Basis of Complex Traits in Sighthounds.

Sighthounds, a distinctive group of hounds comprising numerous breeds, have their origins rooted in ancient artificial selection of dogs. In this study, we performed genome sequencing for 123 sighthounds, including one breed from Africa, six breeds from Europe, two breeds from Russia, and four breeds and 12 village dogs from the Middle East. We gathered public genome data of five sighthounds and 98 other dogs as well as 31 gray wolves to pinpoint the origin and genes influencing the morphology of the sighthound genome. Population genomic analysis suggested that sighthounds originated from native dogs independently and were comprehensively admixed among breeds, supporting the multiple origins hypothesis of sighthounds. An additional 67 published ancient wolf genomes were added for gene flow detection. Results showed dramatic admixture of ancient wolves in African sighthounds, even more than with modern wolves. Whole-genome scan analysis identified 17 positively selected genes (PSGs) in the African population, 27 PSGs in the European population, and 54 PSGs in the Middle Eastern population. None of the PSGs overlapped in the three populations. Pooled PSGs of the three populations were significantly enriched in "regulation of release of sequestered calcium ion into cytosol" (gene ontology: 0051279), which is related to blood circulation and heart contraction. In addition, ESR1, JAK2, ADRB1, PRKCE, and CAMK2D were under positive selection in all three selected groups. This suggests that different PSGs in the same pathway contributed to the similar phenotype of sighthounds. We identified an ESR1 mutation (chr1: g.42,177,149 T > C) in the transcription factor (TF) binding site of Stat5a and a JAK2 mutation (chr1: g.93,277,007 T > A) in the TF binding site of Sox5. Functional experiments confirmed that the ESR1 and JAK2 mutation reduced their expression. Our results provide new insights into the domestication history and genomic basis of sighthounds.

Weak response to selection on stigma-anther distance in a primarily selfing population of yellow monkeyflower.

Stebbins hypothesized that selfing lineages are evolutionary dead ends because they lack adaptive potential. While selfing populations often possess limited nucleotide variability compared with closely related outcrossers, reductions in the genetic variability of quantitative characters remain unclear, especially for key traits determining selfing rates. Yellow monkeyflower (Mimulus guttatus) populations generally outcross and maintain extensive quantitative genetic variation in floral traits. Here, we study the Joy Road population (Bodega Bay, CA, USA) of M. guttatus, where individuals exhibit stigma-anther distances (SAD) typical of primarily selfing monkeyflowers. We show that this population is closely related to nearby conspecifics on the Pacific Coast with a modest 33% reduction in genome-wide variation compared with a more highly outcrossing population. A five-generation artificial selection experiment challenged the hypothesis that the Joy Road population harbours comparatively low evolutionary potential in stigma-anther distance, a critical determinant of selfing rate in Mimulus. Artificial selection generated a weak phenotypic response, with low realized heritabilities (0.020-0.028) falling 84% below those measured for floral characters in more highly outcrossing M. guttatus. These results demonstrate substantial declines in evolutionary potential with a transition toward selfing. Whether these findings explain infrequent reversals to outcrossing or general limits on adaptation in selfers requires further investigation.

A novel miR160a-GmARF16-GmMYC2 module determines soybean salt tolerance and adaptation.

Salt stress is a major challenge that has a negative impact on soybean growth and productivity. Therefore, it is important to understand the regulatory mechanism of salt response to ensure soybean yield under such conditions. In this study, we identified and characterized a miR160a-GmARF16-GmMYC2 module and its regulation during the salt-stress response in soybean. miR160a promotes salt tolerance by cleaving GmARF16 transcripts, members of the Auxin Response Factor (ARF) family, which negatively regulates salt tolerance. In turn, GmARF16 activates GmMYC2, encoding a bHLH transcription factor that reduces salinity tolerance by down-regulating proline biosynthesis. Genomic analysis among wild and cultivated soybean accessions identified four distinct GmARF16 haplotypes. Among them, the GmARF16H3 haplotype is preferentially enriched in localities with relatively saline soils, suggesting GmARF16H3 was artificially selected to improve salt tolerance. Our findings therefore provide insights into the molecular mechanisms underlying salt response in soybean and provide valuable genetic targets for the molecular breeding of salt tolerance.

Effects of long-term voluntary wheel running and selective breeding for wheel running on femoral nutrient canals.

The nutrient artery provides ~50%-70% of the total blood volume to long bones in mammals. Studying the functional characteristics of this artery in vivo can be difficult and expensive, so most researchers have measured the nutrient foramen, an opening on the outer surface of the bone that served as the entry point for the nutrient artery during development and bone ossification. Others have measured the nutrient canal (i.e., the passage which the nutrient artery once occupied), given that the external dimensions of the foramen do not necessarily remain uniform from the periosteal surface to the medullary cavity. The nutrient canal, as an indicator of blood flow to long bones, has been proposed to provide a link to studying organismal activity (e.g., locomotor behavior) from skeletal morphology. However, although external loading from movement and activity causes skeletal remodeling, it is unclear whether it affects the size or configuration of nutrient canals. To investigate whether nutrient canals can exhibit phenotypic plasticity in response to physical activity, we studied a mouse model in which four replicate high runner (HR) lines have been selectively bred for high voluntary wheel-running behavior. The selection criterion is the average number of wheel revolutions on days 5 and 6 of a 6-day period of wheel access as young adults (~6-8 weeks old). An additional four lines are bred without selection to serve as controls (C). For this study, 100 female mice (half HR, half C) from generation 57 were split into an active group housed with wheels and a sedentary group housed without wheels for 12 weeks starting at ~24 days of age. Femurs were collected, soft tissues were removed, and femora were micro-computed tomography scanned at a resolution of 12 µm. We then imported these scans into AMIRA and created 3D models of femoral nutrient canals. We tested for evolved differences in various nutrient canal traits between HR and C mice, plastic changes resulting from chronic exercise, and the selection history-by-exercise interaction. We found few differences between the nutrient canals of HR versus C mice, or between the active and sedentary groups. We did find an interaction between selection history and voluntary exercise for the total number of nutrient canals per femur, in which wheel access increased the number of canals in C mice but decreased it in HR mice. Our results do not match those from an earlier study, conducted at generation 11, which was prior to the HR lines reaching selection limits for wheel running. The previous study found that mice from the HR lines had significantly larger total canal cross-sectional areas compared to those from C lines. However, this discrepancy is consistent with studies of other skeletal traits, which have found differences between HR and C mice to be somewhat inconsistent across generations, including the loss of some apparent adaptations with continued selective breeding after reaching a selection limit for wheel-running behavior.

Drivers of genomic diversity and phenotypic development in early phases of domestication in Hermetia illucens.

The black soldier fly (BSF), Hermetia illucens, has the ability to efficiently bioremediate organic waste into usable bio-compounds. Understanding the impact of domestication and mass rearing on fitness and production traits is therefore important for sustainable production. This study aimed to assess patterns of genomic diversity and its association to phenotypic development across early generations of mass rearing under two selection strategies: selection for greater larval mass (SEL lines) and no direct artificial selection (NS lines). Genome-wide single nucleotide polymorphism (SNP) data were generated using 2bRAD sequencing, while phenotypic traits relating to production and population fitness were measured. Declining patterns of genomic diversity were observed across three generations of captive breeding, with the lowest diversity recorded for the F3 generation of both selection lines, most likely due to founder effects. The SEL cohort displayed statistically significantly greater larval weight com the NS lines with pronounced genetic and phenotypic directional changes across generations. Furthermore, lower genetic and phenotypic diversity, particularly for fitness traits, were evident for SEL lines, illustrating the trade-off between selecting for mass and the resulting decline in population fitness. SNP-based heritability was significant for growth, but was low or non-significant for fitness traits. Genotype-phenotype correlations were observed for traits, but individual locus effect sizes where small and very few of these loci demonstrated a signature for selection. Pronounced genetic drift, due to small effective population sizes, is likely overshadowing the impacts of selection on genomic diversity and consequently phenotypic development. The results hold particular relevance for genetic management and selective breeding for BSF in future.

Limits to sustained energy intake. XXXIV. Can the heat dissipation limit (HDL) theory explain reproductive aging?

According to the heat dissipation limit (HDL) theory, reproductive performance is limited by the capacity to dissipate excess heat. We tested the novel hypotheses that (1) the age-related decline in reproductive performance is due to an age-related decrease of heat dissipation capacity and (2) the limiting mechanism is more severe in animals with high metabolic rates. We used bank voles (Myodes glareolus) from lines selected for high swim-induced aerobic metabolic rate, which have also increased basal metabolic rate, and unselected control lines. Adult females from three age classes - young (4 months), middle-aged (9 months) and old (16 months) - were maintained at room temperature (20°C), and half of the lactating females were shaved to increase heat dissipation capacity. Old females from both selection lines had a decreased litter size, mass and growth rate. The peak-lactation average daily metabolic rate was higher in shaved than in unshaved mothers, and this difference was more profound among old than young and middle-aged voles (P=0.02). In females with large litters, milk production tended to be higher in shaved (least squares mean, LSM±s.e.: 73.0±4.74 kJ day-1) than in unshaved voles (61.8±4.78 kJ day-1; P=0.05), but there was no significan"t effect of fur removal on the growth rate [4.47±2.29 g (4 days-1); P=0.45]. The results provide mixed support of the HDL theory and no support for the hypotheses linking the differences in reproductive aging with either a deterioration in thermoregulatory capability or genetically based differences in metabolic rate.

Impact of hindlimb length variation on jumping dynamics in the Longshanks mouse.

Distantly related mammals (e.g. jerboa, tarsiers, kangaroos) have convergently evolved elongated hindlimbs relative to body size. Limb elongation is hypothesized to make these species more effective jumpers by increasing their kinetic energy output (through greater forces or acceleration distances), thereby increasing take-off velocity and jump distance. This hypothesis, however, has rarely been tested at the population level, where natural selection operates. We examined the relationship between limb length, muscular traits and dynamics using Longshanks mice, which were selectively bred over 22 generations for longer tibiae. Longshanks mice have approximately 15% longer tibiae and 10% longer femora compared with random-bred Control mice from the same genetic background. We collected in vivo measures of locomotor kinematics and force production, in combination with behavioral data and muscle morphology, to examine how changes in bone and muscle structure observed in Longshanks mice affect their hindlimb dynamics during jumping and clambering. Longshanks mice achieved higher mean and maximum lunge-jump heights than Control mice. When jumping to a standardized height (14 cm), Longshanks mice had lower maximum ground reaction forces, prolonged contact times and greater impulses, without significant differences in average force, power or whole-body velocity. While Longshanks mice have longer plantarflexor muscle bodies and tendons than Control mice, there were no consistent differences in muscular cross-sectional area or overall muscle volume; improved lunge-jumping performance in Longshanks mice is not accomplished by simply possessing larger muscles. Independent of other morphological or behavioral changes, our results point to the benefit of longer hindlimbs for performing dynamic locomotion.

Testing personality-pace-of-life associations via artificial selection: insights from selected lines of rainbow trout on the context-dependence of correlations.

More than a decade of study since the personality pace-of-life syndrome (POLS) hypotheses were first proposed, there is little support for it within species. Lack of experimental control, insufficient sampling in the face of highly labile behavioural and metabolic traits, and context dependency of trait correlations are suggested as reasons. Here, I argue that artificial selection and/or use of existing selected lines represents a powerful but under-used approach to furthering our understanding of the POLS. To illustrate this potential, I conducted a focussed review of studies that compared the behaviour, metabolism, growth and survival of an artificially selected fast-growing rainbow trout relative to wild unselected strains, under varying food and risk conditions in the laboratory and field. Resting metabolic rate, food intake, and behaviours that enhance feeding but increase energy expenditure (activity, aggression, boldness), were all higher in the fast strain in paired contrasts, under all food and risk conditions, both in the laboratory and the field. Fast-strain fish grew faster in almost every food and risk situation except where food was highly limited (or absent), had higher survival under low or zero predation risk, but had lower survival under high risk. Several other traits rarely considered in POLS studies were also higher in the fast strain, including maximum swimming speed, and hormones (growth hormone (GH), thyroid hormone (T3) and insulin-like growth factor (IGF-1)). I conclude: (i) assumptions and predictions of the POLS hypothesis are well supported, and (ii) context-dependency was largely absent, but when present revealed trade-offs between food acquisition and predation risk. This focused review highlights the potential of artificial selection in testing POLS ideas, and will hopefully motivate further studies using other animals.

Domestication is associated with increased interspecific hybrid compatibility in landfowl (order: Galliformes).

Some species are able to hybridize despite being exceptionally diverged. The causes of this variation in accumulation of reproductive isolation remain poorly understood, and domestication as an impetus or hindrance to reproductive isolation remains to be characterized. In this study, we investigated the role of divergence time, domestication, and mismatches in morphology, habitat, and clutch size among hybridizing species on reproductive isolation in the bird order Galliformes. We compiled and analyzed hybridization occurrences from literature and recorded measures of postzygotic reproductive isolation. We used a text-mining approach leveraging a historical aviculture magazine to quantify the degree of domestication across species. We obtained divergence time, morphology, habitat, and clutch size data from open sources. We found 123 species pairs (involving 77 species) with known offspring fertility (sterile, only males fertile, or both sexes fertile). We found that divergence time and clutch size were significant predictors of reproductive isolation (McFadden's Pseudo-R2 = 0.59), but not habitat or morphological mismatch. Perhaps most interesting, we found a significant relationship between domestication and reproductive compatibility after correcting for phylogeny, removing extreme values, and addressing potential biases (F1,74 = 5.43, R2 = 0.06, P-value = 0.02). We speculate that the genetic architecture and disruption in selective reproductive regimes associated with domestication may impact reproductive isolation, causing domesticated species to be more reproductively labile.

A Principal Components Analysis and Functional Annotation of Differentially Expressed Genes in Brain Regions of Gray Rats Selected for Tame or Aggressive Behavior.

The process of domestication, despite its short duration as it compared with the time scale of the natural evolutionary process, has caused rapid and substantial changes in the phenotype of domestic animal species. Nonetheless, the genetic mechanisms underlying these changes remain poorly understood. The present study deals with an analysis of the transcriptomes from four brain regions of gray rats (Rattus norvegicus), serving as an experimental model object of domestication. We compared gene expression profiles in the hypothalamus, hippocampus, periaqueductal gray matter, and the midbrain tegmental region between tame domesticated and aggressive gray rats and revealed subdivisions of differentially expressed genes by principal components analysis that explain the main part of differentially gene expression variance. Functional analysis (in the DAVID (Database for Annotation, Visualization and Integrated Discovery) Bioinformatics Resources database) of the differentially expressed genes allowed us to identify and describe the key biological processes that can participate in the formation of the different behavioral patterns seen in the two groups of gray rats. Using the STRING- DB (search tool for recurring instances of neighboring genes) web service, we built a gene association network. The genes engaged in broad network interactions have been identified. Our study offers data on the genes whose expression levels change in response to artificial selection for behavior during animal domestication.

Exploring the mechanism of artificial selection signature in Chinese indigenous pigs by leveraging multiple bioinformatics database tools.

BACKGROUND: Chinese indigenous pigs in Yunnan exhibit considerable phenotypic diversity, but their population structure and the biological interpretation of signatures of artificial selection require further investigation. To uncover population genetic diversity, migration events, and artificial selection signatures in Chinese domestic pigs, we sampled 111 Yunnan pigs from four breeds in Yunnan which is considered to be one of the centres of livestock domestication in China, and genotyped them using Illumina Porcine SNP60K BeadChip. We then leveraged multiple bioinformatics database tools to further investigate the signatures and associated complex traits. RESULTS: Population structure and migration analyses showed that Diannanxiaoer pigs had different genetic backgrounds from other Yunnan pigs, and Gaoligongshan may undergone the migration events from Baoshan and Saba pigs. Intriguingly, we identified a possible common target of sharing artificial selection on a 265.09 kb region on chromosome 5 in Yunnan indigenous pigs, and the genes on this region were associated with cardiovascular and immune systems. We also detected several candidate genes correlated with dietary adaptation, body size (e.g., PASCIN1, GRM4, ITPR2), and reproductive performance. In addition, the breed-sharing gene MMP16 was identified to be a human-mediated gene. Multiple lines of evidence at the mammalian genome, transcriptome, and phenome levels further supported the evidence for the causality between MMP16 variants and the metabolic diseases, brain development, and cartilage tissues in Chinese pigs. Our results suggested that the suppression of MMP16 would directly lead to inactivity and insensitivity of neuronal activity and skeletal development in Chinese indigenous pigs. CONCLUSION: In this study, the population genetic analyses and identification of artificial selection signatures of Yunnan indigenous pigs help to build an understanding of the effect of human-mediated selection mechanisms on phenotypic traits in Chinese indigenous pigs. Further studies are needed to fully characterize the process of human-mediated genes and biological mechanisms.

A Cryptic Sex-Linked Locus Revealed by the Elimination of a Master Sex-Determining Locus in Medaka Fish.

AbstractSex chromosomes rapidly turn over in several taxonomic groups. Sex chromosome turnover is generally thought to start with the appearance of a new sex-determining gene on an autosome while an old sex-determining gene still exists, followed by the fixation of the new one. However, we do not know how prevalent the transient state is, where multiple sex-determining loci coexist within natural populations. Here, we removed a Y chromosome with a master male-determining gene DMY from medaka fish using high temperature-induced sex-reversed males. After four generations, the genomic characteristics of a sex chromosome were found on one chromosome, which was an autosome in the original population. Thus, the elimination of a master sex-determining locus can reveal a cryptic locus with a possible sex-determining effect, which can be the seed for sex chromosome turnover. Our results suggest that populations that seem to have a single-locus XY system may have other chromosomal regions with sex-determining effects. In conclusion, the coexistence of multiple sex-determining genes in a natural population may be more prevalent than previously thought. Experimental elimination of a master sex-determining locus may serve as a promising method for finding a locus that can be a protosex chromosome.

Genomic region and origin for selected traits during differentiation of small-fruit cultivars in Japanese apricot (Prunus mume).

The Japanese apricot (Prunus mume) is a popular fruit tree in Japan. However, the genetic factors associated with fruit trait variations are poorly understood. In this study, we investigated nine fruit-associated traits, including harvesting time, fruit diameter, fruit shape, fruit weight, stone (endocarp) weight, ratio of stone weight to fruit weight, and rate of fruit gumming, using 110 Japanese apricot accessions over four years. A genome-wide association study (GWAS) was performed for these traits and strong signals were detected on chromosome 6 for harvesting time and fruit diameters. These peaks were shown to undergo strong artificial selection during the differentiation of small-fruit cultivars. The genomic region defined by the GWAS and XP-nSL analyses harbored several candidate genes associated with plant hormone regulation. Furthermore, the alleles of small-fruit cultivars in this region were shown to have genetic proximity to some Chinese cultivars of P. mume. These results indicate that the small-fruit trait originated in China; after being introduced into Japan, it was preferred and selected by the Japanese people, resulting in the differentiation of small-fruit cultivars.

Artificial selection for reversal learning reveals limited repeatability and no heritability of cognitive flexibility in great tits (Parus major).

Cognitive flexibility controls how animals respond to changing environmental conditions. Individuals within species vary considerably in cognitive flexibility but the micro-evolutionary potential in animal populations remains enigmatic. One prerequisite for cognitive flexibility to be able to evolve is consistent and heritable among-individual variation. Here we determine the repeatability and heritability of cognitive flexibility among great tits (Parus major) by performing an artificial selection experiment on reversal learning performance using a spatial learning paradigm over three generations. We found low, yet significant, repeatability (R = 0.15) of reversal learning performance. Our artificial selection experiment showed no evidence for narrow-sense heritability of associative or reversal learning, while we confirmed the heritability of exploratory behaviour. We observed a phenotypic, but no genetic, correlation between associative and reversal learning, showing the importance of prior information on reversal learning. We found no correlation between cognitive and personality traits. Our findings emphasize that cognitive flexibility is a multi-faceted trait that is affected by memory and prior experience, making it challenging to retrieve reliable values of temporal consistency and assess the contribution of additive genetic variation. Future studies need to identify what cognitive components underlie variation in reversal learning and study their between-individual and additive genetic components.