Ellen D. Ketterson - US grants

This proposal addresses the evolution of male reproductive behavior in birds. In general, male vertebrates mate promiscuously and provide little help in rearing offspring. However, males are usually monogamous and they often provide care to their young. It has long been assumed that monogamy and parental care evolved in birds because male care was necessary for the successful rearing of offspring. Recent research, however, has shown that female birds whose mates have been taken from them are often able to comensate for mate absence. Other recent research has examined the hormonal basis of mating and parental care behaviors in birds. By manipulation of male physiology through testosterone implants, workers have found that increased testosterone leads to both an increase in the tendency for males to mate polygynously and a decrease in their tendency to care for offspring. If females are able to rear young without male help and if polygyny requires only a relatively small physiological alteration, then the question arises, why has male care evolved and what constrains males to monogamy? This proposal is designed to answer this question; it proposes an alternative explanation for male monogamy and a test of this explanation. Ketterson and Nolan will treat males with testosterone and attempt to quantify any effect of testosterone-altered behavior on male survivorship. They will then observe the responses of females toward males, in particular any tendencies to mate initially with altered males and to maintain or not to maintain the association over the course of the full breeding season. Their results should contribute to our general understanding of the evolution of behavior by permitting a quantitative estimate of costs and benefits associated with alternative reproductive physiologies. One potential applied aspect of this work is the greater insight into the evolution and regulation of human parenting behavior it may provide. Divorce is common in our society and the costs to children are high. Human males differ from most other mammals in the extent to which they provide parental care to their young; their behavior more closely resembles that of birds than the most commonly studied mammal, the rat. Although it is always risky to draw comparisons between human behavior and that of other animals, it is also true that an increased understanding of the physiological and ecological basis for mating and parental behavior may help society take a more objective look at its own behavior. Because of the scarcity of mammal models for male parental care, birds may provide the best alternative.

9014276 Ketterson We propose a Research Training Group in the inter disciplinary study of Animal Behavior. We are a collaborative group of Biologists, Psychologists, and Neuroscientists concerned that the study of behavior is being hampered by overspecialization. By training students in multidisciplinary approaches to common research problems, we can show the benefits of combining evolutionary thinking and the analysis of mechanism in the study of behavior. The primary component of our training will be the opportunity to address research problems from several perspectives and under the direction of more than one mentor. Our theme, "Choice, Transitions, and Constraints," reflects the importance we attach to (1) proximate assessment of the immediate environment, (2) learning and development, and (3) evolution, in understanding behavior. We request support for eight graduate students and two postdoctoral students. These trainees will rotate through at least two laboratories. Our research programs will include (1) the Study of Communication, (2) Sexual Behavior and Mate Choice, (3) Orientation and Migration, (4) Learning Mechanisms, Ecology, and Behavioral Plasticity, and (5) Parental Behavior and Ontogenetic Transitions. We will establish a Center for Animal Behavior at Indiana University that will provide a setting to promote our objective: the focussed, interdisciplinarv investigation of animal behavior. ***

The interests of the sexes are not always coincident, and parents may not agree for at least two reasons on how to distribute parental care. Relatedness between offspring and parents, and offspring sex may affect the relative contributions of male and female care givers, as well as how they invest in sons and daughters. This study addresses the effects of parentage and offspring sex on parental provisioning in Mountain Bluebirds (Scialia currucoides). Preliminary results suggest that Mountain Bluebird males but not females bias their feedings toward male-biased broods. In addition, male care givers appear to feed less, on average, than female care givers do, suggesting that male certainty of parentage is lower than that of females. Patterns of allocation of parental care will be determined by 1) observations in the field, 2) video-taping of activities in the nest, 3) manipulation of number of males and females in the nest (sex-ratio), 4) manipulation of proximate factors, such as nestling postion. Parentage will be assigned using DNA fingerprinting. Mountain Bluebirds are particularly instructive in terms of the evolution of parental care because this species is a member of a clade (the genus Siala) in which the other two members show opposite trends in sex-biased provisioning of offspring.

9408061 Ketterson Animal species differ greatly in body-size, life span, and numbers of offspring produced. Large-bodied species tend to die younger but have fewer offspring, while smaller- bodied species tend to die younger but to produce more offspring per reproductive attempt. Patterns in which one component of fitness is gained at the cost of another (long life or large broods, but not both) are referred to as trade- offs in order to strive to understand the physiological bases for trade-offs in order to understand species differences in aging and reproduction. Another kind of trade-off is observed in reproduction. Adults, particularly males, can gain in fitness either by mating with multiple females or by behaving parentally towards their offspring by a single female. Because time is limiting and behavior that leads to successful mate attraction often differs from behavior that favors offspring survival, male animals often allocate time to parenting but not to both simultaneously. This too is a trade-off, and there is evidence to suggest that the male hormone testosterone controls whether males specialize in one form of reproduction or the other. Birds are unusual among vertebrates in that almost all species of birds provide some kind of parental care, and male care of offspring is much more common among birds than it is among other vertebrates. Presumably this is true because males that are parental leave more offspring than those that are not, but this needs to be tested experimentally. It is also important to understand the physiological underpinnings of male parental care. To address the mechanisms of trade-offs in avian life- histories, and how they are maintained, free-living male dark-eyed juncos (Junco hyemalis) are treated with implants of testosterone. Treated males are then compared with controls. Because hormones affect so many attributes of the animal, some of the induced alterations may be detrimental to fitness, othe r beneficial. Only by considering the net effect of testosterone on an array of traits and a variety of fitness measures can the maintenance of existing phenotypes be understood.

9413220 Ketterson This award renews support of a joint effort of 14 faculty that provides education and research training for undergraduate, graduate and postdoctoral students. This Research Training Group (RTG) is a central focus of the recently-established Center for the Integrative Study of Animal Behavior at Indiana University. The faculty, who come from the Departments of Biology, Psychology, Law, and Medical Sciences, bring six subdisciplinary perspectives to the RTG: sensory physiology, behavioral ecology, evolutionary biology, developmental psychobiology, animal learning, and behavioral neuroscience. Graduate students who participate in the RTG are drawn from three graduate programs: biology, psychology and an interdisciplinary program in neuroscience. The RTG provides students with academic year and summer assistantships, travel support or research support. The courses, seminars and other educational activities of the RTG result from the integration across 5 areas of research: communication, sex and reproductive behavior, learning and motivation, ontogeny and evolution, and spatial organization of behavior. As well as representing natural combinations of faculty research interests, these areas provide foci for the development of student research projects. Several of the courses required of RTG trainees have been newly developed by RTG faculty; students must also participate in a year-long seminar on scientific integrity developed jointly by the RTG and the Poynter Center for Study of Ethics and American Institutions. In addition to the core faculty, there are 10 adjunct faculty in five other departments whose have related research interests and whose students benefit from the courses and seminars and other educational activities of the RTG. ***

9707602 Ketterson This award will provide funds that will be matched by the American Ornithologist's Union (AOU) and used to help defray part of the travel costs of U.S. scientists to attend the XXIInd International Ornithological Congress to be held in Durban, South Africa, in August of 1998. The award will help allow U.S. scientists to attend numerous plenary lectures and symposia, and to participate as speakers in these and other sessions (e.g., contributed papers, round-table discussions) at the Congress. In addition, participation by U.S. scientists will facilitate exchange of new information and foster collaborative-international agreements. Selection procedures for awards from the AOU are in place, and attempts will be made to support younger scientists. Because awards by the AOU to individual scientists are fairly small, the NSF award could be used to help defray tavel costs for more than 30 scientists.

9722823 Schoech The primary goal of this project is to determine the hormonal mechanisms that mediate parental and alloparental behavior in a social species, the Florida scrub-jay (Aphelocoma coerulescens). Because prolactin is associated with parental behavior in many species, including Florida scrub-jays, the thrust of the project will address the role of prolactin in promoting helping behavior by nonbreeders. Previous correlative studies have linked helping and prolactin in this species. This proposal uses a protocol whereby manipulations of hormone levels allow more definitive assessment of hormone function. It has long been known that until a hormone is removed and replaced experimentally, the causal nature of the compound can only be inferred. Recent advances in our understanding of the compounds 'upstream' from the pituitary that control the secretion of prolactin make it possible to remove prolactin with biological elements. Using a prolactin releasing agent, and by treating Florida scrub-jays with anti-bodies against this agent, prolactin can be immunoneutralized. Following removal of the hormone of interest (prolactin), focal observations of treated individuals will allow the determination of whether prolactin is responsible for helping behavior. Similarly, modern field endocrine techniques allow enhancement of an organism's endogenous hormone titres. By removing and then supplementing prolactin in free-living cooperatively breeding Florida scrub-jays, this proposal will pioneer the use of these valuable techniques under field conditions. In addition to greatly enhancing our understanding of hormonal mechanisms that mediate social behavior, the methods used in this proposal may prove to be useful in manipulating behavior, and may set the stage for future research in a variety of species.

Ketterson 9701334 Recent investigations have revealed the presence of maternally-derived steroid hormones in the yolks of avian eggs. However, many questions concerning both the functional significance and the mechanism of action of yolk steroids remain unanswered. By concentrating on the interactions between hormones, physiology, and behavior, the proposed research will elucidate the role yolk steroids in the development and survival of offspring in the context of hatching asynchrony. In doing so, the mechanisms by which yolk steroids facilitate growth and survival will be identified. Preliminary research on the eggs of red-winged blackbirds, Agelaius phoeniceus, has shown that the concentration of yolk testosterone varies with laying order in such a way that later-laid eggs contain more hormone. Therefore, this research will focus on the developmental and fitness effects of yolk testosterone by: (1) Establishing whether yolk testosterone levels are correlated with growth rate, muscle development, or begging intensity. (2) Demonstrating causation between yolk testosterone levels and these physiological and behavioral variables by injecting testosterone into yolks. (3) Investigating the effects of female age on yolk testosterone levels in a system where older females produce a greater number of fledglings than first-time breeders.

Ketterson 97-28384 The primary objective of this research is to explore how the hormone testosterone influences behavior and physiology in a male bird including any effects on a male's ability to survive and reproduce in the wild. The results will enhance understanding of adaptation and constraint and help us to predict how animals respond evolutionarily to changing environments. The Principal Investigators (PIs) treat free-living male dark-eyed juncos (Junco hyemalis, commonly known as the snowbird) with slow-release implants of testosterone (T). Controls receive empty implants. The PIs measure an array of phenotypic traits and compare fitness of the experimental phenotype with that of controls. Only by considering the whole array and the net effect of experimentally induced changes on fitness can they hope to understand what maintains existing harmonal phenotypes. Specific objectives include: 1. continued investigation of direct effects of testosterone on fitness-related traits in male juncos, including a) behavior towards nestlings and b) behavior and physiology during the non-breeding season; 2. quantification of indirect effects of T on the male's extended phenotype by measuring die impact of male T on the behavior and physiology of females (egg steroids, sex of offspring, use of space while fertile); 3. completion of analysis of an already completed l0-year demographic study.

Surprisingly little is known about the hormonal basis of species differences in avian parental care. Of particular interest is how parental males integrate song and territorial behavior with parental behavior, since time spent on one activity usually precludes time spent on the other. The objective of the proposed research is to determine whether circulating levels of the hormones testosterone and prolactin are responsible for the difference in paternal behaviors of two related species of songbird, the blue-headed vireo (BHV) and the red-eyed vireo (REV). Male BHVs incubate eggs, while male REVs are far more typical among songbirds in that they do not. Thus the species differ greatly in the degree to which males contribute to parental care. Using hormone manipulations in free-living males of both species, this research will test whether alterations in an individual's circulating hormone levels will lead to corresponding alterations in its song and parental behaviors.
The mechanisms of parental care in Passeriformes, the largest order of birds, have not been examined as closely as those in other avian orders. The proposed research provides a strong test of the predicted functions of testosterone and prolactin in parental males.

Disseration Research: The Influences of Ecological Condtions on Maternal Antibody Transmission: Consequences for Offspring Immunity and Growth

Dr. Ellen D. Ketterson & Jennifer Grindstaff

The ability to resist disease is an important component of survival and reproductive success. Previous research has addressed the influences of genes and physiology on adult immune function. However, early in life, offspring immunity may be determined largely by antibodies transmitted by the mother during egg production, gestation, or lactation. The amount and types of antibodies transmitted to offspring may be determined by the mother's physiological condition. Through experimental manipulation of two ecological factors, resource availability and disease exposure, the effect of maternal physiological condition on offspring immunity and growth will be addressed. The PIs will examine the influence of resource limitation on immunity in adult females of a well-studied songbird species, the great tit (Parus major), by asking whether food supplementation enhances antibody transmission to offspring. In a second experiment, they will vary pathogen exposure in females prior to egg laying. They anticipate that food shortage will reduce the ability of females to protect their offspring and that exposure to specific diseases will render offspring better able to withstand infection. Both findings would suggest a larger role of the mother's environment in offspring development than has previously been appreciated.

LAY SUMMARY

Males and females typically resemble one another in some ways and differ in others. Differences may arise because one sex is subject to greater reproductive competition than the other (sexual selection) or because the sexes differ in their ecology (natural selection). Because males and females share a common genome, they are also subject to correlated selection, which occurs when genetic variants directly favored by selection in one sex ('like father, like son') are carried along in the other sex ('like father, like daughter'). Correlated selection can hasten evolution if attributes directly favored in one sex also benefit the other, or it may serve as a constraint when the attributes are disadvantageous to the second sex.

Proximate mechanisms underlying sex differences and resemblances include developmental programs, steroid hormones, and steroid-independent differences in gene expression that lead to sexually differentiated tissues. Gonadal steroids can act early in development to organize tissues so that they are or are (or are not) responsive to activation by a steroid later in life. Gonadal steroids can also affect the sexes as adults, and, when their effects are similar, there is potential for correlated selection.

The focus of this proposal is the steroid hormone, testosterone, and its integrating effect on sex differences and resemblances in adult males and females of a songbird species, the dark-eyed junco. The research has four objectives:

(1) to assess the role of testosterone in sexual integration by comparing natural profiles of male and female hormone levels, hormonal response of females to stimuli known to increase T in males, and hormone levels in females that do and do not produce young by extra-pair fertilizations (EPFs). Resemblance between the sexes will reveal the potential for correlated selection.
(2) to alter the level of circulating testosterone experimentally and compare effects of altered testosterone in females to previous results from males. Individual experiments will address whether elevated testosterone in females alters attractiveness to males (sexual selection) and whether it affects parental behavior, social status, or immune function (natural selection). They will also address interactions between testosterone, corticosterone, corticosteroid binding globulins.
(3) to determine whether female responses to altered testosterone are beneficial, neutral, or detrimental in the field. If the sexes constrain one another, alterations that are beneficial in one sex should be detrimental in the other. If the sexes reinforce one another, alterations may be beneficial in both sexes. If the sexes are independent, then alterations already known to be beneficial or detrimental in males may have no fitness consequences for females.
(4) to compare how degree of relatedness affects a sexually selected trait and two hormone-related traits. Juncos of known genetic relatedness will be assessed for family resemblance in measures of body size and a plumage trait that affects attractiveness. Additional juncos will be reared and compared for three hormonally mediated traits. The presence of a genetic correlation would predict correlated selection; the absence of one would predict sexual independence.

Research described in this proposal will provide opportunities for training future scientists of diverse backgrounds in the conduct of laboratory and field experiments that address the integration of proximate and ultimate explanations for biological differences. It will also enrich graduate and undergraduate courses taught at Indiana University. Potential societal implications of the findings to be expected include (1) importance of hormonally active agents in the environment, (2) improved methods for breeding captive songbirds for conservation, and (3) a greater understanding of the relationship between sex and gender.

Fast-paced environmental changes currently threaten bird populations. To predict how birds will respond to these changes it will be essential to acquire new knowledge about environmental influences on individual reproductive decisions (e.g., whether, where, and when to reproduce) and how organisms respond to their environments as physiological and behavioural systems. While some species are flexible with respect to laying date, clutch size, mating system, etc., others are less flexible. If scientists are to predict which populations will be able to adjust or adapt and how rapidly, they will require far greater knowledge of the mechanisms underlying reproductive decisions than they currently possess.

Obtaining that knowledge will require the expertise of reproductive endocrinologists, physiological ecologists, behavioral ecologists, comparative biologists, population biologists, and quantitative geneticists (or, more simply, endocrinologists and ecologists).

This award will support an exploratory workshop that will convene ~30 European and North American scientists. The workshop will foster the exchange of knowledge, techniques, and data between endocrinologists and ecologists to address this pressing and topical issue. It will lead in turn to the formation of a research network that will foster international collaborations and opportunities for international training of graduate and postdoctoral students including students from groups underrepresented in science and developing countries.

? DESCRIPTION (provided by applicant): This proposal requests a second renewal of a highly successful T32 Institutional National Research Service Award to Indiana University, entitled Common Themes in Reproductive Diversity (CTRD). The award will support broadly integrative training in the areas of sexual reproduction and development. Training will focus on behavior in both humans and other animals and will address key questions in three related themes: (1) genetic, epigenetic, environmental, and parental contributions to reproductive and social behavior; (2) the origins and expression of differences among the sexes; and (3) sex and immunity in health and disease. Indiana University's excellent support for research and its globally recognized strengths in animal behavior, endocrinology, human sexual health, and evolution of development will ensure high quality training. The 20 Training Faculty and 3 Affiliated Resource Faculty are drawn from 5 departments (Anthropology, Biology, Chemistry, Gender Studies, Psychological and Brain Sciences) and 2 additional degree-granting programs (Program in Medical Sciences, Program in Neuroscience). They are also associated with 1 or more of 6 research centers, most importantly the Center for the Integrative Study of Animal Behavior (CISAB); the Kinsey Institute for Research on Sex, Gender and Reproduction; the Institute for Pheromone Research; and the Center for Genomics and Bioinformatics. Support is requested for five years to enable training of 4 pre-doctoral and 2 post-doctoral students each year to be drawn from a reliable pool of talented applicants. In addition to course work in the fundamentals and intensive research training, pre-doctoral trainees will participate in (1) a research-based course focused on Concepts in Reproductive Diversity, (2) an interdisciplinary, hands-on methods course, Techniques in Reproductive Diversity, and 3) a Research Ethics course, all co-taught by the training faculty and enhanced by a tradition of inviting distinguished visiting scientists to contribute to courses. Trainees will actively participate in a monthly breakfast research forum, will organize and present research at a highly successful and broadly attended conference, and will have additional opportunities and training to prepare them for more than one career outcome. Pre-doctoral trainees will be drawn from the most highly qualified applicants to the degree-granting programs of the Training Faculty. Post-doctoral trainees will be recruited nationally, will be chosen based on their accomplishments and the potential for CTRD training to broaden their skills and perspectives, and will be expected to foster research collaboration among CTRD trainees and laboratories. Intense effort will be made to enhance recruitment and retention of diverse trainees, to provide mentoring opportunities through a highly effective undergraduate program for underrepresented minorities, and to recruit and support individuals with disabilities or from disadvantaged backgrounds.

Males and females are similar in some ways and different in others. Differences may arise because one sex is subject to greater reproductive competition (sexual selection) or because the sexes differ in their ecology (natural selection). Because males and females share a common genome, they are also subject to correlated responses to selection, which occur when genetic variants favored by selection in one sex ('like father, like son') are carried along in the other sex ('like father, like daughter'). Correlated responses may serve as an evolutionary constraint if the attributes that are beneficial to one sex are disadvantageous to the other sex.
Steroid hormones are among the proximate mechanisms that lead to sex differences. They may act early in development to organize tissues or later in life to activate trait expression. When hormonal effects are similar in males and females, there is potential for correlated responses to selection. The focus of this proposal is the steroid hormone testosterone (T) and its integrating effect on sex differences in adult males and females of a songbird species, the dark-eyed junco. The research has four objectives. The first is to experimentally elevate testosterone (T) in females and measure its effect on the female's phenotype (e.g. appearance, behavior). The second is to determine whether elevated T is adaptive or maladaptive in females. The third is to assess the potential for correlated responses within and between the sexes by measuring change in T after exposure to a fixed dose of a hormonal stimulant (GnRH). The fourth is to assess sexual independence by comparing the role of T in the activation of aggressive behavior in males and females.
Research described in this proposal will add to an already impressive 20-year database, provide opportunities for training future scientists of diverse backgrounds in the conduct of cybernetics, and laboratory and field experiments that address the integration of proximate and ultimate explanations for biological differences. This extensive database will provide fodder for continued synthesis, compilation, and cyber exploitation, and will make a significant contribution to this principle investigator's work, her collaborators, and others who will have access to this gold mine. This cyber research program will also enrich courses taught at Indiana University and elsewhere. Potential societal implications of the findings to be expected include: (1) the impact of hormonally active agents in the environment; and (2) a greater understanding of the relationship between sex and gender.

Adults are predicted to increase reproductive investment with age. This is thought to occur because as adults age, they necessarily have fewer future reproductive attempts. Several studies support this expectation, however, we currently have little information about the physiological mechanisms mediating this commonly observed pattern. One physiological mechanism that may be important in regulating age-related changes in parental investment is the attenuation of the stress response and decreased sensitivity to elevated glucocorticoid levels. In response to stress, vertebrates release glucocorticoids. Elevated glucocorticoid levels inhibit reproductive behaviors and enhance self-maintenance behaviors. Therefore, adults are predicted to suppress the stress response and to be less behaviorally sensitive to elevated glucocorticoid levels with age. This study will investigate whether changes in the stress response and glucocorticoid sensitivity mediate age-related changes in parental investment in a long-lived seabird, the common tern Sterna hirundo. This research will enhance our understanding of the relationship between stress physiology and age as well as our understanding of the physiological mechanisms mediating age-related life-history trade-offs.

Broader Impacts: Undergraduate researchers will be involved in all aspects of this research project. In addition, this research may have important conservation implications. Knowledge about how age influences the stress response may inform wildlife managers about which segments of the population are the most vulnerable to environmental disturbance.

Males of many species use multiple ornamental traits and behaviors to compete for and attract mates. If these interact to determine mating success, selection may lead to the evolution of genetically integrated suites of sexual traits and behaviors, known as mating phenotypes. Because hormones can coordinate the expression of suites of traits, they may play a central role in stabilizing these mating phenotypes. This study examines the evolution of the mating phenotype in a common songbird, the dark-eyed junco. Correlations will be measured between morphology used in acquiring mates (size and plumage), the sexual hormone testosterone, and the sexual behaviors mediated by testosterone. The role of current selection in maintaining these correlations will be quantified by associating trait combinations with genetic measurements of mating success. Experiments will examine whether testosterone acts to stabilize the mating phenotype by direct developmental effects on morphology.

This research should lead to a greater understanding of the evolution of groups of functionally related traits and may suggest important roles for hormones in maintaining integration. All work will involve the extensive participation of undergraduates, both as research assistants and collaborators. Undergraduates will be encouraged to develop their own independent projects related to this work.

This project examines extra-pair mating in birds from the female perspective. The research asks whether females that mate with more than one male benefit directly, by producing more offspring than they would if they mated with just one male, or indirectly, by producing offspring of higher quality. Conclusions will be based on DNA samples collected over 18 years from more than 4000 individuals and on long-term records of population dynamics and reproductive success. The study species, the dark-eyed junco, is a socially monogamous songbird. Males and females form a long-term pair bond and jointly raise offspring, but ~25% of all offspring are sired by a male other than the socially bonded male.

Extra-pair mating is easily understood from the male?s perspective; a male?s reproductive success will increase if he mates with multiple females. Less clear is why females participate in this behavior, and past research has not resolved the question. The duration and scope of this study provide a unique opportunity to examine the influence of mating patterns on lifetime reproductive success of females. Results will clarify the potential for sexual selection in a species with a complex mating system.

Hormones integrate behavior and physiology by responding to environmental signals and altering gene expression in target tissues in the brain and the body. However, when the environment of a species changes, the most adaptive response might be to lose hormone sensitivity at some of the target tissues. An open question is whether hormone-mediated traits can be decoupled in such a way. This research will focus on the steroid hormone testosterone and its integrating effect on the behavior and physiology of males and females of a songbird species, the dark-eyed junco. The relative importance of phenotypic integration or independence of hormonally mediated traits has significant implications for the evolution of life histories, sex differences, and the formation of new species.

The research will help to train future scientists of diverse backgrounds in the conduct of laboratory and field research that combines techniques and perspectives from multiple disciplines. Potential societal implications of the research include greater knowledge of the impact of hormonally active agents in the environment, and the ability of natural populations to respond effectively to environments altered by climate change.

When an animal?s environment changes, interrelated biological characteristics of the animal may also change. Hormones such as testosterone [T], the secretion of which is influenced by the environment, coordinate the expression of many traits including sexual, aggressive, and parental behavior, ornamentation, and immune function. Together these traits form the animal?s phenotype, and linkages between hormones and phenotype may play a central role in facilitating or constraining an animal?s ability to respond to environmental change. This research takes advantage of the colonization of an urban environment by a songbird population to compare hormone-phenotype linkages in the new urban environment to those observed in the nearby ancestral environment. This comparison will test whether traits observed in the two environments indicate ?evolutionarily integrated? or ?evolutionarily independent? linkages between T and target traits. The research also will evaluate whether observed changes in traits represent non-genetic developmental alteration or rapid (genetic) evolution.

In an age of rapid global change and habitat alteration, understanding how individuals and populations respond to novel environments has become critical. The research described in this proposal will add to the basic understanding of how and whether vertebrates adapt to novel environments over very rapid time scales (<30yrs). The research will also enhance the training of more than a dozen future scientists, including those from underrepresented groups.

When the optimal phenotype differs by sex, the evolution of sex differences, or sexual dimorphism, is expected. In many species, however, the degree of dimorphism is mild, and numerous individuals express traits, e.g. body size or behavior, that fall between male and female norms. Females in this group, known as androgynous females, are of interest because they offer a natural experiment for understanding the sources and consequences of sexual dimorphism. Variation in hormone exposure, especially androgens like testosterone, is a primary mechanism for the development of sex differences in vertebrates. This variation may also mediate the expression of androgynous traits within a sex, leading to covariation among male-like traits, e.g. big females may also be aggressive females. If male-like females differ from typical females in survival or reproduction, the degree of sexual dimorphism may reduced or enhanced. This project seeks to determine whether the degree androgyny expressed in one trait (morphology or behavior) is related to the level of androgyny in other traits, which would indicate a common mechanism controlling trait expression. Additionally this project will strive to determine whether androgen exposure during development or in adulthood is driving this covariation, and also whether androgynous females vary from typical females in survival or reproductive success. Answers to these questions will inform our understanding of the potential links between social behavior, morphology and hormone exposure levels in females as well as the evolution of complex traits that are mediated by hormones. This work will be done with free-living songbirds and serve to train undergraduates to conduct research as part of training programs such as NSF's Research Experience for Undergraduates (REU) and Indiana University's Student Achievement in Research and Scholarship (STARS) programs.

Hormones simultaneously mediate numerous traits of plants and animals thus coordinating expression of size, physiology and behavior. When environments change or new environments are colonized, this common control of traits by hormones may or may not facilitate adaptation to the altered environment, depending on the details of hormonal control. In vertebrate animals, the hormone testosterone (T) has particular importance for traits related to reproduction, such as ornamentation and aggression. The common control of traits by T may facilitate adaptation by preserving favorable combinations (e.g., aggression coupled with ornamentation to simultaneously deter rivals and attract mates) or delay adaptation if control of a potentially beneficial trait is linked by the hormone to another trait that would be detrimental. This interplay has important implications for species evolution and response to climate change, yet we know surprisingly little about its actual nature. This project will support field and laboratory research to address whether relationships between T and various behavioral and morphological traits observed in one sub-species of a songbird, the dark-eyed junco, can be used to predict the same relationships in another subspecies that lives in a very different environment. The project will also examine the underlying physiological mechanisms that may contribute to potential differences in these relationships, asking whether they vary among individuals and subspecies. Specifically, hormone manipulations and neural studies with captive birds will examine hormonal cascades that trigger T release, as well as hormone receptors that affect T's influence on traits. Consistent co-variation between T, traits, and physiological mechanisms across subspecies will suggest that hormone systems evolve as a unit. Alternatively, absence of similarity across populations will suggest high potential for independent evolution, allowing hormone systems to diverge more freely or less consistently. The research will provide numerous opportunities for effective mentorship of diverse undergraduates, and the findings are expected to contribute to an understanding of hormone system evolution.

The formation of new species can be promoted by variation in acoustic signals that play an important role in mating behavior and thus can influence interbreeding between populations. The relationship between signals and speciation has been studied extensively in loud, long-range signals, but many species including birds, bats, and insects also produce quiet, short-range signals that are difficult to observe and record. New technology now makes it possible to obtain high quality field recordings of quiet signals through the use of newly developed radiotransmitters containing miniature microphones. Research supported by this proposal will address the function of short- and long-range song in the dark-eyed junco, by employing this new technology along with song playback experiments and laboratory preference tests to assess variation among populations. It is predicted that short-range song will function predominantly in courtship and differ more between populations than long-range song, establishing short-range song a potential contributor to the development of new species. This research will contribute to the mentorship and training of diverse undergraduates through Indiana University and the NSF-funded Research Experience for Undergraduates program at Mountain Lake Biological Station in Pembroke, Virginia. Much of the research will take place in public areas such as national forests and parks, allowing researchers to interact with youth groups and vacationers. This study will also strengthen scientific collaborations between Indiana University and the Universities of Melbourne (Australia), Illinois, Montana, and California at Davis. All recordings generated in this study will be contributed to the Macaulay Library of Natural Sounds (http://macaulaylibrary.org/index.do) to allow access to both the public and future researchers. The results of this project will be disseminated broadly by public lectures promoted by Audubon societies, publications in scholarly journals, and presentations at national and international conferences.

This award will synthesize research conducted over 40 years on a songbird, the dark-eyed junco. The junco has been an important research subject in three areas: the origin of new species, the role of the environment in timing reproduction, and the role of testosterone in adaptive behavior and physiology. The award will lead to three products. The first is a book that will describe the outcomes of research on the junco, including new knowledge about its migration, its reproduction, its historical response to glacial retreat, its response to climate change and the rise of cities, and finally its role as a model system for how testosterone mediates numerous behavioral and physiological attributes. The second product will be a web-based data archive, and the third will be a series of media projects, including a documentary film and classroom videos.

Together, these products will help to promote public understanding of science, and will contribute to ongoing research in ecology and evolution of birds and other organisms. The book, which will be aimed at a professional audience, will synthesize four decades of research. The web-based archive will allow other researchers to take advantage of extensive data sets on physiology and behavior. The media projects will convey key findings to diverse audiences including public consumers of scientific research, young and old, in the classroom, in museums, and on public television. Together, the resulting synthesis will be of strong interest to students, educators, fellow scientists, and the public.

Males and females have nearly identical genomes, yet males and females can differ dramatically in appearance, physiology, and behavior. The proximate explanation for sex differences in phenotype is differences in gene expression, i.e., which genes are ?turned on or off? in males and females. But exactly how gender affects gene expression is an open question where much remains to be learned. Hormones such as testosterone are likely candidates for controlling differences in gene expression of genes in males and females. To date much of the research in this area has focused on experimental manipulation of hormones, rather than on the role of natural, individual variation in levels of circulating hormone. The research supported by this award asks two primary questions: (1) what genes and gene networks are affected by natural, individual variation in testosterone phenotype, and (2) how do males and females differ in affected genes and gene networks? The dark-eyed junco (Junco hyemalis), a North-American songbird, will be the subject of study because of the recent development of genomic tools in this system and the wealth of historical data on its natural history, response to testosterone, and sex differences in behavior; all studied in a wild population. Successful completion of the aims of this project will enhance our understanding of the genes and gene networks that are related to natural variation in testosterone. The research proposed here will have broader impacts because it will provide unique opportunities for undergraduate research: the preliminary stages of this research have already led to undergraduate research opportunities for four individuals from underrepresented groups.

This research asks how animals know whether and when to migrate and when to breed. The focal animal is a songbird in which populations with separate breeding ranges winter together but differ in whether or not they migrate. When spring arrives the non-migrants begin to breed, while the non-migrants delay breeding despite living in the same environment. Research on the mechanisms that account for different responses to identical environments could help to explain how animals monitor the environment and adjust their physiology to their needs. The dark-eyed junco is a songbird and an ideal model to address these questions.

This project will investigate plasticity in hormone systems using hormonal "challenges" and pharmacological manipulations. It will also explore differences in gene expression between resident and migratory juncos from recently diverged populations (California) and longer diverged populations (in Virginia) that over-winter together but breed in different places at different times. The prediction is that migrants will differ from residents in their response to challenges and that recently diverged populations will differ less than populations that diverged longer ago. Another prediction is that gene expression in a common garden will differ between longer diverged populations as compared with recently diverged populations.

Broader impacts
The research will enhance understanding of why and how some animals are able to thrive in changing environments, while other animals' ranges retreat, and some species are lost. This research will provide numerous opportunities to train graduate and undergraduate students, and knowledge gained will be disseminated broadly. The project will enable promotion of a documentary film starring the study species and designed for use in high schools and public venues for adult learners.

Data Management.
Data supporting results in published papers will be made available on Genbank and/or Dryad. All data generated from this research will be archived in an online repository maintained by the Indiana University Library (https://scholarworks.iu.edu/dspace/handle/2022/7911).

As cities grow larger and more species of animals make their homes in urban habitats, it becomes increasingly important to understand the effects of city life on animal health. Parasite infections can have significant impacts on animal survival and physiology, and urban habitats may change parasite infections in several ways. For example, urban habitats may change both the stressors that animals experience and their immune resistance to parasites. However, little is known about parasites in urban vs non-urban populations of vertebrate animals, including how they affect animal health and whether urban animals have different levels of stress hormones or immunity to parasites. This research will provide critical knowledge about the parasites that birds must withstand in order to survive in urban areas, which will inform future conservation efforts in urban habitats. This work will also help protect human health, since urban birds are potential reservoirs of human diseases; for example, urban birds are more likely to be infected with West Nile Virus than non-urban birds. Understanding what parasites urban birds are infected by, and what physiological states are associated with these infections, can help us better prevent disease spread from birds to humans.

In order to better understand the effects of urbanization on parasitism and host physiology, the researchers will study a songbird, the Dark-eyed Junco (Junco hyemalis), from multiple cities and non-urban habitats across California. Previous research has found considerable variation in phenotype and parasitism between an urban and neighboring non-urban population of juncos. In a non-urban population, there was higher prevalence of parasites that require vectors for transmission. In the neighboring urban population, there was greater prevalence and intensity of directly and environmentally transmitted parasites, including an intestinal parasite, coccidia (Isospora sp.). Urban hosts also differed in endocrine physiology from non-urban birds. This project will combine extensive field surveys and aviary experiments to test 1) whether urbanization consistently results in these same patterns for environmentally transmitted versus vector-borne parasites; and 2) whether the observed difference in coccidia infection intensity between the urban and non-urban populations is due to traits of the host (relating to stress response and immune function) or traits of the parasite. By combining approaches from disease ecology and eco-immunology, this project will address the critical links among habitat characteristics, parasitism, and host phenotypes.

The goal of this project is to develop, test, and deploy novel sensors that will enable the study of activity and migration of small songbirds (20-50 grams). The sensors required for such small animals require weight and energy budgets (1 gram and 1 micro Watt, respectively) below most low-energy devices and must survive in challenging environments and collect useful data over many months. This project has the potential to significantly impact the science of animal behavior because many important species cannot be effectively monitored with existing technologies -- for example roughly half of bird species weigh less than 50 grams.

While the focus of this project is to develop the technologies that will enable other researchers to study the behavior of small animals, the project will test these devices on a specific model species (Dark-eyed Juncos) in order to enhance the understanding of the variation of timing on reproduction, migration, and biodiversity. Finally, while the target application for this project is animal biology, many of the fundamental technical problems are common to general low-energy sensing problems. For example, a key problem is detecting the onset of interesting events using extremely low energy levels prior to expending the significantly higher levels of energy needed to collect high resolution data.

Bird feathers are one of the most striking examples of diversity on the planet. The color and shape of feathers are of fundamental importance in bird courtship rituals, camouflage, and other social and ecological interactions. Feathers can evolve rapidly - populations of the same species often show striking differences in feather color and patterns. The mechanisms that are responsible for these differences, however, are poorly understood. Abolins-Abols investigates how and why feather color differs between individuals and populations. In this dissertation improvement project Abolins-Abols will investigate the hormonal mechanisms that regulate the development of two rapidly evolving feather traits - head and tail color - across multiple populations of the Dark-eyed Junco, a small songbird that is a common visitor to feeders across the United States. This research will increase our understanding of the evolution of bird color diversity, because head and tail feathers are hotspots for variation across songbirds. This project will also train undergraduate students and facilitate outreach to the general public using the newly founded bird banding station where Abolins-Abols will characterize color variation across a variety of migratory bird species. The affordability and simplicity of this study system make it a prime candidate for future research programs in development and evolution, especially at smaller institutions such as liberal arts colleges.

Some feather ornaments are used as honest signals, suggesting that their development is integrated with other phenotypic traits such as behavior. A candidate mechanism for this integration is hormones, which regulate both neural and peripheral processes. While hormones have been shown to mediate sex differences coloration, we do not yet understand the role of hormones in mediating individual differences and population divergence in feather color. Abolins-Abols proposes to investigate the genetic and hormonal mechanisms that mediate the development of two rapidly evolving social plumage ornaments - head and tail color in Dark-eyed Juncos - across populations of varying coloration. These honest feather ornaments have been shown to be related to individual differences in testosterone physiology, suggesting a causal link between hormones and feather color. To gain an explicit understanding of whether and how hormones regulate this variation, Abolins-Abols will analyze hormone receptor gene and protein expression in developing feathers in combination with experimental manipulations of testosterone and luteinizing hormone. By understanding the hormonal regulation of these ornaments we will gain insight into how these ornaments are linked to behavior as well as a better understanding of how hormones mediate the divergence of social feather ornaments.