Heidi S. Fisher

Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species

The ability to efficiently and accurately determine genotypes is a keystone technology in modern genetics, crucial to studies ranging from clinical diagnostics, to genotype-phenotype association, to reconstruction of ancestry and the detection of selection. To date, high capacity, low cost genotyping has been largely achieved via “SNP chip” microarray-based platforms which require substantial prior knowledge of both genome sequence and variability, and once designed are suitable only for those targeted variable nucleotide sites. This method introduces substantial ascertainment bias and inherently precludes detection of rare or population-specific variants, a major source of information for both population history and genotype-phenotype association. Recent developments in reduced-representation genome sequencing experiments on massively parallel sequencers (commonly referred to as RAD-tag or RADseq) have brought direct sequencing to the problem of population genotyping, but increased cost and procedural and analytical complexity have limited their widespread adoption. Here, we describe a complete laboratory protocol, including a custom combinatorial indexing method, and accompanying software tools to facilitate genotyping across large numbers (hundreds or more) of individuals for a range of markers (hundreds to hundreds of thousands). Our method requires no prior genomic knowledge and achieves per-site and per-individual costs below that of current SNP chip technology, while requiring similar hands-on time investment, comparable amounts of input DNA, and downstream analysis times on the order of hours. Finally, we provide empirical results from the application of this method to both genotyping in a laboratory cross and in wild populations. Because of its flexibility, this modified RADseq approach promises to be applicable to a diversity of biological questions in a wide range of organisms.

Alteration of the chemical environment disrupts communication in a freshwater fish.

It is well established that changes to the chemical environment can impair development, physiology and reproductive biology; by contrast, impacts on communication have not been widely reported. This is surprising given that chemical communication is the most widely used sensory modality in nature, and that variation in the chemical composition of the environment is the rule, not the exception. Here, we show that chemically mediated species recognition in a swordtail fish, Xiphophorus birchmanni, can be hindered by anthropogenic disturbance to the signalling environment. Females have a strong preference for conspecific male chemical cues, yet they hybridize in nature with the congener X. malinche. Wild-caught females showed a strong preference for conspecifics when tested in clean water, but failed to show a preference when tested in stream water subject to sewage effluent and agricultural runoff. We hypothesized that this was due to the interaction between chemical communication systems and humic acid (HA), a ubiquitous, natural product elevated to high levels by anthropogenic processes. When exposed to elevated concentrations of HA, female X. birchmanni again lost their preference for conspecific male chemical cues, while visual mating preferences and motivation to mate were retained. Sub-lethal concentrations of seemingly benign substances can thus have a drastic effect on natural populations through their specific impact on communication systems.

Competition drives cooperation among closely related sperm of deer mice

Among the extraordinary adaptations driven by sperm competition is the cooperative behaviour of spermatozoa. By forming cooperative groups, sperm can increase their swimming velocity and thereby gain an advantage in intermale sperm competition. Accordingly, selection should favour cooperation of the most closely related sperm to maximize fitness. Here we show that sperm of deer mice (genus Peromyscus) form motile aggregations, then we use this system to test predictions of sperm cooperation. We find that sperm aggregate more often with conspecific than heterospecific sperm, suggesting that individual sperm can discriminate on the basis of genetic relatedness. Next, we provide evidence that the cooperative behaviour of closely related sperm is driven by sperm competition. In a monogamous species lacking sperm competition, Peromyscus polionotus, sperm indiscriminately group with unrelated conspecific sperm. In contrast, in the highly promiscuous deer mouse, Peromyscus maniculatus, sperm are significantly more likely to aggregate with those obtained from the same male than with sperm from an unrelated conspecific donor. Even when we test sperm from sibling males, we continue to see preferential aggregations of related sperm in P. maniculatus. These results suggest that sperm from promiscuous deer mice discriminate among relatives and thereby cooperate with the most closely related sperm, an adaptation likely to have been driven by sperm competition.

Female swordtail fish use chemical cues to select well-fed mates

Selection favours females that attend to reliable information about male health or genetic quality. Male nutritional state can be an important indicator of mate quality since poor nutrition can be related to reduced sperm quality, low sperm quantity and sexually transmitted diseases. Here we show that female swordtail fish, Xiphophorus birchmanni, preferred the chemical cues of males that were well fed over those that had been food deprived. However, focal females did not discriminate among the odour of other females with respect to nutritional state, suggesting that they were responding to a sex-specific chemical cue, not simply to food excretions. It is therefore likely that nutritional condition is related to the production of sexual pheromones in males. Our results suggest that chemical cues convey information about male nutritional state, and that females attend to these cues during mate choice. By doing so, females are able to discriminate among males that may differ in important aspects of quality.

Replicated hybrid zones of Xiphophorus swordtails along an elevational gradient

Natural hybrid zones provide opportunities to study a range of evolutionary phenomena from speciation to the genetic basis of fitness‐related traits. We show that widespread hybridization has occurred between two neo‐tropical stream fishes with partial reproductive isolation. Phylogenetic analyses of mitochondrial sequence data showed that the swordtail fish Xiphophorus birchmanni is monophyletic and that X. malinche is part of an independent monophyletic clade with other species. Using informative single nucleotide polymorphisms in one mitochondrial and three nuclear intron loci, we genotyped 776 specimens collected from twenty‐three sites along seven separate stream reaches. Hybrid zones occurred in replicated fashion in all stream reaches along a gradient from high to low elevation. Genotyping revealed substantial variation in parental and hybrid frequencies among localities. Tests of FIS and linkage disequilibrium (LD) revealed generally low FIS and LD except in five populations where both parental species and hybrids were found suggesting incomplete reproductive isolation. In these locations, heterozygote deficiency and LD were high, which suggests either selection against early generation hybrids or assortative mating. These data lay the foundation to study the adaptive basis of the replicated hybrid zone structure and for future integration of behaviour and genetics to determine the processes that lead to the population genetic patterns observed in these hybrid zones.

Male swordtails court with an audience in mind

Females are usually considered to be the target of male courtship behaviour. In nature, however, social interactions rarely occur without other observers; thus, it is conceivable that some male courtship behaviours are directed not towards females, but rather towards male rivals. The northern swordtail, Xiphophorus birchmanni, is a freshwater fish found in high densities in natural streams. Males court by swimming close to and in parallel with the female, raising their large sail-like dorsal fin, and quivering briefly. Here, we show that females prefer males that display small dorsal fins to those with large ones, and that males are less aggressive to other males with large dorsal fins. Male swordtails also raise their dorsal fins more frequently when courting in the presence of other males. These results suggest that, despite female avoidance of large dorsal fins, males that raise their fin during courtship benefit by intimidating potential competitors; the intended receivers of this signal are thus males, not females. Intrasexual selection can therefore offset the forces of intersexual selection, even in a courtship display.

Odor familiarity and female preferences for males in a threatened primate, the pygmy loris Nycticebus pygmaeus: applications for genetic management of small populations

Here we use sexual selection theory to develop a logistically simple, yet effective, method for the manipulation of female reproductive behavior for conservation goals. Mate choice leading to nonrandom mating patterns can exacerbate the loss of genetic diversity in small populations. On theoretical grounds, females should choose high-quality mates. A prediction stemming from chemical communication theory is that competitive males will be better able to saturate an area with scent marks. If this is true, females should mate preferentially with males whose odors they encounter most frequently. We tested this hypothesis with the pygmy loris, Nycticebus pygmaeus, a threatened and poorly studied nocturnal prosimian. For several weeks females were exposed repeatedly to the urine from a particular male, and were then allowed to choose between a male whose odors were familiar and one whose odors were novel. Females showed an unusually strong preference for the familiar-odor male, as indicated by several behavioral measures of mate preference. Conservation managers can use this method as a tool to obtain reproductive pairings that will maximize genetic compatibility and diversity. For example, unsuccessful males may be given the opportunity to reproduce. In captive populations, studbook managers often select pairs in order to optimize outbreeding, but these selected pairings may not coincide with the preferences of the individual animals involved. Although several authors have made theoretical arguments for manipulating mate choice for conservation, this is a novel test of a proximate mechanism that can be manipulated, cultivating applications rather than mere implications.

Multivariate male traits misalign with multivariate female preferences in the swordtail fish, Xiphophorus birchmanni

Mate choice represents the outcome of a complex decision that is most often based on the integration of information on multiple traits expressed by potential mates. Multiple traits, however, can show phenotypic correlations, which may coincide with, or oppose, female preference for individual traits. Thus, if multivariate preferences are misaligned with trait covariation, variation in single traits can persist because of weakened net sexual selection. Male swordtails, Xiphophorus birchmanni, show positive covariation among three sexually dimorphic visual traits: body size, vertical bars and dorsal fin size. Using synthetic video animations, we isolated each trait and found that females preferred males with large bodies and vertical bars, but small dorsal fins, thus conflicting with male trait covariation. We then tested females using live wild-caught males and found that females preferred smaller dorsal fins on larger males. We suggest that multivariate discordance between traits and preferences results in weak sexual selection on male visual traits, and may help explain the maintenance of variation in sexually dimorphic phenotypes.

Tactical Release of a Sexually-Selected Pheromone in a Swordtail Fish

Background Chemical communication plays a critical role in sexual selection and speciation in fishes; however, it is generally assumed that most fish pheromones are passively released since most fishes lack specialized scent glands or scent-marking behavior. Swordtails (genus Xiphophorus) are widely used in studies of female mate choice, and female response to male chemical cues is important to sexual selection, reproductive isolation, and hybridization. However, it is unclear whether females are attending to passively produced cues, or to pheromones produced in the context of communication. Methodology/Principal Findings We used fluorescein dye injections to visualize pulsed urine release in male sheepshead swordtails, Xiphophorus birchmanni. Simultaneous-choice assays of mating preference showed that females attend to species- and sex-specific chemical cues emitted in male urine. Males urinated more frequently in the presence and proximity of an audience (conspecific females). In the wild, males preferentially courted upstream of females, facilitating transmission of pheromone cues. Conclusions/Significance Males in a teleost fish have evolved sophisticated temporal and spatial control of pheromone release, comparable to that found in terrestrial animals. Pheromones are released specifically in a communicative context, and the timing and positioning of release favors efficient signal transmission.

The dynamics of sperm cooperation in a competitive environment

Sperm cooperation has evolved in a variety of taxa and is often considered a response to sperm competition, yet the benefit of this form of collective movement remains unclear. Here, we use fine-scale imaging and a minimal mathematical model to study sperm aggregation in the rodent genus Peromyscus. We demonstrate that as the number of sperm cells in an aggregate increase, the group moves with more persistent linearity but without increasing speed. This benefit, however, is offset in larger aggregates as the geometry of the group forces sperm to swim against one another. The result is a non-monotonic relationship between aggregate size and average velocity with both a theoretically predicted and empirically observed optimum of six to seven sperm per aggregate. To understand the role of sexual selection in driving these sperm group dynamics, we compared two sister-species with divergent mating systems. We find that sperm of Peromyscus maniculatus (highly promiscuous), which have evolved under intense competition, form optimal-sized aggregates more often than sperm of Peromyscus polionotus (strictly monogamous), which lack competition. Our combined mathematical and experimental study of coordinated sperm movement reveals the importance of geometry, motion and group size on sperm velocity and suggests how these physical variables interact with evolutionary selective pressures to regulate cooperation in competitive environments.