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.     

The genetic basis of an adaptive radiation: warning colour in two Heliconius species

Mimetic colour pattern races of Heliconius butterflies provide a striking example of adaptive radiation and numerous crossing experiments have investigated the genetics of these racial differences. However, colour pattern differentiation between closely related Heliconius species has not been previously studied. Here we present data from crosses between H. erato cyrbia and its sister species, H. himera. The genetic architecture underlying colour pattern divergence between these species is identical to that observed between races of H. erato. As in inter-racial crosses, colour pattern differences resulted from segregation at a few major loci. Evidence from 1321 offspring in 4 F₁, 17 backcross, 7 F₂ and 21 further crosses showed that two major loci controlled most of the colour pattern differences between H. erato and H. himera. There were strong interactions between these loci in their patterns of expression and evidence for other loci with relatively minor phenotypic effects. More importantly, based on patterns of expression within broods and linkage with Aconitase, we conclude that these major loci were homologous with those known to be responsible for colour pattern differences within H. erato. Our crosses also permit a re-evaluation of the relationships between colour pattern races of H. erato. This suggests that H. e. hydara, which occurs across a major mtDNA break, is the ancestral phenotype from which other races have evolved. Based on this assumption, we find no evidence to support the recent suggestion that apparently homologous colour pattern alleles have arisen multiple times.     

Issue Information

Front Cover: The bright warning patterns of the sister species H. himera (top) and H. erato (bottom) are also used as mating cues. Original images by Riccardo Papa. Photo reproduced by permission of Richard M. Merrill, Department of Zoology, University of Cambridge, Cambridge, U.K.     

Divergence in brain composition during the early stages of ecological specialization in Heliconius butterflies

During speciation across ecological gradients, diverging populations are exposed to contrasting sensory and spatial information that present new behavioural and perceptive challenges. These challenges may be met by heritable or environmentally induced changes in brain function which mediate behaviour. However, few studies have investigated patterns of neural divergence at the early stages of speciation, inhibiting our understanding of the relative importance of these processes. Here, we provide a novel case study. The incipient species pair, Heliconius erato and H. himera, are parapatric across an environmental and altitudinal gradient. Despite ongoing gene flow, these species have divergent ecological, behavioural and physiological traits. We demonstrate that these taxa also differ significantly in brain composition, in particular in the relative levels of investment in structures that process sensory information. These differences are not explained solely by environmentally‐induced plasticity, but include heritable, nonallometric shifts in brain structure. We suggest these differences reflect divergence to meet the demands of contrasting sensory ecologies. This conclusion would support the hypothesis that the evolution of brain structure and function play an important role in facilitating the emergence of ecologically distinct species.     

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.     

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.     

STRONG NATURAL SELECTION IN A WARNING-COLOR HYBRID ZONE

Frequency-dependent selection on warning color can maintain narrow hybrid zones between unpalatable prey taxa. To measure such selection, we transferred marked Heliconius erato (Lepidoptera: Nymphalidae) in both directions across a 10-km-wide hybrid zone between Peruvian races differing in color pattern. These experimental H. erato were released at four sites, along with control H. erato of the phenotype native to each site. Survival of experimental butterflies was significantly lower than that of controls at two sites and overall. Most selection, measured as differences in survival, occurred soon after release. Selection against foreign morphs was 52% (confidence limits: 25–71 %) and was probably due to bird attacks on unusual warning-color morphs (more than 10% of the recaptures had beak marks). Since only three major loci determine the color-pattern differences, this suggests an average selection coefficient of 0.17 per locus, sufficient to maintain the narrow clines in H. erato.     

Divergent warning patterns contribute to assortative mating between incipient Heliconius species

Theoretical models suggest that traits under divergent ecological selection, which also contribute to assortative mating, will facilitate speciation with gene flow. Evidence for these so‐called “magic traits” now exists across a range of taxa. However, their importance during speciation will depend on the extent to which they contribute to reproductive isolation. Addressing this requires experiments to determine the exact cues involved as well as estimates of assortative mating in the wild. Heliconius butterflies are well known for their diversity of bright warning color patterns, and their amenability to experimental manipulation has provided an excellent opportunity to test their role in reproductive isolation. Here, we reveal that divergent color patterns contribute to mate recognition between the incipient species Heliconius himera and H. erato, a taxon pair for which assortative mating by color pattern has been demonstrated among wild individuals: First, we demonstrate that males are more likely to attempt to mate conspecific females; second, we show that males are more likely to approach pinned females that share their own warning pattern. These data are valuable as these taxa likely represent the early stages of speciation, but unusually also allow comparisons with rates of interbreeding between divergent ecologically relevant phenotypes measured in the wild.     

Context-dependent pollinator behavior: an explanation for patterns of hybridization among three species of Indian paintbrush

In some areas of sympatry, reproductively compatible plant species hybridize, but in other areas of sympatry, they do not and they remain reproductively isolated from one another. Explanations offered to explain patterns of hybridization that vary by population have usually focused on genetic or environmental factors. Instead, we examined whether different community contexts might change pollinator preference and constancy and thus influence the likelihood of hybridization among three Indian paintbrush species (Castilleja miniata, C. rhexifolia, and C. sulphurea). To determine whether visitation was context‐dependent, we observed pollinator behavior in experimental arrays (constructed using flowering stems of the three Indian paintbrush species) in different contexts. Contexts were defined by which Castilleja species occurred in the immediate neighborhood of the arrays. Specifically, we asked, does visitation to particular species in the arrays depend on context? In general, each Castilleja species was preferred when it matched the surrounding community context, as is predicted by optimal foraging theory. More interestingly, pollinator constancy was weakened in the hybrid context (an area where the three species co‐occurred with morphologically intermediate plants), which is likely to increase pollen flow among the species. Reduced pollinator constancy in hybrid zones could set up a positive feedback loop in which more flower diversity is created through hybridization, decreasing pollinator constancy, and leading to more hybridization. This self‐reinforcing mechanism could lead to “hybridization hot spots” and to a patchy distribution of hybrid populations. We expect that this mechanism may be important in other animal‐pollinated plant hybrid zones.     

Hybridization and mitochondrial genome introgression between Rana chensinensis and R. kukunoris

Mitochondrial genome (mito‐genome) introgression among metazoans is commonplace, and several biological processes may promote such introgression. We examined two proposed processes for the mito‐genome introgression between Rana chensinensis and R. kukunoris: natural hybridization and sex‐biased dispersal. We sampled 477 individuals from 28 sites in the potential hybrid zone in the western Tsinling Mountains. Mitochondrial gene (cyt‐b) trees were used to examine the introgression events. Microsatellite DNA loci, cyt‐b and morphological data were used to identify hybrids and to examine the extent of natural hybridization. We detected rampant bidirectional introgressions, both ancient and recent, between the two species. Furthermore, we found a wide hybrid zone, and frequent and asymmetric hybridization. The hybrid zone cline analysis revealed a clear mitochondrial–nuclear discordance; while most nuclear markers displayed similar and steep clines, cyt‐b had a displaced cline centre and a more gradual and wider cline. We also detected strong and asymmetric historical maternal gene flow across the hybrid zone. This widespread hybridization and detected low mito‐nuclear conflicts may, at least partially, explain the high frequency of introgression. Lastly, microsatellite data and population genetic methods were used to assess sex‐biased dispersal. A weak pattern of female‐biased dispersal was detected in both species, suggesting it may not play an important role in the observed introgression. Our data are consistent with the hybridization hypothesis, but support for the sex‐biased dispersal hypothesis is weak. We further suggest that selective advantages of the R. kukunoris‐type mito‐genome in thermal adaptation may also contribute to the introgression between the two species.     

Sympatry between white-handed gibbons (Hylobates lar) and pileated gibbons (H. pileatus) in Southeastern Thailand

White-handed gibbons (Hylobates lar) are not known to occur to the east or southeast of Bangkok. The reliably documented localities ofH. lar nearest to this area are about 120 km northeast of Bangkok. There, in the Kao Yai National Park, is the only known zone of contact betweenH. lar and the pileated gibbon (H. pileatus), another species of the so-calledlar group. Unpublished documents dating from 1925 indicate, however, that sympatry between these two species may also have existed in the region of Sriracha, about 80 km southeast of Bangkok. Therefore, a large zone of overlap in the distribution of the two species may originally have existed. In most parts of this hypothetical zone, gibbon habitat appears to have been destroyed, with the Khao Yai Park possibly representing the last remnant of the once large contact zone.     

Divergence in drought‐response traits between sympatric species of Mimulus

Differential adaptation to local environmental conditions is thought to be an important driver of speciation. Plants, whose sedentary lifestyle necessitates fine‐tuned adaptation to edaphic conditions such as water availability, are often distributed based on these conditions. Populations occupying water‐limited habitats may employ a variety of strategies, involving numerous phenotypes, to prevent and withstand desiccation. In sympatry, two closely related Mimulus species—M. guttatus and M. nasutus—occupy distinct microhabitats that differ in seasonal water availability. In a common garden experiment, we characterized natural variation within and between sympatric M. guttatus and M. nasutus in the ability to successfully set seed under well‐watered and drought conditions. We also measured key phenotypes for drought adaptation, including developmental timing, plant size, flower size, and stomatal density. Consistent with their microhabitat associations in nature, M. nasutus set seed much more successfully than M. guttatus under water‐limited conditions. This divergence in reproductive output under drought was due to differences in mortality after the onset of flowering, with M. nasutus surviving at a much higher rate than M. guttatus. Higher seed set in M. nasutus was mediated, at least in part, by a plastic increase in the rate of late‐stage development (i.e., fruit maturation), consistent with the ability of this species to inhabit more ephemeral habitats in the field. Our results suggest adaptation to water availability may be an important factor in species maintenance of these Mimulus taxa in sympatry.     

Effect of competition on habitat utilization in two temperate climate gecko species

Competition over spatial niche utilisation is one of most common competitive interactions between species in sympatry. Moreover, competitive interactions may involve age classes, and can fluctuate temporally. Consequently, evasive strategies that enable co-existence are likely to be important in the evolution of species assemblages. Here we investigate a system of two co-existing species of temperate geckos with similar ecologies (the house gecko, Hemidactylus turcicus and the wall gecko, Tarentola mauritanica), providing an opportunity to study the effect of species interactions. Juveniles and adults of both species were investigated throughout their daily and annual cycle to explore the effect of inter- and intra-specific interactions on microhabitat use. The two species showed differences in habitat use for both age classes in sympatry. In sympatry, T. mauritanica uses more open habitats and is more active. In contrast, H. turcicus is found in more closed habitats, closer to the ground and to vegetation cover. In allopatry, H. turcicus was observed in more open habitats, closer to the ground, and to vegetation cover, when compared to the population in sympatry with T. mauritanica. These differences in habitat usage were significant for both age classes. Moreover, there were differences, both in sympatry and in allopatry, between age classes that were dependent on season. In conclusion, the presence of a competitor induces a spatial shift in individuals of both age classes of H. turcicus. Observed plasticity in habitat utilisation in both age classes of H. turcicus is used to argue for the invasive potential of this species.     

Reinforcement and a cline in mating behaviour evolve in response to secondary contact and hybridization in shield‐back katydids (Orthoptera: Tettigoniidae)

In a dispersal‐limited species that has evolved reproductive character displacement at a contact zone, a cline in mating behaviour may result if gene flow diffuses alleles out of the contact zone into allopatric populations. Prior work has found such a clinal pattern in the shield‐back katydid Aglaothorax morsei, in which the male calling songs in a sympatric population have a displaced, short interpulse interval that increases in length with increasing distance from the contact zone. In this study, molecular phylogenetic and female preference data show that (1) sympatric populations result from secondary contact, (2) hybridization in sympatry has resulted in unidirectional mitochondrial introgression and (3) female preferences are consistent with reproductive character displacement and could generate a cline in mating behaviour. These data together suggest a history of reinforcement, generally considered rare in acoustically communicating insects; thus, Aglaothorax represents an important example of a rarely documented evolutionary process.     

Movement of a Heliconius hybrid zone over 30 years: A Bayesian approach

Hybrid zones have long been of interest to biologists as natural laboratories where we can gain insight into the processes of adaptation and speciation. Repeated sampling of individual hybrid zones has been particularly useful in elucidating the dynamic balance between selection and dispersal that maintains most hybrid zones. Here, we revisit a hybrid zone between Heliconius erato butterflies in Panamá for a third time over more than 30 years. We combine a novel Bayesian extension of stepped‐cline hybrid zone models with environmental data to understand the genetic and environmental causes of cline dynamics in this species. The cline has continued to move west, likely due to dominance drive, but has slowed and broadened. Environmental analyses suggest that widespread deforestation in Panamá could be leading to decreased avian predation and relaxed selection, causing the observed changes in cline dynamics.     

Estimating the relative fitness of local adaptive peaks: the aerodynamic costs of flight in mimetic passion-vine butterflies Heliconius

In a related paper, we demonstrated that mimetic Heliconius butterflies have converged in wing-beat frequency and degree of asymmetry in the wing motion, whereas sister species are dissimilar in these same traits. Warning signals of sympatric, distasteful species converge in evolutionary models in order to educate their predators more efficiently that the signal is associated with unprofitable prey. Barring other constraints, the behaviours of the different co-mimetic pairs should ultimately converge on that behaviour which minimizes the energetic cost of flight. We estimated the energetic cost of each mimic''s flight behaviour in order to predict the difference in height of each fitness peak and the direction of convergent selection qualitatively. Following adjustments for body mass, mimetic Heliconius melpomene and Heliconius erato required more aerodynamic power than Heliconius cydno and Heliconius sapho. This difference was attributed to the slower flight speeds and higher wing-beat frequencies of H. melpomene and H. erato. Consequently, H. melpomene and H. erato expended more energy per unit distance per unit body mass than H. cydno and H. sapho. However, differences in body mass may equalize energy budgets and stabilize the sympatric coexistence of the two pairs of co-mimics.     

Hybridization and asymmetric introgression across a narrow zone of contact between Neotoma fuscipes and N. macrotis (Rodentia: Cricetidae)

When lineages diverge in allopatry and come into secondary contact, we have a unique opportunity to examine the degree to which they have become reproductively isolated from one another and the mechanisms that contribute to rates of interspecific gene flow. If hybridization and introgression have occurred in the past or are ongoing, examining patterns of variation in differentially inherited genetic markers can provide insight into underlying mechanisms determining interspecific reproductive interactions. We investigated genetic variation in a recently discovered contact zone between two species of woodrats (Neotoma fuscipes and N. macrotis) in central coastal California. Previous studies have found evidence of historic hybridization between these species, but an active site of sympatry had yet to be discovered. Here, we describe the first known area of sympatry between the two species and present evidence of ongoing hybridization at this site. We intensively sampled throughout the narrow area of interspecific contact and continuously into the adjacent areas where each taxon existed largely in isolation of the other. We genotyped 851 individuals sampled in a single year at 15 nuclear microsatellite loci and sequenced a portion of the cytochrome b gene in a subset of these to examine patterns of introgression. We find that the area of sympatry is less than 1 km in length, but hybridization appeared to be fairly common, with 15% of the woodrats being of hybrid origin. At least some hybrids are fertile because introgression was evident. However, introgression appeared to be asymmetric with backcrosses toward N. macrotis being more common, a pattern we suspect is due in part to directional mate choice related to differential body size. This study adds to our growing understanding of the nature of species boundaries, especially between lineages that are far along the continuum towards speciation. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 115 , 162–172.     

PARALLEL RACE FORMATION AND THE EVOLUTION OF MIMICRY IN HELICONIUS BUTTERFLIES: A PHYLOGENETIC HYPOTHESIS FROM MITOCHONDRIAL DNA SEQUENCES

Mimicry has been a fundamental focus of research since the birth of evolutionary biology yet rarely has been studied from a phylogenetic perspective beyond the simple recognition that mimics are not similar due to common descent. The difficulty of finding characters to discern relationships among closely related and convergent taxa has challenged systematists for more than a century. The phenotypic diversity of wing pattens among mimetic Heliconius adds an additional twist to the problem, because single species contain more than a dozen radically different-looking geographical races even though the mimetic advantage is theoretically highest when all individuals within and between species appear the same. Mitochondrial DNA (mtDNA) offers an independent way to address these issues. In this study, Cytochrome Oxidase I and II sequences from multiple, parallel races of Heliconius erato and Heliconius melpomene are examined, to estimate intraspecific phylogeny and gauge sequence divergence and ages of clades among races within each species. Although phenotypes of sympatric races exhibit remarkable concordance between the two species, the mitochondrial cladograms show that the species have not shared a common evolutionary history. H. erato exhibits a basal split between trans- and cis-Andean groups of races, whereas H. melpomene originates in the Guiana Shield. Diverse races in either species appear to have evolved within the last 200,000 yr, and convergent phenotypes have evolved independently within as well as between species. These results contradict prior theories of the evolution of mimicry based on analysis of wing-pattern genetics.     

CALLING SONG DISPLACEMENT IN A ZONE OF OVERLAP AND HYBRIDIZATION

The ground crickets Allonemobius fasciatus and A. socius meet in a mosaic zone of overlap and hybridization stretching from the East Coast to at least Illinois. To test whether male calling song differences were enhanced in sympatry, we analyzed the songs of crickets from inside and outside the zone of overlap along two transects. No evidence of calling song displacement was found in A. socius males from populations within the zone of overlap. On the other hand, A. fasciatus displayed calling song displacement in three populations. Our results are consistent with the hypothesis that the selective pressure exerted by the challenge from a related species is frequency dependent. While not a conclusive demonstration, the observed shifts in calling song are strongly suggestive of reproductive character displacement.     

Sleeping with the ‘enemy’: hybridization of an endangered tree weta

While hybridization is an important part of the evolutionary process, for rare species mating with more common species hybridization can increase the risks of extinction. By mating with heterospecifics rare species waste valuable reproductive resources and as a result population sizes may decline. If introgression occurs, the rare species can become genetically swamped by alleles from the more common species, rendering it effectively extinct. As a consequence of these risks, hybridization with the more common species Hemideina femorata (Canterbury tree weta) may lead to the extinction of the range restricted species H. ricta (Banks Peninsula tree weta) on Banks Peninsula. The current study uses spatial interpolation to model the distribution of each species and the potential sympatric zone to guide sampling efforts. Polymorphic microsatellite markers and mitochondrial sequence data were used to determine the extent of hybridization between H. ricta and H. femorata. The results confirm that hybridization is possible between these species. However, hybrids and introgression appear to be very rare, suggesting that reproductive isolating barriers are present but incomplete. The threat of extinction to H. ricta via hybridization with H. femorata is low but extreme loss of habitat may cause changes to population densities that could increase the risks of hybridization. Therefore, landowners should be encouraged to conserve native bush.