Female-Based Patterns and Social Function in Avian Chemical Communication.

Much of the growing interest in avian chemical signals has focused on the role of kin recognition or mate attraction, often with an emphasis on males, with uropygial gland secretions perhaps providing information about an individual's identity and quality. Yet, data collected to date suggest sexual dimorphism in uropygial glands and secretions are often emphasized in female, rather than in male birds. That is, when a sexual difference occurs (often during the breeding season only), it is the female that typically exhibits one of three patterns: (1) a larger uropygial gland, (2) a greater abundance of volatile or semi-volatile preen oil compounds and/or (3) greater diversity of preen oil compounds or associated microbes. These patterns fit a majority of birds studied to date (23 of 30 chemically dimorphic species exhibit a female emphasis). Multiple species that do not fit are confounded by a lack of data for seasonal effects or proper quantitative measures of chemical compounds. We propose several social functions for these secretions in female-based patterns, similar to those reported in mammals, but which are largely unstudied in birds. These include: (1) intersexual advertisement of female receptivity or quality, including priming effects on male physiology, (2) intrasexual competition, including scent marking and reproductive suppression or (3) parental behaviors, such as parent-offspring recognition and chemical protection of eggs and nestlings. Revisiting the gaps of chemical studies to quantify the existence of female social chemosignals and any fitness benefit(s) during breeding are potentially fruitful but overlooked areas of future research.

Experimental evidence that symbiotic bacteria produce chemical cues in a songbird.

Symbiotic microbes that inhabit animal scent glands can produce volatile compounds used as chemical signals by the host animal. Though several studies have demonstrated correlations between scent gland bacterial community structure and host animal odour profiles, none have systematically demonstrated a causal relationship. In birds, volatile compounds in preen oil secreted by the uropygial gland serve as chemical cues and signals. Here, we tested whether manipulating the uropygial gland microbial community affects chemical profiles in the dark-eyed junco (Junco hyemalis). We found an effect of antibiotic treatment targeting the uropygial gland on both bacterial and volatile profiles. In a second experiment, we cultured bacteria from junco preen oil, and found that all of the cultivars produced at least one volatile compound common in junco preen oil, and that most cultivars produced multiple preen oil volatiles. In both experiments, we identified experimentally generated patterns in specific volatile compounds previously shown to predict junco reproductive success. Together, our data provide experimental support for the hypothesis that symbiotic bacteria produce behaviourally relevant volatile compounds within avian chemical cues and signals.

Songbird chemical signals reflect uropygial gland androgen sensitivity and predict aggression: implications for the role of the periphery in chemosignaling.

Chemical signals can provide useful information to potential mates and rivals. The production mechanisms of these signals are poorly understood in birds, despite emerging evidence that volatile compounds from preen oil may serve as chemosignals. Steroid hormones, including testosterone (T), may influence the production of these signals, yet variation in circulating T only partly accounts for this variation. We hypothesized that odor is a T-mediated signal of an individual's phenotype, regulated in part by androgen sensitivity in the uropygial gland. We quantified natural variation in chemosignals, T, uropygial gland androgen sensitivity, and aggressive behavior in dark-eyed juncos (Junco hyemalis). The interaction between circulating T and androgen receptor transcript abundance significantly correlated with volatile concentrations in male, but not female, preen oil. In both sexes, odorant variables correlated with aggressive response to an intruder. Our results suggest that preen oil volatiles could function as signals of aggressive intent, and, at least in males, may be regulated by local androgen receptor signaling in the uropygial gland. Because these behavioral and chemical traits have been linked with reproductive success, local regulation of androgen sensitivity in the periphery has the potential to be a target of selection in the evolution of avian olfactory signaling.

Communication Value of Mistakes in Dark-Eyed Junco Song.

Sexual signals contain information on individual quality or motivation, and most explanations for their reliability are based on signal costs. A recent suggestion is that signaling mistakes, defined as deviations from typical signal design, provide cues on individual quality, contributing to reliable communication even when signal design is not costly. We describe several atypical song traits in dark-eyed juncos (Junco hyemalis) that may be mistakes during song production or development and occur in up to 6% of songs. These putative mistakes were more frequent in an urban versus a wildland population, and individuals differed in their frequency of mistakes. Some atypical signals were more frequent in younger males or were negatively related to paternity success, supporting the hypothesis that fewer mistakes indicate individual quality. We also discuss unexpected results, such as some atypical signals being more frequent in more ornamented males and in songs with lower performance demands. Song consistency (similarity across syllable renditions) was positively related to male age and paternity success; nonetheless, relations with paternity were stronger when looking at the most deviant syllable renditions, suggesting that the perceptual salience of large mistakes may mediate receiver responses to song consistency. Results indicate that signaling mistakes reveal relevant information to play a role in communication.

Hormonal, behavioral, and life-history traits exhibit correlated shifts in relation to population establishment in a novel environment.

Climate change, habitat alteration, range expansions, and biological invasions are all predicted to require rapid shifts in multiple traits including behavior and life history, both for initial population establishment and subsequent adaptation. Hormonal mechanisms likely play a key role in facilitating or constraining plastic and genetic responses for suites of traits, but few studies have evaluated their role in shaping contemporary adaptation or diversification. We examined multiple phenotypic adjustments and associated hormonal changes following a recent (early 1980s) colonization event, in which a temperate-breeding songbird, the dark-eyed junco (Junco hyemalis), became established in the Mediterranean climate of San Diego, California. The milder climate has led to an extended breeding season and year-round residency, and we document shifts in multiple sexually selected behaviors and plumage traits. Testosterone titers in San Diego were elevated for longer but with a lower peak value compared to a nearby native-range population, and correlations between testosterone and related traits were similar within and among populations. A common garden study indicated that changes in testosterone likely represent plastic responses to the less seasonal environment of the city, providing the context against which subsequent genetic changes in morphology likely occurred. We argue that correlated shifts in multiple traits, organized by underlying physiology, may be a generally important element of many successful adjustments to changing environments.

De novo transcriptome sequencing in a songbird, the dark-eyed junco (Junco hyemalis): genomic tools for an ecological model system.

BACKGROUND: Though genomic-level data are becoming widely available, many of the metazoan species sequenced are laboratory systems whose natural history is not well documented. In contrast, the wide array of species with very well-characterized natural history have, until recently, lacked genomics tools. It is now possible to address significant evolutionary genomics questions by applying high-throughput sequencing to discover the majority of genes for ecologically tractable species, and by subsequently developing microarray platforms from which to investigate gene regulatory networks that function in natural systems. We used GS-FLX Titanium Sequencing (Roche/454-Sequencing) of two normalized libraries of pooled RNA samples to characterize a transcriptome of the dark-eyed junco (Junco hyemalis), a North American sparrow that is a classically studied species in the fields of photoperiodism, speciation, and hormone-mediated behavior. RESULTS: From a broad pool of RNA sampled from tissues throughout the body of a male and a female junco, we sequenced a total of 434 million nucleotides from 1.17 million reads that were assembled de novo into 31,379 putative transcripts representing 22,765 gene sets covering 35.8 million nucleotides with 12-fold average depth of coverage. Annotation of roughly half of the putative genes was accomplished using sequence similarity, and expression was confirmed for the majority with a preliminary microarray analysis. Of 716 core bilaterian genes, 646 (90 %) were recovered within our characterized gene set. Gene Ontology, orthoDB orthology groups, and KEGG Pathway annotation provide further functional information about the sequences, and 25,781 potential SNPs were identified. CONCLUSIONS: The extensive sequence information returned by this effort adds to the growing store of genomic data on diverse species. The extent of coverage and annotation achieved and confirmation of expression, show that transcriptome sequencing provides useful information for ecological model systems that have historically lacked genomic tools. The junco-specific microarray developed here is allowing investigations of gene expression responses to environmental and hormonal manipulations - extending the historic work on natural history and hormone-mediated phenotypes in this system.

Robust behavioral effects of song playback in the absence of testosterone or corticosterone release.

Some species of songbirds elevate testosterone in response to territorial intrusions while others do not. The search for a general explanation for this interspecific variation in hormonal response to social challenges has been impeded by methodological differences among studies. We asked whether song playback alone is sufficient to bring about elevation in testosterone or corticosterone in the dark-eyed junco (Junco hyemalis), a species that has previously demonstrated significant testosterone elevation in response to a simulated territorial intrusion when song was accompanied by a live decoy. We studied two populations of juncos that differ in length of breeding season (6-8 vs. 14-16 weeks), and conducted playbacks of high amplitude, long-range song. In one population, we also played low amplitude, short-range song, a highly potent elicitor of aggression in juncos and many songbirds. We observed strong aggressive responses to both types of song, but no detectable elevation of plasma testosterone or corticosterone in either population. We also measured rise in corticosterone in response to handling post-playback, and found full capacity to elevate corticosterone but no effect of song class (long-range or short-range) on elevation. Collectively, our data suggest that males can mount an aggressive response to playback without a change in testosterone or corticosterone, despite the ability to alter these hormones during other types of social interactions. We discuss the observed decoupling of circulating hormones and aggression in relation to mechanisms of behavior and the cues that may activate the HPA and HPG axes.

Role of testosterone in stimulating seasonal changes in a potential avian chemosignal.

Songbird preen oil contains volatile and semivolatile compounds that may contain information about species, sex, individual identity, and season. We examined the relationship between testosterone (T) and the amounts of preen oil volatile and semivolatile compounds in wild and captive dark-eyed juncos (Junco hyemalis). In wild males and females, we observed an increase in volatile compound relative concentration early in the breeding season. This increase mirrored previously described seasonal elevation in T levels in wild males and females, suggesting a positive relationship between hormone levels and preen gland secretions, and a possible role for these secretions in signaling receptivity. In females, the greatest relative concentrations of most compounds were observed close to egg laying, a time when steroid hormones are high and also the only time that females respond to an injection of gonadotropin-releasing hormone with a short-term increase in T. In a study of captive juncos held on short days, we asked whether the seasonal increases observed in the wild could be induced with experimental elevation of T alone. We found that exogenous T stimulated the production of some volatile compounds in non-breeding individuals of both sexes. However, of the 15 compounds known to increase during the breeding season, only four showed an increase in relative concentration in birds that received T implants. Our results suggest that testosterone levels likely interact with other seasonally induced physiological changes to affect volatile compound amounts in preen oil.

Natural selection on testosterone production in a wild songbird population.

Because of their role in mediating life-history trade-offs, hormones are expected to be strongly associated with components of fitness; however, few studies have examined how natural selection acts on hormonal variation in the wild. In a songbird, the dark-eyed junco (Junco hyemalis), field experiments have shown that exogenous testosterone alters individuals' resolution of the survival-reproduction trade-off, enhancing reproduction at the expense of survival. Here we used standardized injections of gonadotropin-releasing hormone (GnRH) to assay variation in the testosterone production of males. Using measurements of annual survival and reproduction, we found evidence of strong natural selection acting on GnRH-induced increases in testosterone. Opposite to what would be predicted from the survival-reproduction trade-off, patterns of selection via survival and reproduction were remarkably similar. Males with GnRH-induced testosterone production levels that were slightly above the population mean were more likely to survive and also produced more offspring, leading to strong stabilizing selection. Partitioning reproduction into separate components revealed positive directional selection via within-pair siring success and stabilizing selection via extrapair mating success. Our data represent the most complete demonstration of natural selection on hormones via multiple fitness components, and they complement previous experiments to illuminate testosterone's role in the evolution of life-history trade-offs.

Chemosignaling diversity in songbirds: chromatographic profiling of preen oil volatiles in different species.

Large foraging seabirds are known to navigate to food sources using their excellent sense of smell, but much less is known about the use of olfaction by the songbirds (passerine birds). Some evidence of individual recognition based on the bird preen oil volatile organic compound (VOC) compositions, which is the main odor source in birds, have been reported for dark-eyed junco and house finch. In this study we have investigated preen oil VOCs in 16 different songbird species and two other small bird species in order to determine whether the VOC compositions follow phylogenetic and evolutionary relatedness. We have used the stir bar sorptive extraction (SBSE) methodology followed by gas chromatography-mass spectrometry (GC-MS) to determine preen oil VOCs during the long light summer conditions for mostly wild caught birds. Large diversity among the VOC compositions was observed, while some compound classes were found in almost all species. The divergent VOC profiles did not follow the phylogenetic family lines among the bird species. This suggests that songbirds may use VOC odors as a mate recognition cue.

Boldness behavior and stress physiology in a novel urban environment suggest rapid correlated evolutionary adaptation.

Novel or changing environments expose animals to diverse stressors that likely require coordinated hormonal and behavioral adaptations. Predicted adaptations to urban environments include attenuated physiological responses to stressors and bolder exploratory behaviors, but few studies to date have evaluated the impact of urban life on codivergence of these hormonal and behavioral traits in natural systems. Here, we demonstrate rapid adaptive shifts in both stress physiology and correlated boldness behaviors in a songbird, the dark-eyed junco, following its colonization of a novel urban environment. We compared elevation in corticosterone (CORT) in response to handling and flight initiation distances in birds from a recently established urban population in San Diego, California to birds from a nearby wildland population in the species' ancestral montane breeding range. We also measured CORT and exploratory behavior in birds raised from early life in a captive common garden study. We found persistent population differences for both reduced CORT responses and bolder exploratory behavior in birds from the colonist population, as well as significant negative covariation between maximum CORT and exploratory behavior. Although early developmental effects cannot be ruled out, these results suggest contemporary adaptive evolution of correlated hormonal and behavioral traits associated with colonization of an urban habitat.

Maintenance of MHC Class IIB diversity in a recently established songbird population.

We examined variation at MHC Class IIB genes in a recently established population of dark-eyed juncos (Junco hyemalis) in a coastal urban environment in southern California, USA relative to an ancestral-range population from a nearby species-typical montane environment. The founding population is estimated to have been quite small, but we predicted that variation at the major histocompatibility complex (MHC) among the founders would nevertheless be preserved owing to the high functional significance of MHC. Previous studies of MHC in songbirds have had varying degrees of success in isolating loci, as passerines show extensive MHC gene duplication. In order to compare diversity in the two populations, we employed two published approaches to sequencing MHC Class II exon 2: direct sequencing with exon-based primers, and traditional cloning and sequencing with intron-based primers. Results from both methods show that the colonist population has maintained high levels of variation. Our results also indicate varying numbers of alleles across individuals, corroborating evidence for gene duplication in songbird MHC. While future studies in songbirds may need to take a genomic approach to fully understand the structure of MHC in this lineage, our results show that it is possible to use traditional methods to reveal functional variation across populations.

Songbird chemosignals: volatile compounds in preen gland secretions vary among individuals, sexes, and populations.

Chemical signaling has been documented in many animals, but its potential importance in avian species, particularly songbirds, has received far less attention. We tested whether volatile compounds in the preen oil of a songbird (Junco hyemalis) contain reliable information about individual identity, sex, or population of origin by repeated sampling from captive male and female juncos originating from 2 recently diverged junco populations in southern California. One of the populations recently colonized an urban environment; the other resides in a species-typical montane environment. The birds were field-caught as juveniles, housed under identical conditions, and fed the same diet for 10 months prior to sampling. We used capillary gas chromatography-mass spectrometry to quantify the relative abundance of 19 volatile compounds previously shown to vary seasonally in this species. We found individual repeatability as well as significant sex and population differences in volatile profiles. The persistence of population differences in a common environment suggests that preen oil chemistry likely has a genetic basis and may thus evolve rapidly in response to environmental change. These finding suggest that songbird preen oil odors have the potential to function as chemosignals associated with mate recognition or reproductive isolation.

Resolution of the Hylobates phylogeny: congruence of mitochondrial D-loop sequences with molecular, behavioral, and morphological data sets.

Gibbons of the genus Hylobates likely speciated very rapidly following isolation by rising sea levels during the Pleistocene. We sequenced the hypervariable region I (HV-I) of the mitochondrial D-loop to reconstruct the phylogeny of this group. Although the results clearly supported monophyly of each of the six species, the relationships among them were not clearly resolved by these data alone. A homogeneity test against published data sets of a coding mitochondrial locus (ND3-ND4 region), behavioral characters (vocalizations), and morphological traits (including skeletal and soft tissue anatomy) revealed no significant incongruence, and combining them resulted in a phylogenetic tree with much stronger support. The Kloss's gibbon (H. klossii), long considered a primitive taxon based on morphology, shares many molecular and vocal characteristics with the Javan gibbon (H. moloch), and appear as the most recently derived species. The northernmost species (H. lar and H. pileatus) are the most basal taxa. These data suggest that ancestral gibbons radiated from north to south. Unlike other markers, the HV-I region can accurately identify members of different gibbon species much like a DNA barcode, with potential applications to conservation.