Genetic incompatibilities in killifish and the role of environment

Measuring reproductive isolation across multiple generations and environments is a key endeavor in speciation research because it indicates which isolating barriers currently prevent introgression and the extent to which they are intrinsic versus environmentally dependent. Here, I present data from several crosses (parental crosses, F1s, F2s, back-crosses) between two species of killifish (Lucania goodei and L. parva) that have diverged along a salinity gradient (L. goodei--freshwater, L. parva--euryhaline). Offspring were raised under high and low salinity to test for (1) extrinsic isolation, (2) intrinsic isolation manifested through genetic incompatibilities, and (3) environmentally dependent genetic incompatibilities. I found evidence for both intrinsic and extrinsic isolation, but no evidence for environmentally dependent genetic incompatibilities. The presence of extrinsic and intrinsic isolation varied among fitness measures, and all forms of reproductive isolation were asymmetric. Early egg survival was independent of salinity, but demonstrated pronounced intrinsic isolation. Both extrinsic and intrinsic isolation existed for egg hatching and survival of fry to the eating stage. Unfortunately, the order in which extrinsic and intrinsic isolation arose is unresolved. Understanding the extent to which adaptation to salinity creates multiple forms of reproductive isolation is critical for understanding diversification in many fish taxa.     

Genomic landscape of reproductive isolation in Lucania killifish: The role of sex loci and salinity

Adaptation to different environments can directly and indirectly generate reproductive isolation between species. Bluefin killifish (Lucania goodei) and rainwater killifish (L. parva) are sister species that have diverged across a salinity gradient and are reproductively isolated by habitat, behavioural, extrinsic and intrinsic post‐zygotic isolation. We asked if salinity adaptation contributes indirectly to other forms of reproductive isolation via linked selection and hypothesized that low recombination regions, such as sex chromosomes or chromosomal rearrangements, might facilitate this process. We conducted QTL mapping in backcrosses between L. parva and L. goodei to explore the genetic architecture of salinity tolerance, behavioural isolation and intrinsic isolation. We mapped traits relative to a chromosome that has undergone a centric fusion in L. parva (relative to L. goodei). We found that the sex locus appears to be male determining (XX‐XY), was located on the fused chromosome and was implicated in intrinsic isolation. QTL associated with salinity tolerance were spread across the genome and did not overly co‐localize with regions associated with behavioural or intrinsic isolation. This preliminary analysis of the genetic architecture of reproductive isolation between Lucania species does not support the hypothesis that divergent natural selection for salinity tolerance led to behavioural and intrinsic isolation as a by‐product. Combined with previous studies in this system, our work suggests that adaptation as a function of salinity contributes to habitat isolation and that reinforcement may have contributed to the evolution of behavioural isolation instead, possibly facilitated by linkage between behavioural isolation and intrinsic isolation loci on the fused chromosome.     

A TEST FOR ENVIRONMENTAL EFFECTS ON BEHAVIORAL ISOLATION IN TWO SPECIES OF KILLIFISH

Behavioral isolation is a common and potent mechanism of reproductive isolation. Determining the extent to which behavioral isolation varies with environmental conditions is critical to understanding speciation and the maintenance of species boundaries. Here, we tested the effect of salinity on behavioral isolation (female species recognition, male–male competition, and male species recognition) between two closely related killifish (Lucania goodei and L. parva) that differ in salinity tolerance. We performed no‐choice assays and behavioral trials where males could compete and court females in fresh water (0 ppt) and brackish water (15 ppt). We found high levels of behavioral isolation that did not vary as a function of salinity. In behavioral trials, male species recognition of females was strong and asymmetric between the two species. Lucania goodei males preferred conspecifics and rarely courted or mated with L. parva females. Lucania parva males preferred conspecifics but readily courted and mated with L. goodei females. This asymmetry matches previously documented asymmetries in hybrid offspring fitness. Crosses between L. parva males and L. goodei females produce fully viable/fertile hybrids, but crosses between L. goodei males and L. parva females produce males with reduced fertility. Hence, behavioral isolation may have evolved in part due to reinforcement.     

A Mathematical Technique for Estimating Blastodisc:Yolk Volume Ratios instead of Egg Sizes

Egg size alone is a poor and misleading variable in life-history studies. A mathematical technique for estimating yolk and blastodisc volume ratios in fishes, a much more meaningful character, is generated from first principles. The technique is demonstrated with an example of early ontogeny in fishes of the genus Lucania (Pisces: Cyprinodontidae). Wild, adult rainwater killifish, Lucania parva, and bluefin killifish, L. goodei, were collected in Florida and transported to the laboratory, where offspring were reared under controlled conditions. Offspring were sampled at the onset of cleavage, for simple measurements of yolk and blastodisc morphology. Application of mathematical equations allowed estimates of yolk and blastodisc volumes in the two species. No significant differences were found in clutch size, blastodisc volume, or egg density; however, significant differences existed in the absolute yolk investments, and blastodisc:yolk volume ratios. These differences in reproductive investment within the genus Lucania are interpreted by the altricial-precocial life-history model as a possible causal mechanism in the evolution of species within this genus. The mathematical equations presented in this study enabled us to partition reproductive investment into components that are more biologically meaningful than simple egg size.     

Evidence of prezygotic isolation,but not assortative mating,between locally adapted populations of Fundulus heteroclitus across a salinity gradient

Selection along environmental gradients can drive reproductive isolation and speciation. Among fishes, salinity is a major factor limiting species distributions, and despite its importance in generating species diversity, speciation events between marine and freshwater are rare. Here, we tested for mechanisms of reproductive isolation between locally adapted freshwater and brackish water-native populations of killifish, Fundulus heteroclitus, from either side of a hybrid zone along a salinity gradient. There was evidence for pre-zygotic endogenous reproductive isolation with reduced fertilization success between crosses of freshwater-native males and brackish water-native females. Exogenous pre-zygotic isolation was also present where females had highest fertilization in their native salinity. We used a replicated mass spawning design to test for mate choice in both brackish and fresh water. After genotyping 187 parents and 2523 offspring at 2347 SNPs across the genome, 85% of offspring were successfully assign to their parents. However, no reinforcing mate choice was observed. These results therefore demonstrate emerging, yet limited, reproductive isolation and incipient speciation across a marine to freshwater salinity gradient and suggest that both endogenous and exogenous mechanisms, but not assortative mating, contribute to divergence.     

Fine-scale genetic analysis of species-specific female preference in Drosophila simulans

Behavioural differences are thought to be the first components to contribute to species isolation, yet the precise genetic basis of behavioural isolation remains poorly understood. Here, we used a combination of behaviour assays and genetic mapping to provide the first refined map locating candidate genes for interspecific female preference isolating Drosophila simulans from D. melanogaster. First, we tested whether two genes identified as affecting D. melanogaster female intraspecific mate choice also affect interspecific mate choice; neither of these genes was found to contribute to species‐specific female preference. Next, we used deficiency mapping to locate genes on the right arm of the third chromosome for species‐specific female preference and identified five small significant regions that contain candidate genes contributing to behavioural isolation. All five regions were located in areas that would have low interspecific recombination, which mirrors the results of other behavioural isolation studies that used quantitative trait locus (QTL) mapping, but without the potential concern of bias towards regions of low recombination that QTL mapping may have. As this model system may be refined to the individual gene level using the same methodology, this initial map we provide may potentially serve as a ready template for the identification and characterization of the first behavioural isolation genes.     

THE BIOLOGY OF SPECIATION

Since Darwin published the “Origin,” great progress has been made in our understanding of speciation mechanisms. The early investigations by Mayr and Dobzhansky linked Darwin's view of speciation by adaptive divergence to the evolution of reproductive isolation, and thus provided a framework for studying the origin of species. However, major controversies and questions remain, including: When is speciation nonecological? Under what conditions does geographic isolation constitute a reproductive isolating barrier? and How do we estimate the “importance” of different isolating barriers? Here, we address these questions, providing historical background and offering some new perspectives. A topic of great recent interest is the role of ecology in speciation. “Ecological speciation” is defined as the case in which divergent selection leads to reproductive isolation, with speciation under uniform selection, polyploid speciation, and speciation by genetic drift defined as “nonecological.” We review these proposed cases of nonecological speciation and conclude that speciation by uniform selection and polyploidy normally involve ecological processes. Furthermore, because selection can impart reproductive isolation both directly through traits under selection and indirectly through pleiotropy and linkage, it is much more effective in producing isolation than genetic drift. We thus argue that natural selection is a ubiquitous part of speciation, and given the many ways in which stochastic and deterministic factors may interact during divergence, we question whether the ecological speciation concept is useful. We also suggest that geographic isolation caused by adaptation to different habitats plays a major, and largely neglected, role in speciation. We thus provide a framework for incorporating geographic isolation into the biological species concept (BSC) by separating ecological from historical processes that govern species distributions, allowing for an estimate of geographic isolation based upon genetic differences between taxa. Finally, we suggest that the individual and relative contributions of all potential barriers be estimated for species pairs that have recently achieved species status under the criteria of the BSC. Only in this way will it be possible to distinguish those barriers that have actually contributed to speciation from those that have accumulated after speciation is complete. We conclude that ecological adaptation is the major driver of reproductive isolation, and that the term “biology of speciation,” as proposed by Mayr, remains an accurate and useful characterization of the diversity of speciation mechanisms.     

Testing alternative hypotheses for evolutionary diversification in an African songbird: rainforest refugia versus ecological gradients

Geographic isolation in rainforest refugia and local adaptation to ecological gradients may both be important drivers of evolutionary diversification. However, their relative importance and the underlying mechanisms of these processes remain poorly understood because few empirical studies address both putative processes in a single system. A key question is to what extent is divergence in signals that are important in mate and species recognition driven by isolation in rainforest refugia or by divergent selection across ecological gradients? We studied the little greenbul, Andropadus virens, an African songbird, in Cameroon and Uganda, to determine whether refugial isolation or ecological gradients better explain existing song variation. We then tested whether song variation attributable to refugial or ecological divergence was biologically meaningful using reciprocal playback experiments to territorial males. We found that much of the existing song variation can be explained by both geographic isolation and ecological gradients, but that divergence across the gradient, and not geographic isolation, affects male response levels. These data suggest that ecologically divergent traits, independent of historical isolation during glacial cycles, can promote reproductive isolation. Our study provides further support for the importance of ecology in explaining patterns of evolutionary diversification in ecologically diverse regions of the planet.     

Immigrant reproductive dysfunction facilitates ecological speciation

The distributions of species are not only determined by where they can survive – they must also be able to reproduce. Although immigrant inviability is a well‐established concept, the fact that immigrants also need to be able to effectively reproduce in foreign environments has not been fully appreciated in the study of adaptive divergence and speciation. Fertilization and reproduction are sensitive life‐history stages that could be detrimentally affected for immigrants in non‐native habitats. We propose that “immigrant reproductive dysfunction” is a hitherto overlooked aspect of reproductive isolation caused by natural selection on immigrants. This idea is supported by results from experiments on an externally fertilizing fish (sand goby, Pomatoschistus minutus). Growth and condition of adults were not affected by non‐native salinity whereas males spawning as immigrants had lower sperm motility and hatching success than residents. We interpret these results as evidence for local adaptation or acclimation of sperm, and possibly also components of paternal care. The resulting loss in fitness, which we call “immigrant reproductive dysfunction,” has the potential to reduce gene flow between populations with locally adapted reproduction, and it may play a role in species distributions and speciation.     

Sexual imprinting on ecologically divergent traits leads to sexual isolation in sticklebacks

During sexual imprinting, offspring learn parental phenotypes and then select mates who are similar to their parents. Imprinting has been thought to contribute to the process of speciation in only a few rare cases; this is despite imprinting's potential to generate assortative mating and solve the problem of recombination in ecological speciation. If offspring imprint on parental traits under divergent selection, these traits will then be involved in both adaptation and mate preference. Such 'magic traits' easily generate sexual isolation and facilitate speciation. In this study, we show that imprinting occurs in two ecologically divergent stickleback species (benthics and limnetics: Gasterosteus spp.). Cross-fostered females preferred mates of their foster father's species. Furthermore, imprinting is essential for sexual isolation between species; isolation was reduced when females were raised without fathers. Daughters imprinted on father odour and colour during a critical period early in development. These traits have diverged between the species owing to differences in ecology. Therefore, we provide the first evidence that imprinting links ecological adaptation to sexual isolation between species. Our results suggest that imprinting may facilitate the evolution of sexual isolation during ecological speciation, may be especially important in cases of rapid diversification, and thus play an integral role in the generation of biodiversity.     

Mating asymmetry and the direction of evolution in the Hawaiian cricket genus Laupala

Based on studies from native Hawaiian Drosophila, a model was proposed to explain sexual isolation and mating asymmetry, from which one could potentially infer the 'direction of evolution'. We examined sexual isolation between allopatric cricket species of the genus Laupala, another endemic Hawaiian insect with an elaborate mating system, to begin to explore the nature of sexual isolation and mating asymmetry in closely related Hawaiian organisms. We studied sexual isolation and mating asymmetry in two contrasts. First, an inter-island comparison, including L. makaio from the older island of Maui and L. paranigra from the younger island of Hawaii, and second, an intra-island (Hawaii) comparison, including L. nigra from the older volcano of Mauna Kea and L. paranigra with a primary distribution on the younger volcanoes of Mauna Loa and Kilauea. We used a 'no-choice' experimental design, pairing individual males and females in homospecific or heterospecific combinations. Several behavioural aspects of courtship (proportion of male singing, latency to male singing, production of spermatophores and courtship initiation speed) were quantified as well as the success or failure of matings. We demonstrate asymmetry in sexual isolation between reciprocal combinations of L. makaio and L. paranigra. This result is examined in light of the differences in courtship behaviour manifest in the experiments with these two species. We did not find evidence of asymmetry in sexual isolation between L. nigra and L. paranigra, although differences in courtship initiation speed were evident between reciprocal combinations of these two species. In addition to the geological argument that species on older islands and older volcanoes give rise to species on younger islands and younger volcanoes, we discuss phylogenetic evidence consistent with these biogeographic hypotheses of relationships among the focal taxa. The patterns of asymmetrical sexual isolation and mating asymmetry are consistent with those found in the native Hawaiian Drosophila.     

The effects of water depth and light on oviposition and egg cannibalism in the bluefin killifish Lucania goodei

This study showed that sex and depth had strong effects on egg cannibalism, whereas water clarity (clear v. tea-stained) had no effect on cannibalism or oviposition in the bluefin killifish Lucania goodei. These results are consistent with the extreme levels of iteroparity in L. goodei where females appear to spread their eggs across multiple locations and depths presumably to avoid egg predation.     

DOES THE DESATURASE‐2 LOCUS IN DROSOPHILA MELANOGASTER CAUSE ADAPTATION AND SEXUAL ISOLATION?

The desaturase-2 (desat2) locus of Drosophila melanogaster has two alleles whose frequencies vary geographically: one (the "Z" allele) is found primarily in east Africa and the Caribbean, and the other (the "M" allele) occurs in other parts of the world. It has been suggested that these alleles not only cause sexual isolation between races, but that their distribution reflects differential adaptation to climate: Z alleles are supposedly adapted to tropical conditions and M alleles to temperate ones. This has thus been viewed as a case of reproductive isolation evolving as a pleiotropic byproduct of adaptation. Here we reinvestigate this presumed climatic adaptation, using transgenic lines differing in the nature of their desat2 alleles. We were unable to replicate earlier results showing that carriers of M alleles are uniformly more cold resistant and less starvation resistant than carriers of Z alleles. It is thus doubtful whether the distribution of these alleles reflects natural selection involving climate. Mating studies of transgenic lines show some evidence for sexual isolation due to desat2. However, work on other, wild-type lines, as well as observations on the nature of sexual isolation, suggest that this conclusion--and thus the relationship between this locus and mating discrimination between races of D. melanogaster--may also be doubtful.     

Behavioural mechanisms of sexual isolation involving multiple modalities and their inheritance

Speciation research dissects the genetics and evolution of reproductive barriers between parental species. Hybrids are the “gatekeepers” of gene flow, so it is also important to understand the behavioural mechanisms and genetics of any potential isolation from their parental species. We tested the role of multiple behavioural barriers in reproductive isolation among closely related field crickets and their hybrids (Teleogryllus oceanicus and Teleogryllus commodus). These species hybridize in the laboratory, but the behaviour of hybrids is unusual and there is little evidence for gene flow in the wild. We found that heterospecific pairs exhibited reduced rates of courtship behaviour due to discrimination by both sexes, and that this behavioural isolation was symmetrical. However, hybrids were not sexually selected against and exhibited high rates of courtship behaviour even though hybrid females are sterile. Using reciprocal hybrid crosses, we characterized patterns of interspecific divergence and inheritance in key sexual traits that might underlie the mating patterns we found: calling song, courtship song and cuticular hydrocarbons (CHCs). Song traits exhibited both sex linkage and transgressive segregation, whereas CHCs exhibited only the latter. Calculations of the strength of isolation exerted by these sexual traits suggest that close‐range signals are as important as long‐distance signals in contributing to interspecific sexual isolation. The surprisingly weak mating barriers observed between hybrids and parental species highlight the need to examine reproductive isolating mechanisms and their genetic bases across different potential stages of introgressive hybridization.     

Ecological selection maintains cytonuclear incompatibilities in hybridizing sunflowers

Despite the recent renaissance in studies of ecological speciation, the connection between ecological selection and the evolution of reproductive isolation remains tenuous. We tested whether habitat adaptation of cytoplasmic genomes contributes to the maintenance of reproductive barriers in hybridizing sunflower species, Helianthus annuus and Helianthus petiolaris. We transplanted genotypes of the parental species, reciprocal F1 hybrids and all eight possible backcross combinations of nuclear and cytoplasmic genomes into the contrasting xeric and mesic habitats of the parental species. Analysis of survivorship across two growing seasons revealed that the parental species' cytoplasms were strongly locally adapted and that cytonuclear interactions (CNIs) significantly affected the fitness and architecture of hybrid plants. A significant fraction of the CNIs have transgenerational effects, perhaps due to divergence in imprinting patterns. Our results suggest a common means by which ecological selection may contribute to speciation and have significant implications for the persistence of hybridizing species.     

Developmental effects of visual environment on species‐assortative mating preferences in Lake Victoria cichlid fish

Local adaptation can be a potent force in speciation, with environmental heterogeneity leading to niche specialization and population divergence. However, local adaption often requires nonrandom mating to generate reproductive isolation. Population divergence in sensory properties can be particularly consequential in speciation, affecting both ecological adaptation and sexual communication. Pundamilia pundamila and Pundamilia nyererei are two closely related African cichlid species that differ in male coloration, blue vs. red. They co‐occur at rocky islands in southern Lake Victoria, but inhabit different depth ranges with different light environments. The species differ in colour vision properties, and females exert species‐specific preferences for blue vs. red males. Here, we investigated the mechanistic link between colour vision and preference, which could provide a rapid route to reproductive isolation. We tested the behavioural components of this link by experimentally manipulating colour perception – we raised both species and their hybrids under light conditions mimicking shallow and deep habitats – and tested female preference for blue and red males under both conditions. We found that rearing light significantly affected female preference: shallow‐reared females responded more strongly to P. pundamilia males and deep‐reared females favoured P. nyererei males – implying that visual development causally affects mate choice. These results are consistent with sensory drive predictions, suggesting that the visual environment is key to behavioural isolation of these species. However, the observed plasticity could also make the species barrier vulnerable to environmental change: species‐assortative preferences were weaker in females that were reared in the other species’ light condition.     

A test for negative frequency-dependent mating success as a function of male colour pattern in the bluefin killifish

Rare male mating advantage (a form of negative frequency dependence) is frequently proposed as a mechanism for the maintenance of genetic variation within populations. This hypothesis is attractive for systems with pronounced male colour polymorphism because it can maintain particularly high levels of variation. We tested for negative frequency-dependent mating success between yellow and red male colour patterns in bluefin killifish, Lucania goodei . Lucania goodei populations harbour substantial colour pattern polymorphism, and a large proportion of this variation has a genetic basis. We established outdoor mesocosms with red and yellow males in three different ratios: yellow rare (one yellow ♂ : five red ♂), even (three yellow ♂ : three red ♂), and red rare (five yellow ♂ : one red ♂). We obtained eggs and used microsatellites to determine paternity. By contrast to expectations, we found no support for a rare male mating advantage. Red males had slightly higher spawning success than yellow males, particularly in replicates with large clutches and when red males were rare. However, yellow males did not have higher mating success when rare. We discuss alternative mechanisms for the maintenance of the polymorphism as well as the potential reasons for the lack of a rare male mating advantage.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 489–500.     

Incompatibility between X chromosome factor and pericentric heterochromatic region causes lethality in hybrids between Drosophila melanogaster and its sibling species

The Dobzhansky-Muller model posits that postzygotic reproductive isolation results from the evolution of incompatible epistatic interactions between species: alleles that function in the genetic background of one species can cause sterility or lethality in the genetic background of another species. Progress in identifying and characterizing factors involved in postzygotic isolation in Drosophila has remained slow, mainly because Drosophila melanogaster, with all of its genetic tools, forms dead or sterile hybrids when crossed to its sister species, D. simulans, D. sechellia, and D. mauritiana. To circumvent this problem, we used chromosome deletions and duplications from D. melanogaster to map two hybrid incompatibility loci in F(1) hybrids with its sister species. We mapped a recessive factor to the pericentromeric heterochromatin of the X chromosome in D. simulans and D. mauritiana, which we call heterochromatin hybrid lethal (hhl), which causes lethality in F(1) hybrid females with D. melanogaster. As F(1) hybrid males hemizygous for a D. mauritiana (or D. simulans) X chromosome are viable, the lethality of deficiency hybrid females implies that a dominant incompatible partner locus exists on the D. melanogaster X. Using small segments of the D. melanogaster X chromosome duplicated onto the Y chromosome, we mapped a dominant factor that causes hybrid lethality to a small 24-gene region of the D. melanogaster X. We provide evidence suggesting that it interacts with hhl(mau). The location of hhl is consistent with the emerging theme that hybrid incompatibilities in Drosophila involve heterochromatic regions and factors that interact with the heterochromatin.     

Panmixia across elevation in thermally sensitive Andean dung beetles

Janzen's seasonality hypothesis predicts that organisms inhabiting environments with limited climatic variability will evolve a reduced thermal tolerance breadth compared with organisms experiencing greater climatic variability. In turn, narrow tolerance breadth may select against dispersal across strong temperature gradients, such as those found across elevation. This can result in narrow elevational ranges and generate a pattern of isolation by environment or neutral genetic differentiation correlated with environmental variables that are independent of geographic distance. We tested for signatures of isolation by environment across elevation using genome‐wide SNP data from five species of Andean dung beetles (subfamily Scarabaeinae) with well‐characterized, narrow thermal physiologies, and narrow elevational distributions. Contrary to our expectations, we found no evidence of population genetic structure associated with elevation and little signal of isolation by environment. Further, elevational ranges for four of five species appear to be at equilibrium and show no decay of genetic diversity at range limits. Taken together, these results suggest physiological constraints on dispersal may primarily operate outside of a stable realized niche and point to a lower bound on the spatial scale of local adaptation.