Differential patterns of hybridization and introgression between the swallowtails Papilio machaon and P. hospiton from Sardinia and Corsica islands (Lepidoptera,Papilionidae)

Proportions of hybridization and introgression between the swallowtails Papilio hospiton, endemic to Sardinia and Corsica, and the holarctic Papilio machaon, were characterized using nine fully diagnostic and two differentiated allozyme loci and a mitochondrial DNA marker. Very low frequencies of F1 hybrids were detected in both Sardinia (0-4%, average 1.4%) and Corsica (0-3%, average 0.5%), as well as of first generation backcrosses (B1). No F2 were observed, in agreement with the hybrid breakdown detected in laboratory crosses. In spite of this minimal current gene exchange, specimens carrying introgressed alleles were found in high proportions in P. machaon but in lower proportions in P. hospiton. Introgression apparently occurred through past hybridization and repeated backcrossing, as evidenced by hybrid index scores and Bayesian assignment tests. Levels of introgression were low (0-1%) at two sex-linked loci and mitochondrial DNA, limited (0.4-2%) at three autosomal loci coding for dimeric enzymes, and high (up to 43%) at four autosomal loci coding for monomeric enzymes. Accordingly, selective filters are acting against foreign alleles, with differential effectiveness depending on the loci involved. The low levels of introgression at sex-linked loci and mitochondrial DNA are in agreement with Haldane's rule and suggest that introgression in P. machaon proceeds mainly through males, owing to a lower fitness of hybrid females. Papilio machaon populations showed higher levels of introgression in Sardinia than in Corsica. The role of reinforcement in the present reproductive isolation between P. machaon and P. hospiton is examined, as well as the evolutionary effects of introgressive hybridization between the two species.     

Speciation problems in the Papilio machaon group of butterflies (Lepidoptera: Papilionidae)

Abstract. The taxonomic and genetic relationships between P.machaon rathjensi Warnecke from the west side of southern Arabia and P.machaon muetingi Seyer from the east are discussed, together with suggestions of their relationship to P.machaon saharae Oberthiir from the northern Hejaz, Sinai and desert North Africa. It is concluded that though P.m.rathjensi and P.m.saharae are subspecifically distinct, they are very closely related to each other, and both should be regarded as specifically distinct from the rest of the machaon complex.
The remainder of the machaon complex in its turn presents a somewhat similar problem. Thus P.machaon gorganus Fruhstorfer and P.hospiton Géné are normally regarded as good species, but Fl caterpillars have been found in the wild and the cross can be readily produced in captivity with no upset in the sex ratio. Furthermore, recently we have obtained sparse F2 offspring between the two species.
We therefore suggest that the entire P.machaon complex is in a labile state as regards speciation throughout its range and the relationships between the various forms are not easily expressed through the traditional species and subspecies concepts. Our tentative conclusions are that the status of P.machaon and P.hospiton as distinct species probably remains valid. The saharaelrathjensi complex might be considered distinct from P.machaon using the biological species concept, while their genetics indicate a more conservative approach. However, more important than the exact status of the taxa in question is the fact that the P.machaon complex illustrates evoluton in action, the end result of which cannot be predicted.     

Morphology of foretarsal ventral surfaces of Japanese Papilio butterflies and relations between these morphology, phylogeny and hostplant preferring hierarchy

Foretarsal ventral surface observation of Japanese Papilio butterflies showed that the shapes of fifth foretarsi and numbers and localization of contact chemosensilla and spines in these areas are closely related to both phylogeny and behavior in these species. My results basically supported the classification that Japanese Papilio divides into five subgenera -- Papilio (P. machaon), Princeps (P. xuthus and P. demoleus), Achillides (P. maackii and P. bianor), Menelaides (P. helenus, P. polytes, P. protenor and P. macilentus) and Iliades (P. memnon). Moreover, female foretarsal morphology also corresponded to the physical features of their preferring host plant leaves. The specificity of female P. machaon, female P. macilentus and female P. maackii seemed to also relate to the geographical diversities of these species and their hostplant, and their co-evolution.     

Effects of linear furanocoumarins on an adapted specialist insect (Papilio polyxenes)

Abstract. 1. Examination of phytochemical literature reveals that a disproportionately large number of hostplants for species in the genus Papilio Section II, and in particular the machaon complex, contain linear furanocoumarins.
2. Although the linear furanocoumarin xanthotoxin is known to be toxic to generalist lepidopterous larvae, it failed to affect adversely the growth and survivorship of Papilio polyxenes , a member of the P.machaon complex that feeds primarily on Umbelliferae containing furanocoumarins, when incorporated into an artificial diet.
3. On the contrary, growth rate and weight gain were significantly improved in the presence of xanthotoxin.
4. It is proposed, based on this experimental evidence and on hostplant utilization patterns, that furanocoumarins play a significant role in the behavioural and biochemical adaptation of Papilio species to their umbelliferous hosts.     

The phylogeny of some Papilio species based on interspecific hybridization data

Abstract. Adults of different species of the genus Papilio can easily be mated by hand-pairing. The data on egg hatchability, adult formation and F₁ fertility in these interspecific hybrids show notable variations according to the crosses. Using these data, values of a differentiation index for each cross were calculated. These estimates of genetic distance were then used to study the phylo-genetic relationships of the species involved. The data are consistent with the existence of the following groups, each consisting of closely related species: the black Papilio supergroup, the Papilio memnon group, the P.demoleus group, the P.xuthus group, the P.machaon group, the P.bianor group, the P.glaucus group and the P.troilus group. The phylogenetic relationships revealed by these data generally coincide with the relationships established by classical taxonomy. In cases where there are differences of opinion among taxonomists, the writer's data usually support one or other of them.     

Papilio phylogeny based on mitochondrial cytochrome oxidase I and II genes

Butterflies of the genus Papilio have served as the basis for numerous studies in insect physiology, genetics, and ecology. However, phylogenetic work on relationships among major lineages in the genus has been limited and inconclusive. We have sequenced 2.3 kb of DNA from the mitochondrial cytochrome oxidase I and II genes (COI and COII) for 23 Papilio taxa and two outgroups, Pachliopta neptunus and Eurytides marcellus, in order to assess the potential of these genes for use in Papilio phylogenetics and to examine patterns of gene evolution across a broad taxonomic range. Nucleotide and amino acid variation is distributed heterogeneously, both within and between genes. Structural features of the proteins are not always reliable predictors of variation. In a combined analysis, these sequences support a nearly fully resolved topology within subgenera and species groups, though higher level relationships among species groups require additional study. The most noteworthy findings are that neither Papilio alexanor nor P. xuthus belongs in the machaon group and that the subgenus Pterourus is paraphyletic with respect to the subgenus Pyrrhosticta. We leave relationships among members of the phorcas species group as a trichotomy. These two protein coding genes, particularly COI, show excellent performance in resolving relationships at the level of species and species groups among Papilionidae. We strongly endorse a similar approach for future studies aimed at these levels.     

Enemy-free space and habitat-specific host specialization in a butterfly

The majority of herbivorous insects have relatively specialized food habits. This suggests that specialization has some advantage(s) over generalization. Traditionally, feeding specialization has been thought to be linked to digestive or other food-related physiological advantages, but recent theory suggests that generalist natural enemies of herbivorous insects can also provide a major selective pressure for restricted host plant range. The European swallowtail butterfly Papilio machaon utilizes various plants in the Apiaceae family as hosts, but is an ecological specialist being monophagous on Angelica archangelica in southern Sweden. This perennial monocarp grows in three seaside habitat types: (1) on the barren rocky shore in the absence of any surrounding vegetation, (2) on the rocky shore with some surrounding vegetation, and (3) on species-rich meadows. The rocky shore habitat harbors few invertebrate generalist predators, whereas a number of invertebrate predators abound in the meadowland habitat. Here, we test the importance of enemy-free space for feeding specialization in Papilio machaon by assessing survival of larvae placed by hand on A. archangelica in each of the three habitat types, and by assessing the habitat-specificity of adult female egg-laying behavior by recording the distribution of eggs laid by free-flying adult females among the three habitat types. Larval survival was substantially higher in the rocky shore habitat than in the meadowland and significantly higher on host plants without surrounding vegetation on the rocky shore. Eggs laid by free-flying females were found in all three habitat types, but were significantly more frequent in the rocky shore habitat, suggesting that females prefer to lay eggs in the habitat type where offspring survival is highest. These results show that larval survivorship on the same host plant species can be strongly habitat-specific, and suggest that enemy-free space is an underlying factor that drives feeding specialization in Papilio machaon.     

MITOCHONDRIAL DNA VARIATION WITHIN AND BETWEEN SPECIES OF THE PAPILIO MACHAON GROUP OF SWALLOWTAIL BUTTERFLIES

Species limits and phylogenetic relationships in the Papilio machaon species group are potentially confounded by a complex pattern of Pleistocene range fragmentation, hybridization, and ecological race formation. Mitochondrial DNA (mtDNA) restriction-site analysis has been used to define genetic affinities and genetic population structure within this species group. The distribution of mtDNA haplotypes generally confirms prior phylogenetic hypotheses and species delineations, but there is poor correspondence between ecological races and mtDNA haplotypes. The amount and distribution of mtDNA sequence variation within species vary among species, reflecting differences in current patterns of gene flow and/or historical population structure. In spite of wing pattern characters that ally them with P. polyxenes, both P. joanae and P. brevicauda have mtDNA that is closely related to that of P. machaon. We suggest that P. joanae and P. brevicauda are of hybrid origin.     

Testing hypotheses on the dispersal and evolutionary history of freshwater mussels (Mollusca: Bivalvia: Unionidae)

The relationship between dispersal and differentiation of the European freshwater mussel Unio pictorum (Linnaeus, 1758) was studied with molecular genetic methods. Forty‐two populations from France, Italy and central Europe were analysed. Genetic relationships were assessed from the geographical distribution of allele frequencies at 17 enzyme loci. Neighbouring groups of populations show small to moderate mean genetic distances (0.020 < D_mean < 0.263). With a few exceptions the genetic affinities of the populations are the closest within the same drainage basin. In central Europe and Northern Italy genetic differences between drainage systems are relatively large. Populations from north‐eastern Italy are genetically similar to Danubian populations. Mussels from the islands of Corsica and Sardinia are more closely related to populations from the Italian peninsula than to French populations from the Rhône drainage system. Genetic relationships within U. pictorum from central Europe reflect palaeogeographical relationships between river systems during the Pliocene and Pleistocene. Literature data on two North American unionid species and one European fish species show the same relationship between genetic diversity and the history of drainage systems, although the correlations are less strong. In France and Italy this correspondence is much less evident. Population dynamic processes and human activities leading to populational bottlenecks might have obscured it.     

Satyrinae butterflies from Sardinia and Corsica show a kaleidoscopic intraspecific biogeography (Lepidoptera,Nymphlidae)

The Mediterranean islands of Sardinia and Corsica are known for their multitude of endemics. Butterflies in particular have received much attention. However, no comprehensive studies aiming to compare populations of butterflies from Sardinia and Corsica with those from the neighbouring mainland and Sicily have been carried out. In the present study, the eleven Satyrinae species inhabiting Sardinia and Corsica islands were examined and compared with continental and Sicilian populations by means of geometric morphometrics of male genitalia. Relative warp computation, discriminant analyses, hierarchical clustering, and cross‐validation tests were used to identify coherent distributional patterns including both islands and mainland populations. The eleven species showed multifaceted distributional patterns, although three main conclusions can be drawn: (1) populations from North Africa and Spain are generally different from those belonging to the Italian Peninsula; (2) populations from Sardinia and Sicily often resemble the North Africa/Spain ones; Corsica shows transitional populations similar to those from France; and (3) sea barriers represent filters to dispersal, although their efficacy appears to be unrelated to their extension. Indeed, the short sea straits between Sardinia and Corsica and between Sicily and the Italian Peninsula revealed a strong effectiveness with respect to preventing faunal exchanges; populations giving onto sea channels between Corsica and Northern Italy and between Sicily and Tunisia showed a higher similarity. A comparison of island and mainland distributions of the eleven taxa have helped to unravel the complex co‐occurrence of historical factors, refugial dynamics, and recent (post‐glacial) dispersal with respect to shaping the populations of Mediterranean island butterflies. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 195–212.     

On humans and wildlife in Mediterranean islands

Aim To investigate the effects of human‐induced landscape changes in Mediterranean islands on the ecological and evolutionary responses of bird communities and populations. The combination of mass extinction of large mammals and massive deforestation by humans was hypothesized to produce new selection regimes to which organisms were likely to respond. Habitat selection and niche breadth have been investigated at the scale of species, and phenotypic variation at the scale of local populations. Location The study was carried out along habitat gradients and in habitat mosaics at different spatial scales on the island of Corsica and in areas of similar size and structure in continental France. Methods Two sets of gradients have been used for investigating habitat selection and niche breadth: gradients of altitude, and gradients of vegetation structure. Population studies focused on the blue tit, Cyanistes caeruleus. Large samples of breeding attempts by this species in 10 habitats provided detailed data on phenotypic variation of fitness‐related traits both on Corsica and on the mainland. Results The extent of niche space used by birds differed substantially depending on which habitat gradient was considered. Many species have been found to contract their habitat niche along the elevation gradient on Corsica compared with the mainland, whereas all species in the vegetation gradient broadened their niche on the island. Breeding patterns of the blue tit differed considerably depending on whether they settle in deciduous oaks (Quercus humilis) or in evergreen sclerophyllous oaks (Quercus ilex). Phenotypic variation of breeding traits was much higher on the island, where more populations were correctly timed for the best breeding period than on the mainland, a pattern that is likely to result from lower dispersal of organisms on the island. Main conclusions The differences in observed niche breadth between the two series of habitat gradients is explained both by the species‐specific ecology of the species and the human‐induced environmental history of Corsica. Large‐scale landscape changes provided new opportunities for island colonization by non‐forest species, which are isolated as small, ‘fugitive’ local populations. In both gradients, forest species that are typical components of the Corsican bird fauna definitely expanded their niche and occupied a wider range of habitats on Corsica than on the mainland. At the population scale, landscapes included habitat patches with contrasted selection regimes, which resulted in high phenotypic variation for many fitness‐related traits. Reduced dispersal of birds on the island resulted in a much higher degree of local differentiation on Corsica than on the mainland.     

Purification and Properties of Isocitrate Lyase from Pupas of the Butterfly Papilio machaon L.

Key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were identified in pupas of the butterfly Papilio machaon L. The activities of these enzymes in pupas were 0.056 and 0.108 unit per mg protein, respectively. Isocitrate lyase was purified by a combination of various chromatographic steps including ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl, and gel filtration. The specific activity of the purified enzyme was 5.5 units per mg protein, which corresponded to 98-fold purification and 6% yield. The enzyme followed Michaelis-Menten kinetics (Km for isocitrate, 1.4 mM) and was competitively inhibited by succinate (Ki = 1.8 mM) and malate (Ki = 1 mM). The study of physicochemical properties of the enzyme showed that it is a homodimer with a subunit molecular weight of 68 +/- 2 kD and a pH optimum of 7.5 (in Tris-HCl buffer).     

Population genetic structure of Tomicus piniperda L. (Curculionidae: Scolytinae) on different pine species and validation of T. destruens (Woll.)

Genetic diversity and population structure of Tomicus piniperda was assessed using mitochondrial sequences on 16 populations sampled on 6 pine species in France. Amplifications of Internal transcribed space 1 (ITS1) were also performed. Our goals were to determine the taxonomic status of the Mediterranean ecotype T. piniperda destruens, and to test for host plant or geographical isolation effect on population genetic structure. We showed that T. piniperda clusters in two mtDNA haplotypic groups. Clade A corresponds to insects sampled in continental France on Pinus sylvestris, P. pinaster and P. uncinata, whereas clade B gathers the individuals sampled in Corsica on P. pinaster and P. radiata and in continental France on P. pinea and P. halepensis. Insects belonging to clade A and clade B also consistently differ in the length of ITS1. Individuals belonging to both clades were found once in sympatry on P. pinaster. Genetic distances between clades are similar to those measured between distinct species of Tomicus. We concluded that clade B actually corresponds to the destruens ecotype and forms a good species, T. destruens. Analyses of molecular variance ( amova ) were conducted separately on T. destruens and T. piniperda to test for an effect of either geographical isolation or host species. Interestingly, the effect of host plant was significant for T. piniperda only, while the effect of geographical isolation was not. Pine species therefore seems to act as a significant barrier to gene flow, even if host race formation is not observed. These results still need to be confirmed by nuclear markers.     

Beta-globin gene cluster haplotypes in the Corsican and Sardinian populations

The distribution of beta-globin cluster haplotypes has been studied in the populations of Corsica (France) and Sardinia (Italy). The analysis was carried out using five restriction fragment length polymorphism markers on chromosome 11 inside the beta-globin cluster using the restriction enzymes HincII and HindIII. The results show a remarkable heterogeneity within the two islands. However, the presence of rare haplotypes common to the most conservative areas (Nuoro and Corte) of the two islands is particularly interesting. These data support the hypothesis of a common origin of the populations of Sardinia and Corsica during the middle and upper Paleolithic periods and could be interpreted as a founder effect.     

Ghloroplast DNA and nuclear DNA variation in the sympatric alder species, Alnus cordata (Lois.) Duby and A. glutinosa (L.) Gaertn.

The European alder species, Alnus cordata and A. glutinosa have sympatric distributions on Corsica and southern Italy, where they often grow in mixed populations. An assessment was made of the levels of genetic variation in the chloroplast and nuclear genomes of individual trees from 22 alder populations from Corsica and Italy. The distribution of 10 chloroplast DNA polymorphisms generated by the PCR-RFLP method shows strong geographic structuring of cpDNA haplotypes. Conversely, variation in the nuclear genome, as determined by ISSR analysis, is strongly correlated to the taxonomic affinities of the individual trees analysed. Approximately 50% of ISSR variation (P= 0.00, AMOVA) could be attributed to differences between species. Possible reasons for the incongruence between variation in the chloroplast and nuclear genomes are discussed.     

Origin identification of Pinus nigra populations in southwestern Europe using terpene composition variations

The geographical variation of terpenes of Pinus nigra populations from southwestern Europe was studied. Terpenes from the foliage of 16 populations from Corsica, Herault (France) and the East Pyrenees (France and Spain) were analyzed. A total of 42 terpenes were detected, with -pinene the dominant monoterpene and germacrene-d and caryophyllene the dominant sesquiterpenes. The differences in quantitative content of selected compounds clearly divide populations into two basic geographical groups: on one side the populations from Herault and the East Pyrenees and on the other the populations from Corsica. -Phellandrene and -cadinene have the greatest influence on this global discrimination. Some trees and populations show a similarity although they belong to different geographic locations. The similarity of some trees from Herault and the East Pyrenees and trees from Corsica points to their common origin (Corsica). Our results confirm the hypothesis that the afforestation of Herault and the East Pyrenees was also performed with black pine from Corsica.     

A new perspective on the zoogeography of the sibling mouse-eared bat species Myotis myotis and Myotis blythii: morphological, genetical and ecological evidence

The actual geographic distribution of the two sibling mouse-eared bat species Myotis myotis and Myotis blythii , which occur widely sympatrically in the western Palaearctic region, remains largely controversial. This concerns particularly the specific attribution of marginal populations from the Mediterranean islands and from adjacent areas of North Africa and Asia, which are morphologically intermediate between continental M. myotis and M. blythii from Europe. This study attempts to clarify this question by using four different approaches: cranial morphology, external morphology, genetics and trophic ecology. The three latter methods show unambiguously that North Africa, Malta, Sardinia and Corsica are presently inhabited by monospecific populations of M. myotis . In contrast, cranial morphometrics do not yield conclusive results. These results contradict all recent studies, which attribute North African and Maltese mouse-eared bats to M. blythii and consider that Sardinia and Corsica harbour sympatric populations of the two species. As concerns south-eastem populations, doubts are also expressed about the attribution of the subspecific taxon omari which may actually refer to M. myotis instead of M. blythii. Protein electrophoresis is presently the only absolute method available for determining M. myotis and M. blythii throughout their distribution ranges. However, species identification may be approached by relying on less sophisticated morphometrical methods as presented in this study. Species-specific habitat specializations are probably responsible for the differences observed between the geographic distributions of M. myotis and M. blythii , as they provide a logical groundwork for a coherent model of speciation for these two bat species.     

Adaptive inter-population differences in blue tit life-history traits on Corsica

Few studies of natural populations have investigated how phenotypic variation across populations relates to key factors in the environment and landscape structure. In the blue tits of southern France, inter-population differences in reproductive life-history traits (e.g. laying date and clutch size) are small, whatever the timing of maximum caterpillar availability, a key factor for offspring survival in tits. These small differences are attributed to gene flow between local populations occupying different habitat types. In contrast, in blue tits on the island of Corsica, we noted large differences in reproductive life-history traits between two populations, where each population is synchronized with the peak-date of caterpillar abundance. These occur over a short geographical distance (25km). Considering our study within a framework of long-term population studies in tits, our results support the hypothesis that different blue tit populations on Corsica show adaptive differences in life-history traits, and suggest that landscape structure at a small spatial scale can have profound effects on adaptive between-population differentiation in life-history traits that are closely linked with fitness.     

Characterization of eight new microsatellite loci in the invading maritime pine bast scale Matsucoccus feytaudi (Hemiptera: Coccoidea: Margarodidae)

The bast scale Matsucoccus feytaudi is a specific pest of maritime pine that is endemic in the Iberian Peninsula and southwestern France, and invasive in southeastern France, Italy and Corsica where it causes heavy damages. We developed eight microsatellite polymorphic markers using an enrichment protocol in order to study the invasion pathways and infer the genetic diversity of the populations. All loci could be successfully amplified with no evidence of null alleles. Cross‐species amplifications failed to amplify the congeneric Matsucoccus josephi.     

Sex chromosome mosaicism and hybrid speciation among tiger swallowtail butterflies

Hybrid speciation, or the formation of a daughter species due to interbreeding between two parental species, is a potentially important means of diversification, because it generates new forms from existing variation. However, factors responsible for the origin and maintenance of hybrid species are largely unknown. Here we show that the North American butterfly Papilio appalachiensis is a hybrid species, with genomic admixture from Papilio glaucus and Papilio canadensis. Papilio appalachiensis has a mosaic phenotype, which is hypothesized to be the result of combining sex-linked traits from P. glaucus and P. canadensis. We show that P. appalachiensis' Z-linked genes associated with a cooler thermal habitat were inherited from P. canadensis, whereas its W-linked mimicry and mitochondrial DNA were inherited from P. glaucus. Furthermore, genome-wide AFLP markers showed nearly equal contributions from each parental species in the origin of P. appalachiensis, indicating that it formed from a burst of hybridization between the parental species, with little subsequent backcrossing. However, analyses of genetic differentiation, clustering, and polymorphism based on molecular data also showed that P. appalachiensis is genetically distinct from both parental species. Population genetic simulations revealed P. appalachiensis to be much younger than the parental species, with unidirectional gene flow from P. glaucus and P. canadensis into P. appalachiensis. Finally, phylogenetic analyses, combined with ancestral state reconstruction, showed that the two traits that define P. appalachiensis' mosaic phenotype, obligatory pupal diapause and mimicry, evolved uniquely in P. canadensis and P. glaucus, respectively, and were then recombined through hybridization to form P. appalachiensis. These results suggest that natural selection and sex-linked traits may have played an important role in the origin and maintenance of P. appalachiensis as a hybrid species. In particular, ecological barriers associated with a steep thermal cline appear to maintain the distinct, mosaic genome of P. appalachiensis despite contact and occasional hybridization with both parental species.