Thermal acclimation in a non-migratory songbird occurs via changes to thermogenic capacity, but not conductance.

Thermoregulatory performance can be modified through changes in various subordinate traits, but the rate and magnitude of change in these traits is poorly understood. We investigated flexibility in traits that affect thermal balance between black-capped chickadees (Poecile atricapillus) acclimated for 6 weeks to cold (-5°C) or control (23°C) environments (n=7 per treatment). We made repeated measurements of basal and summit metabolic rates via flow-through respirometry and of body composition using quantitative magnetic resonance of live birds. At the end of the acclimation period, we measured thermal conductance of the combined feathers and skins. Cold-acclimated birds had a higher summit metabolic rate, reflecting a greater capacity for endogenous heat generation, and an increased lean mass. However, birds did not alter their thermal conductance. These results suggest that chickadees respond to cold stress by increasing their capacity for heat production rather than increasing heat retention, an energetically expensive strategy.

Conspecific olfactory preferences and interspecific divergence in odor cues in a chickadee hybrid zone.

Understanding how mating cues promote reproductive isolation upon secondary contact is important in describing the speciation process in animals. Divergent chemical cues have been shown to act in reproductive isolation across many animal taxa. However, such cues have been overlooked in avian speciation, particularly in passerines, in favor of more traditional signals such as song and plumage. Here, we aim to test the potential for odor to act as a mate choice cue, and therefore contribute to premating reproductive isolation between the black-capped (Poecile atricapillus) and Carolina chickadee (P. carolinensis) in eastern Pennsylvania hybrid zone populations. Using gas chromatography-mass spectrometry, we document significant species differences in uropygial gland oil chemistry, especially in the ratio of ester to nonester compounds. We also show significant preferences for conspecific over heterospecific odor cues in wild chickadees using a Y-maze design. Our results suggest that odor may be an overlooked but important mating cue in these chickadees, potentially promoting premating reproductive isolation. We further discuss several promising avenues for future research in songbird olfactory communication and speciation.

Maladaptive learning and memory in hybrids as a reproductive isolating barrier.

Selection against hybrid offspring, or postzygotic reproductive isolation, maintains species boundaries in the face of gene flow from hybridization. In this review, we propose that maladaptive learning and memory in hybrids is an important, but overlooked form of postzygotic reproductive isolation. Although a role for learning in premating isolation has been supported, whether learning deficiencies can contribute to postzygotic isolation has rarely been tested. We argue that the novel genetic combinations created by hybridization have the potential to impact learning and memory abilities through multiple possible mechanisms, and that any displacement from optima in these traits is likely to have fitness consequences. We review evidence supporting the potential for hybridization to affect learning and memory, and evidence of links between learning abilities and fitness. Finally, we suggest several avenues for future research. Given the importance of learning for fitness, especially in novel and unpredictable environments, maladaptive learning and memory in hybrids may be an increasingly important source of postzygotic reproductive isolation.

Hybrid chickadees are deficient in learning and memory.

Identifying the phenotypes underlying postzygotic reproductive isolation is crucial for fully understanding the evolution and maintenance of species. One potential postzygotic isolating barrier that has rarely been examined is learning and memory ability in hybrids. Learning and memory are important fitness-related traits, especially in scatter-hoarding species, where accurate retrieval of hoarded food is vital for winter survival. Here, we test the hypothesis that learning and memory ability can act as a postzygotic isolating barrier by comparing these traits among two scatter-hoarding songbird species, black-capped (Poecile atricapillus) and Carolina chickadees (Poecile carolinensis), and their naturally occurring hybrids. In an outdoor aviary setting, we find that hybrid chickadees perform significantly worse on an associative learning spatial task and are worse at solving a novel problem compared to both parental species. Deficiencies in learning and memory abilities could therefore contribute to postzygotic reproductive isolation between chickadee species. Given the importance of learning and memory for fitness, our results suggest that these traits may play an important, but as yet overlooked, role in postzygotic reproductive isolation.

Genetic variation during range expansion: effects of habitat novelty and hybridization.

How species' ranges evolve remains an enduring problem in ecology and evolutionary biology. Species' range limits are potentially set by the inability of peripheral populations to adapt to range-edge habitat. Indeed, peripheral populations are often assumed to have reduced genetic diversity and population sizes, which limit evolvability. However, support for this assumption is mixed, possibly because the genetic effects of range expansion depend on two factors: the extent that habitat into which expansion occurs is novel and sources of gene flow. Here, we used spadefoot toads, Spea bombifrons, to contrast the population genetic effects of expansion into novel versus non-novel habitat. We further evaluated gene flow from conspecifics and from heterospecifics via hybridization with a resident species. We found that range expansion into novel habitat, relative to non-novel habitat, resulted in higher genetic differentiation, lower conspecific gene flow and bottlenecks. Moreover, we found that hybridizing with a resident species introduced genetic diversity in the novel habitat. Our results suggest the evolution of species' ranges can depend on the extent of differences in habitat between ancestral and newly occupied ranges. Furthermore, our results highlight the potential for hybridization with a resident species to enhance genetic diversity during expansions into novel habitat.

Population differentiation at a regional scale in spadefoot toads: contributions of distance and divergent selective environments.

The causes of population differentiation can provide insight into the origins of early barriers to gene flow. Two key drivers of population differentiation are geographic distance and local adaptation to divergent selective environments. When reproductive isolation arises because some populations of a species are under selection to avoid hybridization while others are not, population differentiation and even speciation can result. Spadefoot toad populations Spea multiplicata that are sympatric with a congener have undergone reinforcement. This reinforcement has resulted not only in increased reproductive isolation from the congener, but also in the evolution of reproductive isolation from nearby and distant conspecific allopatric populations. We used multiple approaches to evaluate the contributions of geographic distance and divergent selective environments to population structure across this regional scale in S. multiplicata, based on genotypes from six nuclear microsatellite markers. We compared groups of populations varying in both geographic location and in the presence of a congener. Hierarchical F-statistics and results from cluster analyses and discriminant analyses of principal components all indicate that geographic distance is the stronger contributor to genetic differentiation among S. multiplicata populations at a regional scale. However, we found evidence that adaptation to divergent selective environments also contributes to population structure. Our findings highlight how variation in the balance of evolutionary forces acting across a species' range can lead to variation in the relative contributions of geographic distance and local adaptation to population differentiation across different spatial scales.

Differential effects of climate and species interactions on range limits at a hybrid zone: potential direct and indirect impacts of climate change.

The relative contributions of climate versus interspecific interactions in shaping species distributions have important implications for closely related species at contact zones. When hybridization occurs within a contact zone, these factors regulate hybrid zone location and movement. While a hybrid zone's position may depend on both climate and interactions between the hybridizing species, little is known about how these factors interact to affect hybrid zone dynamics. Here, we utilize SDM (species distribution modeling) both to characterize the factors affecting the current location of a moving North American avian hybrid zone and to predict potential direct and indirect effects of climate change on future distributions. We focus on two passerine species that hybridize where their ranges meet, the Black-capped (Poecile atricapillus) and Carolina (P. carolinensis) chickadee. Our contemporary climate models predict the occurrence of climatically suitable habitat extending beyond the hybrid zone for P. atricapillus only, suggesting that interspecific interactions primarily regulate this range boundary in P. atricapillus, while climatic factors regulate P. carolinensis. Year 2050 climate models predict a drastic northward shift in suitable habitat for P. carolinensis. Because of the greater importance of interspecific interactions for regulating the southern range limit of P. atricapillus, these climate-mediated shifts in the distribution of P. carolinensis may indirectly lead to a range retraction in P. atricapillus. Together, our results highlight the ways climate change can both directly and indirectly affect species distributions and hybrid zone location. In addition, our study lends support to the longstanding hypothesis that abiotic factors regulate species' poleward range limits, while biotic factors shape equatorial range limits.

Reinforcement generates reproductive isolation between neighbouring conspecific populations of spadefoot toads.

Reproductive character displacement is the adaptive evolution of traits that minimize deleterious reproductive interactions between species. When arising from selection to avoid hybridization, this process is referred to as reinforcement. Reproductive character displacement generates divergence not only between interacting species, but also between conspecific populations that are sympatric with heterospecifics versus those that are allopatric. Consequently, such conspecific populations can become reproductively isolated. We compared female mate preferences in, and evaluated gene flow between, neighbouring populations of spadefoot toads that did and did not occur with heterospecifics (mixed- and pure-species populations, respectively). We found that in mixed-species populations females significantly preferred conspecifics. Such females also tended to prefer a conspecific call character that was dissimilar from heterospecifics. By contrast, females from pure-species populations did not discriminate conspecific from heterospecific calls. They also preferred a more exaggerated conspecific call character that resembles heterospecific males. Moreover, gene flow was significantly reduced between mixed- and pure-species population types. Thus, character displacement (and, more specifically, reinforcement) may initiate reproductive isolation between conspecific populations that differ in interactions with heterospecifics.

Estimation of linkage disequilibrium and interspecific gene flow in Ficedula flycatchers by a newly developed 50k single-nucleotide polymorphism array.

With the access to draft genome sequence assemblies and whole-genome resequencing data from population samples, molecular ecology studies will be able to take truly genome-wide approaches. This now applies to an avian model system in ecological and evolutionary research: Old World flycatchers of the genus Ficedula, for which we recently obtained a 1.1 Gb collared flycatcher genome assembly and identified 13 million single-nucleotide polymorphism (SNP)s in population resequencing of this species and its sister species, pied flycatcher. Here, we developed a custom 50K Illumina iSelect flycatcher SNP array with markers covering 30 autosomes and the Z chromosome. Using a number of selection criteria for inclusion in the array, both genotyping success rate and polymorphism information content (mean marker heterozygosity = 0.41) were high. We used the array to assess linkage disequilibrium (LD) and hybridization in flycatchers. Linkage disequilibrium declined quickly to the background level at an average distance of 17 kb, but the extent of LD varied markedly within the genome and was more than 10-fold higher in 'genomic islands' of differentiation than in the rest of the genome. Genetic ancestry analysis identified 33 F1 hybrids but no later-generation hybrids from sympatric populations of collared flycatchers and pied flycatchers, contradicting earlier reports of backcrosses identified from much fewer number of markers. With an estimated divergence time as recently as <1 Ma, this suggests strong selection against F1 hybrids and unusually rapid evolution of reproductive incompatibility in an avian system.

Optimizing the trade-off between offspring number and quality in unpredictable environments: testing the role of differential androgen transfer to collared flycatcher eggs.

According to the brood reduction hypothesis, parents adjust their brood size in response to current environmental conditions. When resources are abundant, parents can successfully raise all hatched offspring, but when resources are scarce, brood reduction, i.e., the sacrifice of some siblings to secure the quality of a subset of offspring, may maximize fitness. Differential transfer of maternal androgens is one potential proximate mechanism through which female birds may facilitate brood reduction because it may alter the relative competitive ability of sibling nestlings. We tested the hypothesis that female collared flycatchers (Ficedula albicollis) manipulate sibling competition by transferring less androgens to eggs late in the laying sequence. We experimentally elevated androgen levels in i) whole clutches and ii) only the two last laid eggs, and compared growth and begging behavior of offspring from these treatments with a control treatment. By using three treatments and video assessment of begging, we examined the effects of within-clutch patterns of yolk androgen transfer on levels of sibling competition in situ. When androgens were elevated in only the two last laid eggs, begging was more even among siblings compared to control nests. We also found that female nestlings receiving additional yolk androgens showed higher mass gain later in the breeding season, while their male counterparts did not. Our results suggest that females may improve reproductive success in unpredictable environments by altering within-clutch patterns of yolk androgen transfer. We discuss the possibility that life-history divergence between the co-occurring collared and pied flycatcher (Ficedula hypoleuca) is amplified by patterns of yolk androgen transfer.

Low fertility of wild hybrid male flycatchers despite recent divergence.

Postzygotic isolation may be important for maintaining species boundaries, particularly when premating barriers are incomplete. Little is known about the course of events leading from minor environmental mismatches affecting hybrid fitness to severe genetic incompatibilities causing sterility or inviability. We investigated whether reduced reproductive success of hybrid males was caused by suboptimal sperm traits or by more severe genetic incompatibilities in a hybrid zone of pied (Ficedula hypoleuca) and collared flycatchers (F. albicollis) on the island of Öland, Sweden. About 4 per cent hybridization is observed in this population and all female hybrids are sterile. We found no sperm in the ejaculates of most sampled hybrid males, and sperm with abnormal morphology in two hybrids. Furthermore, none of the hybrids sired any offspring because of high levels of hatching failure and extra-pair paternity in their nests. These results from a natural hybrid zone suggest that the spermatogenesis of hybrid males may become disrupted despite little genetic divergence between the parental species.

Positive feedback between ecological and reproductive character displacement in a young avian hybrid zone.

Character displacement can reduce costly interspecific interactions between young species. We investigated the mechanisms behind divergence in three key traits-breeding habitat choice, timing of breeding, and plumage coloration-in Ficedula flycatchers. We found that male pied flycatchers became expelled from the preferred deciduous habitat into mixed forest as the superior competitor, collared flycatchers, increased in numbers. The peak in food abundance differs between habitats, and the spatial segregation was paralleled by an increased divergence in timing of breeding between the two species. Male pied flycatchers vary from brown to black with brown coloration being more frequent in sympatry with collared flycatchers, a pattern often proposed to result from selection against hybridization, that is, reinforcement. In contrast to this view, we show that brown male pied flycatchers more often hybridize than black males. Male pied flycatcher plumage coloration influenced the territory obtained in areas of co-occurrence with collared flycatchers, and brown male pied flycatchers experienced higher relative fitness than black males when faced with heterospecific competition. We suggest that allopatric divergence in resource defense ability causes a feedback loop at secondary contact where male pied flycatchers with the most divergent strategy compared to collared flycatchers are favored by selection.

A guide to the genomics of ecological speciation in natural animal populations.

Interest in ecological speciation is growing, as evidence accumulates showing that natural selection can lead to rapid divergence between subpopulations. However, whether and how ecological divergence can lead to the buildup of reproductive isolation remains under debate. What is the relative importance of natural selection vs. neutral processes? How does adaptation generate reproductive isolation? Can ecological speciation occur despite homogenizing gene flow? These questions can be addressed using genomic approaches, and with the rapid development of genomic technology, will become more answerable in studies of wild populations than ever before. In this article, we identify open questions in ecological speciation theory and suggest useful genomic methods for addressing these questions in natural animal populations. We aim to provide a practical guide for ecologists interested in incorporating genomic methods into their research programs. An increased integration between ecological research and genomics has the potential to shed novel light on the origin of species.

Speciation in Ficedula flycatchers.

Speciation in animals often requires that population divergence goes through three major evolutionary stages, i.e. ecological divergence, development of sexual isolation and the build-up of genetic incompatibility. There is theoretical consensus regarding favourable conditions required for speciation to reach its final and irreversible stage, but empirical tests remain rare. Here, we review recent research on processes of speciation, based on studies in hybrid zones between collared (Ficedula albicollis) and pied flycatchers (Ficedula hypoleuca). A major advantage of this study system is that questions concerning all three major sources of reproductive isolation and their interconnections can be addressed. We conclude that (i) ecological divergence is caused by divergence in life-history traits, (ii) females prefer mates of their own species based on differences in both plumage and song characteristics, (iii) male plumage characteristics have diverged but their song has converged in sympatry, (iv) there is genetic incompatibility in accordance with Haldane's rule, and (v) the Z-chromosome appears to be a hotspot for genes involved in sexual isolation and genetic incompatibility. We discuss how identification of the genes underlying the three major sources of reproductive isolation can be used to draw conclusions about links between the processes driving their evolution.

Parallel evolution and ecological selection: replicated character displacement in spadefoot toads.

Ecological character displacement--trait evolution stemming from selection to lessen resource competition between species--is most often inferred from a pattern in which species differ in resource-use traits in sympatry but not in allopatry, and in which sympatric populations within each species differ from conspecific allopatric populations. Yet, without information on population history, the presence of a divergent phenotype in multiple sympatric populations does not necessarily imply that there has been repeated evolution of character displacement. Instead, such a pattern may arise if there has been character displacement in a single ancestral population, followed by gene flow carrying the divergent phenotype into multiple, derived, sympatric populations. Here, we evaluate the likelihood of such historical events versus ongoing ecological selection in generating divergence in trophic morphology between multiple populations of spadefoot toad (Spea multiplicata) tadpoles that are in sympatry with a heterospecific and those that are in allopatry. We present both phylogenetic and population genetic evidence indicating that the same divergent trait, which minimizes resource competition with the heterospecific, has arisen independently in multiple sympatric populations. These data, therefore, provide strong indirect support for competition's role in divergent trait evolution.

An experimental test of character displacement's role in promoting postmating isolation between conspecific populations in contrasting competitive environments.

Ecological character displacement takes place when two closely related species co-occur in only part of their geographical range, and selection to minimize competition between them promotes divergence in resource-use traits in sympatry but not in allopatry. Because populations sympatric with the heterospecific competitor will experience a different competitive environment than conspecific populations in allopatry, conspecific populations from these two competitive environments will also diverge in resource traits as an indirect consequence of interspecific ecological character displacement. Ultimately, ecologically dependent postmating isolation may arise between conspecific populations from these divergent competitive environments if offspring produced by matings between them are competitively inferior in either type of competitive environment. Yet, there are no direct tests of character displacement's role in initiating such postmating isolation. Here, we present a test by comparing the phenotypes and performances of spadefoot toad tadpoles produced from between-competitive-environment (BCE) matings versus those produced from within-competitive-environment (WCE) matings. When raised with naturally occurring competitors, BCE offspring grew significantly less and were significantly smaller than WCE offspring. BCE offspring generally performed worse even when raised alone, suggesting that they may have harbored intrinsic genetic incompatibilities. Moreover, the difference in growth and body size of BCE versus WCE offspring was significantly greater when each was raised with competitors than when each was raised alone, suggesting that BCE tadpoles were competitively inferior to WCE tadpoles. Presumably, this enhanced difference arose because BCE tadpoles produced an intermediate resource-use phenotype that is less well adapted to either competitive environment. Because larval size is under strong, positive, directional selection, reduced growth and size of BCE offspring may diminish gene flow between populations in divergent competitive environments, thereby generating postmating isolation. Thus, postmating isolation between conspecific populations, and possibly even speciation, may arise as a by-product of interactions between species.

Field and experimental evidence for competition's role in phenotypic divergence.

Resource competition has long been viewed as a major cause of phenotypic divergence within and between species. Theory predicts that divergence arises because natural selection favors individuals that are phenotypically dissimilar from their competitors. Yet, there are few conclusive tests of this key prediction. Drawing on data from both natural populations and a controlled experiment, this paper presents such a test in tadpoles of two species of spadefoot toads (Spea bombifrons and S. multiplicata). These two species show exaggerated divergence in trophic morphology where they are found together (mixed-species ponds) but not where each is found alone (pure-species ponds), suggesting that they have undergone ecological character displacement. Moreover, in pure-species ponds, both species exhibit resource polymorphism. Using body size as a proxy for fitness, we found that in pure-species ponds disruptive selection favors extreme trophic phenotypes in both species, suggesting that intraspecific competition for food promotes resource polymorphism. In mixed-species ponds, by contrast, we found that trophic morphology was subject to stabilizing selection in S. multiplicata and directional selection in S. bombifrons. A controlled experiment revealed that the more similar an S. multiplicata was to its S. bombifrons tankmate in resource use, the worse was its performance. These results indicate that S. multiplicata individuals that differ from S. bombifrons would be selectively favored in competition. Our data therefore demonstrate how resource competition between phenotypically similar individuals can drive divergence between them. Moreover, our results indicate that how competition contributes to such divergence may be influenced not only by the degree to which competitors overlap in resource use, but also by the abundance and quality of resources. Finally, our finding that competitively mediated disruptive selection may promote resource polymorphism has potentially important implications for understanding how populations evolve in response to heterospecific competitors. In particular, once a population evolves resource polymorphism, it may be more prone to undergo ecological character displacement.

Ecological opportunity and phenotypic plasticity interact to promote character displacement and species coexistence.

We investigated the roles of resource availability and phenotypic plasticity in promoting ecological character displacement (i.e., trait evolution stemming from resource competition between species). Because ecological character displacement generates new populations that differ in resource use, this process should only occur when exploitable resources are available. We tested this hypothesis in two species of spadefoot toads (Spea bombifrons and S. multiplicata) whose tadpoles use phenotypic plasticity to develop into either an omnivore morph, which specializes on detritus, or a physically distinctive carnivore morph, which specializes on shrimp. Both species grow best on shrimp, but when reared together, S. bombifrons outcompetes S. multiplicata for shrimp and S. multiplicata outcompetes S. bombifrons for detritus. We found that when each species occurred alone in the field, they produced similar proportions of omnivores and carnivores. When the two species occurred together, however, they underwent ecological character displacement in larval development, with S. multiplicata producing mostly omnivores, and S. bombifrons producing mostly carnivores. We combined observations of natural populations with experiments to evaluate whether such character displacement was only possible when both shrimp and detritus were relatively abundant. Mixed-species ponds contained abundant detritus and shrimp, in contrast with nearby pure-species ponds, which were deficient in one resource. Experiments revealed that S. multiplicata competed poorly when detritus was rare and that S. bombifrons competed poorly when shrimp was rare. In nature, when one of these two resources was scarce, one species was missing, perhaps through competitive exclusion by the species that was the superior competitor for the remaining resource. Thus, ecological character displacement and, therefore, coexistence of close competitors, was only possible when diverse resources were available. Finally, even if exploitable resources are available, character displacement is not guaranteed to transpire if species cannot utilize such resources expeditiously. Phenotypic plasticity provides a general and important mechanism for facilitating resource partitioning. Thus, by facilitating shifts in resource use, phenotypic plasticity and ecological opportunity may often interact to promote divergence and coexistence of competitors.

Fisheries: mislabelling of a depleted reef fish.

Any fish species that appears to be readily available in the marketplace will create an impression among the public that there is a plentiful supply of that fish in the sea, but this may belie the true state of the fisheries' stock. Here we use molecular genetic analysis to show that some three-quarters of the fish sold in the United States as 'red snapper'--the US Food and Drug Administration's legally designated common name for Lutjanus campechanus--belong to another species. Mislabelling to this extent not only defrauds consumers but could also adversely affect estimates of stock size if it influences the reporting of catch data that are used in fisheries management.

Patterns and mechanisms of selection on a family-diagnostic trait: evidence from experimental manipulation and lifetime fitness selection gradients.

Plant traits that show little variation across higher taxa are often used as diagnostic traits, but the reason for the stasis of such traits remains unclear. Wild radish, Raphanus raphanistrum, exhibits tetradynamous stamens (four long and two short, producing a dimorphism in anther height within each flower), as do the vast majority of the more than 3,000 species in the Brassicaceae. Here we examine the hypothesis that selection maintains the stasis of dimorphic anther height by investigating the effects of this trait on pollen removal, seed siring success, and seed set in R. raphanistrum using both experimental and observational methods. Observational selection gradient analysis based on lifetime seed siring success provided evidence for an optimum dimorphism that was greater than zero in one of three years. In both experimentally manipulated and unmanipulated flowers, more pollen was removed in single visits from flowers with less dimorphism. There was no significant effect of anther dimorphism on female fitness (seed set). Therefore, there is some evidence to suggest that selection is maintaining anther dimorphism in wild radish, and that higher male fitness might result from restriction of single-visit pollen removal. We discuss these results in light of pollen presentation theory.