Sex diversity in the 21st century: Concepts, frameworks, and approaches for the future of neuroendocrinology.

Sex is ubiquitous and variable throughout the animal kingdom. Historically, scientists have used reductionist methodologies that rely on a priori sex categorizations, in which two discrete sexes are inextricably linked with gamete type. However, this binarized operationalization does not adequately reflect the diversity of sex observed in nature. This is due, in part, to the fact that sex exists across many levels of biological analysis, including genetic, molecular, cellular, morphological, behavioral, and population levels. Furthermore, the biological mechanisms governing sex are embedded in complex networks that dynamically interact with other systems. To produce the most accurate and scientifically rigorous work examining sex in neuroendocrinology and to capture the full range of sex variability and diversity present in animal systems, we must critically assess the frameworks, experimental designs, and analytical methods used in our research. In this perspective piece, we first propose a new conceptual framework to guide the integrative study of sex. Then, we provide practical guidance on research approaches for studying sex-associated variables, including factors to consider in study design, selection of model organisms, experimental methodologies, and statistical analyses. We invite fellow scientists to conscientiously apply these modernized approaches to advance our biological understanding of sex and to encourage academically and socially responsible outcomes of our work. By expanding our conceptual frameworks and methodological approaches to the study of sex, we will gain insight into the unique ways that sex exists across levels of biological organization to produce the vast array of variability and diversity observed in nature.

Novel insights into symbiont population structure: Globe-trotting avian feather mites contradict the specialist-generalist variation hypothesis.

Researchers often examine symbiont host specificity as a species-level pattern, but it can also be key to understanding processes occurring at the population level, which are not as well understood. The specialist-generalist variation hypothesis (SGVH) attempts to explain how host specificity influences population-level processes, stating that single-host symbionts (specialists) exhibit stronger population genetic structure than multi-host symbionts (generalists) because of fewer opportunities for dispersal and more restricted gene flow between populations. However, this hypothesis has not been tested in systems with highly mobile hosts, in which population connectivity may vary temporally and spatially. To address this gap, we tested the SGVH on proctophyllodid feather mites found on migratory warblers (family Parulidae) with contrasting host specificities, Amerodectes protonotaria (a host specialist of Protonotaria citrea) and A. ischyros (a host generalist of 17 parulid species). We used a pooled-sequencing approach and a novel workflow to analyse genetic variants obtained from whole genome data. Both mite species exhibited fairly weak population structure overall, and contrary to predictions of the SGVH, the generalist was more strongly structured than the specialist. These results may suggest that specialists disperse more freely among conspecifics, whereas generalists sort according to geography. Furthermore, our results may reflect an unexpected period for mite transmission - during the nonbreeding season of migratory hosts - as mite population structure more closely reflects the distributions of hosts during the nonbreeding season. Our findings alter our current understanding of feather mite biology and highlight the potential for studies to explore factors driving symbiont diversification at multiple evolutionary scales.

Sex and Biology: Broader Impacts Beyond the Binary.

What are the implications of misunderstanding sex as a binary, and why is it essential for scientists to incorporate a more expansive view of biological sex in our teaching and research? This roundtable will include many of our symposium speakers, including biologists and intersex advocates, to discuss these topics and visibilize the link between ongoing reification of dyadic sex within scientific communities and the social, political, and medical oppression faced by queer, transgender, and especially intersex communities. As with the symposium as a whole, this conversation is designed to bring together empirical research and implementation of equity, inclusion, and justice principles, which are often siloed into separate rooms and conversations at academic conferences. Given the local and national attacks on the rights of intersex individuals and access to medical care and bodily autonomy, this interdisciplinary discussion is both timely and urgent.

Multivariate Models of Animal Sex: Breaking Binaries Leads to a Better Understanding of Ecology and Evolution.

"Sex" is often used to describe a suite of phenotypic and genotypic traits of an organism related to reproduction. However, these traits-gamete type, chromosomal inheritance, physiology, morphology, behavior, etc.-are not necessarily coupled, and the rhetorical collapse of variation into a single term elides much of the complexity inherent in sexual phenotypes. We argue that consideration of "sex" as a constructed category operating at multiple biological levels opens up new avenues for inquiry in our study of biological variation. We apply this framework to three case studies that illustrate the diversity of sex variation, from decoupling sexual phenotypes to the evolutionary and ecological consequences of intrasexual polymorphisms. We argue that instead of assuming binary sex in these systems, some may be better categorized as multivariate and nonbinary. Finally, we conduct a meta-analysis of terms used to describe diversity in sexual phenotypes in the scientific literature to highlight how a multivariate model of sex can clarify, rather than cloud, studies of sexual diversity within and across species. We argue that such an expanded framework of "sex" better equips us to understand evolutionary processes, and that as biologists, it is incumbent upon us to push back against misunderstandings of the biology of sexual phenotypes that enact harm on marginalized communities.

Comparative phylogeography reveals widespread cryptic diversity driven by ecology in Panamanian birds.

Widespread species often harbor unrecognized genetic diversity, and investigating the factors associated with such cryptic variation can help us better understand the forces driving diversification. Here, we identify potential cryptic species based on a comprehensive dataset of COI mitochondrial DNA barcodes from 2,333 individual Panamanian birds across 429 species, representing 391 (59%) of the 659 resident landbird species of the country, as well as opportunistically sampled waterbirds. We complement this dataset with additional publicly available mitochondrial loci, such as ND2 and cytochrome b, obtained from whole mitochondrial genomes from 20 taxa. Using barcode identification numbers (BINs), we find putative cryptic species in 19% of landbird species, highlighting hidden diversity in the relatively well-described avifauna of Panama. Whereas some of these mitochondrial divergence events corresponded with recognized geographic features that likely isolated populations, such as the Cordillera Central highlands, the majority (74%) of lowland splits were between eastern and western populations. The timing of these splits are not temporally coincident across taxa, suggesting that historical events, such as the formation of the Isthmus of Panama and Pleistocene climatic cycles, were not the primary drivers of cryptic diversification. Rather, we observed that forest species, understory species, insectivores, and strongly territorial species-all traits associated with lower dispersal ability-were all more likely to have multiple BINs in Panama, suggesting strong ecological associations with cryptic divergence. Additionally, hand-wing index, a proxy for dispersal capability, was significantly lower in species with multiple BINs, indicating that dispersal ability plays an important role in generating diversity in Neotropical birds. Together, these results underscore the need for evolutionary studies of tropical bird communities to consider ecological factors along with geographic explanations, and that even in areas with well-known avifauna, avian diversity may be substantially underestimated.

Especies extendidas frecuentemente tiene diversidad genética no reconocida, y investigando los factores asociados con esta variación críptica puede ayudarnos a entender las fuerzas que impulsan la diversificación. Aquí, identificamos especies crípticas potenciales basadas en un conjunto de datos de códigos de barras de ADN mitocondrial de 2,333 individuos de aves de Panama en 429 especies, representando 391 (59%) de las 659 especies de aves terrestres residentes del país, además de algunas aves acuáticas muestreada de manera oportunista. Adicionalmente, complementamos estos datos con secuencias mitocondriales disponibles públicamente de otros loci, tal como ND2 o citocroma b, obtenidos de los genomas mitocondriales completos de 20 taxones. Utilizando los números de identificación de código de barras (en ingles: BINs), un sistema taxonómico numérico que proporcina una estimación imparcial de la diversidad potencial a nivel de especie, encontramos especies crípticas putativas en 19% de las especies de aves terrestres, lo que destaca la diversidad oculta en la avifauna bien descrita de Panamá. Aunque algunos de estos eventos de divergencia conciden con características geográficas que probablemente aislaron las poblaciones, la mayoría (74%) de la divergencia en las tierras bajas se encuentra entre las poblaciones orientales y occidentales. El tiempo de esta divergencia no coincidió entre los taxones, sugiriendo que eventos históricos tales como la formación del Istmo de Panamá y los ciclos climáticos del pleistoceno, no fueron los principales impulsores de la especiación. En cambio, observamos asociaciones fuertes entre las características ecológicas y la divergencia mitocondriale: las especies del bosque, sotobosque, con una dieta insectívora, y con territorialidad fuerte mostraton múltiple BINs probables. Adicionalmente, el índice mano-ala, que está asociado a la capacidad de dispersión, fue significativamente menor en las especies con BINs multiples, sugiriendo que la capacidad de dispersión tiene un rol importamente en la generación de la diversidad de las aves neotropicales. Estos resultos demonstran la necesidad de que estudios evolutivos de las comunidades de aves tropicales consideren los factores ecológicos en conjunto con las explicaciones geográficos.

How Female-Female Competition Affects Male-Male Competition: Insights into Postcopulatory Sexual Selection from Socially Polyandrous Species.

AbstractSexual selection is a major driver of trait variation, and the intensity of male competition for mating opportunities has been linked with sperm size across diverse taxa. Mating competition among females may also shape the evolution of sperm traits, but the effect of the interplay between female-female competition and male-male competition on sperm morphology is not well understood. We evaluated variation in sperm morphology in two species with socially polyandrous mating systems, in which females compete to mate with multiple males. Northern jacanas (Jacana spinosa) and wattled jacanas (J. jacana) vary in their degree of social polyandry and sexual dimorphism, suggesting species differences in the intensity of sexual selection. We compared mean and variance in sperm head, midpiece, and tail length between species and breeding stages because these measures have been associated with the intensity of sperm competition. We found that the species with greater polyandry, northern jacana, has sperm with longer midpieces and tails as well as marginally lower intraejaculate variation in tail length. Intraejaculate variation was also significantly lower in copulating males than in incubating males, suggesting flexibility in sperm production as males cycle between breeding stages. Our results indicate that stronger female-female competition for mating opportunities may also shape more intense male-male competition by selecting for longer and less variable sperm traits. These findings extend frameworks developed in socially monogamous species to reveal that sperm competition may be an important evolutionary force layered atop female-female competition for mates.

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.

How thermal challenges change gene regulation in the songbird brain and gonad: Implications for sexual selection in our changing world.

In a rapidly warming world, exposure to high temperatures may impact fitness, but the gene regulatory mechanisms that link sublethal heat to sexually selected traits are not well understood, particularly in endothermic animals. Our experiment used zebra finches (Taeniopygia guttata), songbirds that experience extreme temperature fluctuations in their native Australia. We exposed captive males to an acute thermal challenge (43°C) compared with thermoneutral (35°C) and lower (27°C) temperatures. We found significantly more heat dissipation behaviours at 43°C, a temperature previously shown to reduce song production and fertility, and more heat retention behaviours at 27°C. Next, we characterized transcriptomic responses in tissues important for mating effort-the posterior telencephalon, for its role in song production, and the testis, for its role in fertility and hormone production. Differential expression of hundreds of genes in the testes, but few in the brain, suggests the brain is less responsive to extreme temperatures. Nevertheless, gene network analyses revealed that expression related to dopaminergic signalling in the brain covaried with heat dissipation behaviours, providing a mechanism by which temporary thermal challenges may alter motivational circuits for song production. In both brain and testis, we observed correlations between thermally sensitive gene networks and individual differences in thermoregulatory behaviour. Although we cannot directly relate these gene regulatory changes to mating success, our results suggest that individual variation in response to thermal challenges could impact sexually selected traits in a warming world.

Nesting strategy shapes territorial aggression but not testosterone: A comparative approach in female and male birds.

Our understanding of the proximate and ultimate mechanisms shaping competitive reproductive phenotypes primarily stems from research on male-male competition for mates, even though competition is widespread in both sexes. We evaluate the hypothesis that the restricted nature of a resource required for reproduction, i.e. nest site, is a key variable driving territorial competition and testosterone secretion in female and male birds. Obligate secondary cavity-nesting has evolved repeatedly across avian lineages, providing a useful comparative context to explore how competition over limited nest cavities shapes aggression and its underlying mechanisms across species. Although evidence from one or another cavity-nesting species suggests that territorial aggression is adaptive in both females and males, this has not yet been tested in a comparative framework. We predicted that cavity-nesting generates more robust territorial aggression, in comparison to close relatives with less restrictive nesting strategies. Our focal species were two obligate secondary cavity-nesting species and two related species with more flexible nesting strategies in the same avian family: tree swallow (Tachycineta bicolor) vs. barn swallow (Hirundo rustica); Eastern bluebird (Sialia sialis) vs. American robin (Turdus migratorius). We assayed conspecific aggression using simulated territorial intrusion and found that cavity-nesting species displayed greater territorial aggression than their close relatives. This pattern held for both females and males. Because territorial aggression is often associated with elevated testosterone, we also hypothesized that cavity-nesting species would exhibit higher testosterone levels in circulation. However, cavity-nesting species did not have higher testosterone in circulation for either sex, despite some correlative evidence that testosterone is associated with higher rates of physical attack in female tree swallows. Our focus on a context that is relevant to both sexes - competition over essential breeding resources - provides a useful framework for co-consideration of proximate and ultimate drivers of reproductive competition in females and males.

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.

Neuroendocrinology of Sex-Role Reversal.

Females of some species are considered sex-role reversed, meaning that they face stronger competition for mates compared to males. While much attention has been paid to behavioral and morphological patterns associated with sex-role reversal, less is known about its physiological regulation. Here, we evaluate hypotheses relating to the neuroendocrine basis of sex-role reversal. We refute the most widely tested activational hypothesis for sex differences in androgen secretion; sex-role reversed females do not have higher levels of androgens in circulation than males. However, we find some evidence that the effects of androgens may be sex-specific; circulating androgen levels correlate with some competitive phenotypes in sex-role reversed females. We also review evidence that sex-role reversed females have higher tissue-specific sensitivity to androgens than males, at least in some species and tissues. Organizational effects may explain these relationships, considering that early exposure to sex steroids can shape later sensitivity to hormones, often in sex-specific ways. Moving forward, experimental and correlative studies on the ontogeny and expression of sex-role reversal will further clarify the mechanisms that generate sex-specific behaviors and sex roles.

Testosterone secretion varies in a sex- and stage-specific manner: Insights on the regulation of competitive traits from a sex-role reversed species.

Testosterone (T) mediates a variety of traits that function in competition for mates, including territorial aggression, ornaments, armaments, and gametogenesis. The link between T and mating competition has been studied mainly in males, but females also face selection pressures to compete for mates. Sex-role reversed species, in which females are the more competitive sex, provide a unique perspective on the role of T in promoting competitive traits. Here, we examine patterns of T secretion in sex-role reversed northern jacanas (Jacana spinosa) during breeding, when females are fertile and males are either seeking copulations or conducting parental care. We measured baseline levels of T in circulation along with a suite of behavioral and morphological traits putatively involved in mating competition. We evaluated hypotheses that levels of T track gonadal sex and parental role, and we begin to investigate whether T and competitive traits co-vary in a sex- and stage- specific manner. Although females had higher expression of competitive traits than males at either breeding stage, we found that females and incubating males had similar levels of T secretion, which were lower than those observed in copulating males. T was correlated with wing spur length in females and testes mass in copulating males, but was otherwise uncorrelated with other competitive traits. These findings suggest that levels of T in circulation alone do not predict variation in competitive traits across levels of analysis, including gonadal sex and parental role. Instead, our findings coupled with prior research indicate that selection for female mating competition and male care may generate different physiological regulation of competitive traits.

Differential introgression of a female competitive trait in a hybrid zone between sex-role reversed species.

Mating behavior between recently diverged species in secondary contact can impede or promote reproductive isolation. Traditionally, researchers focus on the importance of female mate choice and male-male competition in maintaining or eroding species barriers. Although female-female competition is widespread, little is known about its role in the speciation process. Here, we investigate a case of interspecific female competition and its influence on patterns of phenotypic and genetic introgression between species. We examine a hybrid zone between sex-role reversed, Neotropical shorebird species, the northern jacana (Jacana spinosa) and wattled jacana (J. jacana), in which female-female competition is a major determinant of reproductive success. Previous work found that females of the more aggressive and larger species, J. spinosa, disproportionately mother hybrid offspring, potentially by monopolizing breeding territories in sympatry with J. jacana. We find a cline shift of female body mass relative to the genetic center of the hybrid zone, consistent with asymmetric introgression of this competitive trait. We suggest that divergence in sexual characteristics between sex-role reversed females can influence patterns of gene flow upon secondary contact, similar to males in systems with more typical sex roles.

Interspecific competition, hybridization, and reproductive isolation in secondary contact: missing perspectives on males and females.

Research on sexual selection and hybridization has focused on female mate choice and male-male competition. While the evolutionary outcomes of interspecific female preference have been well explored, we are now gaining a better understanding of the processes by which male-male competition between species in secondary contact promotes reproductive isolation versus hybridization. What is relatively unexplored is the interaction between female choice and male competition, as they can oppose one another or align with similar outcomes for reproductive isolation. The role of female-female competition in hybridization is also not well understood, but could operate similarly to male-male competition in polyandrous and other systems where costs to heterospecific mating are low for females. Reproductive competition between either sex of sympatric species can cause the divergence and/or convergence of sexual signals and recognition, which in turn influences the likelihood for interspecific mating. Future work on species interactions in secondary contact should test the relative influences of both mate choice and competition for mates on hybridization outcomes, and should not ignore the possibilities that females can compete over mating resources, and males can exercise mate choice.

Behavioural response to song and genetic divergence in two subspecies of white-crowned sparrows (Zonotrichia leucophrys).

Divergence in sexual signals may drive reproductive isolation between lineages, but behavioural barriers can weaken in contact zones. Here, we investigate the role of song as a behavioural and genetic barrier in a contact zone between two subspecies of white-crowned sparrows (Zonotrichia leucophrys). We employed a reduced genomic data set to assess population structure and infer the history underlying divergence, gene flow and hybridization. We also measured divergence in song and tested behavioural responses to song using playback experiments within and outside the contact zone. We found that the subspecies form distinct genetic clusters, and demographic inference supported a model of secondary contact. Song phenotype, particularly length of the first note (a whistle), was a significant predictor of genetic subspecies identity and genetic distance along the hybrid zone, suggesting a close link between song and genetic divergence in this system. Individuals from both parental and admixed localities responded significantly more strongly to their own song than to the other subspecies song, supporting song as a behavioural barrier. Putative parental and admixed individuals were not significantly different in their strength of discrimination between own and other songs; however, individuals from admixed localities tended to discriminate less strongly, and this difference in discrimination strength was explained by song dissimilarity as well as genetic distance. Therefore, we find that song acts as a reproductive isolating mechanism that is potentially weakening in a contact zone between the subspecies. Our findings also support the hypothesis that intraspecific song variation can reduce gene flow between populations.

Patterns of Song across Natural and Anthropogenic Soundscapes Suggest That White-Crowned Sparrows Minimize Acoustic Masking and Maximize Signal Content.

Soundscapes pose both evolutionarily recent and long-standing sources of selection on acoustic communication. We currently know more about the impact of evolutionarily recent human-generated noise on communication than we do about how natural sounds such as pounding surf have shaped communication signals over evolutionary time. Based on signal detection theory, we hypothesized that acoustic phenotypes will vary with both anthropogenic and natural background noise levels and that similar mechanisms of cultural evolution and/or behavioral flexibility may underlie this variation. We studied song characteristics of white-crowned sparrows (Zonotrichia leucophrys nuttalli) across a noise gradient that includes both anthropogenic and natural sources of noise in San Francisco and Marin counties, California, USA. Both anthropogenic and natural soundscapes contain high amplitude low frequency noise (traffic or surf, respectively), so we predicted that birds would produce songs with higher minimum frequencies in areas with higher amplitude background noise to avoid auditory masking. We also anticipated that song minimum frequencies would be higher than the projected lower frequency limit of hearing based on site-specific masking profiles. Background noise was a strong predictor of song minimum frequency, both within a local noise gradient of three urban sites with the same song dialect and cultural evolutionary history, and across the regional noise gradient, which encompasses 11 urban and rural sites, several dialects, and several anthropogenic and natural sources of noise. Among rural sites alone, background noise tended to predict song minimum frequency, indicating that urban sites were not solely responsible for driving the regional pattern. These findings support the hypothesis that songs vary with local and regional soundscapes regardless of the source of noise. Song minimum frequency from five core study sites was also higher than the lower frequency limit of hearing at each site, further supporting the hypothesis that songs vary to transmit through noise in local soundscapes. Minimum frequencies leveled off at noisier sites, suggesting that minimum frequencies are constrained to an upper limit, possibly to retain the information content of wider bandwidths. We found evidence that site noise was a better predictor of song minimum frequency than territory noise in both anthropogenic and natural soundscapes, suggesting that cultural evolution rather than immediate behavioral flexibility is responsible for local song variation. Taken together, these results indicate that soundscapes shape song phenotype across both evolutionarily recent and long-standing soundscapes.

Genetic and phenotypic characterization of a hybrid zone between polyandrous Northern and Wattled Jacanas in Western Panama.

BACKGROUND: Hybridization provides a unique perspective into the ecological, genetic and behavioral context of speciation. Hybridization is common in birds, but has not yet been reported among bird species with a simultaneously polyandrous mating system; a mating system where a single female defends a harem of males who provide nearly all parental care. Unlike simple polyandry, polyandrous mating is extremely rare in birds, with only 1% of bird species employing this mating system. Although it is classically held that females are "choosy" in avian hybrid systems, nearly-exclusive male parental care raises the possibility that female selection against heterospecific matings might be reduced compared to birds with other mating systems. RESULTS: We describe a narrow hybrid zone in southwestern Panama between two polyandrous freshwater waders: Northern Jacana, Jacana spinosa and Wattled Jacana, J. jacana. We document coincident cline centers for three phenotypic traits, mtDNA, and one of two autosomal introns. Cline widths for these six markers varied from seven to 142 km, with mtDNA being the narrowest, and five of the six markers having widths less than 100 km. Cline tails were asymmetrical, with greater introgression of J. jacana traits extending westward into the range of J. spinosa. Likewise, within the hybrid zone, the average hybrid index of phenotypic hybrids was significantly biased towards J. spinosa. Species distribution models indicate that the hybrid zone is located at the edge of a roughly 100 km wide overlap where habitat is predicted to be suitable for both species, with more westerly areas suitable only for spinosa and eastward habitats suitable only for J. jacana. CONCLUSION: The two species of New World jacanas maintain a narrow, and persistent hybrid zone in western Panama. The hybrid zone may be maintained by the behavioral dominance of J. spinosa counterbalanced by unsuitable habitat for J. spinosa east of the contact zone. Although the two parental species are relatively young, mitochondrial cline width was extremely narrow. This result suggests strong selection against maternally-inherited markers, which may indicate either mitonuclear incompatibilities and/or female choice against heterospecific matings typical of avian hybrid systems, despite jacana sex role reversal.