Gene regulation and speciation in a migratory divide between songbirds.

Behavioral variation abounds in nature. This variation is important for adaptation and speciation, but its molecular basis remains elusive. Here, we use a hybrid zone between two subspecies of songbirds that differ in migration - an ecologically important and taxonomically widespread behavior---to gain insight into this topic. We measure gene expression in five brain regions. Differential expression between migratory states was dominated by circadian genes in all brain regions. The remaining patterns were largely brain-region specific. For example, expression differences between the subspecies that interact with migratory state likely help maintain reproductive isolation in this system and were documented in only three brain regions. Contrary to existing work on regulatory mechanisms underlying species-specific traits, two lines of evidence suggest that trans- (vs. cis) regulatory changes underlie these patterns - no evidence for allele-specific expression in hybrids and minimal associations between genomic differentiation and expression differences. Additional work with hybrids shows expression levels were often distinct (transgressive) from parental forms. Behavioral contrasts and functional enrichment analyses allowed us to connect these patterns to mitonuclear incompatibilities and compensatory responses to stress that could exacerbate selection on hybrids and contribute to speciation.

The pace of mitochondrial molecular evolution varies with seasonal migration distance.

Animals that engage in long-distance seasonal migration experience strong selective pressures on their metabolic performance and life history, with potential consequences for molecular evolution. Species with slow life histories typically show lower rates of synonymous substitution (dS) than "fast" species. Previous research suggests long-distance seasonal migrants have a slower life history strategy than short-distance migrants, raising the possibility that rates of molecular evolution may covary with migration distance. Additionally, long-distance migrants may face strong selection on metabolically-important mitochondrial genes due to their long-distance flights. Using over 1,000 mitochondrial genomes, we assessed the relationship between migration distance and mitochondrial molecular evolution in 39 boreal-breeding migratory bird species. We show that migration distance correlates negatively with dS, suggesting that the slow life history associated with long-distance migration is reflected in rates of molecular evolution. Mitochondrial genes in every study species exhibited evidence of purifying selection, but the strength of selection was greater in short-distance migrants, contrary to our predictions. This result may indicate effects of selection for cold tolerance on mitochondrial evolution among species overwintering at high latitudes. Our study demonstrates that the pervasive correlation between life history and molecular evolutionary rates exists in the context of differential adaptations to seasonality.

Genetic evidence for widespread population size expansion in North American boreal birds prior to the Last Glacial Maximum.

Pleistocene climate cycles are well documented to have shaped contemporary species distributions and genetic diversity. Northward range expansions in response to deglaciation following the Last Glacial Maximum (LGM; approximately 21 000 years ago) are surmised to have led to population size expansions in terrestrial taxa and changes in seasonal migratory behaviour. Recent findings, however, suggest that some northern temperate populations may have been more stable than expected through the LGM. We modelled the demographic history of 19 co-distributed boreal-breeding North American bird species from full mitochondrial gene sets and species-specific molecular rates. We used these demographic reconstructions to test how species with different migratory strategies were affected by glacial cycles. Our results suggest that effective population sizes increased in response to Pleistocene deglaciation earlier than the LGM, whereas genetic diversity was maintained throughout the LGM despite shifts in geographical range. We conclude that glacial cycles prior to the LGM have most strongly shaped contemporary genetic diversity in these species. We did not find a relationship between historic population dynamics and migratory strategy, contributing to growing evidence that major switches in migratory strategy during the LGM are unnecessary to explain contemporary migratory patterns.

Beyond a biased binary: A perspective on the misconceptions, challenges, and implications of studying females in avian behavioral endocrinology.

For decades, avian endocrinology has been informed by male perspectives and male-focused research, leaving significant gaps in our understanding of female birds. Male birds have been favored as research subjects because their reproductive behaviors are considered more conspicuous and their reproductive physiology is presumably less complex than female birds. However, female birds should not be ignored, as female reproductive behavior and physiology are essential for the propagation of all avian species. Endocrine research in female birds has made much progress in the last 20 years, but a substantial disparity in knowledge between male and female endocrinology persists. In this perspective piece, we provide examples of why ornithology has neglected female endocrinology, and we propose considerations for field and laboratory techniques to facilitate future studies. We highlight recent advances that showcase the importance of female avian endocrinology, and we challenge historic applications of an oversimplified, male-biased lens. We further provide examples of species for which avian behavior differs from the stereotypically described behaviors of male and female birds, warning investigators of the pitfalls in approaching endocrinology with a binary bias. We hope this piece will inspire investigators to engage in more comprehensive studies with female birds, to close the knowledge gap between the sexes, and to look beyond the binary when drawing conclusions about what is 'male' versus 'female' biology.

Plasticity in female timing may explain earlier breeding in a North American songbird.

Many species have shifted their breeding phenology in response to climate change. Identifying the magnitude of phenological shifts and whether climate-mediated selection drives these shifts is key for determining species' resilience to climate change. Birds are a strong model for studying phenological shifts due to numerous long-term research studies; however, generalities pertaining to drivers of phenological shifts will emerge only as we add study species that differ in life history and geography. We investigated 32 years of reproductive timing in a non-migratory population of dark-eyed juncos Junco hyemalis. We predicted that plasticity in reproductive timing would allow females to breed earlier in warmer springs. We also predicted that selection would favour earlier breeding and asked whether the temperatures throughout the breeding season would predict the strength of selection. To test these predictions, we examined temporal changes in the annual median date for reproductive onset (i.e. first egg date) and we used a sliding window analysis to identify spring temperatures driving these patterns. Next, we explored plasticity in reproductive timing and asked whether selection favoured earlier breeding. Lastly, we used a sliding window analysis to identify the time during the breeding season that temperature was most associated with selection favouring earlier breeding. First egg dates occurred earlier over time and strongly covaried with April temperatures. Furthermore, individual females that bred in at least 3 years typically bred earlier in warmer Aprils, exhibiting plastic responses to April temperature. We also found significant overall selection favouring earlier breeding (i.e. higher relative fitness with earlier first egg dates) and variation in selection for earlier breeding over time. However, temperature across diverse climatic windows did not predict the strength of selection. Our findings provide further evidence for the role of phenotypic plasticity in shifting phenology in response to earlier springs. We also provide evidence for the role of selection favouring earlier breeding, regardless of temperature, thus setting the stage for adaptive changes in female breeding phenology. We suggest for multi-brooded birds that advancing first egg dates likely increase the length of the breeding season, and therefore, reproductive success.

Circulating Hormones and Dominance Status Predict Female Behavior during Courtship in a Lekking Species.

Female competitive behaviors during courtship can have substantial fitness consequences, yet we know little about the physiological and social mechanisms underlying these behaviors-particularly for females of polygynous lek mating species. We explored the hormonal and social drivers of female intersexual and intrasexual behavior during courtship by males in a captive population of Indian peafowl. We investigated whether (1) female non-stress induced circulating estradiol (E2) and corticosterone (CORT) levels or (2) female dominance status in a dyad predicts female solicitation behavior. We also tested whether female circulating E2 and CORT predict dominant females' aggressive behaviors toward subordinate females in the courtship context. Our findings demonstrate that females with higher levels of circulating E2 as well as higher levels of circulating CORT solicit more courtships from males. Dominant females also solicit more courtships from males than subordinate females. Female intrasexual aggressive behaviors during courtship, however, were not associated with circulating levels of E2 or CORT. Overall, we conclude that circulating steroid hormones in conjunction with social dominance might play a role in mediating female behaviors associated with competition for mates. Experimental manipulation and measures of hormonal flexibility throughout the breeding season in relation to competitive and sexual behaviors will be necessary to further examine the link between hormonal mechanisms and female behavior in polygynous lekking systems.

Estimating hybridization rates in the wild: Easier said than done?

Hybridization has important effects on the evolutionary trajectories of natural populations but estimates of this process in the wild and at the individual-level are lacking. Justyn et al. attempted to fill this gap using the citizen science database eBird but there are limitations to this approach. Here, we outline and directly test these limitations using literature searches, case studies, and a comparison between eBird and Birds of North America (BNA), a database that documents hybridization using the scientific literature. We use a hybrid zone between Lazuli and Indigo buntings to highlight the importance of considering geographic range when estimating rates of hybridization and two literature searches to show the importance of considering cryptic hybrids (those that cannot be identified using phenotypic traits) when quantifying these rates. We also use BNA and a case study of hybrid White-faced and Glossy Ibises to show that citizen scientists are underreporting hybrids compared with experts. Justyn et al. highlighted an important gap in the literature, but their results likely represent the lower limit of hybridization between birds and a more nuanced interpretation of their results (e.g., considering extrinsic postzygotic selection) is needed.

It Takes Two to Tango: Including a Female Perspective in Reproductive Biology.

Like many scientific disciplines, the field of reproductive biology is subject to biases in terminology and research foci. For example, females are often described as coy and passive players in reproductive behaviors and are termed "promiscuous" if they engage in extra-pair copulations. Males on the other hand are viewed as actively holding territories and fighting with other males. Males are termed "multiply mating" if they mate with multiple females. Similarly, textbooks often illustrate meiosis as it occurs in males but not females. This edition of Integrative and Comparative Biology (ICB) includes a series of papers that focus on reproduction from the female perspective. These papers represent a subset of the work presented in our symposium and complementary sessions on female reproductive biology. In this round table discussion, we use a question and answer format to leverage the diverse perspectives and voices involved with the symposium in an exploration of theoretical, cultural, pedagogical, and scientific issues related to the study of female biology. We hope this dialog will provide a stepping-stone toward moving reproductive science and teaching to a more inclusive and objective framework.

Females as the Gatekeepers to Seasonal Breeding: What We Can Learn by Studying Reproductive Mechanisms in Both Sexes.

Seasonal reproduction is a widespread adaptation in vertebrates, such that individuals time their reproductive efforts to match peak resource abundance. Individuals rely on environmental cues to regulate hormonal mechanisms governing timing of breeding. Historically, studies on physiological mechanisms of seasonal reproduction, specifically in birds, have disproportionately focused on males compared to females. For this review, I conducted a literature search of the last decade of avian research and found a persistent sex bias in the field of physiological mechanisms of seasonal reproduction. Using work conducted with the dark-eyed junco (Junco hyemalis) as a case study, I present a possible solution to combat the sex bias: natural comparisons of populations that differ in reproductive timing to investigate mechanisms of reproduction in both sexes. Populations of dark-eyed juncos that differ in migratory behavior (i.e., migrant and resident) exhibit overlapping ranges during winter and early spring; residents begin breeding in early spring prior to the departure of migrants. This system, and others like it, provides an opportunity to compare mechanisms of reproduction in populations that differ in reproductive timing despite experiencing the same environmental conditions in early spring. In juncos, migrant and resident females and males exhibit similar patterns of hypothalamic regulation of reproduction in early spring, but sex differences in gonadal sensitivity between the populations could be an important distinction that partially explains sex differences in reproductive development. Comparing mechanisms of reproduction in free-living populations and in captivity can reveal important mechanisms that determine the onset of reproductive development, as well as potential sex differences in these mechanisms. Understanding the mechanisms of reproductive phenology has important implications for understanding how species will survive and reproduce in a changing climate.

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.

Seasonally sympatric songbirds that differ in migratory strategy also differ in neuroendocrine measures.

Seasonally breeding animals initiate gonadal recrudescence when mechanisms that suppress reproduction give way to mechanisms that stimulate it. However, knowledge of mechanistic changes in hormonal regulation during this transition is limited. Further, most studies of reproductive timing have focused on males, despite the critical role of females in determining breeding phenology. Closely related populations that live in the same environment but differ in reproductive timing provide an opportunity to examine differences in mechanisms during the transition from the pre-reproductive to reproductive state. We studied closely related migrant and resident populations of dark-eyed juncos (Junco hyemalis) that reside in the same environment in spring but differ in breeding phenology. Residents initiate breeding earlier than migrants, which do not breed until after they have migrated. To directly study differences in the hypothalamic mechanisms of reproduction, we captured 16 migrant and 13 resident females from the field on March 25-April 11. We quantified expression of mRNA transcripts and show that resident females had higher abundance of gonadotropin-releasing hormone transcripts than migrant females, indicating greater reproductive development in resident than migrant females living in the same environment. We also found higher transcript abundance of estrogen receptor and androgen receptor in migrant than resident females, suggesting that negative feedback may delay reproductive development in migrant females until after they migrate. These differences in hypothalamic mechanisms may help to explain differences in reproductive timing in populations that differ in migratory strategy.

Male courtship preference during seasonal sympatry may maintain population divergence.

Animal migration can lead to a population distribution known as seasonal sympatry, in which closely-related migrant and resident populations of the same species co-occur in sympatry during part of the year, but are otherwise allopatric. During seasonal sympatry in early spring, residents may initiate reproduction before migrants depart, presenting an opportunity for gene flow. Differences in reproductive timing between migrant and resident populations may favor residents that exhibit preferences for potential mates of similar migratory behavior and reproductive timing, thus maintaining population divergence. We studied dark-eyed juncos (Junco hyemalis), a songbird that exhibits seasonal sympatry. We conducted simulated courtship interactions in which we presented free-living resident males with either a caged migrant or resident female and quantified courtship behavior prior to the departure of the migrants. We found that resident males preferred to court resident females: they sang more short-range songs and exhibited more visual displays associated with courtship when presented with resident females. We conclude that males distinguish between migrant and resident females during seasonal sympatry when the risk of interacting with non-reproductive, migrant females is high. Male mate choice in seasonal sympatry is likely adaptive for male reproductive success. As a secondary effect, male mating preference could act to maintain or promote divergence between populations that differ in migratory strategy.