ESTIMATING THE MATING BEHAVIOR OF A PAIR OF HYBRIDIZING HELICONIUS SPECIES IN THE WILD

Premating isolation between incipient species is rarely studied in nature, even though mating tests in captivity may give an inaccurate picture of natural hybridization. We studied premating barriers between the warningly colored butterflies Heliconius erato and H. himera (Lepidoptera) in a narrow contact zone in Ecuador, where hybrids are found at low frequency. Eggs obtained from wild-mated females, supplemented with eggs and young larvae collected from the wild, were reared to adulthood. Adult color patterns of these progeny were then used to infer how their parents must have mated. Likelihood was used to estimate both the frequencies of potential parental genotypes from adult phenotypes collected in the wild, and the degree of assortative mating from the inferred parents. The frequencies of parental genotypes varied across the hybrid zone, but our statistical method allowed estimates of hybrid deficit and assortative mating to be integrated across all sites sampled. The best estimate of the frequency of F₁ and backcross hybrid adults in the center of the hybrid zone was 10%, with support limits (7.1%, 13.0%; support limits are asymptotically equivalent to 95% confidence limits). Mating was highly assortative: in the center of the hybrid zone the cross-mating probability between H. erato and H. himera was only 5% (0.3%, 21.4%). Wild hybrids themselves mated with both pure forms, and the probabilities that they mated in any direction were not significantly lower than those among conspecifics. These results are consistent with earlier laboratory studies on mate choice, and suggest that selection against hybrids must be strong to prevent formation of a hybrid swarm. Unfortunately, the wide support limits on mating behavior precluded a measure of the strength of selection from these data alone. Our statistical approach provides a useful general method for estimating mate choice in the wild.     

Behavioral and Physiological Differences between Two Parapatric Heliconius Species1

The behavior and physiology of two parapatric sibling species, i Heliconius erato cyrbia Godt. and H. himera Hew., were investigated to assess if environmental adaptation enabled stable morphological, genetic, and ecological differences to exist in the face of hybridization. Morning and evening activity, egg production, and larval development time of H. himera and H. erato in insectaries were recorded; individuals were collected in allopatry and in sympatry from a hybrid zone in which the species overlapped. Studies were performed at ambient conditions within the natural range of H. himera. H. himera was considerably more active than H, erato flying earlier in the morning and later in the evening, even when both species were collected in sympatry. Similarly, H. himera laid more eggs, and the hatched larvae developed more rapidly. The results suggest that physiological constraints are an important selective force that may have been important in speciation and counteracts hybridization in the maintenance of the H. himeral H. erato contact zone. Ecological selection, arising from adaptation to low temperatures, may help explain the competitive exclusion of H. erato by H. himera in the drier, cooler montane habitat favored by the latter species.     

Spatial patterns of variation in color and spine shape in the sea urchin Heliocidaris erythrogramma

Abstract. Here we report on the first quantitative survey of morphological variation in the sea urchin Heliocidaris erythrogramma within Western Australia and distinguish between two subspecies found to co‐occur in this region. We surveyed urchins at multiple spatial scales along the Western Australian coastline to assess variation in dermis and spine color and, using landmark‐based geometric morphometrics, spine morphology. Both color and morphology proved to be useful for separating subspecies within Western Australia. There were four major color morphs: red dermis/violet spines (56%), red/violet‐green (23%), red/green (7%), and white/green (10%). Members of the first two color morphs had bulbous spines with wide, flattened tips, a morphology that is unique to Western Australia and characteristic of H. e. armigera, and members of the latter two consistently exhibited the narrow, pointed spines typical of specimens of H. e. erythrogramma, which has a broader distribution. In Western Australia, H. e. armigera was relatively abundant both within and among sites, but H. e. erythrogramma was found only in a few localized patches. Shifts in the relative abundance of these two subspecies occurred at fine spatial scales (<5 km), although environmental correlates of these transitions were unclear. Contrary to expectations, neither dermis color nor spine morphology varied with relative wave exposure: individuals with a red dermis or thickened spine morphology occurred at most sites regardless of exposure, and while white dermis and thinner spines only occurred at high‐exposure sites, these features were not common across the majority of exposed sites. Both color morph frequencies and spine morphology remained stable within sites over the 3‐year duration of this study. While the ecological significance of this morphological variation remains unclear, the consistency of the association between color and spine morphology, occurring across fine spatial scales, suggests that strong environmental or genetic factors are involved in maintaining morphological differentiation between these two subspecies.     

FLOWER COLOR POLYMORPHISM AND POLLINATION BIOLOGY OF PLATYSTEMON CALIFORNICUS BENTH. (PAPAVERACEAE)

Five genetically controlled flower color morphs in Platystemon californicus tend to occur in distinct geographic regions, suggesting regional selection of morphs. I examined the reproductive biology of P. californicus to determine whether color morphs are subject to selection due to differences in pollinator attractiveness. Plants of P. californicus have a high pollen: ovule ratio and are highly self-incompatible. Solitary bees (Andrenidae and Halictidae) are the most effective pollen vectors, but show inconsistent discrimination among color morphs. Platystemon californicus is unusual among annual self-incompatible plants in that wind is an important pollen vector. As a result of wind pollination, most polymorphic populations show no intermorph seed-set differences. Therefore, the geographic distribution of flower color morphs cannot be explained by differential attractiveness of color morphs to pollinators in different regions. Any selective value of color morphs must be due to linkage of flower color with as yet undetected morphologically or physiologically adaptive characters.     

AN EXPERIMENTAL EVALUATION OF SELECTION ON COLOR MORPHS OF THE POLYMORPHIC SPIDER ENOPLOGNATHA OVATA (ARANEAE: THERIDIIDAE)

The annual theridiid spider Enoplognatha ovata exhibits a genetically based color polymorphism of red and nonred phenotypes. We evaluated fitness differences between red and nonred spiders by manipulating morph frequencies in a population in which red morphs were rare (≤5%). Broods from red females were introduced to open experimental plots from which natural aggregations of spiders had been repeatedly removed. Control plots in which spiders were removed but not replaced were used to estimate spider immigration from surrounding vegetation into experimental plots. Morph frequencies observed in experimental plots one year following the manipulation were adjusted by immigration estimates and tested against frequencies predicted with the hypothesis of no selection. We found no evidence of selection against red morphs: female morph frequencies in experimental plots did not differ significantly from expected frequencies assuming no selection; female frequencies did not change significantly between subadult and adult stages; and red and nonred spiders exhibited similar fecundities. We conclude that 1) selection on E. ovata color morphs is not likely to be detected easily within a single population because of the swamping effect of dispersal and 2) local patterns of morph-frequency variation may arise more from dispersal and drift than from selection on the color phenotypes.     

Facultative pupal mating in Heliconius erato: Implications for mate choice,female preference,and speciation

Mating systems have broad impacts on how sexual selection and mate choice operate within a species, but studies of mating behavior in the laboratory may not reflect how these processes occur in the wild. Here, we examined the mating behavior of the neotropical butterfly Heliconius erato in the field by releasing larvae and virgin females and observing how they mated. H. erato is considered a pupal‐mating species (i.e., males mate with females as they emerge from the pupal case). However, we observed only two teneral mating events, and experimentally released virgins were almost all mated upon recapture. Our study confirms the presence of some pupal‐mating behavior in H. erato, but suggests that adult mating is likely the prevalent mating strategy in this species. These findings have important implications for the role of color pattern and female mate choice in the generation of reproductive isolation in this diverse genus.     

Spatial analyses of two color polymorphisms in an alpine grasshopper reveal a role of small‐scale heterogeneity

Discrete color polymorphisms represent a fascinating aspect of intraspecific diversity. Color morph ratios often vary clinally, but in some cases, there are no marked clines and mixes of different morphs occur at appreciable frequencies in most populations. This poses the questions of how polymorphisms are maintained. We here study the spatial and temporal distribution of a very conspicuous color polymorphism in the club‐legged grasshopper Gomphocerus sibiricus. The species occurs in a green and a nongreen (predominately brown) morph, a green–brown polymorphism that is common among Orthopteran insects. We sampled color morph ratios at 42 sites across the alpine range of the species and related color morph ratios to local habitat parameters and climatic conditions. Green morphs occurred in both sexes, and their morph ratios were highly correlated among sites, suggesting shared control of the polymorphism in females and males. We found that in at least 40 of 42 sites green and brown morphs co‐occurred with proportions of green ranging from 0% to 70% with significant spatial heterogeneity. The proportion of green individuals tended to increase with decreasing summer and winter precipitations. Nongreen individuals can be further distinguished into brown and pied individuals, and again, this polymorphism is shared with other grasshopper species. We found pied individuals at all sites with proportions ranging from 3% to 75%, with slight, but significant variation between years. Pied morphs show a clinal increase in frequency from east to west and decreased with altitude and lower temperatures and were more common on grazed sites. The results suggest that both small‐scale and large‐scale spatial heterogeneity affects color morph ratios. The almost universal co‐occurrence of all three color morphs argues against strong effects of genetic drift. Instead, the data suggest that small‐scale migration–selection balance and/or local balancing selection maintain populations polymorphic.     

WING SHAPE VARIATION ASSOCIATED WITH MIMICRY IN BUTTERFLIES

Mimetic resemblance in unpalatable butterflies has been studied by evolutionary biologists for over a century, but has largely focused on the convergence in wing color patterns. In Heliconius numata, discrete color‐pattern morphs closely resemble comimics in the distantly related genus Melinaea. We examine the possibility that the shape of the butterfly wing also shows adaptive convergence. First, simple measures of forewing dimensions were taken of individuals in a cross between H. numata morphs, and showed quantitative differences between two of the segregating morphs, f. elegans and f. silvana. Second, landmark‐based geometric morphometric and elliptical Fourier outline analyses were used to more fully characterize these shape differences. Extension of these techniques to specimens from natural populations suggested that, although many of the coexisting morphs could not be discriminated by shape, the differences we identified between f. elegans and f. silvana hold in the wild. Interestingly, despite extensive overlap, the shape variation between these two morphs is paralleled in their respective Melinaea comimics. Our study therefore suggests that wing‐shape variation is associated with mimetic resemblance, and raises the intriguing possibility that the supergene responsible for controlling the major switch in color pattern between morphs also contributes to wing shape differences in H. numata.     

Stable Heliconius butterfly hybrid zones are correlated with a local rainfall peak at the edge of the Amazon basin

Multilocus clines between Müllerian mimetic races of Heliconius butterflies provide a classic example of the maintenance of hybrid zones and their importance in speciation. Concordant hybrid zones in the mimics Heliconius erato and H. melpomene in northern Peru were carefully documented in the 1980s, and this prior work now permits a historical analysis of the movement or stasis of the zones. Previous work predicted that these zones might be moving toward the Andes due to selective asymmetry. Extensive deforestation and climate change might also be expected to affect the positions and widths of the hybrid zones. We show that the positions and shapes of these hybrid zones have instead remained remarkably stable between 1985 and 2012. The stability of this interaction strongly implicates continued selection, rather than neutral mixing following secondary contact. The stability of cline widths and strong linkage disequilibria (gametic correlation coefficients R_max = 0.35–0.56 among unlinked loci) over 25 years suggest that mimetic selection pressures on each color pattern locus have remained approximately constant (s ≈ 0.13–0.40 per locus in both species). Exceptionally high levels of precipitation at the edge of the easternmost Andes may act as a population density trough for butterflies, trapping the hybrid zones at the foot of the mountains, and preventing movement. As such, our results falsify one prediction of the Pleistocene Refugium theory: That the ranges of divergent species or subspecies should be centered on regions characterized by maxima of rainfall, with hybrid zones falling in more arid regions between them.     

Can different species of medicinal leeches (Hirudo spp.) interbreed?

Abstract. Since the 18th century, the medicinal leech Hirudo medicinalis has been thought to comprise a single species with several different color morphs, but recently some of these color morphs have been assigned to separate species based on morphology, geographical distribution, and molecular sequence data. This research was aimed at testing the ability of three of these species, H. medicinalis, Hirudo verbana, and Hirudo orientalis, to interbreed. We found that in the laboratory, all three species were able to mate with each other and produce hybrid offspring. This suggests that the reproductive isolation is not strong among these species of the genus Hirudo. However, fewer offspring were produced from interspecific crosses compared with intraspecific crosses. This decrease of fecundity (and in some cases, offspring viability) indicates some degree of reproductive isolation between H. medicinalis, H. verbana, and H. orientalis.     

Wing patterning genes and coevolution of Müllerian mimicry in Heliconius butterflies: Support from phylogeography,cophylogeny, and divergence times

Examples of long‐term coevolution are rare among free‐living organisms. Müllerian mimicry in Heliconius butterflies had been suggested as a key example of coevolution by early genetic studies. However, research over the last two decades has been dominated by the idea that the best‐studied comimics, H. erato and H. melpomene, did not coevolve at all. Recently sequenced genes associated with wing color pattern phenotype offer a new opportunity to resolve this controversy. Here, we test the hypothesis of coevolution between H. erato and H. melpomene using Bayesian multilocus analysis of five color pattern genes and five neutral genetic markers. We first explore the extent of phylogenetic agreement versus conflict between the different genes. Coevolution is then tested against three aspects of the mimicry diversifications: phylogenetic branching patterns, divergence times, and, for the first time, phylogeographic histories. We show that all three lines of evidence are compatible with strict coevolution of the diverse mimicry wing patterns, contrary to some recent suggestions. Instead, these findings tally with a coevolutionary diversification driven primarily by the ecological force of Müllerian mimicry.     

Hybridization promotes color polymorphism in the aposematic harlequin poison frog,Oophaga histrionica

Whether hybridization can be a mechanism that drives phenotypic diversity is a widely debated topic in evolutionary biology. In poison frogs (Dendrobatidae), assortative mating has been invoked to explain how new color morphs persist despite the expected homogenizing effects of natural selection. Here, we tested the complementary hypothesis that new morphs arise through hybridization between different color morphs. Specifically, we (1) reconstructed the phylogenetic relationships among the studied populations of a dart‐poison frog to provide an evolutionary framework, (2) tested whether microsatellite allele frequencies of one putative hybrid population of the polymorphic frog O. histrionica are intermediate between O. histrionica and O. lehmanni, and (3) conducted mate‐choice experiments to test whether putatively intermediate females prefer homotypic males over males from the other two populations. Our findings are compatible with a hybrid origin for the new morph and emphasize the possibility of hybridization as a mechanism generating variation in polymorphic species. Moreover, because coloration in poison frogs is aposematic and should be heavily constrained, our findings suggest that hybridization can produce phenotypic novelty even in systems where phenotypes are subject to strong stabilizing selection.     

Direct and indirect selection on floral pigmentation by pollinators and seed predators in a color polymorphic South African shrub

The coexistence of different color morphs is often attributed to variable selection pressures across space, time, morph frequencies, or selection agents, but the routes by which each morph is favored are rarely identified. In this study we investigated factors that influence floral color polymorphisms on a local scale in Protea, within which approximately 40% of species are polymorphic. Previous work shows that seed predators and reproductive differences likely contribute to maintaining polymorphism in four Protea species. We explored whether selection acts directly or indirectly on floral color in two populations of Protea aurea, using path analysis of pollinator behavior, nectar production, seed predation, color, morphology, and maternal fecundity fitness components. We found that avian pollinators spent more time on white morphs, likely due to nectar differences, but that this had no apparent consequences for fecundity. Instead, the number of flowers per inflorescence underpinned many of the reproductively important differences between color morphs. White morphs had more flowers per inflorescence, which itself was positively correlated with nectar production, seed predator occurrence, and total long-term seed production. The number of seeds per plant to survive predation, in contrast, was not directly associated with color or any other floral trait. Thus, although color differences may be associated with conflicting selection pressures, the selection appears to be associated with the number of flowers per inflorescence and its unmeasured correlates, rather than with inflorescence color itself.     

The maintenance of phenotypic divergence through sexual selection: An experimental study in barn swallows Hirundo rustica

Previous studies have shown that sexual signals can rapidly diverge among closely related species. However, we lack experimental studies to demonstrate that differences in trait‐associated reproductive performance maintain sexual trait differences between closely related populations, in support for a role of sexual selection in speciation. Populations of Northern Hemisphere distributed barn swallows Hirundo rustica are closely related, yet differ in two plumage‐based traits: ventral color and length of the outermost tail feathers (streamers). Here we provide experimental evidence that manipulations of these traits result in different reproductive consequences in two subspecies of barn swallow: (H. r. erythrogaster in North America and H. r. transitiva in the East Mediterranean). Experimental results in Colorado, USA, demonstrate that males with (1) darkened ventral coloration and (2) shortened streamers gained paternity between two successive reproductive bouts. In contrast, exaggeration of both traits improved reproductive performance within H. r. transitiva in Israel: males with a combination treatment of darkened ventral coloration and elongated streamers gained paternity between two successive reproductive bouts. Collectively, these experimental results fill an important gap in our understanding for how divergent sexual selection maintains phenotype differentiation in closely related populations, an important aspect of the speciation process.     

The role of vision and color in the close proximity foraging behavior of four coccinellid species

The role of vision and color in close-proximity foraging behavior was investigated for four species of lady beetles: Coccinella septempunctata, Hippodamia convergens, Harmonia axyridis, and Coleomegilla maculata. The effect of light level and color cues on consumption rates varied among the four predator species. The consumption rates of these predators on the pea aphid Acyrthosiphon pisum (Harris) was measured under light and dark conditions. C. septempunctata,H. convergens, and Ha. axyridis consumed significantly more aphids in the light than in the dark, while the consumption rate of Col. maculata was not affected by light level. Foraging ability was also measured on red and green color morphs of the pea aphid on red, green, and white backgrounds. C. septempunctata consumed significantly more of the aphid morph that contrasted with the background color, and showed no difference between morphs on the white background. H. axyridis consumed significantly more red morph aphids regardless of background. The remaining two species showed no difference in consumption rates on the two color morphs. The variation in the use of visual cues demonstrates how different species of predators can exhibit different foraging behaviors when searching for the same prey. Received: 4 August 1997 / Accepted: 3 February 1998     

Male mate choice and the potential for complex mating dynamics in the tree lizard (Urosaurus ornatus)

A growing body of literature is recognizing that males may also play a role in the mating process by behaving non‐randomly toward potential female mates during courtship. In numerous species, discrete color polymorphisms in males are inferred to represent alternative mating tactics, which often correspond with concomitant asymmetries in ecology and behavior. In terms of their mating behavior, these ecological outcomes of a color polymorphism should affect a morph's likelihood and frequency of encountering females in a population, possibly favoring the evolution of morph‐specific mating preferences. Knowledge of how male morphs contribute to a species’ overall mating dynamics will improve our understanding of how sexual selection shapes phenotypic diversity in color polymorphic systems. We conducted a mate choice experiment to evaluate the extent and morph specificity of non‐random mating preferences by male ornate tree lizards, Urosaurus ornatus. We observed the behavior of blue and yellow males in an experimental arena in response to a choice between an orange or yellow female. We found that blue males preferred yellow females over orange females, and although yellow males visited females more often than blue males overall, their attention was not morph‐specific. Given male morph differences in choosiness, and their differences in social dominance, we conclude that female throat color may be partly under sexual selection in U. ornatus. However, a lack of concordance between male and female mating preferences (drawn from an earlier study) suggests that overall mating dynamics may serve to maintain, rather than enhance, color morph differences in this species.     

Müllerian mimicry of a quantitative trait despite contrasting levels of genomic divergence and selection

Hybrid zones, where distinct populations meet and interbreed, give insight into how differences between populations are maintained despite gene flow. Studying clines in genetic loci and adaptive traits across hybrid zones is a powerful method for understanding how selection drives differentiation within a single species, but can also be used to compare parallel divergence in different species responding to a common selective pressure. Here, we study parallel divergence of wing colouration in the butterflies Heliconius erato and H. melpomene, which are distantly related Müllerian mimics which show parallel geographic variation in both discrete variation in pigmentation, and quantitative variation in structural colour. Using geographic cline analysis, we show that clines in these traits are positioned in roughly the same geographic region for both species, which is consistent with direct selection for mimicry. However, the width of the clines varies markedly between species. This difference is explained in part by variation in the strength of selection acting on colour traits within each species, but may also be influenced by differences in the dispersal rate and total strength of selection against hybrids between the species. Genotyping‐by‐sequencing also revealed weaker population structure in H. melpomene, suggesting the hybrid zones may have evolved differently in each species, which may also contribute to the patterns of phenotypic divergence in this system. Overall, we conclude that multiple factors are needed to explain patterns of clinal variation within and between these species, although mimicry has probably played a central role.     

Multi-Allelic Major Effect Genes Interact with Minor Effect QTLs to Control Adaptive Color Pattern Variation in Heliconius erato

Recent studies indicate that relatively few genomic regions are repeatedly involved in the evolution of Heliconius butterfly wing patterns. Although this work demonstrates a number of cases where homologous loci underlie both convergent and divergent wing pattern change among different Heliconius species, it is still unclear exactly how many loci underlie pattern variation across the genus. To address this question for Heliconius erato, we created fifteen independent crosses utilizing the four most distinct color pattern races and analyzed color pattern segregation across a total of 1271 F2 and backcross offspring. Additionally, we used the most variable brood, an F2 cross between H. himera and the east Ecuadorian H. erato notabilis, to perform a quantitative genetic analysis of color pattern variation and produce a detailed map of the loci likely involved in the H. erato color pattern radiation. Using AFLP and gene based markers, we show that fewer major genes than previously envisioned control the color pattern variation in H. erato. We describe for the first time the genetic architecture of H. erato wing color pattern by assessing quantitative variation in addition to traditional linkage mapping. In particular, our data suggest three genomic intervals modulate the bulk of the observed variation in color. Furthermore, we also identify several modifier loci of moderate effect size that contribute to the quantitative wing pattern variation. Our results are consistent with the two-step model for the evolution of mimetic wing patterns in Heliconius and support a growing body of empirical data demonstrating the importance of major effect loci in adaptive change.     

Variable Selection and the Coexistence of Multiple mimetic forms of the Butterfly Heliconius numata

Polymorphism in aposematic animals and coexistence of multiple mimicry rings within a habitat are not predicted by classical Müllerian mimicry. The butterfly Heliconius numata Cramer (Lepidoptera: Nymphalidae; Heliconiinae) is both polymorphic and aposematic. The polymorphism is due to variation at a single locus (or `supergene') which determines colour patterns involved in Müllerian mimicry. We sampled 11 sites in a small area (approx. 60×30km) of North-eastern Peru for H. numata and its co-mimics in the genus Melinaea and Athyrtis (Ithomiinae), and examined the role of temporal and spatial heterogeneity in the maintenance of polymorphism. Colour-patterns of Melinaea communities, which constitute the likely `mimetic environment' for H. numata, are differentiated on a more local scale than morphs of H. numata, but the latter do show a strong and significant response to local selection for colour-pattern. In contrast, analysis of enzyme polymorphism in H. numata across the region revealed no spatial structure, which is consistent with a high mobility of this species. Differences in spatial variability in the two taxa may have caused H. numata to become polymorphic, while temporal variability, not significant in this study, probably has a lesser effect. The mimetic polymorphism is therefore explained by means of multiple selection-migration clines at a single locus, a similar process to that which explains narrow hybrid zones between geographic races of other Heliconius butterflies. This revised version was published online in July 2006 with corrections to the Cover Date.