Rapid evolutionary divergence of a songbird population following recent colonization of an urban area.

Colonization of a novel environment by a few individuals can lead to rapid evolutionary change, yet there is scarce evidence of the relative contributions of neutral and selective factors in promoting divergence during the early stages of colonization. Here we explore the role of neutral and selective forces in the divergence of a unique urban population of the dark-eyed junco (Junco hyemalis), which became established on the campus of the University of California at San Diego (UCSD) in the early 1980s. Previous studies based on microsatellite loci documented significant genetic differentiation of the urban population as well as divergence in phenotypic traits relative to nearby montane populations, yet the geographical origin of the colonization and the contributing factors remained uncertain. Our genome-wide single nucleotide polymorphism data set confirmed the marked genetic differentiation of the UCSD population, and we identified the coastal subspecies pinosus from central California as its sister group instead of the neighbouring mountain population. Demographic inference recovered a separation from pinosus as recent as 20-32 generations ago after a strong bottleneck, suggesting a role for drift in genetic differentiation. However, we also found significant associations between habitat variables and genome-wide variants linked to functional genes, some of which have been reported as potentially adaptive in birds inhabiting modified environments. These results suggest that the interplay between founder events and selection may result in rapid shifts in neutral and adaptive loci across the genome, and reveal the UCSD junco population as a case of contemporary evolutionary divergence in an anthropogenic environment.

Temperature heterogeneity correlates with intraspecific variation in physiological flexibility in a small endotherm.

Phenotypic flexibility allows individuals to reversibly modify trait values and theory predicts an individual's relative degree of flexibility positively correlates with the environmental heterogeneity it experiences. We test this prediction by integrating surveys of population genetic and physiological variation with thermal acclimation experiments and indices of environmental heterogeneity in the Dark-eyed Junco (Junco hyemalis) and its congeners. We combine field measures of thermogenic capacity for 335 individuals, 22,006 single nucleotide polymorphisms genotyped in 181 individuals, and laboratory acclimations replicated on five populations. We show that Junco populations: (1) differ in their thermogenic responses to temperature variation in the field; (2) harbor allelic variation that also correlates with temperature heterogeneity; and (3) exhibit intra-specific variation in thermogenic flexibility in the laboratory that correlates with the heterogeneity of their native thermal environment. These results provide comprehensive support that phenotypic flexibility corresponds with environmental heterogeneity and highlight its importance for coping with environmental change.

Sex steroids modulate circadian behavioral rhythms in captive animals, but does this matter in the wild?

Nearly all organisms alter physiological and behavioral activities across the twenty-four-hour day. Endogenous timekeeping mechanisms, which are responsive to environmental and internal cues, allow organisms to anticipate predictable environmental changes and time their daily activities. Among-individual variation in the chronotype, or phenotypic output of these timekeeping mechanisms (i.e. timing of daily behaviors), is often observed in organisms studied under naturalistic environmental conditions. The neuroendocrine system, including sex steroids, has been implicated in the regulation and modulation of endogenous clocks and their behavioral outputs. Numerous studies have found clear evidence that sex steroids modulate circadian and daily timing of activities in captive animals under controlled conditions. However, little is known about how sex steroids influence daily behavioral rhythms in wild organisms or what, if any, implication this may have for survival and reproductive fitness. Here we review the evidence that sex steroids modulate daily timing in vertebrates under controlled conditions. We then discuss how this relationship may be relevant for the reproductive success and fitness of wild organisms and discuss the limited evidence that sex steroids modulate circadian rhythms in wild organisms.

Migrant and resident female songbirds differ in gonadal response to upstream stimulation during seasonal sympatry.

Timing of seasonal reproduction is driven by environmental cues acting on the hypothalamic-pituitary-gonadal (HPG) axis. Groups of individuals, or populations, of the same species can exhibit different phenology despite facing similar environmental cues or living in the same habitat (i.e., seasonal sympatry). The mechanisms giving rise to population-level differences in reproductive timing are not fully understood, particularly for females. We studied the dark-eyed junco, a songbird with migratory and sedentary (i.e., resident) populations that live in overlapping distributions during winter. In early spring, residents initiate breeding and associated behaviors, including territory establishment and formation of pair bonds, while migrants prepare to depart for their breeding grounds. We tested whether migrant and resident hormonal response to upstream hormonal stimulation differed during this time period. We collected blood from free-living females in early spring, and challenged them with repeated gonadotropin-releasing hormone (GnRH) injections to measure testosterone (T) response. We predicted that if migrants are less sensitive to upstream stimulation than residents, then they would exhibit lower response to the repeated GnRH challenges in migrants. We found that migrant and resident females both responded to an initial challenge by elevating T, but residents responded more robustly, indicating that the ovary plays a role in population-level differences in reproductive timing. We also found that migrants and residents attenuated their response to repeated challenges, and did not differ from one another in final T levels. We speculate that the explanation for the generally reduced T response after repeated GnRH injections need not be the same for migrants and residents, but possible explanations include suppression of upstream stimulation owing to negative feedback after the initial injection oraromatization of T to estradiol between sampling time points. We suggest that future studies experimentally explore how the ovarian response to upstream stimulation changes during the transition to reproduction.

The World Is Not Flat: Accounting for the Dynamic Nature of the Environment as We Move Beyond Static Experimental Manipulations.

Although we have long understood that environmental variation affects both physiology and behavior, historically, most studies have limited or simplified environmental variation to focus more directly on traits of interest. Recently, a number of investigators have turned their focus toward attempting to incorporate such variation into studies of physiology and behavior, and not surprisingly, are finding that the results from studies that include more realistic variation, both from the environment as well as in physiological processes within individuals, can differ substantially from those of studies that attempt to hold the parameters constant. Understanding the role that this dynamic variation plays in shaping phenotypes is critical given that, under most predictions from future climate change models, increased variability in factors such as temperature and rainfall are predicted.

Onset of Daily Activity in a Female Songbird Is Related to Peak-Induced Estradiol Levels.

Research in captive birds and mammals has demonstrated that circadian (i.e., daily) behavioral rhythms are altered in response to increases in sex-steroid hormones. Recently, we and others have demonstrated a high degree of individual repeatability in peak (gonadotropin-releasing hormone [GnRH]-induced sex) steroid levels, and we have found that these GnRH-induced levels are highly correlated with their daily (night-time) endogenous peak. Whether or not individual variation in organization and activity of the reproductive endocrine axis is related to daily timing in wild animals is not well known. To begin to explore these possible links, we tested the hypothesis that maximal levels of the sex steroid hormone estradiol (E2) and onset of daily activity are related in a female songbird, the dark-eyed junco (Junco hyemalis). We found that females with higher levels of GnRH-induced E2 departed from their nest in the morning significantly earlier than females with lower stimulated levels. We did not observe a relationship between testosterone and this measure of onset of activity. Our findings suggest an interaction between an individual's reproductive endocrine axis and the circadian system and variation observed in an individuals' daily activity onset. We suggest future studies examine the relationship between maximal sex-steroid hormones and timing of daily activity onset.

Corticosterone negative feedback is weaker during spring vs. autumn migration in a songbird (Junco hyemalis).

Birds face many challenges during seasonal migrations and must make important decisions about whether to accelerate, maintain, or delay travel to their final destinations. Spring migration is likely more challenging than autumn migration as spring journeys are completed more quickly and weather conditions are harsher during this time. These differential challenges may be reflected in the endocrine stress response, as the hypothalamic-pituitary-adrenal (HPA) axis is important for both daily energetic needs and coping with stressors. Indeed, most avian studies have found that both baseline and stress-induced corticosterone (CORT) levels tend to be higher in spring migrants than in autumn migrants. We hypothesized that CORT negative feedback efficacy also differs across the season, and is likely weaker during times of year when birds must be most sensitive to stressors. We therefore predicted that CORT negative feedback efficacy would be weaker during spring vs. autumn migration as spring migrants are more likely to encounter situations where they must decide whether to turn back or delay their travel. We examined male dark-eyed juncos (Junco hyemalis) during their spring and autumn stopovers in Fargo, ND, USA. Our prediction was met as we did find that negative feedback efficacy was weaker during spring vs. autumn, although we notably did not find any seasonal differences in baseline and stress-induced CORT. We also found that spring migrants were heavier, had greater subcutaneous fat stores, and had slightly higher hematocrit compared to autumn migrants. These findings suggest that CORT negative feedback sensitivity may help migrating birds effectively cope with the differential challenges of autumn and spring migration.

Changes in processes downstream of the hypothalamus are associated with seasonal follicle development in a songbird, the dark-eyed junco (Junco hyemalis).

Mechanisms related to seasonal reproductive timing in vertebrates have received far more study in males than in females, despite the fact that female timing decisions dictate when rearing of offspring will occur. Production and release of gonadotropin-releasing hormone (GnRH) by the hypothalamus stimulates the pituitary to secrete gonadotropins, initiating the beginning stages of gonadal recrudescence and production of the sex steroids, testosterone and estradiol, which are necessary to prime the liver for secretion of yolk precursors in breeding female birds. While stimulation by the hypothalamus can occur during the pre-breeding period, egg development itself is likely regulated downstream of the hypothalamus. We used GnRH challenges to examine variation in breeding-stage-specific patterns of pituitary and ovarian responsiveness in free-living female dark-eyed juncos (Junco hyemalis) and also examined the ovary and liver for variation in mRNA expression of candidate genes. Baseline LH levels increased during the transition from pre-breeding to egg-development, however no significant difference was observed in post-GnRH injection levels for LH or sex steroids (testosterone and estradiol). Interestingly, a stage by time-point interaction was observed, with post-GnRH LH levels increasing over baseline during the pre-breeding stage, but not during the egg-development stage. We observed a decrease in liver mRNA expression of estradiol receptor-alpha, and glucocorticoid and mineralocorticoid receptors and a decrease in glucocorticoid receptor expression levels in the ovary. A decline in FSH receptor expression across stages was also observed in the ovary. Combined, our data suggest seasonal variation in female's sensitivity to signals of HPG activity and energetic or stress signals. These data provide additional insight into the physiological mechanisms regulating onset of clutch initiation.

Early-breeding females experience greater telomere loss.

Annual reproductive success is often highest in individuals that initiate breeding early, yet relatively few individuals start breeding during this apparently optimal time. This suggests that individuals, particularly females who ultimately dictate when offspring are born, incur costs by initiating reproduction early in the season. We hypothesized that increases in the ageing rate of somatic cells may be one such cost. Telomeres, the repetitive DNA sequences on the ends of chromosomes, may be good proxies of biological wear and tear as they shorten with age and in response to stress. Using historical data from a long-term study population of dark-eyed juncos (Junco hyemalis), we found that telomere loss between years was greater in earlier breeding females, regardless of chronological age. There was no relationship between telomere loss and the annual number of eggs laid or chicks that reached independence. However, telomere loss was greater when temperatures were cooler, and cooler temperatures generally occur early in the season. This suggests that environmental conditions could be the primary cause of accelerated telomere loss in early breeders.

Endotoxin rapidly desensitizes the gonads to kisspeptin-induced luteinizing hormone release in male Siberian hamsters (Phodopus sungorus).

Activation of the immune system induces rapid reductions in hypothalamic-pituitary-gonadal (HPG) axis activity, which in turn decreases secretion of sex steroids. This response is likely adaptive for survival by temporarily inhibiting reproduction to conserve energy; however, the physiological mechanisms controlling this response remain unclear. The neuropeptide kisspeptin is a candidate to mediate the decrease in sex hormones seen during sickness through its key regulation of the HPG axis. In this study, the effects of acute immune activation on the response to kisspeptin were assessed in male Siberian hamsters (Phodopus sungorus). Specifically, an immune response was induced in animals by a single treatment of lipopolysaccharide (LPS), and reproductive hormone concentrations were determined in response to subsequent injections of exogenous kisspeptin. Saline-treated controls showed a robust increase in circulating testosterone in response to kisspeptin; however, this response was blocked in LPS-treated animals. Circulating luteinizing hormone (LH) levels were elevated in response to kisspeptin in both LPS- and saline-treated groups and, thus, were unaffected by LPS treatment, suggesting gonad-level inhibition of testosterone release despite central HPG activation. In addition, blockade of glucocorticoid receptors by mifepristone did not attenuate the LPS-induced inhibition of testosterone release, suggesting that circulating glucocorticoids do not mediate this phenomenon. Collectively, these findings reveal that acute endotoxin exposure rapidly renders the gonads less sensitive to HPG stimulation, thus effectively inhibiting sex hormone release. More broadly, these results shed light on the effects of immune activation on the HPG axis and help elucidate the mechanisms controlling energy allocation and reproduction.

Seasonally sympatric but allochronic: differential expression of hypothalamic genes in a songbird during gonadal development.

Allochrony, the mismatch of reproductive schedules, is one mechanism that can mediate sympatric speciation and diversification. In songbirds, the transition into breeding condition and gonadal growth is regulated by the hypothalamic-pituitary-gonadal (HPG) axis at multiple levels. We investigated whether the difference in reproductive timing between two seasonally sympatric subspecies of dark-eyed juncos (Junco hyemalis) was related to gene expression along the HPG axis. During the sympatric pre-breeding stage, we measured hypothalamic and testicular mRNA expression of candidate genes via qPCR in captive male juncos. For hypothalamic mRNA, we found our earlier breeding subspecies had increased expression of gonadotropin-releasing hormone (GnRH) and decreased expression of androgen receptor, oestrogen receptor alpha and mineralocorticoid receptor (MR). Subspecies did not differ in expression of hypothalamic gonadotropin-inhibitory hormone (GnIH) and glucocorticoid receptor (GR). While our earlier breeding subspecies had higher mRNA expression of testicular GR, subspecies did not differ in testicular luteinizing hormone receptor, follicle-stimulating hormone receptor or MR mRNA expression levels. Our findings indicate increased GnRH production and decreased hypothalamic sensitivity to sex steroid negative feedback as factors promoting differences in the timing of gonadal recrudescence between recently diverged populations. Differential gene expression along the HPG axis may facilitate species diversification under seasonal sympatry.

Chronological and Biological Age Predict Seasonal Reproductive Timing: An Investigation of Clutch Initiation and Telomeres in Birds of Known Age.

Female vertebrates that breed earlier in the season generally have greater reproductive success. However, evidence suggests that breeding early may be costly, thus leading to the prediction that females with fewer future reproductive events will breed earlier in the season. While chronological age is a good indicator of remaining life span, telomere lengths may also be good biomarkers of longevity as they potentially reflect lifetime wear and tear (i.e., biological age). We examined whether variation in the timing of the first seasonal clutch was related to age and telomere length in female dark-eyed juncos (Junco hyemalis), predicting that older females and those with shorter telomeres would breed earlier. Both predictions held true and were independent of each other, as telomere length did not significantly vary with age. These results suggest that females may adjust their reproductive effort based on both chronological and biological age.

Repeated immune challenges affect testosterone but not sperm quality.

Mounting an immunological response is energetically demanding and necessarily redirects allocation of resources toward immune system activation and away from other energetically expensive processes, such as reproduction. Lipopolysaccharide (LPS), a major component of the outer membrane of the cell wall of Gram-negative bacteria Escherichia coli, mimics a bacterial infection without producing the cost of replicating the pathogen and is one of the most commonly used agents to induce an acute phase immune response. Here, we ask if a trade-off can be induced between activation of the acute phase immune response and sperm function, a key indicator of sperm competitive ability. Further, we ask whether repeated exposure to this endotoxin in a social species such as the house sparrow (Passer domesticus), where repeated pathogen exposure may be common, may have a more pronounced effect. To address our questions, we exposed individuals to two rounds of LPS treatment or control, to mimic a repeated pathogen exposure in the wild. We predicted that repeated pathogen exposure would have detrimental effects on sperm quality, and therefore, reproductive success. We compared a measure of sperm quality (straight-line velocity) in captive male house sparrows between LPS-treated and control individuals. We found that although LPS treatment impaired circulating testosterone and induced a hypothermic state when compared with controls, it did not affect sperm quality within days or weeks following a single or repeated LPS exposure.

Consistent individual variation in day, night, and GnRH-induced testosterone concentrations in house sparrows (Passer domesticus).

The hypothalamic-pituitary-gonadal (HPG) axis, with gonadotropin-releasing hormone (GnRH) initiating the endocrine cascade, regulates testosterone secretion. Testosterone, through its pleiotropic effects, plays a crucial role in coordinating morphology, physiology and behavior in a reproductive context. The concentration of circulating testosterone, however, varies over the course of the day and in response to other internal or external stimuli, potentially making it difficult to relate testosterone sampled at one time point with traits of interest. Many researchers now utilize the administration of exogenous GnRH to elicit a standardized stimulation of testosterone secretion. However, it has remained unclear if and how this exogenously stimulated activation of the HPG axis is related with endogenously regulated testosterone that is capable of influencing testosterone related traits. Repeated measures of a hormone can uncover consistent individual variation in hormonal differences at the HPG axis level, variation that potentially stems from underlying genetic variation in a population experiencing identical environmental cues. Thus, we asked, using the house sparrow (Passer domesticus), how daily endogenous variation in testosterone profiles relates to GnRH-induced testosterone secretion. Further, we explore the relationship between endogenous daily testosterone peaks and GnRH-induced testosterone with badge size, a morphological trait related with status within a social group. We found that GnRH-induced testosterone levels reflect a highly repeatable hormonal phenotype that is strongly correlated with nighttime testosterone levels. The results demonstrate the usefulness of GnRH-induced testosterone in studies aimed at understanding individual variation and selection on endogenously regulated testosterone levels and the potential importance of nighttime testosterone levels to physiology and behavior.

Estimating heritable genetic contributions to innate immune and endocrine phenotypic correlations: A need to explore repeatability.

The immune system plays an important role in enhancing an individual's ability to survive in a world inhabited by pathogens and parasites. The innate immune system is regulated by processes encoded in an individual's genome, providing an avenue for selection to act on this system, as well as the phenotypic relationships generated between this system and other traits of interest. While relationships between innate immunity and endocrine traits (e.g. testosterone) have been reported often in the literature, these relationships are complex and may differ under varying environmental conditions. To better understand the relative contribution of innate immunity (or an endocrine or behavioral trait) to a phenotypic correlation with another trait, an estimation of the underlying heritable genetic variation of the trait of interest is needed. An upper level estimate of the heritability of such traits can be obtained from calculating its repeatability. We conducted a literature review to determine how often repeated samples of measures of innate immune function were conducted and repeatability estimates obtained. This review revealed a very limited number of repeatability estimates, with a large range (0.0-0.9); estimates were exclusively from livestock that have undergone strong artificial selection. This observation of the present literature suggests more work is needed in non-domesticated and free-living animals to begin to understand the underlying genetic contribution of innate immune function to phenotypic correlations of interest (e.g. testosterone and immunity) to behavioral ecologists, evolutionary physiologists and ecoimmunologists.

Early spring sex differences in luteinizing hormone response to gonadotropin releasing hormone in co-occurring resident and migrant dark-eyed juncos (Junco hyemalis).

To optimally time reproduction, animals must coordinate changes in the hypothalamo-pituitary-gonadal (HPG) axis. The extent of intra-species variation in seasonal timing of reproductive function is considerable, both within and among populations. Dark-eyed junco (Junco hyemalis) populations are known to differ in their reproductive timing response to cues experienced in the same habitat in late winter/early spring. Specifically in juncos cohabitating on shared wintering grounds, residents initiate breeding and reproductive activity but migrants delay reproductive development and prepare to migrate before breeding. Here, we test the hypothesis that the pituitary gland acts as a 'control point' to modulate differential HPG axis activity across populations. We sampled free-living resident and migrant juncos on their shared over-wintering grounds in March, thus all individuals were experiencing the same environmental cues, including photoperiod. We predicted that during this critical time of transition, residents would more readily respond to repeated gonadotropin releasing hormone (GnRH) stimulation with increases in luteinizing hormone (LH), in contrast to migrants, which should delay full reproductive activity. Our data indicate that migrant females, while still on the overwintering grounds, have a reduced LH response to repeated GnRH injections compared to resident females. Male migrant and resident birds did not differ in their responsiveness to repeated GnRH. Our results suggest a sex difference in the costs of mistimed activation of the HPG axis, with female migrants being less responsive than residents females and males to repeated stimulation. Further, our data implicate a key role for the pituitary in regulating appropriate reproductive timing responses.

Reproductive Allochrony in Seasonally Sympatric Populations Maintained by Differential Response to Photoperiod: Implications for Population Divergence and Response to Climate Change.

Reproductive allochrony presents a potential barrier to gene flow and is common in seasonally sympatric migratory and sedentary birds. Mechanisms mediating reproductive allochrony can influence population divergence and the capacity of populations to respond to environmental change. We asked whether reproductive allochrony in seasonally sympatric birds results from a difference in response to supplementary or photoperiodic cues and whether the response varies in relation to the distance separating breeding and wintering locations as measured by stable isotopes. We held seasonally sympatric migratory and sedentary male dark-eyed juncos (Junco hyemalis) in a common garden in early spring under simulated natural changes in photoperiod and made measurements of reproductive and migratory physiology. On the same dates and photoperiods, sedentary juncos had higher testosterone (initial and gonadotropin-releasing hormone induced), more developed cloacal protuberances, and larger testes than migrants. In contrast, migratory juncos had larger fat reserves (fuel for migration). We found a negative relationship between testis mass and feather hydrogen isotope ratios, indicating that testis growth was more delayed in migrants making longer migrations. We conclude that reproductive allochrony in seasonally sympatric migratory and sedentary birds can result from a differential response to photoperiodic cues in a common garden, and as a result, gene flow between migrants and residents may be reduced by photoperiodic control of reproductive development. Further, earlier breeding in response to future climate change may currently be constrained by differential response to photoperiodic cues.

Differential gene expression in seasonal sympatry: mechanisms involved in diverging life histories.

In an era of climate change, understanding the genetic and physiological mechanisms underlying flexibility in phenology and life history has gained greater importance. These mechanisms can be elucidated by comparing closely related populations that differ in key behavioural and physiological traits such as migration and timing of reproduction. We compared gene expression in two recently diverged dark-eyed Junco ( Junco hyemalis) subspecies that live in seasonal sympatry during winter and early spring, but that differ in behaviour and physiology, despite exposure to identical environmental cues. We identified 547 genes differentially expressed in blood and pectoral muscle. Genes involved in lipid transport and metabolism were highly expressed in migrant juncos, while genes involved in reproductive processes were highly expressed in resident breeders. Seasonal differences in gene expression in closely related populations residing in the same environment provide significant insights into mechanisms underlying variation in phenology and life history, and have potential implications for the role of seasonal timing differences in gene flow and reproductive isolation.

Hypothalamic-pituitary-adrenal axis activity is not elevated in a songbird (Junco hyemalis) preparing for migration.

During spring, increasing daylengths stimulate gonadal development in migratory birds. However, late-stage reproductive development is typically postponed until migration has been completed. The hypothalamic-pituitary-adrenal (HPA) axis regulates the secretion of glucocorticoids, which have been associated with pre-migratory hyperphagia and fattening. The HPA-axis is also known to suppress the hypothalamic-pituitary-gonadal (HPG) axis, suggesting the possibility that final transition into the breeding life history stage may be slowed by glucocorticoids. We hypothesized that greater HPA-axis activity in individuals preparing for migration may foster preparation for migration while simultaneously acting as a "brake" on the development of the HPG-axis. To test this hypothesis, we sampled baseline corticosterone (CORT), stress-induced CORT, and negative feedback efficacy of Dark-eyed Juncos (Junco hyemalis) in an overwintering population that included both migratory (J.h. hyemalis) and resident (J.h. carolinensis) individuals. We predicted that compared to residents, migrants would have higher baseline CORT, higher stress-induced CORT, and weaker negative feedback. Juncos were sampled in western Virginia in early March, which was about 2-4wk before migratory departure for migrants and 4-5wk before first clutch initiation for residents. Contrary to our predictions, we found that migrants had lower baseline and stress-induced CORT and similar negative feedback efficacy compared with residents, which suggests that delayed breeding in migrants is influenced by other physiological mechanisms. Our findings also suggest that baseline CORT is not elevated during pre-migratory fattening, as migrants had lower baseline CORT and were fatter than residents.