Phylogeographic structure across one of the largest intact tropical savannahs: Molecular and morphological analysis of Australia's iconic frilled lizard Chlamydosaurus kingii.

The spectacular threat display of the savannah specialist Australo-Papuan frilled lizards has made them one of the world's most iconic reptiles. They are increasingly used as a model system for research in evolutionary biology and ecology but little is known of their population structure. Their distribution across northern Australia and southern New Guinea also provides an opportunity to examine biogeographic patterns as they relate to the large-scale movement of savannah habitat during the Plio/Pleistocene and the associated increase in aridity. We generated sequence data for one mitochondrial and four nuclear DNA loci (5052 base pairs) for 83 frilled lizards sampled throughout their range. We also quantified body proportion variation for 279 individuals. Phylogenetic analyses based on maximum likelihood and Bayesian species-tree methods revealed three shallow clades that replace each other across the monsoon tropics. We found the expected pattern of male biased sexual size dimorphism in both maximum body size and head size but there was no sexual dimorphism in overall body shape or in frill size, relative to head size, supporting the hypothesis that the frill is used primarily as a threat display rather than a sexual display. The genetic clades are broadly consistent with known clinal variation in frill color that gradually shifts from west to east (red, orange, yellow/white) but otherwise show little morphological differentiation in body proportion measures. The biogeographic breaks between clades occur at the Carpentaria Gap and the lowlands surrounding the Ord River, and our ecological niche modeling predicts lower habitat suitability for C. kingii in these regions. While this biogeographic pattern is consistent with numerous other taxonomic groups in northern Australia, the overall low genetic diversity in frilled lizards across the entire monsoon tropics and southern New Guinea contrasts starkly to patterns seen in other terrestrial vertebrates. Extremely low intra-clade genetic diversity over vast geographic areas is indicative of recent gene flow that would likely have been facilitated by widespread savannah during interglacials, or alternatively may reflect population bottlenecks induced by extreme aridity during Pleistocene glacials. The shallow divergence between Australian and New Guinean samples is consistent with recent connectivity between Australia and New Guinea that would have been possible via a savannah corridor across the Torres Strait. Based on our molecular and morphological data, we do not support taxonomic recognition of any of the frilled lizard clades and instead consider C. kingii a single species with shallow phylogeographic structure and clinal variation in frill color.

Testosterone production in response to exogenous gonadotropin releasing hormone (GnRH challenge) depends on social environment and color polymorphism.

Testosterone is an important mediator of behavior, morphology and physiology. A cascade of signals regulates the amount of testosterone (T) circulating in the plasma; in response to stimulus the hypothalamus releases gonadotropin-releasing hormone (GnRH), which triggers secretion of gonadotropins from the pituitary, stimulating the synthesis and release of T from the gonads. Previous work has shown that changes to the social environment can alter circulating T-levels, which may have important fitness consequences, but it is currently unclear whether these changes are due to alterations in the signal from the brain, or changes in the ability of the pituitary and gonads to respond to this signal. Further, the strength and direction of response to a changing environment may differ according to life-history strategy. Species with genetically determined alternative strategies offer a pathway for examining these differences. Here we use a finch with a genetically determined polymorphism, the Gouldian finch (Erythrura gouldiae), to determine whether T-levels change in response to social environment. We also use injections of GnRH to determine whether these changes are due to alterations in the ability of the pituitary and gonads to respond to this signal. We found that social environment (presence of females) had a rapid effect on male circulating T-levels, and that this difference was reflected in responsiveness to GnRH. We observed no overall morph differences in T-levels, but we did observe morph differences in the pattern of T secretion across environments, and morph differences in the repeatability of T-levels across time and environment.

The hawk-dove game in a sexually reproducing species explains a colourful polymorphism of an endangered bird.

The hawk-dove game famously introduced strategic game theory thinking into biology and forms the basis of arguments for limited aggression in animal populations. However, aggressive 'hawks' and peaceful 'doves', with strategies inherited in a discrete manner, have never been documented in a real animal population. Thus, the applicability of game-theoretic arguments to real populations might be contested. Here, we show that the head-colour polymorphism of red and black Gouldian finches (Erythrura gouldiae) provides a real-life example. The aggressive red morph is behaviourally dominant and successfully invades black populations, but when red 'hawks' become too common, their fitness is severely compromised (via decreased parental ability). We also investigate the effects of real-life deviations, particularly sexual reproduction, from the simple original game, which assumed asexual reproduction. A protected polymorphism requires mate choice to be sufficiently assortative. Assortative mating is adaptive for individuals because of genetic incompatibilities affecting hybrid offspring fitness, but by allowing red 'hawks' to persist, it also leads to significantly reduced population sizes. Because reductions in male contributions to parental care are generally known to lead to lower population productivity in birds, we expect zero-sum competition to often have wide ranging population consequences.

Covariation in life-history traits: differential effects of diet on condition, hormones, behavior, and reproduction in genetic finch morphs.

The relative contribution of genetic and environmental factors in determining variation in life-history traits is of central interest to evolutionary biologists, but the physiological mechanisms underlying these traits are still poorly understood. Here we experimentally demonstrate opposing effects of nutritional stress on immune function, endocrine physiology, parental care, and reproduction between red and black head-color morphs of the Gouldian finch (Erythrura gouldiae). Although the body condition of black morphs was largely unaffected by diet manipulation, red birds were highly sensitive to dietary changes, exhibiting considerable within-individual changes in condition and immune function. Consequently, nutritionally stressed red birds delayed breeding, produced smaller broods, and reared fewer and lower-quality foster offspring than black morphs. Differences in offspring quality were largely due to morph-specific differences in parental effort: red morphs reduced parental provisioning, whereas black morphs adaptively elevated their provisioning effort to meet the increased nutritional demands of their foster brood. Nutritionally stressed genetic morphs also exhibited divergent glucocorticoid responses. Black morphs showed reduced corticosterone-binding globulin (CBG) concentrations and increased levels of free corticosterone, whereas red morphs exhibited reduced free corticosterone levels and elevated CBG concentrations. These opposing glucocorticoid responses highlight intrinsic differences in endocrine sensitivities and plasticity between genetic morphs, which may underlie the morph-specific differences in condition, behavior, and reproduction and thus ultimately contribute to the evolution and maintenance of color polymorphism.

Mothers adjust offspring sex to match the quality of the rearing environment.

Theory predicts that mothers should adjust offspring sex ratios when the expected fitness gains or rearing costs differ between sons and daughters. Recent empirical work has linked biased offspring sex ratios to environmental quality via changes in relative maternal condition. It is unclear, however, whether females can manipulate offspring sex ratios in response to environmental quality alone (i.e. independent of maternal condition). We used a balanced within-female experimental design (i.e. females bred on both low- and high-quality diets) to show that female parrot finches (Erythrura trichroa) manipulate primary offspring sex ratios to the quality of the rearing environment, and not to their own body condition and health. Individual females produced an unbiased sex ratio on high-quality diets, but over-produced sons in poor dietary conditions, even though they maintained similar condition between diet treatments. Despite the lack of sexual size dimorphism, such sex ratio adjustment is in line with predictions from sex allocation theory because nutritionally stressed foster sons were healthier, grew faster and were more likely to survive than daughters. These findings suggest that mothers may adaptively adjust offspring sex ratios to optimally match their offspring to the expected quality of the rearing environment.

In the eye of the beholder: visual mate choice lateralization in a polymorphic songbird.

Birds choose mates on the basis of colour, song and body size, but little is known about the mechanisms underlying these mating decisions. Reports that zebra finches prefer to view mates with the right eye during courtship, and that immediate early gene expression associated with courtship behaviour is lateralized in their left hemisphere suggest that visual mate choice itself may be lateralized. To test this hypothesis, we used the Gouldian finch, a polymorphic species in which individuals exhibit strong, adaptive visual preferences for mates of their own head colour. Black males were tested in a mate-choice apparatus under three eye conditions: left-monocular, right-monocular and binocular. We found that black male preference for black females is so strongly lateralized in the right-eye/left-hemisphere system that if the right eye is unavailable, males are unable to respond preferentially, not only to males and females of the same morph, but also to the strikingly dissimilar female morphs. Courtship singing is consistent with these lateralized mate preferences; more black males sing to black females when using their right eye than when using their left. Beauty, therefore, is in the right eye of the beholder for these songbirds, providing, to our knowledge, the first demonstration of visual mate choice lateralization.

Constrained mate choice in social monogamy and the stress of having an unattractive partner.

In socially monogamous animals, mate choice is constrained by the availability of unpaired individuals in the local population. Here, we experimentally investigate the physiological stress endured by a female (the choosy sex) when pairing with a non-preferred social partner. In two experimental contexts, female Gouldian finches (Erythrura gouldiae) socially paired with poor-quality mates had levels of circulating corticosterone that were three to four times higher than those observed in females that were paired with preferred mates. The elevated level of this stress hormone in response to partner quality was observed within 12 h of the experimental introduction and maintained over a period of several weeks. Our findings demonstrate the extent of intra-individual conflict that occurs when individuals are forced to make mate-choice decisions that are not perfectly aligned with mate-choice preferences. The elevated level of corticosterone also suggests a mechanistic route through which females might adaptively manage their responses to intersexual conflict over reproductive investment.

Interference from long-tailed finches constrains reproduction in the endangered Gouldian finch.

1. Interspecific interference competition for nest-sites among cavity-nesting birds can have important effects on reproductive fitness and the distribution of competing species. 2. We observed interference at nest-sites in free-living populations of the endangered Gouldian finch (Erythrura gouldiae) and sympatric long-tailed finch (Poephila acuticauda), and also experimentally tested the relative strength and effect of interference at nest-sites in captive populations. 3. Levels of competitive interference at nest-sites in the wild were high for Gouldian, but not long-tailed finches, and interference frequency was inversely related to Gouldian finch reproductive success. High levels of interference conferred reduced fledging success but did not affect offspring condition. 4. Captive experiments corroborated the field data, also demonstrating fitness costs of interspecific competition, and that long-tailed finches dominated resources under standardized conditions. 5. Such asymmetrical competition dynamics are likely to constrain reproduction in Gouldian finch populations, potentially affecting recruitment and hindering the recovery of remaining populations of this endangered species.

Socially mediated trade-offs between aggression and parental effort in competing color morphs.

Individuals often face trade-offs between investment in parental care and alternative investments of time, energy, and resources into other life-history components, such as dominance, attractiveness, and health. Selection is thought to promote the optimal balance between the costs and the benefits of these conflicting activities by favoring individuals that adopt different tactics to maximize their overall evolutionary fitness in different environments. To test this, we experimentally manipulate both aggression (i.e., competitive environment) and parental effort (i.e., brood size) in red and black morphs of the Gouldian finch (Erythrura gouldiae). Although aggressive red males provide parental effort comparable to that of black males in environments where competition is low, irrespective of their relative brood size, they severely reduce or abandon parental investment in highly competitive environments. In contrast, nonaggressive black males are largely unaffected by the competitive environment and instead adaptively adjust their provisioning effort to the relative demands of their brood. Consequently, in highly competitive environments, although dominant red males defend higher-quality nest sites, they produce fewer and lower-quality offspring (in terms of mass and immunocompetence) than black males do. These opposing effects of frequency-dependent competitive environments on red and black males underlie their differential trade-offs between the costs and the benefits of aggression and parental effort.

Red dominates black: agonistic signalling among head morphs in the colour polymorphic Gouldian finch.

Recent sexual selection studies on the evolution of bird colouration have mainly focused on signals with a high level of condition-dependent variation, with much less attention given to colour traits whose expression is genetically controlled. Here, we experimentally tested the relative importance of a genetic colour polymorphism in determining male dominance in the Gouldian finch (Erythrura gouldiae), a species displaying three completely discrete but naturally co-occurring genetically inherited phenotypes; yellow-, red- (carotenoid) and black-headed (melanin) morphs. First, in staged dominance contests between unfamiliar birds of different head morphs, red-headed males dominated black-headed males, both of which dominated the yellow-headed birds. Second, within morphs, the intensity and size of the strongly ultraviolet-blue collar determined the outcome of these contests, and among the red-headed males, redder males dominated less chromatic birds. Lastly, when the dominance signal of red-headed birds was experimentally destabilized (i.e. blackened or reddened), naturally red-headed morphs continued to dominate both the black-and yellow-headed morphs. Together, these results suggest that intrinsic dominance-related behavioural differences between the three colour morphs, which are likely to influence the relative fitness of each morph, contribute to the complex selective patterns maintaining these three discrete phenotypes in relatively stable frequencies in wild populations.

Multiple receivers, multiple ornaments, and a trade-off between agonistic and epigamic signaling in a widowbird.

Sexual displays often involve several different ornamental traits. Yet most indicator models of sexual selection based on a single receiver (usually a choosy female) find that multiple handicap signals should be unstable. Here we study reasons for this contradiction, analyzing signal function, signal content, and trade-offs between signals in the polygynous red-collared widowbird Euplectes ardens. Males have both a long, graduated tail and a red carotenoid collar badge. Territory-holding "residents" have slightly shorter tails than the nonbreeding "floaters," but their carotenoid collars are 40% larger, and they have (on the basis of reflectance spectrometry and objective colorimetry) a 23-nm more long-wave ("redder") hue than floaters. This corroborates experimental evidence that the red collar is selected by male contest competition, whereas female choice is based almost exclusively on male tail length. Tail length is negatively correlated with the carotenoid signal, which together with body size and condition explains 55% of the variation in tail length. The trade-off in tail length and carotenoid investment is steeper among residents, suggesting an interaction with costs of territory defense. We propose that the "multiple receiver hypothesis" can explain the coexistence of multiple handicap signals. Furthermore, the trade-off between signal expressions might contribute to the inverse relation between nuptial tail elongation and coloration in the genus Euplectes (bishops and widowbirds).

A generalized female bias for long tails in a short-tailed widowbird.

Tail elongation in the polygynous widowbirds (Euplectes spp.) has evoked both adaptive and non-adaptive explanations. Female choice has been shown in the three longest tailed species (20-50 cm), whereas an agonistic function was proposed for a medium-tailed (10 cm) widowbird. To test the generality and directionality of sexual selection on tail length in widowbirds, we experimentally investigated selection in the relatively short-tailed (7 cm) red-shouldered widowbirds (E. axillaris). Prior to territory establishment, males were assigned to four tail-treatment groups; control, short, long and supernormal (similar to a sympatric long-tailed congener). No effects on male competition were detected as the groups were equally successful in acquiring territories of similar size and quality. However, mating success among the 92 territorial males was strongly skewed in favour of supernormal-tailed males (62% of active nests; 5.2 +/- 1.3 nests per territory). Long-tailed males also acquired more nests (1.9 +/- 0.7) than control (0.7 +/- 0.5) and short-tailed (0.5 +/- 0.3) males, while the latter two groups did not differ significantly. These results support a general, open-ended female preference for long tails in widowbirds and may represent a receiver bias that arose early in their divergence from the short-tailed weaverbirds (Ploceinae).

The role of the Ord Arid Intrusion in the historical and contemporary genetic division of long-tailed finch subspecies in northern Australia.

The effect of separation by biogeographic features followed by secondary contact can blur taxonomic boundaries and produce complex genetic signatures. We analyzed population structure and gene flow across the range of the long-tailed finch (Poephila acuticauda) in northern Australia (1) to test the hypothesis that Ord Arid Intrusion acted as the causative barrier that led to divergence of P. acuticauda subspecies, (2) to determine whether genetic data support the presence of a gradual cline across the range or a sudden shift, both of which have been suggested based on morphological data, and (3) to estimate levels of contemporary gene flow within this species complex. We collected samples from 302 individuals from 10 localities. Analyses of 12 microsatellite loci and sequence data from 333 base pairs of the mitochondrial control region were used to estimate population structure and gene flow, using analysis of molecular variance (AMOVA), haplotype network analysis, frequency statistics, and clustering methods. Mitochondrial sequence data indicated the presence of three genetic groups (regions) across the range of P. acuticauda. Genetic diversity was highest in the east and lowest in the west. The Ord Arid Intrusion appears to have functioned as a biogeographic barrier in the past, according to mtDNA evidence presented here and evidence from previous studies. The absence of isolation by distance between adjacent regions and the lack of population genetic structure of mtDNA within regions indicates that genetic changes across the range of P. acuticauda subspecies are characterized by discrete breaks between regions. While microsatellite data indicate a complete absence of genetic structure across this species' range, it appears unlikely that this results from high levels of gene flow. Mitochondrial data do not support the presence of contemporary gene flow across the range of this species.

Context-dependent sex allocation: constraints on the expression and evolution of maternal effects.

Despite decades of research, whether vertebrates can and do adaptively adjust the sex ratio of their offspring is still highly debated. However, this may have resulted from the failure of empirical tests to identify large and predictable fitness returns to females from strategic adjustment. Here, we test the effect of diet quality and maternal condition on facultative sex ratio adjustment in the color polymorphic Gouldian finch (Erythrura gouldiae), a species that exhibits extreme maternal allocation in response to severe and predictable (genetically-determined) fitness costs. On high-quality diets, females produced a relatively equal sex ratio, but over-produced sons in poor dietary conditions. Despite the lack of sexual size dimorphism, nutritionally stressed foster sons were healthier, grew faster, and were more likely to survive than daughters. Although these findings are in line with predictions from sex allocation theory, the extent of adjustment is considerably lower than previously reported for this species. Females therefore have strong facultative control over sex allocation, but the extent of adjustment is likely determined by the relative magnitude of fitness gains and the ability to reliably predict sex-specific benefits from environmental (vs. genetic) variables. These findings may help explain the often inconsistent, weak, or inconclusive empirical evidence for adaptive sex ratio adjustment in vertebrates.

Sex chromosome linkage of mate preference and color signal maintains assortative mating between interbreeding finch morphs.

Assortative mating is a key aspect in the speciation process because it is important for both initial divergence and maintenance of distinct species. However, it remains a challenge to explain how assortative mating evolves when diverging populations are undergoing gene flow (e.g., during hybridization). Here I experimentally test how assortative mating is maintained with frequent gene flow between diverged head-color morphs of the Gouldian finch (Erythrura gouldiae). Contrary to the predominant view on the development of sexual preferences in birds, cross-fostered offspring did not imprint on the phenotype of their conspecific (red or black morphs) or heterospecific (Bengalese finch) foster parents. Instead, the mating preferences of F(1) and F(2) intermorph-hybrids are consistent with inheritance on the Z chromosomes, which are also the location for genes controlling color expression and the genes causing low fitness of intermorph-hybrids. Genetic associations between color signal and preference loci on the sex chromosomes may prevent recombination from breaking down these associations when the morphs interbreed, helping to maintain assortative mating in the face of gene flow. Although sex linkage of reproductively isolating traits is theoretically expected to promote speciation, social and ecological constraints may enforce frequent interbreeding between the morphs, thus preventing complete reproductive isolation.

Females use multiple mating and genetically loaded sperm competition to target compatible genes.

Individuals in socially monogamous species may participate in copulations outside of the pair bond, resulting in extra-pair offspring. Although males benefit from such extra-pair behavior if they produce more offspring, the adaptive function of infidelity to females remains elusive. Here we show that female participation in extra-pair copulations, combined with a genetically loaded process of sperm competition, enables female finches to target genes that are optimally compatible with their own to ensure fertility and optimize offspring viability. Such female behavior, along with the postcopulatory processes demonstrated here, may provide an adaptive function of female infidelity in socially monogamous animals.

Pronounced within-individual plasticity in sperm morphometry across social environments.

Sperm morphometry (i.e., size and shape) and function are important determinants of male reproductive success and are thought to be under stabilizing selection. However, recent studies suggest that sperm morphometry can be a phenotypically plastic trait, which can be adjusted to varying conditions. We tested whether different behavioral strategies in aggression between aggressive red and nonaggressive black males of the color polymorphic Gouldian finch (Erythrura gouldiae) can influence sperm morphometry. We show pronounced within-individual phenotypic plasticity in sperm morphometry of male Gouldian finches in three different social environments. Both red and black males placed in intermediate to high competitive environments (high frequency of red males) increased the relative length of their sperm midpiece. By contrast, red males placed in low to intermediate competitive environments (higher frequency of black males) increased the length of the sperm flagellum. Significant changes in stress and sex steroid hormone levels (in response to the competitive environment) appear to influence sperm traits in red but not in black males, suggesting that changes in hormonal levels are not solely responsible for the observed changes in sperm morphometry. These findings imply that males can adjust sperm morphometry across social environments.

Postzygotic genetic incompatibility between sympatric color morphs.

Alternative genetically determined color morphs within a population or species are believed to successfully interbreed within a population. However, the occurrence of prezygotic or ecological selection in a number of polymorphic systems may lead to nonrandom mating and prevent genetic morphs from fully interbreeding. Here we show that postzygotic incompatibility significantly limits gene flow between the sympatric red and black color morphs of the Gouldian finch (Erythrura gouldiae). Using a balanced within-female experimental design, in which individuals were forced to breed in pure and mixed morph crosses, we found large inviability effects (>30%) in offspring resulting from genetically mixed genotypes. The consistent mortality effects across different stages of development (e.g., prehatching, juvenile, adulthood), unconfounded by environmentally derived parental effects or social environments, reveal an underlying genetic incompatibility between different genotypes. Furthermore, mortality in mixed morph genotypes was particularly severe (43.6%) for the heterogametic sex (daughters), which is consistent with Haldane's rule predicted for postzygotic incompatibilities between hybridizing species. This significant, but incomplete, postzygotic isolation suggests that the sympatric morphs may represent transient stages in the speciation-hybridization process.

Genetic incompatibility drives sex allocation and maternal investment in a polymorphic finch.

Genetic compatibility may drive individual mate choice decisions because of predictable fitness effects associated with breeding with incompatible partners. In Gouldian finches (Erythrura gouldiae), females paired with genetically incompatible males of alternative color morphs overproduce sons, presumably to reduce investment in inviable daughters. We also observed a reduced overall investment in clutch size, egg size, and care to offspring resulting from incompatible matings. Within-female experimental pairings demonstrate that female birds have the ability to adaptively adjust the sex of their eggs and allocate resources on the basis of partner quality. Female Gouldian finches thus make cumulative strategic allocation decisions to minimize the costs of poor-quality pairings when faced with a genetically incompatible partner.

Frequency-dependent physiological trade-offs between competing colour morphs.

Evolutionary theory suggests that alternative colour morphs (i.e. genetically controlled phenotypes) may derive similar fitness under frequency-dependent selection. Here we experimentally demonstrate opposing effects of frequency-dependent social environments on plasma hormone levels (testosterone and corticosterone) and immune function between red- and black-headed male morphs of the Gouldian finch (Erythrura gouldiae). Red-headed males are highly sensitive to changes in the social environment, especially towards the relative density of their own aggressive morph, exhibiting high stress responses and immunosuppression in socially competitive environments. In contrast, the non-aggressive black-headed males follow a more passive strategy that appears to buffer them against social stresses. The differential effect of hormones on aggressive behaviour and immune performance reinforces the contrasting behavioural strategies employed by these colour morphs, and highlights the importance of the social environment in determining the individual basis of behavioural and physiological responses.