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.     

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.     

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.     

基于系统发生探讨绿带翠凤蝶和西番翠凤蝶的分类地位(英文)

绿带翠凤蝶和西番翠凤蝶的分类问题存在一定的争议。应用分子系统学方法对这一问题进行了研究。对6个不同地区的24个绿带翠凤蝶、2个地区的16个西番翠凤蝶个体的COI(579bp)和COII(655bp)基因测序,绿带翠凤蝶与西番翠凤蝶的遗传距离为0至0.6%,共获得了15个单倍型。结果显示这些单倍型不能形成各自独立的单系群,因此认为绿带翠凤蝶和西番翠凤蝶为近期分化的两个种。     

Evolution of the mimetic African swallowtail butterfly Papilio dardanus: molecular data confirm relationships with P. phorcas and P. constantinus

Sister group relationships of the African Mocker Swallowtail, Papilio dardanus, were determined using mitochondrial 16S rRNA and Cytochrome B sequences, and nuclear Elongation Factor EF-la and the ITS-1 region of the ribosomal RNA locus. All four data sets placed P. dardanus as the sister species of P. phorcas, with P. constantinus as the next closest relative, and quite distinct from other African Papilios such as P. nobilis. These data support earlier morphological studies indicating that the only two African Papilio species with multiple female colour forms have a common ancestor not shared with any other living species. This information is important for conclusions about the evolution of female-limited mimicry in swallowtail butterflies, and successful use of the ITS-1 gene opens up new possibilities for studying this phenomenon within P. dardanus.     

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.     

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.     

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.     

四个达摩凤蝶新孵幼虫细胞系的建立及其生物学特性

【目的】建立达摩凤蝶 Papilio demoleus Linnaeus新孵幼虫细胞系,并对其生物学特性进行研究。【方法】使用改良配方的Grace培养基并辅以20%胎牛血清,通过原代和传代培养建立达摩凤蝶新孵幼虫细胞系。通过显微观察、细胞活力分析、核型分析和分子鉴定,获得4个新建细胞系的生物学特性数据。使用Bac-To-Bac杆状病毒表达系统构建重组分泌型碱性磷酸酶杆状病毒(AcMNPV-SEAP)。使用有限稀释法测定达摩凤蝶细胞系对AcMNPV-SEAP的半数组织培养感染剂量(TCID₅₀), 比较达摩凤蝶细胞系对AcMNPV-SEAP的敏感性。【结果】建立了4个贴壁生长的达摩凤蝶新孵幼虫细胞系(RIRI-PaDe-1, RIRI-PaDe-2, RIRI-PaDe-3及RIRI-PaDe-4),且均传至60代以上。形态学方面,细胞系RIRI-PaDe-1和RIRI-PaDe-4较为相近,均由圆形、梭形以及多边形细胞组成,细胞系RIRI-PaDe-2主要为圆形细胞,而RIRI-PaDe-3主要为类似表皮细胞和纤维状细胞。细胞增殖动力学方面,4个细胞系的群体倍增时间分别为69.77, 67.42, 81.48及65.43 h。核型分析显示4个细胞系染色体数量均呈正态分布,其中RIRI-PaDe-2, RIRI-PaDe-3以及RIRI-PaDe-4的染色体数目分布区间比较接近,在45~97条之间,RIRI-PaDe-1的染色体条数相比偏少,在36~90条之间。通过比对4个达摩凤蝶细胞系和虫卵的细胞色素c氧化酶亚基I(COI)基因序列,证明4个新建细胞系来源于达摩凤蝶组织。比较4个细胞系对AcMNPV-SEAP敏感性发现RIRI-PaDe-3对此病毒较为敏感,可作为重组杆状病毒表达的宿主。【结论】虽然4个新建达摩凤蝶细胞系的来源相同,具有相同的遗传背景,但其生物学特征仍有明显差异,具有进一步研究的价值。     

Ecological significance of puddling as a behavioural phenomenon in the life history of butterfly Papilio polytes Linn. (Lepidoptera: Papilionidae)

Puddling in butterflies is an essential physiological phenomenon, by which they gain mineral nutrients required for their reproductive success and flight. The preference given to the puddling by butterflies has been analysed by two hypotheses: butterflies puddle when there is a scarcity of nutritional resource in one hypothesis and competition with other males to increase their probability of mating in the other. In the present investigation, an attempt has been made to verify these hypotheses through experimental evidences in the butterfly Papilio polytes, in which only males are known to puddle. Our investigation revealed the significance of puddling as an alternative source of nutrition in the absence of nectar. However, increase in the number of males in a given vicinity with constant number of females enhances the puddling activity in males. This shows the importance of puddling as a distinct competitive behaviour by which male improves their chances of mating. Additionally, in both cases the puddling is drastically regulated by the environmental temperature. Males prefer to puddle at a temperature ranging from 26 °C to 28 °C, which is considered as optimum for puddling activity. Further, the fecundity and longevity of imago are completely unaffected by puddling activity. All these results are in good agreement with the existing hypotheses and show the behavioural significance of puddling in butterflies.     

Field bioassays show heterospecific mating preference asymmetry between hybridizing North American Papilio butterfly species (Lepidoptera: Papilionidae)

Few studies of interspecific mating preferences of naturally hybridizing species have been done in the field. Yet this is the only way potentially critical habitat-specific factors can be included in mating behavior evaluations. We conducted mate selection (male preference) studies using tethered pairs of heterospecific, size-matched virgin yellow females of Papilio glaucus and P. canadensis with free-flying male P. glaucus populations in south-central Florida, and with free-flying male P. canadensis populations in northern Michigan. Florida males clearly preferred the conspecific P. glaucus females of the pairs, as indicated by the touches/attempted matings and by the actual copulations observed. In 1997, 66.7% of the total attempts (n=168) and 94.2% of all copulations (n=69) were with the conspecific. In 1998 the same pattern of conspecific male preference was observed, with 67.3% of the touches/attempts (n=49) and 100% of the copulations (n=30) being with the P. glaucus females. In contrast, the natural populations of P. canadensis males did not show the expected conspecific preference. In fact, with wild P. canadensis males in 1997 a very strong preference was observed for the heterospecific P. glaucus females of the pairs, accounting for 75.8% of all touches/attempted matings (n=483) and 81.7% of all copulations (n=476). The strong asymmetry in interspecific mating preferences among these hybridizing Papilio species may reflect an ancestral trait in P. glaucus females that elicits strong preference in P. canadensis males, due perhaps to previously possessed male sensory bias or "good genes" indicators in the females. However, reinforcement of reproductive isolation in or near the hybrid zone of species overlap (sympatry) cannot be ruled out. Electronic Publication     

The mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) and related phylogenetic analyses

The nearly complete mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) was sequenced for its nucleotide sequence of 13,964 bp. The genome has a typical gene order identical to other lepidopteran species. All tRNAs showed same stable canonical clover-leaf structure as those of other insects, except for tRNA^Ser (AGN), in which the dihydrouracil arm (DHU arm) could not form stable stem–loop structure. Anomalous initiation codons have been observed for the cox1 gene, where the ATTACG hexa-nucleotide was believed to be involved in the initiation signaling. Twelve mitochondrial protein-coding gene sequence data were used to infer the phylogenetic relationships among the insect orders. Even though the number of insect orders represented by complete mitochondrial genomes is still limited, several well-established relationships are evident in the phylogenetic analysis of the complete sequences. Monophyly of the Homometabola was not supported in this paper. Phylogenetic analyses of the available species of Bombycoidea, Pyraloidea, Papilionoidea and Tortricidea bolstered the current morphology-based hypothesis that Bombycoidea, Pyraloidea and Papilionoidea are monophyletic (Obtectomera). Bombycoidea (Bombyx mandarina and Antheraea pernyi) and Papilionoidea (P. xuthus and Coreana raphaelis) formed a sister group.     

Papilio aegeus Donovan (Lepidoptera: Papilionidae) host plant range evaluated experimentally on ancient angiosperms

Abstract  Chemical similarities among ancient Angiosperms presumably played a role in the ecological and evolutionary diversification of the swallowtail butterflies (Papilionidae). The abilities of neonate larvae of the Citrus swallowtail, Papilio (= Princeps ) aegeus (from Queensland, Australia), to eat, survive and grow on leaves (a choice of young and old) of 34 plant species from families of ancient Angiosperms; 8 Rutaceae, 3 Magnoliaceae, 13 Lauraceae, 3 Monimiaceae, 1 Aristolochiaceae, 2 Apiaceae, 1 Sapotaceae, 1 Winteraceae and 2 Annonaceae were tested. It was apparent that there is genetic variation in populations of Rutaceae-specialised Australian P. aegeus for acceptance, consumption and larval growth, reflecting differential suitability of some native Australian Lauraceae species as food plants (as well as certain Winteraceae, Monimiaceae and non-Australian Magnoliaceae, Lauraceae and Annonaceae). No consumption or survival of P. aegeus was seen on Aristolochia elegans (Aristolochiaceae) or Pouteria australis (Sapotaceae) despite literature records alluding to this possibility. The Rutaceae specialist P. aegeus appears to have the fundamental detoxification capabilities for processing many existing species of the basal Angiosperm families, without having direct ancestors that historically had fed on them.     

Structure and metamorphic changes in the brain of the lemon butterfly Papilio demoleus L. (Lepidoptera: Papilionidae)

The morphology of the larval and adult brain of Papilio demoleus, and changes in the cell population and neuropile morphology during the pupal period have been described. The larval brain has more simple fibre areas than that of the adult. Dividing neuroblasts have been found which form the adult neurones. The larval brain contains the three neuromeres (proto-, deuto-, and tritocerebrum). The protocerebrum has well developed corpora pedunculata, a central body, a pons cerebralis and developing optic centres. The corpora ventralia are joined with each other by paired ventral commissures (single in adult). The deutocerebrum is simple and small, the antennal centres are small and simple (ef. adult). The glomerular tritocerebrum is posteroventral to the deutocerebrum, and fibres from the former travel to the crura cerebri. The cortex of the brain consists of four types of glial cells and of association cells, and large and medium sized motor neurones. The number of mitoses is greatest in the larval and prepupal stages; in the pupa it decreases gradually and in late stages it does not occur. Histolysis and pyknosis begin in the prepupa and decrease considerably in the late pupa. The entire neural lamella is broken down in the early pupa. Numerous haemocytes penetrate the laminae of the neural lambella and envelop the entire brain. In the adult, behind the well-developed central body is an ellipsoid body. The medulla interna is divided into two smaller lobes and the deutocerebral lobes are differentiated into cortical and medullary zones. Chiasmata between optic centres are also formed during the pupal period.     

Identification of odoriferous compounds from adults of a swallowtail butterfly, Papilio machaon (Lepidoptera: Papilionidae)

Adults, particularly males, of a papilionid butterfly, Papilio machaon hippocrates, emit a fairly strong scent perceivable by humans. We have identified a variety of volatile compounds (hydrocarbons, alcohols, aldehydes, ketones, esters, and so on) from the wings and bodies of both sexes of the butterfly. Male wings secreted n-dodecane, linalool and geranylacetone as major components together with small amounts of camphene, limonene, p-cymene, 2-phenylethanol, n-hexanal, n-decanal, isoamyl acetate, p-allylanisole, 2-pyrrolidone and other characteristic volatiles. The overall profile of volatile compounds detected from male body was quite different from that of the wings. Male body was devoid of camphene, 2-phenylethanol, n-hexanal but instead contained limonene, acetoin, a sesquiterpene hydrocarbon (C15H24, methyl n-octanoate, (E,E)-hepta-2,4-dienal, and another isomer of heptadienal as principal components, of which the last four compounds were specific to the body. All these substances seem to concurrently characterize the male odor. The chemical patterns of compounds found from female wings and body were essentially the same in quality as those of male wings and body, respectively, although their quantities in females were generally smaller than in males. Females, however, had a larger amount of acetamide than males. The chemical compositions of volatiles from the fore and hind wings of males were not greatly different from each other, and every component was considered to be present on all parts of the wings. This suggests that the scent-producing organs or scent-emitting pores are widely distributed on the whole wings. EAG responses of both sexes to 12 selected compounds identified from the butterfly were not strong at a dose of 1 microg, while both sexes showed relatively stronger responses to n-nonanal, methyl n-octanoate, D-limonene and linalool at a higher dose (10 microg). Although sexual difference in EAG response was not prominent, females appeared a little more sensitive, and n-nonanal and acetoin evoked significantly higher responses from females at 1 microg.     

A novel biorational pesticide: efficacy of methionine against Heraclides (Papilio) cresphontes, a surrogate of the invasive Princeps (Papilio) demoleus (Lepidoptera: Papilionidae)

ABSTRACT The Southeast Asian citrus-feeding butterfly Princeps (Papilio) demoleus (L.) was recently introduced into the Americas, causing an imminent threat to citrus production and ornamental flora. The human nutrient amino acid methionine has been shown by us to disrupt aminoacid-modulated ion transport systems in caterpillars and other insect larvae that possess an alkaline midgut. Heraclides (Papilio) cresphontes was bioassayed as a United States Department of Agriculture permitted surrogate of the Florida quarantined P. demoleus to test the potential efficacy of methionine. Larvae were allowed to feed ad libitum on wild lime plants with leaves treated with methionine or proline. Methionine caused 100% mortality in first through fourth instars in a time- and dose-dependent manner, as determined by probit analysis whereas proline was not toxic. Wild lime host plants did not exhibit phytotoxicity with methionine treatments during a 14 d test period. It is concluded that methionine is an effective larvicide against H. cresphontes, and therefore may be a candidate environmentally safe biorational pesticide for use against invasive P. demoleus in the Americas.     

Effects of plant odor on oviposition by the black swallowtail butterfly,Papilio polyxenes (Lepidoptera: Papilionidae)

Black swallowtail females laid more eggs on plant models treated with contact stimulants and volatiles from carrot leaves than on models treated only with contact stimulants. The volatiles enhanced landing rates and females alighted more frequently on artificial leaves treated with host volatiles than on adjacent control leaves. Volatiles from cabbage, a nonhost, inhibited landing rates on artificial leaves treated with carrot contact stimulants. Examination of antennae revealed two major types of sensilla, believed to be olfactory in function. Electroantennogram preparations responded more strongly to carrot volatiles than to cabbage volatiles and several shared responses at particular retention times to carrot volatile components eluting from a gas chromatograph. Our results are consistent with a long-standing hypothesis that behavioral responses to essential oil components characteristic of the larval food plants have facilitated host shifts in the genus Papilio.     

Pupation site preference and environmentally cued pupal colour dimorphism in the swallowtail butterfly Papilio polyxenes Fabr. (Lepidoptera: Papilionidae)

Environmentally cued polymorphisms are hypothesized to evolve when the environment is coarsegrained and different genotypes are unable to choose the habitats in which they are most fit. In Papilio polyxenes , which has an environmentally cued pupal colour dimorphism, there is genetic variation in both tendency to produce brown or green pupae and preference for green- or brown-inducing pupation sites, but the two traits are not correlated.     

Morphology,life cycle and management of two invasive subspecies of Papilio demoleus (Lepidoptera: Papilionidae): A review

Papilio demoleus L., also known as the citrus butterfly or chequered swallowtail, is a common species in the Asia-Pacific region, with two of its subspecies (P. d. demoleus and P. d. malayanus) posing significant threats to the citrus industry in Asia and other regions of the world. Its predominant natural host plants are the Rutaceae for the invasive northern subspecies and Fabaceae for the southern subspecies. The morphology and development of this species are reasonably well studied, although further information is required on its natural mortality rates and environmental tolerance in order to accurately predict its future potential spread throughout the Middle East and Central Americas. Synthetic pyrethroids such as deltamethrin are highly effective for the control of P. demoleus larvae. However, better control is likely to be found using an integrated approach to pest management, incorporating techniques such as biocontrol agents, microbial pesticides and phytopesticides, in conjunction with synthetic pesticides where required.