Allochronic speciation, secondary contact, and reproductive character displacement in periodical cicadas (Hemiptera: Magicicada spp.): genetic, morphological, and behavioural evidence

Periodical cicadas have proven useful in testing a variety of ecological and evolutionary hypotheses because of their unusual life history, extraordinary abundance, and wide geographical range. Periodical cicadas provide the best examples of synchronous periodicity and predator satiation in the animal kingdom, and are excellent illustrations of habitat partitioning (by the three morphologically distinct species groups), incipient species (the year classes or broods), and cryptic species (a newly discovered 13-year species, Magicicada neotredecim). They are particularly useful for exploring questions regarding speciation via temporal isolation, or allochronic speciation. Recently, data were presented that provided strong support for an instance of allochronic speciation by life-cycle switching. This speciation event resulted in the formation of a new 13-year species from a 17-year species and led to secondary contact between two formerly separated lineages, one represented by the new 13-year cicadas (and their 17-year ancestors), and the other represented by the pre-existing 13-year cicadas. Allozyme frequency data, mitochondrial DNA (mtDNA), and abdominal colour were shown to be correlated genetic markers supporting the life-cycle switching/allochronic speciation hypothesis. In addition, a striking pattern of reproductive character displacement in male call pitch and female pitch preference between the two 13-year species was discovered. In this paper we report a strong association between calling song pitch and mtDNA haplotype for 101 individuals from a single locality within the M. tredecim/M. neotredecim contact zone and a strong association between abdomen colour and mtDNA haplotype. We conclude by reviewing proposed mechanisms for allochronic speciation and reproductive character displacement.     

Relative abundance and the species-specific reinforcement of male mating preference in the Chrysochus (Coleoptera: Chrysomelidae) hybrid zone

Most studies of reinforcement have focused on the evolution of either female choice or male mating cues, following the long-held view in sexual selection theory that mating mistakes are typically more costly for females than for males. However, factors such as conspecific sperm precedence can buffer females against the cost of mating mistakes, suggesting that in some hybrid zones mating mistakes may be more costly for males than for females. Thus, the historical bias in reinforcement research may underestimate its frequency. In this study, we present evidence that reinforcement has driven the evolution of male choice in a hybrid zone between the highly promiscuous leaf beetles Chrysochus cobaltinus and C. auratus, the hybrids of which have extremely low fitness. In addition, there is evidence for male choice in these beetles and that male mating mistakes may be costly, due to reduced opportunities to mate with conspecific females. The present study combines laboratory and field methods to quantify the strength of sexual isolation, test the hypothesis of reproductive character displacement, and assess the link between relative abundance and the strength of selection against hybridization. We document that, while sexual isolation is weak, it is sufficient to produce positive assortative mating. In addition, reproductive character displacement was only detected in the relatively rare species. The strong postzygotic barriers in this system are sufficient to generate the bimodality that characterizes this hybrid zone, but the weak sexual isolation is not, calling into question whether strong prezygotic isolation is necessary for the maintenance of bimodality. Growing evidence that the cost of mating mistakes is sufficient to shape the evolution of male mate choice suggests that the reinforcement of male mate choice may prove to be a widespread occurrence.     

Quantified reproductive isolation in Heliconius butterflies: Implications for introgression and hybrid speciation

Heliconius butterflies have become a model for the study of speciation with gene flow. For adaptive introgression to take place, there must be incomplete barriers to gene exchange that allow interspecific hybridization and multiple generations of backcrossing. The recent publication of estimates of individual components of reproductive isolation between several species of butterflies in the Heliconius melpomene–H. cydno clade allowed us to calculate total reproductive isolation estimates for these species. According to these estimates, the butterflies are not as promiscuous as has been implied. Differences between species are maintained by intrinsic mechanisms, while reproductive isolation of geographical races within species is mainly due to allopatry. We discuss the implications of this strong isolation for basic aspects of the hybrid speciation with introgression hypothesis.     

Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement

The rapid evolution of sexual isolation in sympatry has long been associated with reinforcement (i.e., selection to avoid maladaptive hybridization). However, there are many species pairs in sympatry that have evolved rapid sexual isolation without known costs to hybridization. A major unresolved question is what evolutionary processes are involved in driving rapid speciation in such cases. Here, we focus on one such system; the Drosophila athabasca species complex, which is composed of three partially sympatric and interfertile semispecies: WN, EA, and EB. To study speciation in this species complex, we assayed sexual and genomic isolation within and between these semispecies in both sympatric and allopatric populations. First, we found no evidence of reproductive character displacement (RCD) in sympatric zones compared to distant allopatry. Instead, semispecies were virtually completely sexually isolated from each other across their entire ranges. Moreover, using spatial approaches and coalescent demographic simulations, we detected either zero or only weak heterospecific gene flow in sympatry. In contrast, within each semispecies we found only random mating and little population genetic structure, except between highly geographically distant populations. Finally, we determined that speciation in this system is at least an order of magnitude older than previously assumed, with WN diverging first, around 200K years ago, and EA and EB diverging 100K years ago. In total, these results suggest that these semispecies should be given full species status and we adopt new nomenclature: WN—D. athabasca, EA—D. mahican, and EB—D. lenape. While the lack of RCD in sympatry and interfertility do not support reinforcement, we discuss what additional evidence is needed to further decipher the mechanisms that caused rapid speciation in this species complex.     

Genital lock-and-key system and premating isolation by mate preference in carabid beetles (Carabus subgenus Ohomopterus)

The shapes and lengths of copulatory pieces and vaginal appendices of the carabid beetle subgenus Ohomopterus (genus Carabus) vary among species. In Japan, the species in the group with a medium body size (C. yaconinus, C. iwawakianus, C. maiyasanus, C. uenoi, C. arrowianus, C. esakii, and C. insulicola) are usually allopatric or parapatric, except at Mt Kongosan, where C. uenoi, C. iwawakianus, and C. yaconinus are sympatrically distributed. The degree of premating isolation by mate preference was high between sympatric populations, irrespective of the genetic distance between them. However, premating isolation was absent between parapatric populations. The degree of premating isolation for allopatric populations spanned a wide range of isolation values. Thus, mate discrimination by males seems to have evolved mostly between sympatric pairs. These results suggest two hypotheses. First, premating isolation has evolved through reinforcement or through reproductive character displacement after sympatric contact. Second, premating isolation has evolved in allopatry, and as a result of premating isolation, the species can coexist in sympatry. We also examined the degree of mechanical isolation between C. uenoi and C. iwawakianus (a sympatric pair), which have a very large difference in the length of the copulatory piece. The insertion success was low and only one female produced viable offspring among 15 crosses; however, death in females due to copulation was rare. For sympatric matings between C. uenoi and C. iwawakianus, a large difference in the genital size might reduce the gene flow with small mating costs. Gene flow that was significantly reduced by genital difference might cause either the evolution of premating isolation through reinforcement/reproductive character displacement or through the maintenance of a high degree of premating isolation following sympatric contact. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87 , 145–154.     

Components of reproductive isolation between the monkeyflowers Mimulus lewisii and M. cardinalis (Phrymaceae)

Evolutionists have long recognized the role of reproductive isolation in speciation, but the relative contributions of different reproductive barriers are poorly understood. We examined the nature of isolation between Mimulus lewisii and M. cardinalis, sister species of monkeyflowers. Studied reproductive barriers include: ecogeographic isolation; pollinator isolation (pollinator fidelity in a natural mixed population); pollen competition (seed set and hybrid production from experimental interspecific, intraspecific, and mixed pollinations in the greenhouse); and relative hybrid fitness (germination, survivorship, percent flowering, biomass, pollen viability, and seed mass in the greenhouse). Additionally, the rate of hybridization in nature was estimated from seed collections in a sympatric population. We found substantial reproductive barriers at multiple stages in the life history of M. lewisii and M. cardinalis. Using range maps constructed from herbarium collections, we estimated that the different ecogeographic distributions of the species result in 58.7% reproductive isolation. Mimulus lewisii and M. cardinalis are visited by different pollinators, and in a region of sympatry 97.6% of pollinator foraging bouts were specific to one species or the other. In the greenhouse, interspecific pollinations generated nearly 50% fewer seeds than intraspecific controls. Mixed pollinations of M. cardinalis flowers yielded >75% parentals even when only one-quarter of the pollen treatment consisted of M. cardinalis pollen. In contrast, both species had similar siring success on M. lewisii flowers. The observed 99.915% occurrence of parental M. lewisii and M. cardinalis in seeds collected from a sympatric population is nearly identical to that expected, based upon our field observations of pollinator behavior and our laboratory experiments of pollen competition. F1 hybrids exhibited reduced germination rates, high survivorship and reproduction, and low pollen and ovule fertility. In aggregate, the studied reproductive barriers prevent, on average, 99.87% of gene flow, with most reproductive isolation occurring prior to hybrid formation. Our results suggest that ecological factors resulting from adaptive divergence are the primary isolating barriers in this system. Additional studies of taxa at varying degrees of evolutionary divergence are needed to identify the relative importance of pre- and postzygotic isolating mechanisms in speciation.     

Assessing reproductive isolation using a contact zone between parapatric lake‐stream stickleback ecotypes

Ecological speciation occurs when populations evolve reproductive isolation as a result of divergent natural selection. This isolation can be influenced by many potential reproductive barriers, including selection against hybrids, selection against migrants and assortative mating. How and when these barriers act and interact in nature is understood for relatively few empirical systems. We used a mark–recapture experiment in a contact zone between lake and stream three‐spined sticklebacks (Gasterosteus aculeatus, Linnaeus) to evaluate the occurrence of hybrids (allowing inferences about mating isolation), the interannual survival of hybrids (allowing inferences about selection against hybrids) and the shift in lake‐like vs. stream‐like characteristics (allowing inferences about selection against migrants). Genetic and morphological data suggest the occurrence of hybrids and no selection against hybrids in general, a result contradictory to a number of other studies of sticklebacks. However, we did find selection against more lake‐like individuals, suggesting a barrier to gene flow from the lake into the stream. Combined with previous work on this system, our results suggest that multiple (most weakly and often asymmetric) barriers must be combining to yield substantial restrictions on gene flow. This work provides evidence of a reproductive barrier in lake–stream sticklebacks and highlights the value of assessing multiple reproductive barriers in natural contexts.     

Low intraspecific variation for genomic isolation between hybridizing sunflower species

Barriers to gene flow between species result from selection against foreign linkage blocks in hybrids. When the geographic ranges of taxa meet at multiple locations, the opportunity exists for variation in the genetic architecture of isolating barriers. Hybrid zones between two sunflower species (Helianthus annuus and H. petiolaris) in Nebraska and California exhibited remarkably similar patterns of introgression of mapped molecular markers. Congruence among hybrid zones may result from limited intraspecific variation at loci contributing to isolation and from similar selective effects of alleles in the heterospecific genetic background. The observed consistency of introgression patterns across distantly separated hybrid zones suggests that intrinsic forces predominate in determining hybrid zone dynamics and boundaries between these sunflower species.     

Fine‐scale geographic patterns of gene flow and reproductive character displacement in Drosophila subquinaria and Drosophila recens

When two species are incompletely isolated, strengthening premating isolation barriers in response to the production of low fitness hybrids may complete the speciation process. Here, we use the sister species Drosophila subquinaria and Drosophila recens to study the conditions under which this reinforcement of species boundaries occurs in natural populations. We first extend the region of known sympatry between these species, and then we conduct a fine‐scale geographic survey of mate discrimination coupled with estimates of gene flow within and admixture between species. Within D. subquinaria, reinforcement is extremely effective: we find variation in mate discrimination both against D. recens males and against conspecific allopatric males on the scale of a few kilometres and in the face of gene flow both from conspecific populations and introgression from D. recens. In D. recens, we do not find evidence for increased mate discrimination in sympatry, even where D. recens is rare, consistent with substantial gene flow throughout the species’ range. Finally, we find that introgression between species is asymmetric, with more from D. recens into D. subquinaria than vice versa. Within each species, admixture is highest in the geographic region where it is rare relative to the other species, suggesting that when hybrids are produced they are of low fitness. In sum, reinforcement within D. subquinaria is effective at maintaining species boundaries, but even when reinforcing selection is strong it may not always result in a pattern of strong reproductive character displacement due to variation in the frequency of hybridization and gene flow from neighbouring populations.     

Ecological divergence plays an important role in strong but complex reproductive isolation in campions (Silene)*

New species arise through the evolution of reproductive barriers between formerly interbreeding lineages. Yet, comprehensive assessments of potential reproductive barriers, which are needed to make inferences on processes driving speciation, are only available for a limited number of systems. In this study, we estimated individual and cumulative strengths of seven prezygotic and six postzygotic reproductive barriers between the recently diverged taxa Silene dioica (L.) Clairv. and S. latifolia Poiret using both published and new data. A combination of multiple partial reproductive barriers resulted in near‐complete reproductive isolation between S. dioica and S. latifolia, consistent with earlier estimates of gene flow between the taxa. Extrinsic barriers associated with adaptive ecological divergence were most important, while intrinsic postzygotic barriers had moderate individual strength but contributed only little to total reproductive isolation. These findings are in line with ecological divergence as driver of speciation. We further found extensive variation in extrinsic reproductive isolation, ranging from sites with very strong selection against migrants and hybrids to intermediate sites where substantial hybridization is possible. This situation may allow for, or even promote, heterogeneous genetic divergence.     

Reproductive isolation between phylogeographic lineages scales with divergence

Phylogeographic studies frequently reveal multiple morphologically cryptic lineages within species. What is not yet clear is whether such lineages represent nascent species or evolutionary ephemera. To address this question, we compare five contact zones, each of which occurs between ecomorphologically cryptic lineages of skinks from the rainforests of the Australian Wet Tropics. Although the contacts probably formed concurrently in response to Holocene expansion from glacial refugia, we estimate that the divergence times (τ) of the lineage pairs range from 3.1 to 11.5 Ma. Multi-locus analyses of the contact zones yielded estimates of reproductive isolation that are tightly correlated with divergence time and, for lineages with older divergence times (τ > 5 Myr), substantial. These results show that phylogeographic splits of increasing depth represent stages along the speciation continuum, even in the absence of overt change in ecologically relevant morphology.     

Genetic variation for postzygotic reproductive isolation between Caenorhabditis briggsae and Caenorhabditis sp. 9

The process of speciation is key to the origins of biodiversity, and yet the Caenorhabditis nematode model system has contributed little to this topic. Genetic studies of speciation in the genus are now feasible, owing to crosses between the recently discovered Caenorhabditis sp. 9 and the well-known C. briggsae producing fertile F(1) hybrid females. We dissected patterns of postzygotic reproductive isolation between these species by crossing eight isogenic strains of C. briggsae reciprocally with six strains of C. sp. 9. We determined that overall patterns of reproductive isolation are robust across these genetic backgrounds. However, we also quantified significant heritable variation within each species for interspecific hybrid incompatibilities for total adult progeny, egg-to-adult viability, and the percentage of male progeny. This demonstrates that intraspecific variation for interspecific hybrid incompatibility occurs despite extensive, albeit incomplete, reproductive isolation. Therefore, this emerging general phenomenon of variable reproductive isolation is not restricted to highly interfertile, early-stage incipient species, but also applies to species in the latest stages of the speciation process. Furthermore, we confirm Haldane's rule and demonstrate strongly asymmetric parent-of-origin effects (Darwin's corollary) that consistently manifest more extremely when hermaphroditic C. briggsae serves as maternal parent. These findings highlight Caenorhabditis as an emerging system for understanding the genetics of general patterns of reproductive isolation.     

Reconstructing asymmetrical reproductive character displacement in a periodical cicada contact zone

Selection against costly reproductive interactions can lead to reproductive character displacement (RCD). We use information from patterns of displacement and inferences about predisplacement character states to investigate causes of RCD in periodical cicadas. The 13-year periodical cicada Magicicada neotredecim exhibits RCD and strong reproductive isolation in sympatry with a closely related 13-year species, Magicicada tredecim. Displacement is asymmetrical, because no corresponding pattern of character displacement exists within M. tredecim. Results from playback and hybridization experiments strongly suggest that sexual interactions between members of these species were possible at initial contact. Given these patterns, we evaluate potential sources of selection for displacement. One possible source is 'acoustical interference', or mate-location inefficiencies caused by the presence of heterospecifics. Acoustical interference combined with the species-specificity of song pitch and preference appears to predict the observed asymmetrical pattern of RCD in Magicicada. However, acoustical interference does not appear to be a complete explanation for displacement in Magicicada, because our experiments suggest a significant potential for direct sexual interactions between these species before displacement. Another possible source of selection for displacement is hybrid failure. We evaluate the attractiveness of inferred hybrid mating signals, and we examine the viability of hybrid eggs. Neither of these shows strong evidence of hybrid inferiority. We conclude by presenting a model of hybrid failure related to life cycle differences in Magicicada.     

The origins of premating reproductive isolation: testing hypotheses in the grasshopper Chorthippus parallelus

There are many proposed routes for the origin of premating reproductive isolation, but few systematic studies aimed at testing their relative importance. Accumulated information about the biogeographical history of the European meadow grasshopper, Chorthippus parallelus, has allowed us to make a planned series of comparisons among populations aimed at distinguishing the contributions of some of these hypotheses. We have compared the effects on assortative mating of long-term isolation in glacial refugia, founder events during postglacial colonization, and sympatry with a closely related species. A likelihood-based analysis allowed us to separate effects of variation in male and female mating propensity among populations from variation in mate choice leading to assortative mating. All three effects contributed significantly to the overall variation in mating pattern in a set of 21 pairwise comparisons among seven populations. Male cuticular composition, but not other candidate signals, was significantly associated with the level of assortative mating. Of the hypotheses for the origin of reproductive isolation, only the predictions of the founder hypothesis explained a significant amount of the variation in assortative mating. This does not rule out the possiblity that there may be some other explanation. Having established the pattern of divergence, it is possible to generate hypotheses that explain our results at least as well as the founder hypothesis. However, because many such post hoc hypotheses are possible, they cannot be tested with this dataset. On this basis, our results favor the hypothesis that some aspect of the colonization process tends to accelerate divergence in mating signals leading to premating reproductive isolation. This could be accomplished through any one of several mechanisms. Colonization involves many bottlenecks as new populations are established at the edge of the range by long-distance migrants. Genetic effects may be important, but these bottlenecks may also alter the conditions under which mates are found and chosen, as suggested by Kaneshiro. At the same time, the colonizing populations may encounter novel environmental challenges.     

Testing Wallace's intuition: water type,reproductive isolation and divergence in an Amazonian fish

Alfred Russel Wallace proposed classifying Amazon rivers based on their colour and clarity: white, black and clear water. Wallace also proposed that black waters could mediate diversification and yield distinct fish species. Here, we bring evidence of speciation mediated by water type in the sailfin tetra (Crenuchus spilurus), a fish whose range encompasses rivers of very distinct hydrochemical conditions. Distribution of the two main lineages concords with Wallace's water types: one restricted to the acidic and nutrient‐poor waters of the Negro River (herein Rio Negro lineage) and a second widespread throughout the remaining of the species’ distribution (herein Amazonas lineage). These lineages occur over a very broad geographical range, suggesting that despite occurring in regions separated by thousands of kilometres, individuals of the distinct lineages fail to occupy each other's habitats, hundreds of metres apart and not separated by physical barrier. Reproductive isolation was assessed in isolated pairs exposed to black‐water conditions. All pairs with at least one individual of the lineage not native to black waters showed significantly lower spawning success, suggesting that the water type affected the fitness and contributed to reproductive isolation. Our results endorse Wallace's intuition and highlight the importance of ecological factors in shaping diversity of the Amazon fish fauna.     

Ecological divergence associated with mating system causes nearly complete reproductive isolation between sympatric Mimulus species

Speciation often involves the evolution of numerous prezygotic and postzygotic isolating barriers between divergent populations. Detailed knowledge of the strength and nature of those barriers provides insight into ecological and genetic factors that directly or indirectly influenced their origin, and may help predict whether they will be maintained in the face of sympatric hybridization and introgression. We estimated the magnitude of pre- and postzygotic barriers between naturally occurring sympatric populations of Mimulus guttatus and M. nasutus. Prezygotic barriers, including divergent flowering phenologies, differential pollen production, mating system isolation, and conspecific pollen precedence, act asymmetrically to completely prevent the formation of F(1) hybrids among seeds produced by M. guttatus (F(1)g), and reduce F(1) hybrid production among seeds produced by M. nasutus (F(1)n) to only about 1%. Postzygotic isolation is also asymmetric: in field experiments, F(1)g but not F(1)n hybrids had significantly reduced germination rates and survivorship compared to parental species. Both hybrid classes had flower, pollen, and seed production values within the range of parental values. Despite the moderate degree of F(1)g hybrid inviability, postzygotic isolation contributes very little to the total isolation between these species in the wild. We also found that F(1) hybrid flowering phenology overlapped more with M. guttatus than M. nasutus. These results, taken together, suggest greater potential for introgression from M. nasutus to M. guttatus than for the reverse direction. We also address problems with commonly used indices of isolation, discuss difficulties in calculating meaningful measures of reproductive isolation when barriers are asymmetric, and propose novel measures of prezygotic isolation that are consistent with postzygotic measures.     

The role of sexual isolation during rapid ecological divergence: Evidence for a new dimension of isolation in Rhagoletis pomonella

The pace of divergence and likelihood of speciation often depends on how and when different types of reproductive barriers evolve. Questions remain about how reproductive isolation evolves after initial divergence. We tested for the presence of sexual isolation (reduced mating between populations due to divergent mating preferences and traits) in Rhagoletis pomonella flies, a model system for incipient ecological speciation. We measured the strength of sexual isolation between two very recently diverged (~170 generations) sympatric populations, adapted to different host fruits (hawthorn and apple). We found that flies from both populations were more likely to mate within than between populations. Thus, sexual isolation may play an important role in reducing gene flow allowed by early-acting ecological barriers. We also tested how warmer temperatures predicted under climate change could alter sexual isolation and found that sexual isolation was markedly asymmetric under warmer temperatures – apple males and hawthorn females mated randomly while apple females and hawthorn males mated more within populations than between. Our findings provide a window into the early speciation process and the role of sexual isolation after initial ecological divergence, in addition to examining how environmental conditions could shape the likelihood of further divergence.     

Geographic variation in hybridization across a reinforcement contact zone of chorus frogs (Pseudacris)

Reinforcement contact zones, which are secondary contact zones where species are diverging in reproductive behaviors due to selection against hybridization, represent natural laboratories for studying speciation‐in‐action. Here, we examined replicate localities across the entire reinforcement contact zone between North American chorus frogs Pseudacris feriarum and P. nigrita to investigate geographic variation in hybridization frequencies and to assess whether reinforcement may have contributed to increased genetic divergence within species. Previous work indicated these species have undergone reproductive character displacement (RCD) in male acoustic signals and female preferences due to reinforcement. We also examined acoustic signal variation across the contact zone to assess whether signal characteristics reliably predict hybrid index and to elucidate whether the degree of RCD predicts hybridization rate. Using microsatellites, mitochondrial sequences, and acoustic signal information from >1,000 individuals across >50 localities and ten sympatric focal regions, we demonstrate: (1) hybridization occurs and (2) varies substantially across the geographic range of the contact zone, (3) hybridization is asymmetric and in the direction predicted from observed patterns of asymmetric RCD, (4) in one species, genetic distance is higher between conspecific localities where one or both have been reinforced than between nonreinforced localities, after controlling for geographic distance, (5) acoustic signal characters strongly predict hybrid index, and (6) the degree of RCD does not strongly predict admixture levels. By showing that hybridization occurs in all sympatric localities, this study provides the fifth and final line of evidence that reproductive character displacement is due to reinforcement in the chorus frog contact zone. Furthermore, this work suggests that the dual action of cascade reinforcement and partial geographic isolation is promoting genetic diversification within one of the reinforced species.     

Breeding ecology and evidence of reproductive isolation of a widespread stickleback fish (Gasterosteidae) in Nova Scotia, Canada

A stickleback with brilliant white dorsal breeding colours is widely distributed in north-eastern Nova Scotia, Canada, where it often breeds sympatrically with the threespine stickleback, Gasterosteus aculeatus. Breeding males are highly conspicuous and visible at distances of 20 m or more whereas sympatric G. aculeatus are cryptic and difficult to detect even at 2 m. The white stickleback nests only above the substrate in filamentous algae, where G. aculeatus nests only on the substrate. The white stickleback is smaller in size and more terete than G. aculeatus , but it is morphologically similar in having a complete row of lateral plates and similar lateral plate and gill raker numbers. The white stickleback occurs only in environments where there are filamentous algae (which appears to be an obligatory nesting substrate) and where the water is clear, saline and relatively still. Female choice tests in the laboratory show that the white stickleback is reproductively isolated from G. aculeatus , and field observations on natural spawning support this conclusion. We suggest that the bright breeding colouration may have evolved through sexual selection and/or to advertise unprofitability to predators.