A genome-wide SNP scan reveals novel loci for egg production and quality traits in white leghorn and brown-egg dwarf layers

Availability of the complete genome sequence as well as high-density SNP genotyping platforms allows genome-wide association studies (GWAS) in chickens. A high-density SNP array containing 57,636 markers was employed herein to identify associated variants underlying egg production and quality traits within two lines of chickens, i.e., White Leghorn and brown-egg dwarf layers. For each individual, age at first egg (AFE), first egg weight (FEW), and number of eggs (EN) from 21 to 56 weeks of age were recorded, and egg quality traits including egg weight (EW), eggshell weight (ESW), yolk weight (YW), eggshell thickness (EST), eggshell strength (ESS), albumen height(AH) and Haugh unit(HU) were measured at 40 and 60 weeks of age. A total of 385 White Leghorn females and 361 brown-egg dwarf dams were selected to be genotyped. The genome-wide scan revealed 8 SNPs showing genome-wise significant (P<1.51E-06, Bonferroni correction) association with egg production and quality traits under the Fisher's combined probability method. Some significant SNPs are located in known genes including GRB14 and GALNT1 that can impact development and function of ovary, but more are located in genes with unclear functions in layers, and need to be studied further. Many chromosome-wise significant SNPs were also detected in this study and some of them are located in previously reported QTL regions. Most of loci detected in this study are novel and the follow-up replication studies may be needed to further confirm the functional significance for these newly identified SNPs.     

Mapping of quantitative trait loci affecting eggshell quality on chromosome 9 in an F2 intercross between two chicken lines divergently selected for eggshell strength

Broken and cracked eggshells are major causes of significant economic losses to the egg production industry. The quantitative trait loci (QTL) on chromosome 9 influencing the quality of eggshells were identified by analysing an intercross between two parent lines developed from the same founder population by a two-way selection for eggshell strength with non-destructive deformation conducted over 14 generations. Chromosome-wide highly significant ( P  <   0.01) QTL associated with egg weight (EW), short length of egg (SLE), long length of egg (LLE) and eggshell weight were mapped to the distal region of chromosome 9. Among the QTL affecting EW, SLE and LLE, ovocalyxin-32 was identified as a potential candidate gene influencing eggshell traits. Marker-assisted selection based on these QTL could be used to develop strategies for reducing the breakage and cracking of eggs in commercial layer houses.     

Association between ovocalyxin-32 gene haplotypes and eggshell quality traits in an F2 intercross between two chicken lines divergently selected for eggshell strength

Broken and cracked eggshells contribute significantly to economic losses in the egg production industry. We previously identified ovocalyxin-32 as a potential gene influencing eggshell traits, by analysing an intercross between two parent lines developed from the same founder population by a two-way selection for eggshell strength with non-destructive deformation (DEF) conducted over 14 generations. We determined the nucleotide sequences of six ovocalyxin-32 exons in the parent individuals and analysed the association between ovocalyxin-32 and eggshell traits in the F₂ individuals. We identified three haplotypes (W, M and S) of ovocalyxin-32 in the parent individuals. A mismatch amplification mutation assay was performed to distinguish six diplotype individuals (WW, MM, SS, WM, MS and WS) in the F₂ population. The egg weight (EW) of SS-diplotype individuals was significantly higher than that of WW-, WM- and WS-diplotypes. Short length of the egg (SLE) of SS-diplotype individuals was significantly higher than that of WW-, WM- and MS-diplotypes. Long length of the egg (LLE) of SS-diplotype individuals was significantly higher than that of WM- and WS-diplotypes. DEF of WW-diplotype individuals was significantly higher than that of SS-, WM, MS and WM-diplotypes. Haplotypic effect analyses showed significant differences between the W-haplotype and the S-haplotypes in the EW, SLE, LLE and DEF. The DEF of M-haplotype was significantly lower than that of W- and S-haplotypes. These results suggest that S- and M-haplotypes are critical for high quality of eggshells in the F₂ population. In conclusion, ovocalyxin-32 is a useful marker of eggshell traits and can be used to develop strategies for improving eggshell traits in commercial layer houses.     

Multivariate analysis of adaptive capacity for upper thermal limits in Drosophila simulans

Thermal tolerance is an important factor influencing the distribution of ectotherms, but our understanding of the ability of species to evolve different thermal limits is limited. Based on univariate measures of adaptive capacity, it has recently been suggested that species may have limited evolutionary potential to extend their upper thermal limits under ramping temperature conditions that better reflect heat stress in nature. To test these findings more broadly, we used a paternal half‐sibling breeding design to estimate the multivariate evolutionary potential for upper thermal limits in Drosophila simulans. We assessed heat tolerance using static (basal and hardened) and ramping assays. Our analyses revealed significant evolutionary potential for all three measures of heat tolerance. Additive genetic variances were significantly different from zero for all three traits. Our G matrix analysis revealed that all three traits would contribute to a response to selection for increased heat tolerance. Significant additive genetic covariances and additive genetic correlations between static basal and hardened heat‐knockdown time, marginally nonsignificant between static basal and ramping heat‐knockdown time, indicate that direct and correlated responses to selection for increased upper thermal limits are possible. Thus, combinations of all three traits will contribute to the evolution of upper thermal limits in response to selection imposed by a warming climate. Reliance on univariate estimates of evolutionary potential may not provide accurate insight into the ability of organisms to evolve upper thermal limits in nature.     

Eggshell coloration and its importance in postmating sexual selection

Avian eggshell color seems to fulfill multiple functions, some of them being structural and others signaling. In this study, we tested whether or not eggshell coloration may play a role in sexual selection of Tree Sparrows (Passer montanus). According to the “Sexually selected eggshell coloration” hypothesis, eggshell coloration signals female, egg or chick quality and males adjust parental investment according to this signal. Eggs of this species are covered with brown spots and patches, and variation between clutches is high. We found that eggshell coloration correlates with both protoporphyrin and biliverdin, but protoporphyrin concentrations are ten times higher. Eggshell coloration reflects egg and offspring quality, but not female quality. Thus, eggshell coloration may signal female postmating investment in offspring rather than female quality. Furthermore, differential allocation in terms of maternal investment is supported by the fact that females lay more pigmented clutches when mated to males with bigger melanin‐based ornaments relative to their own. Moreover, males invested proportionally more to chicks that hatched from more pigmented clutches. Our correlative results thus seem to support a role of sexual selection in the evolution of eggshell coloration in birds laying brown eggs, pigmented mainly by protoporphyrin.     

Adaptation to hot climate and strategies to alleviate heat stress in livestock production

Despite many challenges faced by animal producers, including environmental problems, diseases, economic pressure, and feed availability, it is still predicted that animal production in developing countries will continue to sustain the future growth of the world's meat production. In these areas, livestock performance is generally lower than those obtained in Western Europe and North America. Although many factors can be involved, climatic factors are among the first and crucial limiting factors of the development of animal production in warm regions. In addition, global warming will further accentuate heat stress-related problems. The objective of this paper was to review the effective strategies to alleviate heat stress in the context of tropical livestock production systems. These strategies can be classified into three groups: those increasing feed intake or decreasing metabolic heat production, those enhancing heat-loss capacities, and those involving genetic selection for heat tolerance. Under heat stress, improved production should be possible through modifications of diet composition that either promotes a higher intake or compensates the low feed consumption. In addition, altering feeding management such as a change in feeding time and/or frequency, are efficient tools to avoid excessive heat load and improve survival rate, especially in poultry. Methods to enhance heat exchange between the environment and the animal and those changing the environment to prevent or limit heat stress can be used to improve performance under hot climatic conditions. Although differences in thermal tolerance exist between livestock species (ruminants > monogastrics), there are also large differences between breeds of a species and within each breed. Consequently, the opportunity may exist to improve thermal tolerance of the animals using genetic tools. However, further research is required to quantify the genetic antagonism between adaptation and production traits to evaluate the potential selection response. With the development of molecular biotechnologies, new opportunities are available to characterize gene expression and identify key cellular responses to heat stress. These new tools will enable scientists to improve the accuracy and the efficiency of selection for heat tolerance. Epigenetic regulation of gene expression and thermal imprinting of the genome could also be an efficient method to improve thermal tolerance. Such techniques (e.g. perinatal heat acclimation) are currently being experimented in chicken.     

Variation in eggshell traits between geographically distant populations of pied flycatchers Ficedula hypoleuca

The expression and impact of maternal effects may vary greatly between populations and environments. However, little is known about large‐scale geographical patterns of variation in maternal deposition to eggs. In birds, as in other oviparous animals, the outermost maternal component of an egg is the shell, which protects the embryo, provides essential mineral resources and allows its interaction with the environment in the form of gas exchange. In this study, we explored variation of eggshell traits (mass, thickness, pore density and pigmentation) across 15 pied flycatcher populations at a large geographic scale. We found significant between‐population variation in all eggshell traits, except in pore density, suggesting spatial variation in their adaptive benefits or in the females’ physiological limitations during egg laying. Between‐ population variation in shell structure was not due to geographic location (latitude and longitude) or habitat type. However, eggshells were thicker in populations that experienced higher ambient temperature during egg laying. This could be a result of maternal resource allocation to the shell being constrained under low temperatures or of an adaptation to reduce egg water loss under high temperatures. We also found that eggshell colour intensity was positively associated with biliverdin pigment concentration, shell thickness and pore density. To conclude, our findings reveal large‐ scale between‐population variation of eggshell traits, although we found little environmental dependency in their expression. Our findings call for further studies that explore other environmental factors (e.g. calcium availability and pollution levels) and social factors like sexual selection intensity that may account for differences in shell structure between populations.     

Quantitative trait loci analysis of egg and meat production traits in a red junglefowlxWhite Leghorn cross

Egg and production traits are of considerable economic importance in chickens. Using a White Leghorn x red junglefowl F(2) intercross, standard production measures of liver weight and colour, egg size, eggshell thickness, egg taste and meat quality were taken. A total of 160 markers covering 29 autosomes and the Z chromosome were genotyped on 175-243 individuals, depending on the trait under consideration. A total of nine significant quantitative trait loci (QTL) and three suggestive QTL were found on chicken chromosomes 1, 2, 4, 5, 7, 8, 10, 12, E47W24 and E22C19W28.     

Eggshell Spottiness Reflects Maternally Transferred Antibodies in Blue Tits

Blue-green and brown-spotted eggshells in birds have been proposed as sexual signals of female physiological condition and egg quality, reflecting maternal investment in the egg. Testing this hypothesis requires linking eggshell coloration to egg content, which is lacking for brown protoporphyrin-based pigmentation. As protoporphyrins can induce oxidative stress, and a large amount in eggshells should indicate either high female and egg quality if it reflects the female''s high oxidative tolerance, or conversely poor quality if it reflects female physiological stress. Different studies supported either predictions but are difficult to compare given the methodological differences in eggshell-spottiness measurements. Using the blue tit Cyanistes caeruleus as a model species, we aimed at disentangling both predictions in testing if brown-spotted eggshell could reflect the quality of maternal investment in antibodies and carotenoids in the egg, and at improving between-study comparisons in correlating several common measurements of eggshell coloration (spectral and digital measures, spotted surface, pigmentation indices). We found that these color variables were weakly correlated highlighting the need for comparable quantitative measurements between studies and for multivariate regressions incorporating several eggshell-color characteristics. When evaluating the potential signaling function of brown-spotted eggshells, we thus searched for the brown eggshell-color variables that best predicted the maternal transfer of antibodies and carotenoids to egg yolks. We also tested the effects of several parental traits and breeding parameters potentially affecting this transfer. While eggshell coloration did not relate to yolk carotenoids, the eggs with larger and less evenly-distributed spots had higher antibody concentrations, suggesting that both the quantity and distribution of brown pigments reflected the transfer of maternal immune compounds in egg yolks. As yolk antibody concentrations were also positively related to key proxies of maternal quality (egg volume, number, yellow feather brightness, tarsus length), eggshells with larger spots concentrated at their broad pole may indicate higher-quality eggs.     

An experimental test of the effect of food and antioxidants on blue‐green eggshell coloration in mountain bluebirds Sialia currucoides

Evolutionary biologists have long debated the functional relevance of the ornamental traits that occur in many animal taxa, and yet, female ornaments have received relatively little attention compared to those produced by males. A greater understanding of these traits, particularly those that are unique to females, may shed light on the potential for sexual selection to shape female phenotypes. Recently, blue‐green eggshell colour, derived from the antioxidant pigment biliverdin, has been proposed as a candidate trait that advertises female quality to males in species of birds with biparental care. However, studies have been equivocal in their support for blue‐green eggshell colour being an informative signal, and correlations between eggshell colour and other female characteristics have been inconsistent. We conducted a supplementation experiment to test if improving the access of female birds to food resources and micronutrients, thereby improving their condition prior to egg laying, would intensify the blue‐green colouration of the eggs they laid. We provided mountain bluebirds Sialia currucoides with food and carotenoids during nest building and egg laying in two breeding seasons, and assessed both within‐ and among‐clutch variation in colour. Supplementation did not affect patterns of within‐clutch variation, but did result in differences in colour among clutches. Specifically, we found that food, but not carotenoids, resulted in higher colour saturation, and decreased the brightness of blue‐green eggshells. Although this trend was observed in both years, the effect was statistically significant only in one year. Our results suggest that food supplementation influences eggshell colour, but that conditions, such as weather and natural food availability, which can vary annually, may also determine how female birds allocate pigment to eggshells.     

Plastic and evolutionary responses to heat stress in a temperate dung fly: negative correlation between basal and induced heat tolerance?

Extreme weather events such as heat waves are becoming more frequent and intense. Populations can cope with elevated heat stress by evolving higher basal heat tolerance (evolutionary response) and/or stronger induced heat tolerance (plastic response). However, there is ongoing debate about whether basal and induced heat tolerance are negatively correlated and whether adaptive potential in heat tolerance is sufficient under ongoing climate warming. To evaluate the evolutionary potential of basal and induced heat tolerance, we performed experimental evolution on a temperate source population of the dung fly Sepsis punctum. Offspring of flies adapted to three thermal selection regimes (Hot, Cold and Reference) were subjected to acute heat stress after having been exposed to either a hot‐acclimation or non‐acclimation pretreatment. As different traits may respond differently to temperature stress, several physiological and life history traits were assessed. Condition dependence of the response was evaluated by exposing juveniles to different levels of developmental (food restriction/rearing density) stress. Heat knockdown times were highest, whereas acclimation effects were lowest in the Hot selection regime, indicating a negative association between basal and induced heat tolerance. However, survival, adult longevity, fecundity and fertility did not show such a pattern. Acclimation had positive effects in heat‐shocked flies, but in the absence of heat stress hot‐acclimated flies had reduced life spans relative to non‐acclimated ones, thereby revealing a potential cost of acclimation. Moreover, body size positively affected heat tolerance and unstressed individuals were less prone to heat stress than stressed flies, offering support for energetic costs associated with heat tolerance. Overall, our results indicate that heat tolerance of temperate insects can evolve under rising temperatures, but this response could be limited by a negative relationship between basal and induced thermotolerance, and may involve some but not other fitness‐related traits.     

Associations between novel polymorphisms at the 5'-UTR region of the prolactin gene and egg production and quality in chickens

The objective was to characterize polymorphisms at the 5′-UTR region of the prolactin gene, and determine their association with egg production and egg quality traits in White Leghorn chickens. The study was conducted on four strains of White Leghorn chickens, namely IWH, IWI, IWK, and layer control. Overall, there were three alleles (designated A, B, and C) and five genotypes, with genotypic frequencies of 0.09, 0.75, 0.07, 0.02, and 0.07 for AA, AB, AC, BB, and BC, respectively. There were significant differences among genotypes for egg production up to 52 and 64 wk of age, with maximal egg yields for genotypes AA and AC (144.5 ± 5.06 and 143.2 ± 4.67 eggs, respectively). Furthermore, there were significant differences among genotypes for egg quality traits, including egg weight and Haugh unit at 40 wk of age, Haugh unit at 52 wk, and yolk color index and Haugh unit at 64 wk. Birds with AA or AC genotypes had the best egg quality traits. On the contrary, these genotypes had the lowest prolactin expression, whereas this expression was highest in birds with the BB genotype. In conclusion, polymorphisms at the 5′-UTR of prolactin gene were significantly associated with egg production and egg quality traits in White Leghorn chickens.     

Effects of mating system in Japanese quail

Summary A 17-generation selection experiment was conducted to study direct and correlated responses to mass selection under a mating system with alternating generations of full-sib inbreeding and wide outbreeding (population I) as compared with a mass selected, randomly mated population (population II). The selection criterion was an index of total egg mass to 78 days divided by adult female body weight. Estimates of heritabilities and genetic correlations are reported. Estimated heritabilities for the index were 0.38±0.04 and 0.29±0.05 in population I and II, respectively. Realized heritabilites were 0.10±0.05 and 0.12±0.03. For most traits studied the mean phenotypic values in the cyclic mated population decreased for inbred generations. Increased inbreeding levels also caused outbred generation means of population I to decrease through the first six or seven generations. After this period of adaptation to inbreeding selection response was positive for the index and positively correlated traits. Total response to selection under the cyclic inbred-outbred mating system did not exceed selection response made under random mating. However, the rate of response in the cyclically mated population exceeded that in the randomly mated population in later generations when the cyclically mated population had apparently adapted to inbreeding.Published with approval of the Director of the Montana Agricultural Experiment Station, Journal Series No. 1221     

Improvement of heat and desiccation tolerance in <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis> through cross-breeding of tolerant strains and successive genetic selection

Genetic selection can be a powerful tool to increase beneficial traits in biological control agents. In this study the heat and desiccation tolerance of the entomopathogenic nematode Heterorhabditis bacteriophora Poinar (Rhabditidomorpha: Strongyloidea) were significantly increased by cross breeding tolerant parental strains and successive genetic selection. These strains originated from a prior screening among 60 strains for increased stress tolerance. During genetic selection, the selection pressure was constantly increased and only the most tolerant 10% of the nematode populations were propagated for further selection steps. Assessment of tolerance and selection for both traits was performed with and without prior adaptation to the stress conditions. Eleven selection steps were performed to increase heat tolerance. A final overall increase in mean heat tolerance of 5.5°C was achieved when nematodes had been adapted to heat stress. For non-adapted tolerance an increase of 3.0°C from 40.1°C to 43.1°C was recorded. For comparison, a commercial strain had a mean tolerated temperature after adaptation of 38.2°C and of 36.5°C without adaptation. For assessment of the desiccation tolerance the mean tolerated water activity (a_w-value) of a population was measured. Cross-breeding most tolerant strains reduced the a_w-value from 0.67 to 0.65 after adaptation and from 0.9 to 0.7 without prior adaptation. The following six selection steps could not increase the tolerance whether nematodes had been adapted to stress or not. In comparison, the commercial strain tolerated a mean a_w-value of 0.985 after adaptation and 0.951 without adaptation. Further investigation will have to assess trait stability and possible trade-off effects. This study is a first important step on the road towards domestication of the entomopathogenic nematode H. bacteriophora.     

Response and inbreeding from a genomic selection experiment in layer chickens

Background

Genomic selection (GS) using estimated breeding values (GS-EBV) based on dense marker data is a promising approach for genetic improvement. A simulation study was undertaken to illustrate the opportunities offered by GS for designing breeding programs. It consisted of a selection program for a sex-limited trait in layer chickens, which was developed by deterministic predictions under different scenarios. Later, one of the possible schemes was implemented in a real population of layer chicken.

Methods

In the simulation, the aim was to double the response to selection per year by reducing the generation interval by 50 %, while maintaining the same rate of inbreeding per year. We found that GS with retraining could achieve the set objectives while requiring 75 % fewer reared birds and 82 % fewer phenotyped birds per year. A multi-trait GS scenario was subsequently implemented in a real population of brown egg laying hens. The population was split into two sub-lines, one was submitted to conventional phenotypic selection, and one was selected based on genomic prediction. At the end of the 3-year experiment, the two sub-lines were compared for multiple performance traits that are relevant for commercial egg production.

Results

Birds that were selected based on genomic prediction outperformed those that were submitted to conventional selection for most of the 16 traits that were included in the index used for selection. However, although the two programs were designed to achieve the same rate of inbreeding per year, the realized inbreeding per year assessed from pedigree was higher in the genomic selected line than in the conventionally selected line.

Conclusions

The results demonstrate that GS is a promising alternative to conventional breeding for genetic improvement of layer chickens.     

Genetics of heat tolerance and thyroid function in Athens-Canadian randombred chickens

Summary Five experiments were conducted to assess the genetic variation in thyroid function (T₃, T₄), body weight and heat stress survival time in chickens. Thyroxine (T₄) levels were found to be elevated in response to 4 and 8 g bovine thyroid stimulating hormone (TSH) in experiment I. In experiment II, 4 g of TSH was injected into chickens from 30 sire families of the Athens-Canadian Randombred population. The heritability of T₄ levels after TSH injection was high. In experiment III, families identified as having innate high or low T₄ levels after TSH injection and a group of control birds were subjected to a heat Stressor of 50 °C for up to 240 min at six weeks of age and heat stress survival time was studied. The groups did not differ from each other in heat stress survival time. Experiment IV was similar to experiment I except triiodothyronine (T₃) was also measured after TSH injection. Both T₄ and T₃ levels after TSH injection were moderately heritable. In experiment V birds were reared to six weeks of age and heritability calculated for body weight, T₄, T₃, and heat stress survival time. Heritabilities were high for body weight, moderate for T₄ and heat stress survival time, and low for T₃. Phenotypic correlations were significant and negative for heat stress survival time with body weight and T₄, and for body weight with T₃ after TSH. Significant positive correlations were found for T₄ with T₃ after TSH and also T₄ and body weight. Analysis of genetic correlations suggested that none of the traits studied would be an adequate selection parameter for achieving heat tolerance without reducing body weight.Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia     

Correlated responses of chickens to selection for production of antibodies to sheep erythrocytes

A bi directional selection experiment was conducted to measure 5-day antibody titers to sheep erythrocytes in White Leghorn chickens. There was an immediate response to selection with significant differences between lines for the selected trait found in the S₁ and all subsequent generations. Comparisons of S₆, S₇ and S₈ generation females revealed differences between lines in disease resistance and in certain reproductive traits such as age at first egg, percentage hen-day egg production, percentage fertility and duration of fertility. The implications of these correlated responses are important to selection programs for general disease resistance.     

Thermal stress exposure of pupal oriental fruit fly has strong and trait-specific consequences in adult flies

Global climate change is projected to increase the incidence of heat waves, their magnitude and duration resulting in insects experiencing increasing environmental stress in both natural and managed ecosystems. While studies on insect thermal tolerance are rapidly increasing, variation across developmental or juvenile stress cross-stage effects within and across generations remain largely unexplored. Yet in holometabolous insects, heat stress at an early developmental stage may influence performance and survival during later stages. Here, we investigated the effects of pupal mild heat stress on the performance of laboratory-reared adult Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) measured as longevity, critical thermal maximum (CT_max), critical thermal minima (CT_min), heat knockdown time (HKDT) and chill coma recovery time (CCRT). Pupal heat stress significantly influenced performance of B. dorsalis adults resulting in impaired longevity and heat tolerance (CT_max and HKDT) in both sexes with improved and compromised cold tolerance (CT_min and CCRT) in females and males, respectively. These findings highlight the role of juvenile stages in mediating stress responses at adult stages. For B. dorsalis, pupal heat stress largely compromised thermal tolerance implying that the species has limited potential to shift its geographic range in heat prone areas. Significant benefits in cold tolerance in females following heat stress may help in improving survival in the cold in the short-term despite restricted activity to the same traits in males. This study suggests that basal heat tolerance and not short-term compensatory thermal plasticity following heat stress may have aided the recent invasion of B. dorsalis in African landscapes.     

Complex patterns of local adaptation in heat tolerance in Drosophila simulans from eastern Australia

Latitudinal clines are considered a powerful means of investigating evolutionary responses to climatic selection in nature. However, most clinal studies of climatic adaptation in Drosophila have involved species that contain cosmopolitan inversion polymorphisms that show clinal patterns themselves, making it difficult to determine whether the traits or inversions are under selection. Further, although climatic selection is unlikely to act on only one life stage in metamorphic organisms, a few studies have examined clinal patterns across life stages. Finally, clinal patterns of heat tolerance may also depend on the assay used. To unravel these potentially confounding effects on clinal patterns of thermal tolerance, we examined adult and larval heat tolerance traits in populations of Drosophila simulans from eastern Australia using static and dynamic (ramping 0.06 °C min^?1) assays. We also used microsatellites markers to clarify whether demographic factors or selection are responsible for population differentiation along clines. Significant cubic clinal patterns were observed for adult static basal, hardened and dynamic heat knockdown time and static basal heat survival in larvae. In contrast, static, hardened larval heat survival increased linearly with latitude whereas no clinal association was found for larval ramping survival. Significant associations between adult and larval traits and climatic variables, and low population differentiation at microsatellite loci, suggest a role for climatic selection, rather than demographic processes, in generating these clinal patterns. Our results suggest that adaptation to thermal stress may be species and life‐stage specific, complicating our efforts to understand the evolutionary responses to selection for increasing thermotolerance.     

Avian eggshell pigments are not consistently correlated with colour measurements or egg constituents in two Turdus thrushes

The colourful appearance of avian eggshells is a prominent aspect of maternal reproductive effort in birds. Some differences in eggshell coloration have been reported to co‐vary with various measures of maternal condition and these patterns support the hypothesis that, in some bird species, several aspects of eggshell colour (i.e. primary chroma and brightness) function as a signal of maternal and offspring quality to induce greater paternal investment. We directly quantified eggshell pigment concentrations of blackbird Turdus merula and song thrush T. philomelos eggshells and tested how the two key pigments (protoporphyrin IX and biliverdin) co‐varied with other eggshell traits and egg constituents as measures of maternal reproductive investment, including total yolk carotenoid concentration, total lipid concentration, yolk mass, and shell thickness. Contrary to predictions, we detected few statistical patterns overall. We found that protoporphyrin IX concentration was negatively associated with blue‐green chroma in blackbirds but not in song thrush. The concentration of protoporphyrin IX was significantly greater in blackbirds and also showed different patterns of association with total yolk lipids and yolk carotenoid concentrations between these two species (significant species interaction terms). Our results reveal that it is not appropriate to simply assume in these two avian species that reflectance‐based eggshell colour measures are a suitable proxy for eggshell pigment concentrations or can be used as consistent predictors of maternal reproductive investment. These results highlight the need to assess and validate the strength and direction of the statistical relationships between eggshell colour measures, pigment concentrations, and maternal resource deposition in the egg for other species of birds.