Hybrid queen butterflies from the cross Danaus chrysippus (L.) × D. gilippus (Cramer):confirmation of species status for the parents and further support for Haldane's Rule

A cross between queen butterflies of the Palaeotropical species Danaus chrysippus and the Neotropical D. gilippus was achieved with difficulty in both directions. Only one progeny ( N  = 70) was reared comprising sterile males and inviable females in a precisely 1 : 1 ratio. Both prezygotic and postzygotic barriers to gene flow are strong. The result supports Haldane's Rule, to which we propose a minor amendment. The F₁ hybrids were intermediate for background colour between the brown (genotype BB ) of gilippus and orange (genotype bb ) of chrysippus . Most F₁ pattern characters were also intermediate. In polymorphic chrysippus populations, because Bb heterozygotes are brown, or nearly so, we suggest the B allele may have evolved towards dominance in sympatry. Hybrid males show positive heterosis for body size. The close similarity of male genitalia between the allopatric, genetically distant species chrysippus  and gilippus , compared to their divergence between gilippus and its largely sympatric sister species eresimus , suggest that reinforcement of sexual isolation or reproductive character displacement have evolved in sympatry.  © 2002 The Linnean Societyof London, Biological Journal of the Linnean Society , 2002, 76 , 535–544.     

Cuticular lipid diversification in Lasiommata megera and Lasiommata paramegaera: the influence of species, sex, and population (Lepidoptera: Nymphalidae)

Numerous studies have investigated the presence and the effectiveness of volatile pheromones in Lepidoptera. Conversely, very few studies have focused on the composition and the perception of the relatively low volatile components of cuticular mixtures. Yet, cuticular lipids are implied in the recognition processes of several solitary and social insects. In the present study, the cuticular signatures of the satyrid butterflies Lasiommata megera and Lasiommata paramegaera were examined by gas chromatography. General linear model and discriminant analyses on chemical data clearly revealed large differences between sexes, which showed the same diversification pattern in both species. Moreover, a strong diversification between the two species was found, as were differences among populations. These results represent a first step in demonstrating the communicative function of cuticular compounds in the L. megera / paramegaera complex. Moreover, the discrimination among different species and populations on the basis of cuticular mixtures could represent a platform for studying chemotaxonomy and chemical biogeography in butterflies, as already found in several other insect groups.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 703–710.     

Pyrrolizidine alkaloids from Senecio vulgaris sequestered and stored by Danaus plexippus

Danaus plexippus reared on Asclepias curassavica , sprayed with homogenized leaves of Senecio vulgaris , were found to contain mean pyrrolizidine alkaloid concentrations of 38.8±6.08 μg/male butterfly and 16.5 ± 3–63 μg/female butterfly.     

Transitions in cuticular composition across a hybrid zone: historical accident or environmental adaptation?

Two subspecies of the grasshopper Chorthippus parallelus form a hybrid zone in the Pyrenees. Transitions across the zone, including changes in mating signals and reproductive isolation have been intensively studied. Cuticular pheromones have been identified as likely mate recognition signals. Since the major role of the cuticle is in waterproofing, environmental adaptation of cuticular composition has the interesting potential to generate assortative mating as an incidental by-product. We describe the pattern of variation in cuticular hydrocarbon blend in four transects through the hybrid zone. We find no evidence for a previously observed displaced cline in one blend component. There were differences between subspecies but these varied among transects and were small compared with variation between transects. We examined environmental variation within one transect and found a correlation between vegetation and cuticular composition, suggesting that environment influences the constitution of the cuticle, and hence natural selection may interact with mating signals in this species.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 78 , 193−201.     

Molecular phylogeny of the genus Tibicina (Hemiptera, Cicadidae): rapid radiation and acoustic behaviour

To estimate the potential contribution of ethological and ecological parameters to the mechanisms of species formation and species isolation in the Palearctic cicada genus Tibicina , we constructed a molecular phylogenetic hypothesis of extant Tibicina species. Seven mitochondrial genes and a fragment of a nuclear gene were sequenced (3046 bp). Mitochondrial genes included 547 informative sites but the nuclear gene was too conserved to be included in the analysis. The tree was characterized by a basal polytomy indicating that Tibicina species arose rapidly. Such rapid radiation might explain the low divergence in the acoustic communication observed between species. Parameters describing habitat selection and acoustic communication were mapped onto the tree. A shift in habitat selection accompanied by acoustic changes might have contributed to one speciation event. The stochastic distribution of the same acoustic characters on the other branches of the tree implies, however, that the subtle acoustic differences between species could be the result of previous speciation events and independent evolutionary histories, rather than having contributed themselves in the speciation and isolation processes.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 611–626.     

Long-term stability of hornet cuticular hydrocarbons facilitates chemotaxonomy using museum specimens

Cuticular hydrocarbons are key compounds used for insect chemical communication and their species-specificity makes them of great utility to chemotaxonomists. However, very little is known about their long-term stability in relation to their use as reliable taxonomic tools. We compared the cuticular hydrocarbon profiles of fresh extracts from four hornet ( Vespa ) species with extracts from specimens that were frozen for 1 year and of those stored in insect display boxes for 20 years. Cuticular hydrocarbon profiles were qualitatively very stable, maintaining their species-specific profiles even after 20 years. The long-term stability of cuticular hydrocarbons in hymenopterans opens up the possibility of using museum collections for chemotaxonomy studies and helping with the delineation of species in difficult groups.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 732–737.     

Genetic differentiation between Australian and North American populations of the monarch butterfly Danaus plexippus (L.) (Lepidoptera: Nymphalidae): an exploration using allozyme electrophoresis

Allozyme analysis was used to address the question of the source of the Australian populations of the monarch butterfly Danaus plexippus (L.). The study had three major aims: (1) To compare the levels of diversity of Australian and Hawaiian populations with potential source populations. (2) To determine whether eastern and western North American populations were sufficiently divergent for the Australian populations to be aligned to a source population. (3) To compare the differentiation among regions in Australia and North America to test the prediction of greater genetic structure in Australia, as a consequence of reduced migratory behaviour. The reverse was found, with F _ST values an order of magnitude lower in Australia than in North America. Predictably, Australian and Hawaiian populations had lower allelic diversity, but unexpected higher heterozygosity values than North American populations. It was not possible to assign the Australian populations to a definitive source, although the high levels of similarity of Australian populations to each other suggest a single colonization event. The possibility that the Australian populations have not been here long enough to reach equilibrium is discussed. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 75 , 437–452.     

Electrophoretic characterization of Amaranthus L. seed proteins and its systematic implications

The seed protein profiles of 11 Amaranthus taxa (Amaranthaceae) from Spain were studied. These profiles were evaluated as a chemical character to clarify the taxonomic complexity in the genus. Tricine-sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of the Amaranthus seed proteins studied showed a range of peptides varying from 64 to 12 kDa, with a larger number of protein bands observed between 25.1 and 12 kDa. For the taxonomic study, 14 bands, some of them subdivided into several isoforms, were considered. The similarity analysis based on the SDS-PAGE profile is a useful character for the discrimination of species in Amaranthus , except for A. cruentus and A. hypochondriacus , for which a hybrid population was found. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 57–63.     

Phenotypic divergence and reproductive isolation between sympatric forms of Japanese threespine sticklebacks

The threespine stickleback ( Gasterosteus aculeatus ) species complex is well suited for identifying the types of phenotypic divergence and isolating barriers that contribute to reproductive isolation at early stages of speciation. In the present study, we characterize the patterns of genetic and phenotypic divergence as well as the types of isolating barriers that are present between two sympatric pairs of threespine sticklebacks in Hokkaido, Japan. One sympatric pair consists of an anadromous and a resident freshwater form and shows divergence in body size between the forms, despite the lack of genetic differentiation between them. The second sympatric pair consists of two anadromous forms, which originated before the last glacial period and are currently reproductively isolated. These two anadromous forms have diverged in many morphological traits as well as in their reproductive behaviours. Both sexual isolation and hybrid male sterility contribute to reproductive isolation between the anadromous species pair. We discuss the shared and unique aspects of phenotypic divergence and reproductive isolation in the Japanese sympatric pairs compared with postglacial stickleback species pairs. Further studies of these divergent species pairs will provide a deeper understanding of the mechanisms of speciation in sticklebacks.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 671–685.     

Microsatellite DNA polymorphism confirms reproductive isolation and reveals differences in population genetic structure of cryptic pipistrelle bat species

Previous studies have indicated that the common European pipistrelle bat ( Pipistrellus pipistrellus ) comprises two cryptic species, P. pipistrellus and Pipistrellus pygmaeus , which differ in echolocation call frequency and mitochondrial DNA sequence. However, levels of divergence based on nuclear markers have not been examined, and hence the potential for male-mediated gene flow between the species cannot be discounted. Moreover, little is known about population structure and migration patterns in either species. Here, we describe the use of microsatellites to investigate nuclear DNA differentiation between, and the pattern of population genetic structure within, the two cryptic pipistrelle species. In total, 1300 individuals from 82 maternity colonies were sampled across the British Isles and Continental Europe. We show, using multivariate analyses, that colonies of the same species are generally genetically more similar to each other than to those from the other species regardless of geographical location. Our findings support the hypothesis that the species are reproductively isolated. Significant patterns of genetic isolation by distance were identified in both species, indicating that mating may occur before any long-distance autumnal migration. The presence of a sea channel does not confer higher levels of genetic differentiation among colonies over and above distance alone in either species. Differences in genetic population structure were identified between the species, with P. pipistrellus showing a wider range of levels of genetic differentiation among colonies and a stronger relationship between genetic and geographical distance than P. pygmaeus . Differences in dispersal, mating behaviour, colony size and/or postglacial colonization patterns could contribute to the differences observed.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 539–550.     

Character displacement of male nuptial colour in threespine sticklebacks (Gasterosteus aculeatus)

To study the isolation mechanism of two commonly intermingled louseworts, Pedicularis rhinanthoides and Pedicularis longiflora , pollination biology in three mixed populations with the two species was investigated during a 3-year project. The results indicated that higher flowering density could help to enhance pollinator activity, and thus increase reproductive output. Bumblebees are the exclusive pollinator for the two louseworts and are essential for their reproductive success. Reproductive isolation between the two species is achieved by a combination of pre- and postzygotic isolation mechanisms. Although both species are pollinated by bumblebees, the present study indicates they successfully avoid interspecific pollen transfer due to floral isolation. Mechanical isolation is achieved by the stigma in the two species picking up pollen from different parts of the pollinator's body, whereas ethological isolation occurs due to flower constancy. Additionally, strong postzygotic isolation was demonstrated by non seed set after artificial cross-pollination even with successful pollen tube growth. We describe the hitherto unreported role of variation in the tightness and direction of the twist of the corolla beak in maintaining mechanical isolation between Pedicularis species. Although floral isolation in Pedicularis is incomplete, it is considered to be an important mechanism in maintaining species boundaries in the genus.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 37–48.     

Hybridization patterns and the evolution of reproductive isolation in ducks

Much of our knowledge of the evolution of reproductive isolation comes from studies of Drosophila . This body of work has revealed the following patterns: (1) reproductive isolation increases with phylogenetic distance between hybridizing species; (2) reproductive isolation is greater between sympatric than allopatric species with the same level of divergence; and (3) hybrid crosses conform to Haldane's rule. We tested for the existence of these patterns in ducks (subfamily Anatinae, sensu Livezey, 1997b ) based on 1037 hybrids of known parentage. Our analyses of the number of interspecific crosses in relation to phylogenetic distance found a significant deviation between the observed and expected distribution of crosses controlling for the topology of the Anatinae phylogeny. In particular, we found both an excess of hybrid crosses among closely related species and a scarcity among distantly related species. The number of hybrid males also decreased with increasing phylogenetic distance between parental species, although the number of hybrid females remained low and constant. Sympatric species produced higher numbers of hybrid males than allopatric ones, despite no difference in phylogenetic distance among parental species in compared groups. The number of hybrid males exceeded the number of hybrid females, consistent with Haldane's rule. This was evident even though the analysis was restricted to a reduced set of phylogenetically independent crosses. However, the pattern was no longer significant after correction for the number of hybrid males by the male-biased sex ratio of adult ducks.  © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 77 , 193−200.     

A new species of Astragalus L. (Leguminosae) from India based on morphological and molecular markers

A new species, Astragalus himachalensis , is described and illustrated from the Himalaya in India. The species grows occasionally in different parts of the cold desert of Lahul-Spiti (Himachal Pradesh). It resembles A. himalayanus , but differs chiefly in the small size of the plants (length, 5–15 cm), diadelphous stamens, and small pods (length, 5–6 mm) with the stipe length (2–3 mm) more or less equal to the calyx length. The similarities and significant differences between A. himachalensis and A. himalayanus were confirmed by analyses of random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite DNA (DAMD) markers. RAPD and DAMD methods clearly distinguished between the two species and were in congruence with morphological markers. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 27–34.     

Incipient speciation revealed in Anastrepha fraterculus (Diptera; Tephritidae) by studies on mating compatibility, sex pheromones, hybridization, and cytology

It has long been proposed that the nominal species Anastrepha fraterculus is a species complex and earlier studies showed high levels of pre-zygotic isolation between two laboratory strains from Argentina and Peru. Further experiments were carried out on the same populations and on their reciprocal hybrids, including pre- and post-zygotic isolation studies, pheromone analysis, and mitotic and polytene chromosome analysis. A high level of pre-zygotic isolation had been maintained between the parental strains despite 3 years of laboratory rearing under identical conditions. The level of pre-zygotic isolation was reduced in matings with hybrids. There were also differences in other components of mating behaviour. There were quantitative and qualitative differences in the sex pheromone of the two strains with the hybrids producing a mixture. The pre-zygotic isolation barriers were complemented by high levels of post-zygotic inviability and sex ratio distortion, most likely not due to Wolbachia , although there was evidence of some cytoplasmic factor involved in sex ratio distortion. Analysis of polytene chromosomes revealed a high level of asynapsis in the hybrids, together with karyotypic differences between the parental strains. The combined results of the present study indicate that these two strains belong to different biological entities within the proposed A. fraterculus complex.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 152–165.     

Mandible of the giant panda (Ailuropoda melanoleuca) compared with other Chinese carnivores: functional adaptation

The aim of this study was to understand the mandible of the giant panda in morphometric terms to explore differences between the giant panda and other carnivores distributed in China, in terms of functional adaptation. Twelve mandibular variables were studied using bivariate (allometry) and multivariate (principal components analysis, PCA, and discriminant functional analysis, DFA) tools. When deviations were produced from allometric baselines consisting of all the species studied, the giant panda displayed a much more developed mandibular structure than the bear, leopard, and tiger. This may be related to its specific dietary preference for bamboo, which has very strong fibers. Results also indicate that the mandibular structure among carnivores mainly reflects the differences in their dietary preferences and functional adaptation. Three groups were found referring to dispersal profiles expressed by the first two axes of PCA and DFA: (1) the two panda species – the herbivorous carnivores; (2) the black bear – the omnivorous carnivore; and (3) the tiger and leopard – the hypercarnivores. Nevertheless, a significant separation between the two panda species was also found with the profiles displayed by the first and third axes of DFA. In addition to no close evolutionary relationship and phylogenetic development, a noticeable separation between the two panda species found in DFA analysis may be associated with their variation in consuming different parts of the bamboo plant: the giant panda feeds on stems and the red panda feeds on leaves.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 449–456.     

Genetic and morphological variability of the endangered Syngonanthus mucugensis Giul. (Eriocaulaceae) from the Chapada Diamantina, Brazil: implications for conservation and taxonomy

Allozymic and morphometric studies were carried out on ten populations of Syngonanthus mucugensis (Eriocaulaceae), a species from north-eastern Brazil threatened by extinction. Genetic and morphological variability was low or moderately low in all populations, being lower in populations from Rio de Contas/Catolés ( P _L = 14.3–21.4, A  = 1.1–1.2, H _e = 0.026–0.059, D2M = 26.893–33.157) than in those from Mucugê ( P _L = 28.6–35.7, A  = 1.3–1.5, H _e = 0.078–0.164, D2M = 28.999–45.077). A high coefficient of endogamy ( F _is = 0.257) was found, which can be explained by the reproductive characteristics and distribution of the species. The values for genetic and morphological structuring ( F _st = 0.512 and A _MRPP = 0.175, respectively) were high as a result of the differentiation between populations from the two areas. The mean genetic identity between populations from the two areas (0.812) was much lower than between populations from the same area (Mucugê, 0.980; Rio de Contas/Catolés, 0.997). These results indicate that we are dealing with two distinct taxa and, as a result of the nature of the morphological differences found, a new subspecies is described for the populations of the region of Rio de Contas and Catolés, Syngonanthus mucugensis ssp. riocontensis . Such conclusions raise important implications for the conservation of Syngonanthus mucugensis , and will be used in the drawing up of management plans for its conservation.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 153 , 401–416.     

A danaid mullerian mimic, Euploea core amymone (Cramer) lacking cardenolides in the pupal and adult stages

Euploea core amymone , a Mullerian Mimic, when reared on Serium oleander and Asclepias curassavica , stores emetic cardiac glycosides only in the larval stages, and not as pupa and adult. Danaus plexippus erippus. which exhibits less cardioactivity if tested on the isolated rat heart than E. c. amymone , nevertheless stores them at all three stages of development; polar cardenolides not present in its food plant A. curassavica , were found in the pupa and imago.     

Amana kuocangshanica (Liliaceae), a new species from south-east China

Amana kuocangshanica D. Y. Tan & D. Y. Hong is described as a new species from Mt. Kuocang, Zhejiang Province, south-east China. Morphological observations and a statistical analysis of the new species and its relatives show that it is most closely related to A. anhuiensis and A. erythronioides , but differs in having the lower leaf oblanceolate, the widest at two-thirds length from the base, tunics glabrous inside, and fruit beaks 5–7.5 mm long. The morphologies of the pollen, seeds, and epidermal cells of the leaves of this new species are described.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 435–442.     

Postzygotic isolation between the two European subspecies of the house mouse: estimates from fertility patterns in wild and laboratory-bred hybrids

We assessed the fertility (reproductive success, litter size, testis weight, spermatocyte-to-spermatid ratio) of F₁s and backcrosses between different wild-derived outbred and inbred strains of two mouse subspecies, Mus musculus domesticus and M. m. musculus . A significant proportion of the F₁ females between the outbred crosses did not reproduce, suggesting that female infertility was present. As the spermatocyte-to-spermatid ratio was correlated with testis weight, the latter was used to attribute a sterile vs. fertile phenotype to all males. Segregation proportions in the backcrosses of F₁ females yielded 11 (inbred) to 17% (outbred) sterile males, suggesting the contribution of two to three major genetic factors to hybrid male sterility. Only one direction of cross between the inbred strains produced sterile F₁ males, indicating that one factor was borne by the musculus X-chromosome. No such differences were observed between reciprocal crosses in the outbred strains. The involvement of the X chromosome in male sterility thus could not be assessed, but its contribution appears likely given the limited introgression of X-linked markers through the hybrid zone between the subspecies. However, we observed no sterile phenotypes in wild males from the hybrid zone, although testis weight tended to decrease in the centre of the transect.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 379–393.     

Genetic detection of sperm from forced copulations between sympatric populations of Poecilia reticulata and Poecilia picta

The reproductive success of the sneaky mating tactic is difficult to determine in fish with internal fertilization. We approached this problem by developing an assay that allows the recovery of sperm DNA from the reproductive tract of females. This assay was used to test whether sperm transfer occurs between sympatric populations of the guppy, Poecilia reticulata , and its putative sister species, Poecilia picta . Six hundred females of both species from two sympatric sites in Trinidad were collected, and then the contents of the females' reproductive tracts were analysed using reciprocally diagnostic microsatellite markers. Conspecific sperm occurred in approximately 86% of females, whereas heterospecific sperm were found in only 4% of females. Because females of either species do not mate willingly with heterospecific males, the results indicate that sneaky mating results in the transfer of sperm. The data are consistent with the idea that sexual conflict can result in differences in the strength of behavioural isolation between the sexes, and they suggest that behavioural isolation is unlikely to have driven speciation between the guppy and P. picta .  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 397–402.