Pervasive gene flow despite strong and varied reproductive barriers in swordtails.

One of the mechanisms that can lead to the formation of new species occurs through the evolution of reproductive barriers. However, recent research has demonstrated that hybridization has been pervasive across the tree of life even in the presence of strong barriers. Swordtail fishes (genus Xiphophorus) are an emerging model system for studying the interface between these barriers and hybridization. We document overlapping mechanisms that act as barriers between closely related species, X. birchmanni and X. cortezi, by combining genomic sequencing from natural hybrid populations, artificial crosses, behavioral assays, sperm performance, and developmental studies. We show that strong assortative mating plays a key role in maintaining subpopulations with distinct ancestry in natural hybrid populations. Lab experiments demonstrate that artificial F1 crosses experience dysfunction: crosses with X. birchmanni females were largely inviable and crosses with X. cortezi females had a heavily skewed sex ratio. Using F2 hybrids we identify several genomic regions that strongly impact hybrid viability. Strikingly, two of these regions underlie genetic incompatibilities in hybrids between X. birchmanni and its sister species X. malinche. Our results demonstrate that ancient hybridization has played a role in the origin of this shared genetic incompatibility. Moreover, ancestry mismatch at these incompatible regions has remarkably similar consequences for phenotypes and hybrid survival in X. cortezi × X. birchmanni hybrids as in X. malinche × X. birchmanni hybrids. Our findings identify varied reproductive barriers that shape genetic exchange between naturally hybridizing species and highlight the complex evolutionary outcomes of hybridization.

Genome evolution is surprisingly predictable after initial hybridization.

Over the past two decades, evolutionary biologists have come to appreciate that hybridization, or genetic exchange between distinct lineages, is remarkably common - not just in particular lineages but in taxonomic groups across the tree of life. As a result, the genomes of many modern species harbor regions inherited from related species. This observation has raised fundamental questions about the degree to which the genomic outcomes of hybridization are repeatable and the degree to which natural selection drives such repeatability. However, a lack of appropriate systems to answer these questions has limited empirical progress in this area. Here, we leverage independently formed hybrid populations between the swordtail fish Xiphophorus birchmanni and X. cortezi to address this fundamental question. We find that local ancestry in one hybrid population is remarkably predictive of local ancestry in another, demographically independent hybrid population. Applying newly developed methods, we can attribute much of this repeatability to strong selection in the earliest generations after initial hybridization. We complement these analyses with time-series data that demonstrates that ancestry at regions under selection has remained stable over the past ~40 generations of evolution. Finally, we compare our results to the well-studied X. birchmanni×X. malinche hybrid populations and conclude that deeper evolutionary divergence has resulted in stronger selection and higher repeatability in patterns of local ancestry in hybrids between X. birchmanni and X. cortezi.

Antagonistic selection on body size and sword length in a wild population of the swordtail fish, Xiphophorus multilineatus: Potential for intralocus tactical conflict.

Alternative reproductive tactics (ARTs) have provided valuable insights into how sexual selection and life history trade-offs can lead to variation within a sex. However, the possibility that tactics may constrain evolution through intralocus tactical conflict (IATC) is rarely considered. In addition, when IATC has been considered, the focus has often been on the genetic correlations between the ARTs, while evidence that the ARTs have different optima for associated traits and that at least one of the tactics is not at its optimum is often missing. Here, we investigate selection on three traits associated with the ARTs in the swordtail fish Xiphophorus multilineatus; body size, body shape, and the sexually selected trait for which these fishes were named, sword length (elongation of the caudal fin). All three traits are tactically dimorphic, with courter males being larger, deeper bodied and having longer swords, and the sneaker males being smaller, more fusiform and having shorter swords. Using measures of reproductive success in a wild population we calculated selection differentials, as well as linear and quadratic gradients. We demonstrated that the tactics have different optima and at least one of the tactics is not at its optimum for body size and sword length. Our results provide the first evidence of selection in the wild on the sword, an iconic trait for sexual selection. In addition, given the high probability that these traits are genetically correlated to some extent between the two tactics, our study suggests that IATC is constraining both body size and the sword from reaching their phenotypic optima. We discuss the importance of considering the role of IATC in the evolution of tactical dimorphism, how this conflict can be present despite tactical dimorphism, and how it is important to consider this conflict when explaining not only variation within a species but differences across species as well.

Feeding Rates in the Swordtail Fish Xiphophorus multilineatus: A Model System for Genetic Variation in Nutritional Programming.

A better understanding of the role of appetite regulation in obesity and metabolic disorders requires consideration of both genetic and developmental influences on appetite. Previously we detected genetic differences in responses to nutritional programming (e.g., the permanent influence that nutrition in early life has on the physiological and metabolic states in adults) on a presumed measure of appetite (feeding rate) in the swordtail fish Xiphophorus multilineatus. In this study we validate that feeding rate is a good measure of appetite, by first demonstrating that it is repeatable and correlated with food consumed when controlling for body size. Second, we detected a significant positive correlation between juvenile growth rates and feeding rates measured in adult males, when growth has ceased. In addition, feeding rates explained significant variation in the size of the nuchal hump, a fat deposit that develops after sexual maturity. Finally, we show that the feeding rates of "courter" males were significantly greater than "sneaker" males, alternative reproductive tactics that are influenced by variation in the Mc4r gene on the Y-chromosome. Our results suggest that examining feeding rate in X. multilineatus could provide valuable insights into how nutritional programming influences appetite independently from food intake, as well as insights into the mechanisms that produce the correlation between delayed maturation and faster growth rates of the courter males as compared with the sneaker males that mature early and grow slower in this species.

Condition-dependent female preference for male genitalia length is based on male reproductive tactics.

There is extensive morphological variation of male genitalia across animals with internal fertilization, even among closely related species. Most studies attempting to explain this extraordinary diversity have focused on processes that occur post-copula (e.g. sperm competition, cryptic female choice). Only a few studies have focused on the pre-copula process of female preference. In addition, the extent to which this variation could be associated with the use of different reproductive tactics has yet to be explored. Here, we show that female preference for male genitalia length in two livebearing fishes depends on the type of reproductive tactic of the males being evaluated as well as the body condition of the female. In a species where all males coax females to acquire matings (courters), females preferred males with short genitalia. In a species with genetically influenced alternative reproductive tactics (courter males that only court and produce courter sons, sneaker males that use the coercive tactic of sneak chase and produce sneaker sons), female preference depended on an interaction between male tactic and female condition: females in good condition preferred courter males with short genitalia, and sneaker males with long genitalia. Our results suggest that female preference for male traits favourable to their sons may be an important factor contributing to the diversification of male genitalia. Despite the contrasting selection for genitalia length that our female preference tests suggest, we found no significant differences in genitalia length between coaxing (courters) and coercive (sneakers) males. Our study represents a starting point to more clearly understand the role of alternative reproductive tactics and variation in female mate preference in the evolution of male genitalia.

Transcriptome assembly and candidate genes involved in nutritional programming in the swordtail fish Xiphophorus multilineatus.

BACKGROUND: Nutritional programming takes place in early development. Variation in the quality and/or quantity of nutrients in early development can influence long-term health and viability. However, little is known about the mechanisms of nutritional programming. The live-bearing fish Xiphophorus multilineatus has the potential to be a new model for understanding these mechanisms, given prior evidence of nutritional programming influencing behavior and juvenile growth rate. We tested the hypotheses that nutritional programming would influence behaviors involved in energy homeostasis as well gene expression in X. multilineatus. METHODS: We first examined the influence of both juvenile environment (varied in nutrition and density) and adult environment (varied in nutrition) on behaviors involved in energy acquisition and energy expenditure in adult male X. multilineatus. We also compared the behavioral responses across the genetically influenced size classes of males. Males stop growing at sexual maturity, and the size classes of can be identified based on phenotypes (adult size and pigment patterns). To study the molecular signatures of nutritional programming, we assembled a de novo transcriptome for X. multilineatus using RNA from brain, liver, skin, testis and gonad tissues, and used RNA-Seq to profile gene expression in the brains of males reared in low quality (reduced food, increased density) and high quality (increased food, decreased density) juvenile environments. RESULTS: We found that both the juvenile and adult environments influenced the energy intake behavior, while only the adult environment influenced energy expenditure. In addition, there were significant interactions between the genetically influenced size classes and the environments that influenced energy intake and energy expenditure, with males from one of the four size classes (Y-II) responding in the opposite direction as compared to the other males examined. When we compared the brains of males of the Y-II size class reared in a low quality juvenile environment to males from the same size class reared in high quality juvenile environment, 131 genes were differentially expressed, including metabolism and appetite master regulator agrp gene. DISCUSSION: Our study provides evidence for nutritional programming in X. multilineatus, with variation across size classes of males in how juvenile environment and adult diet influences behaviors involved in energy homeostasis. In addition, we provide the first transcriptome of X. multilineatus, and identify a group of candidate genes involved in nutritional programming.

Maternal investment in the swordtail fish Xiphophorus multilineatus: support for the differential allocation hypothesis.

The differential allocation hypothesis predicts that reproductive investment will be influenced by mate attractiveness, given a cost to reproduction and a tradeoff between current and future reproduction. We tested the differential allocation hypothesis in the swordtail fish Xiphophorus multilineatus, where males have genetically influenced (patroclinous inheritance) alternative mating tactics (ARTs) maintained by a tradeoff between being more attractive to females (mature later as larger courting males) and a higher probability of reaching sexual maturity (mature earlier as smaller sneaker males). Males in X. multilineatus do not provide parental care or other resources to the offspring. Allelic variation and copy number of the Mc4R gene on the Y-chromosome influences the size differences between males, however there is no variation in this gene on the X-chromosome. Therefore, to determine if mothers invested more in offspring of the larger courter males, we examined age to sexual maturity for daughters. We confirmed a tradeoff between number of offspring and female offspring's age to sexual maturity, corroborating that there is a cost to reproduction. In addition, the ART of their fathers significantly influenced the age at which daughters reached sexual maturity, suggesting increased maternal investment to daughters of courter males. The differential allocation we detected was influenced by how long the wild-caught mother had been in the laboratory, as there was a brood order by father genotype (ART) interaction. These results suggest that females can adjust their reproductive investment strategy, and that differential allocation is context specific. We hypothesize that one of two aspects of laboratory conditions produced this shift: increased female condition due to higher quality diet, and/or assessment of future mating opportunities due to isolation from males.

A molecular genetic examination of the mating system of pumpkinseed sunfish reveals high pay-offs for specialized sneakers.

Intrasexual variation in reproductive behaviour and morphology are common in nature. Often, such variation appears to result from conditional strategies in which some individuals (e.g. younger males or those in poor condition) adopt a low pay-off phenotype as a 'best of a bad job'. Alternatively, reproductive polymorphisms can be maintained by balancing selection, with male phenotypes having equal fitnesses at equilibrium, but examples from nature are rare. Many species of sunfish (genus Lepomis) are thought to have alternative male reproductive behaviours, but most empirical work has focused on the bluegill sunfish and the mating systems of other sunfish remain poorly understood. We studied a population of pumpkinseed sunfish (Lepomis gibbosus) in upstate New York. Field observations confirm the existence of two male reproductive strategies: 'parentals' were relatively old and large males that maintained nests, and 'sneakers' were relatively young and small males that fertilize eggs by darting into nests of parentals during spawning. The sneaker and parental male strategies appear to be distinct life-history trajectories. Sneaker males represented 39% of the males observed spawning, and sneakers intruded on 43% of all mating attempts. Microsatellite analyses revealed that sneaker males fertilized an average of 15% of the eggs within a nest. This level of paternity by sneaker males appears to be higher than seen in most other fishes, and preliminary analyses suggest that the two male reproductive strategies are maintained as a balanced polymorphism.

Larger swordtail females prefer asymmetrical males.

Many organisms, including humans, find symmetry more attractive than asymmetry. Is this bias towards symmetry simply a by-product of their detection system? We examined female preference for symmetry of the pigment pattern vertical bars in the swordtail fishes Xiphophorus cortezi and Xiphophorus malinche. We found a relationship between preference for symmetry and female size, with larger and thus older females spending significantly more time with the asymmetrical video animation as compared to the symmetrical video animation. The preference for asymmetry we report demonstrates that even if females can detect symmetrical males better, this does not preclude subsequent selection on females to prefer symmetrical or asymmetrical males. In addition, because the preference was correlated with female size, past studies may have missed preference for either asymmetry and/or symmetry by not examining the relationship between female preference and size/age or by measuring a limited size/age distribution of females. In both of the species of swordtail fishes examined, a high proportion of males are asymmetrical by more than one bar. We suggest that female preference may be maintaining fluctuating asymmetries in these fishes.

Patterns of Parental Investment and Sexual Selection in Teleost Fishes: Do They Support Bateman's Principles?

Bateman demonstrated differences in variance for fertility and mating success between the sexes, with males usually having a greater variance than females. Thus in general, male reproductive success increases with number of mates acquired. These results have been referred to as "Bateman's principles" and taken together with other parameters (e.g., relative parental investment) have been proposed to estimate a component of sexual selection. For this review I examine patterns of parental care and sexual selection in teleost fishes (substrate brooding and with internal fertilization). I present data for the pumpkinseed sunfish Lepomis gibbosus, in which I estimated cost of paternal care and compared direct measures of the intensity of selection on possible sexually selected traits to measures of sexual selection based on Bateman's principles.Despite high levels of paternal care in substrate brooding fishes, sexual selection tends to act more strongly on males than on females, which suggests that maternal investment is higher than paternal investment and that parental care does not limit the reproductive rate for males. In pumpkinseed sunfish, selection favors parents with high levels of defense that may exclude predators more effectively and, as suggested by Bateman's measures, alternative reproductive strategies may decrease the opportunity for sexual selection within the parental strategy. In teleost fishes with internal fertilization, patterns of parental investment and intensity of sexual selection seem to support Bateman's principles, but further studies using these systems and these measures of selection will improve the understanding of factors affecting the intensity of sexual selection and its relation to mating systems.