Phylogenetic relationships of three African swallowtail butterflies,Papilio dardanus,P.phorcas and P.constantinus: A cladistic analysis (Lepidoptera: Papilionidae)

Abstract. The well-known polymorphic mimetic African papilionid butterfly, Papilio dardanus Brown, is demonstrated by cladistic analysis to form a monophyletic group (the phorcas-group) with two other African swallowtails, P.phorcas Cramer and P.constantinus Ward. Within this group, dardanus and phorcas are shown to be sister-species, with constantinus as the stem. The supposed relationship of this group of three Rutaceae-feeding species to the five members of the Canellaceae/Lauraceae-feeding Papilio hesperus-group (including P.nobilis Rogenhofer) is questioned. The significance of this analysis is discussed with special reference to Trimen's original hypothesis on the evolution of mimetic patterns in female Papilio dardanus, the unique male patterns of phorcas and dardanus, and natural and laboratory-produced hybrids. A pathway modelling technique is developed to represent the possible course of evolution of the phorcas-group, in terms of speciation events and the accumulation and decay of the pattern polymorphisms.     

Independent evolution of sexual dimorphism and female‐limited mimicry in swallowtail butterflies (Papilio dardanus and Papilio phorcas)

Several species of swallowtail butterflies (genus Papilio) are Batesian mimics that express multiple mimetic female forms, while the males are monomorphic and nonmimetic. The evolution of such sex‐limited mimicry may involve sexual dimorphism arising first and mimicry subsequently. Such a stepwise scenario through a nonmimetic, sexually dimorphic stage has been proposed for two closely related sexually dimorphic species: Papilio phorcas, a nonmimetic species with two female forms, and Papilio dardanus, a female‐limited polymorphic mimetic species. Their close relationship indicates that female‐limited polymorphism could be a shared derived character of the two species. Here, we present a phylogenomic analysis of the dardanus group using 3964 nuclear loci and whole mitochondrial genomes, showing that they are not sister species and thus that the sexually dimorphic state has arisen independently in the two species. Nonhomology of the female polymorphism in both species is supported by population genetic analysis of engrailed, the presumed mimicry switch locus in P. dardanus. McDonald–Kreitman tests performed on SNPs in engrailed showed the signature of balancing selection in a polymorphic population of P. dardanus, but not in monomorphic populations, nor in the nonmimetic P. phorcas. Hence, the wing polymorphism does not balance polymorphisms in engrailed in P. phorcas. Equally, unlike in P. dardanus, none of the SNPs in P. phorcas engrailed were associated with either female morph. We conclude that sexual dimorphism due to female polymorphism evolved independently in both species from monomorphic, nonmimetic states. While sexual selection may drive male–female dimorphism in nonmimetic species, in mimetic Papilios, natural selection for protection from predators in females is an alternative route to sexual dimorphism.     

Phylogenetic relationships of three African swallowtail butterflies,Papilio dardanus,P.phorcas and P.constantinus: New data from hybrids (Lepidoptera: Papilionidae)

Abstract. Various laboratory-bred hybrids between Papilio dardanus, P.phorcas and P.constantinus are reported. The principal object of the research was to obtain evidence regarding the evolutionary relationships of the three species, with particular reference to understanding the evolution of the mimetic patterns of P.dardanus. With regard to the latter, little progress was made but other findings were interesting, notably those concerning sex ratio, pupal coloration, and differences in male genitalia.     

Evolution of dominance in polymorphic Batesian mimicry

Models of two simple genetic systems of two alleles segregating at two loci are used to study the evolution of dominance of a Batesian mimic maintained in a population by frequency-dependent selection. The alleles at one locus determine the mimetic patterns, and their dominance is modified by the alleles at the other locus. In the model, the modifiers of dominance may themselves be either fully dominant or have additive effects on the dominance of the mimics. When the modifier is fully dominant in its effect on the dominance of a new mimic, the mimic will evolve dominance irrespective of the initial frequency of the modifier. When the modifiers act additively on the dominance of the mimics, a new mimic will evolve either dominance or recessiveness depending on the initial frequency of the modifiers. Unless the modifier is initially at quite a high frequency dominance will not evolve. And dominance will not evolve fully unless the modifiers are more or less selectively neutral in their effects on all other characters except the mimicry. The significance of these results is discussed with reference to the different dominance relations of the mimics in different races of the butterfly Papilio dardanus.     

Perspectives Poulton,Wallace and Jordan: How discoveries in Papilio butterflies led to a new species concept 100 years ago

A hundred years ago, in January 1904, E.B. Poulton gave an address entitled ‘What is a species?’ The resulting article, published in the Proceedings of the Entomological Society of London, is perhaps the first paper ever devoted entirely to a discussion of species concepts, and the first to elaborate what became known as the ‘biological species concept’. Poulton argued that species were syngamic (i.e. formed reproductive communities), the individual members of which were united by synepigony (common descent). Poulton's species concept was informed by his knowledge of polymorphic mimicry in Papilio butterflies: male and female forms were members of the same species, in spite of being quite distinct morphologically, because they belonged to syngamic communities. It is almost certainly not a coincidence that Alfred Russel Wallace had just given Poulton a book on mimicry in December 1903. This volume contained key reprints from the 1860s including the first mimicry papers, by Henry Walter Bates, Wallace himself and Roland Trimen. All these papers deal with species concepts and speciation as well as mimicry, and the last two contain the initial discoveries about mimetic polymorphism in Papilio: strongly divergent female morphs must belong to the same species as non‐mimetic males, because they can be observed in copula in nature. Poulton, together with his contemporaries Karl Jordan and Walter Rothschild, who had monographed world Papilionidae, were strongly influential on the evolutionary synthesis 40 years later. Ernst Mayr, in particular, had collected birds and butterflies for Walter Rothschild, and had visited Tring, where Jordan worked, in the 1920s. The recognition of different kinds of reproductive and geographic isolation, the classification of isolating mechanisms, the use of the term sympatry, and the biological species concept all trace back to Poulton's 1904 paper. Poulton's paper, in turn, inherits much from Wallace's 1865 paper on Asian Papilio contained in the very book Wallace gave Poulton a month earlier. Wallace's gift, and Poulton's subsequent New Year address are thus key events in the history of species concepts, systematics and evolutionary biology.     

The Adaptive Significance of Batesian Mimicry in the Swallowtail Butterfly,Papilio polytes (Insecta,Papilionidae): Associative Learning in a Predator

Experiments were conducted to examine the adaptive significance of Batesian mimicry in the swallowtail butterfly, Papilio polytes, with its model, Pacbliopta aristolochiae, an alkaloidal butterfly. The female of Pap. polytes is polymorphic, whereas the male is monomorphic. Two forms of the female, f. polytes mimic and f. cyrus non-mimic, were used in these experiments. Naive birds, brown-eared bulbuls Hypsipetes amaurotis pryeri, were trained to take food from two feeders in captivity, and then were offered Pach. aristolochiae in one of the feeders. After experiencing an uncomfortable encounter with this species, the birds reduced the frequency of taking regular food from the feeder where the butterfly had been placed. This result suggests that the birds can learn not only the model itself but also the place where they have experienced it. Thereafter, the birds also came to avoid the mimetic form of f. polytes. It is suspected that wild predators behave in the same way. These findings imply that it is adaptive for the mimic to overlap its habitats and daily activity with those of the model species.     

No Evidence that Male Choice Contributes to the Maintenance of a Shared, Sex-Limited Trait in Mimetic and Non-mimetic Female Tiger Swallowtail Butterflies, Papilio glaucus

In animal species that have morphological polymorphisms maintained by unique or divergent selection pressures, understanding the preservation of shared traits is important for identifying the factors that are influencing overall evolutionary processes. In the Eastern tiger swallowtail butterfly, Papilio glaucus, females are dimorphic. One morph (‘dark-morph’) is mostly black and mimics the toxic pipevine swallowtail, Battus philenor. These females have large amounts of blue coloration on the dorsal hind wings that enhances their mimetic resemblance. Conversely, the alternate female type (‘yellow-morph’) is similar to males in coloration with the exception of extensive dorsal blue coloration, comparable to dark-morph females. Such coloration is almost completely absent in males. We examined dorsal blue coloration in P. glaucus to determine if mimetic resemblance in dark morphs is predominantly responsible for the maintenance of dorsal blue color in both female types, or whether mate recognition and/or sexual selection by males has a stronger influence on this trait. We measured the relative amount and variance of dorsal and ventral blue coloration in females of both color morphs, as well as males. We also compared these measurements to similar ones taken in the sister species, P. canadensis (which does not exhibit female dimorphism). Lastly, we investigated mate recognition and preferences of wild males. Our results suggest that mimetic resemblance may be more important than sexual selection for sustaining dorsal blue coloration in dark-morph females and that yellow-morphs could have elevated levels of blue due to currently unknown genetic associations. Although trait correlation between sexes is common, intrasexual trait correlation in a sex-limited, polymorphic species has not been frequently observed.     

Characterising the Phenotypic Diversity of Papilio dardanus Wing Patterns Using an Extensive Museum Collection

The history of 20^th Century evolutionary biology can be followed through the study of mimetic butterflies. From the initial findings of discontinuous polymorphism through the debates regarding the evolution of mimicry and the step-size of evolutionary change, to the studies on supergene evolution and molecular characterisation of butterfly genomes, mimetic butterflies have been at the heart of evolutionary thought for over 100 years. During this time, few species have received as much attention and in-depth study as Papilio dardanus. To assist all aspects of mimicry research, we present a complete data-derived overview of the extent of polymorphism within this species. Using historical samples permanently held by the NHM London, we document the extent of phenotypic variation and characterise the diversity present in each of the subspecies and how it varies across Africa. We also demonstrate an association between “imperfect” mimetic forms and the transitional race formed in the area where Eastern and Western African populations meet around Lake Victoria. We present a novel portal for access to this collection, www.mimeticbutterflies.org, allowing remote access to this unique repository. It is hoped that this online resource can act as a nucleus for the sharing and dissemination of other collections databases and imagery connected with mimetic butterflies.     

GENETICS OF MIMICRY IN THE TIGER SWALLOWTAIL BUTTERFLIES,PAPILIO GLAUCUS AND P. CANADENSIS (LEPIDOPTERA: PAPILIONIDAE)

The tiger swallowtail butterfly, Papilio glaucus, exhibits a female-limited polymorphism for Batesian mimicry; the Canadian tiger swallowtail, Papilio canadensis, lacks the mimetic (dark) form entirely. The species hybridize to a limited extent where their ranges overlap. Field collections and censuses indicate that mimetic females occur throughout the range of P. glaucus but at lowest frequencies in populations at the latitudinal edges of its geographic range such as the southernmost part of Florida and along the entire northern edge of its distribution from Massachusetts to Minnesota. Frequencies of mimetic females have remained relatively stable over time. Inheritance of the mimetic form is controlled primarily by two interacting sex-linked loci. The typical matrilineal pattern of inheritance in P. glaucus can be explained by polymorphism at a Y-linked locus, b. Analysis of P. glaucus × P. canadensis crosses has also revealed an X-linked locus, s, which controls the expression of the mimetic phenotype. The P. canadensis allele, s^can, suppresses the mimetic phenotype in hybrid and backcross females. Results from more than 12 yr of rearing tiger swallowtails, including interspecies hybrids, indicate that the absence of mimetic P. canadensis females is due to both a high frequency of the “suppressing” allele s^can and low frequency of the black-pigment-determining b + allele. The frequency of s^can (or other suppressing alleles of s) in P. glaucus populations outside the hybrid zone is low. Some males heterozygous at the s locus and some suppressed mimetic females occur within the hybrid zone. A simple genetic model predicts the frequency of daughters that differ in phenotype from their mothers.     

Experimental field tests of Batesian mimicry in the swallowtail butterfly Papilio polytes

The swallowtail butterfly Papilio polytes is known for its striking resemblance in wing pattern to the toxic butterfly Pachliopta aristolochiae and is a focal system for the study of mimicry evolution. Papilio polytes females are polymorphic in wing pattern, with mimetic and nonmimetic forms, while males are monomorphic and nonmimetic. Past work invokes selection for mimicry as the driving force behind wing pattern evolution in P. polytes. However, the mimetic relationship between P. polytes and P. aristolochiae is not well understood. In order to test the mimicry hypothesis, we constructed paper replicas of mimetic and nonmimetic P. polytes and P. aristolochiae, placed them in their natural habitat, and measured bird predation on replicas. In initial trials with stationary replicas and plasticine bodies, overall predation was low and we found no differences in predation between replica types. In later trials with replicas mounted on springs and with live mealworms standing in for the butterfly's body, we found less predation on mimetic P. polytes replicas compared to nonmimetic P. polytes replicas, consistent with the predator avoidance benefits of mimicry. While our results are mixed, they generally lend support to the mimicry hypothesis as well as the idea that behavioral differences between the sexes contributed to the evolution of sexually dimorphic mimicry.     

Crossing the threshold: gene flow,dominance and the critical level of standing genetic variation required for adaptation to novel environments

Genetic architecture plays an important role in the process of adaptation to novel environments. One example is the role of allelic dominance, where advantageous recessive mutations have a lower probability of fixation than advantageous dominant mutations. This classic observation, termed ‘Haldane's sieve’, has been well explored theoretically for single isolated populations adapting to new selective regimes. However, the role of dominance is less well understood for peripheral populations adapting to novel environments in the face of recurrent and maladaptive gene flow. Here, we use a combination of analytical approximations and individual‐based simulations to explore how dominance influences the likelihood of adaptation to novel peripheral environments. We demonstrate that in the face of recurrent maladaptive gene flow, recessive alleles can fuel adaptation only when their frequency exceeds a critical threshold within the ancestral range.     

Testosterone Induces Sexual Release Quality in the Song of Female Canaries

We tested the sexual responsiveness of female canaries, Serinus canaria, to two sets of different types of female conspecific songs versus an adult male conspecific song. Female songs were either spontaneously emitted (‘S-songs’) or were testosterone-induced (‘T-songs’). Copulation-solicitation displays (CSD) were used as an index of female sexual response. Playbacks were performed several days before and during egg laying, a period of natural sexual responsiveness of the females to song. We demonstrated that the weaker sexual displays of female canaries were recorded to S-songs, thus suggesting that these types of female songs do not contain fully functional sexual releasers. Three T-songs elicited high levels of sexual displays, thus demonstrating that testosterone treatment may induce sexual release quality in the female songs. Study of the phonology of these three T-songs strongly suggested that special song phrases may be good candidates as powerful sexual releasers. To test the sexual value of these female song phrases, we carried out a third experiment, using hybrid songs where each of these special T-song phrase types was included in a well-known heterospecific context. Two phrases elicited high levels of sexual responses in females. Essential features of the male full song, such as broadband rapid frequency modulations and high repetition rate, are retrieved in both female song phrases. Taken together, these data demonstrate that testosterone treatment not only induces a male-like structure in the songs of females, but also induces functionally ‘male-like’ songs. This result allows features of the vocal control network of testosterone-treated females to be compared with those of adult males singing full songs, to distinguish neural correlates of testosterone-dependent full songs. However, because testosterone does not induce functionally male-like songs in all the females, neuroanatomical structure-function correlations need detailed behavioural analysis.     

Papilio nandina, a probable hybrid between Papilio dardanus and Papilio phorcas

Abstract. Hybrids bred in the laboratory between Papilio dardanus Brown and Papilio phorcas Cramer resulted in male butterflies similar to the two naturally occurring males described as P.nandina Rothschild & Jordan. A single female hybrid insect resembling the female parent was also bred. Male hybrids were also obtained between P.dardanus and P.demodocus/demoleus but not between P.dardanus and P.constantinus .
The phylogenetic relationships of dardanus and phorcas are discussed, and reasons given for preferring the older hypothesis that male-like females are primitive rather than that they represent the most specialized forms. Further information on this point would be forthcoming if backcrosses and F₂ s could be obtained.     

Behaviour before beauty: Signal weighting during mate selection in the butterfly Papilio polytes

Mating displays often contain multiple signals. Different combinations of these signals may be equally successful at attracting a mate, as environment and signal combination may influence relative signal weighting by choosy individuals. This variation in signal weighting among choosy individuals may facilitate the maintenance of polymorphic displays and signalling behaviour. One group of animals known for their polymorphic patterning are Batesian mimetic butterflies, where the interaction of sexual selection and predation pressures is hypothesized to influence the maintenance of polymorphic wing patterning and behaviour. Males in the female‐limited polymorphic Batesian mimetic butterfly Papilio polytes use female wing pattern and female activity levels when determining whom to court. They court stationary females with mimetic wing patterns more often than stationary females with non‐mimetic, male‐like wing patterns and active females more often than inactive females. It is unclear whether females modify their behaviour to increase (or decrease) their likelihood of receiving male courtship, or whether non‐mimetic females spend more time in cryptic environments than mimetic females, to compensate for their lack of mimicry‐driven predation protection (at the cost of decreased visibility to males). In addition, relative signal weighting of female wing pattern and activity to male mate selection is unknown. To address these questions, we conducted a series of observational studies of a polymorphic P. polytes population in a large butterfly enclosure. We found that males exclusively courted active females, irrespective of female wing pattern. However, males did court active non‐mimetic females significantly more often than expected given their relative abundance in the population. Females exhibited similar activity levels, and selected similar resting environments, irrespective of wing pattern. Our results suggest that male preference for non‐mimetic females may play an active role in the maintenance of the non‐mimetic female form in natural populations, where males are likely to be in the presence of active, as well as inactive, mimetic and non‐mimetic females.     

Cranial variation in the European badger Meles meles (Carnivora,Mustelidae) in Scandinavia

Morphometric variation in 30 craniometric characters of 465 skulls of the European badgers (Meles meles) from across Europe was analysed. Multivariate analyses revealed that the populations from Norway, Sweden, and Finland differ from other European populations in having smaller skulls. The analyses also revealed significant differences between the ‘south‐western Norwegian’ and ‘main Fennoscandian’ forms. On average, badgers from south‐west Norway were smaller than those of the remaining Fennoscandia. Morphological differences between the ‘south‐western Norwegian’ and ‘main Fennoscandian’ populations of M. meles suggest a possible in situ semisympatric divergence since the beginning of the Holocene warming, or a complex history of two groups involving at least two colonization routes. The small‐sized Scandinavian badgers may be close to the ancestral form that used to be widespread in Denmark and throughout Europe. The animals from south‐west Norway may instead be descendants of ancestors that were the first to penetrate the southern parts of the Scandinavian Peninsula. The ‘main Fennoscandian’ badgers are likely to have been the descendants of the second wave of recolonization of Scandinavia. Specifically, they might have colonized the Scandinavian Peninsula from the east after the last glaciation.     

Eunotia amazonica sp. nov. (Bacillariophyta), a common stalk-forming species from the Rio Negro basin (Brazilian Amazon)

A new stalk-forming diatom was observed at high relative abundances on periphytic samples collected in the Rio Negro hydrographic basin (Brazilian Amazon) and is here described using light and scanning electron microscopy. We also present ecological preferences of the new species. Eunotia amazonica sp. nov. co-dominated periphytic samples collected during the rainy seasons of 2013 and 2014 near the city of Manaus (Amazonas, Brazil). The main diagnostic feature of the new species is the stalk-forming growth form (‘Cymbella-like’) illustrated here for the first time in the genus Eunotia Ehrenberg. Previous reports on growth forms in common European databases for this genus included colonial (‘ribbon-like’) but also ‘mobile’, ‘fixed by pads’ or ‘without structures of fixation’. We demonstrated that the three dominant Eunotia species in periphytic samples from the Rio Negro all exhibited distinct ‘growth forms’ or habits: Eunotia amazonica sp. nov. (stalked), Eunotia intricans Metzeltin & Lange-Bertalot (ribbon-like) and Eunotia rabenhorstiana (Grunow) Hustedt (branched-arborescent). The high diversity of species, many of which are unknown to science, as well as the poorly known life-forms and habits of the Eunotiaceae in the region, partially prevent the use of metrics based on life-form to assess ecological changes. Generic assumptions based on robust statistical methods may obscure reality and lead to biased conclusions of diatom community changes in poorly explored regions such as Neotropical freshwater rivers, where the Eunotiaceae are largely dominant, particularly in the Amazon basin.     

Female dominance in blue-eyed black lemurs(Eulemur macaco flavifrons)

Female dominance is unusual among mammals and has been described in detail for only a handful of species. Here we present data on the frequency and outcome of dominance interactions in seven semi-free ranging and captive groups of blue-eyed black lemurs (Eulemur macaco flavifrons) housed at the Duke University Primate Center. We collected over 260 hours of focal data during which all occurrences of dominant-subordinate interactions were recorded. We collected data outside the typical breeding and birthing seasons for this species, thus eliminating possible confounding factors and increased aggression associated with these periods. We found that females were dominant over males in all seven groups, with females winning 99% of all dominance interactions.E. m. flavifrons used aggressive dominance (e.g. chase, cuff, bite) in 81% of all interactions, with the remainder of interactions being decided using social dominance (e.g. deference in the form of supplants or cowers). Older females were dominant over younger females in two out of three multi-female groups (in each case, younger females were daughters), and younger males (sons of the dominant female) received less aggression from females than did older males (n = 2 groups). Caging and group size appear to play a minimal role in the expression of female dominance. While confirmation must await further observations on free-ranging groups ofE. m. flavifrons, our data strongly suggest that this subspecies can be characterized as female dominant.     

A method for adjusting allele frequencies in the case of microsatellite allele drop‐out

A new method is proposed to adjust allele frequencies when allelic drop‐out is common. This method assumes Hardy–Weinberg equilibrium (HWE), and treats the problematic alleles as a one‐locus two‐allele system with dominance. By assuming that the homozygote frequency of the ‘recessive’ allele is measured correctly, we can back calculate the allele frequency of the ‘dominant’ allele, and adjust the heterozygote frequency accordingly. The drawback is that multilocus genotypes cannot be constructed and tests that use deviations from Hardy–Weinberg such as tests for bottlenecks become impossible. An example is given where a large homozygote excess (F_IS = 0.44) is adjusted to a reasonable level (F_IS = 0.046). The effect of scoring error was set in relation to sampling error and while F_IS values can be seriously biased, F_ST values are not necessarily so, if scoring error and sample size are both low. As sample size increases, the effect of scoring error increases.     

Mimetic butterflies support Wallace's model of sexual dimorphism

Theoretical and empirical observations generally support Darwin's view that sexual dimorphism evolves due to sexual selection on, and deviation in, exaggerated male traits. Wallace presented a radical alternative, which is largely untested, that sexual dimorphism results from naturally selected deviation in protective female coloration. This leads to the prediction that deviation in female rather than male phenotype causes sexual dimorphism. Here I test Wallace's model of sexual dimorphism by tracing the evolutionary history of Batesian mimicry-an example of naturally selected protective coloration-on a molecular phylogeny of Papilio butterflies. I show that sexual dimorphism in Papilio is significantly correlated with both female-limited Batesian mimicry, where females are mimetic and males are non-mimetic, and with the deviation of female wing colour patterns from the ancestral patterns conserved in males. Thus, Wallace's model largely explains sexual dimorphism in Papilio. This finding, along with indirect support from recent studies on birds and lizards, suggests that Wallace's model may be more widely useful in explaining sexual dimorphism. These results also highlight the contribution of naturally selected female traits in driving phenotypic divergence between species, instead of merely facilitating the divergence in male sexual traits as described by Darwin's model.