Allochronic isolation and incipient hybrid speciation in tiger swallowtail butterflies

Hybridization leading to reproductively isolated, novel genotypes is poorly understood as a means of speciation and few empirical examples have been studied. In 1999, a previously non-existent delayed flight of what appeared to be the Canadian tiger swallowtail butterfly, Papilio canadensis, was observed in the Battenkill River Valley, USA. Allozyme frequencies and morphology suggest that this delayed flight was the product of hybridization between Papilio canadensis and its sibling species Papilio glaucus. The mitochondrial DNA (mtDNA) restriction fragment length polymorphisms presented here indicate that only P. canadensis-like mtDNA occurs in this population, suggesting that introgression likely occurred from hybrid males mating with P. canadensis females. Preliminary studies of this population indicated that delayed post-diapause pupal emergence in this hybrid genotype was the root cause behind the observed delayed flight, which suggests a potential empirical example of a mechanism leading to reproductive isolation. Here we provide further evidence of the role of adult pupal emergence as a reproductive barrier likely leading to reproductive isolation. In particular, we present results from pupal emergence studies using four different spring and two different winter temperature treatments. The results indicate a clear separation of adult emergences between the hybrid population and both parental species. However, our results indicate that exceptionally hot springs are likely to lead to greater potential for overlap between the local parental species, P. canadensis, and this delayed population with hybrid origins. Conversely, our results also show that warmer winters are likely to increase the temporal separation of the hybrid population and the parental species. Finally, we report recently collected evidence that this hybrid population remains morphologically distinct.     

Hot summer temperatures may stop movement of Papilio canadensis butterflies and genetic introgression south of the hybrid zone in the North American Great Lakes Region

The reasons that the northern tiger swallowtail butterfly Papilio canadensis does not move south of the Great Lakes hybrid zone, where it meets P. glaucus , may be largely due to natural temperature-induced stress on diapausing pupae. Temperatures of 36°C for only four days killed all P. canadensis (from northern Michigan) and most of the Papilio troilus , a species that lives south of the hybrid zone (sympatrically with P. glaucus ). In contrast, interspecific hybrids ( P. glaucus mother× P. canadensis father) had significant adult eclosion or pupal survival at both 30° and 36°C. All surviving hybrid pupae were only females (the heterogametic sex, which are known to express the prolonged diapause due to Haldane effects).
The southern species, P. troilus , had almost all (81%) normal (non-deformed) adults eclose at 30°C, whereas only 20% of the P. canadensis females and ca 30% of the P. canadensis males eclosed to produce non-deformed adults. Unlike the case with P. troilus and P. canadensis , no hybrid females eclosed at 30°C (only males did). Unlike P. troilus and P. canadensis pupae, hybrid (female) pupae remain viable, some of which have already successfully emerged after the chamber experiments.
A follow-up study using P. glaucus , P. canadensis (from Vermont), and their hybrids with more normal lower thermal regimes included (27°, 30°, 33°, and 36°C) again showed higher hybrid survival as uneclosed (living) pupae at 36°C. In addition, P. glaucus and P. canadensis showed high mortality and wing deformity of eclosing adults at 36°C, suggesting that geographic source of the P. canadensis may reflect differential tolerances of the extreme 36°C temperature.     

Asymmetrical thermal constraints on the parapatric species boundaries of two widespread generalist butterflies

Abstract.  1. The sibling species Papilio glaucus and Papilio canadensis meet in a narrow hybrid zone believed to be maintained by temperature thresholds acting independently on both species. The present study tests if this assertion is true for the cold-adapted species, P. canadensis , which is presumed to be limited by the effect of high temperatures in late summer and/or autumn on pupal survival.
2. Three experiments were conducted examining the effects of: (i) short periods of high temperature stress in autumn, (ii) prolonged warm temperatures in autumn, and (iii) temperatures simulating warmer winters/longer springs upon the survival of P. canadensis and P. glaucus.
3. Results indicated that short periods of high temperatures did not induce the high mortality rates required to be the key factor limiting the range of P. canadensis . However, P. canadensis did exhibit a considerably lower tolerance to high temperature extremes, prolonged warm temperatures in autumn, and conditions simulating shorter/warmer winters than P. glaucus .
4. Differences in temperature tolerance throughout the pupal stage are likely to be a significant factor in maintaining the southern range limit of P. canadensis . Further warming as may occur during climate change, particularly in winter and spring, will likely affect the dynamics of southerly populations of P. canadensis.     

Microsatellite markers for the hybridizing tiger swallowtails, Papilio glaucus and P. canadensis, and their applicability to historic specimens and congeneric species

We report the development of microsatellite markers for the sister species Papilio glaucus and P. canadensis (Papilionidae: Lepidoptera). All 16 markers displayed a high degree of variation in both species, ranging from eight to 24 alleles per locus. Substantial heterozygote deficits were observed for several loci, indicating the presence of null alleles. All markers were successfully used to genotype dried samples from a historical collection. Cross-species amplification with six additional Papilio species showed that most loci can be used to study genetic variation in other closely related species of tiger swallowtails.     

Divergence in the ovipositional behavior of the Papilio glaucus group

This study contrasts the ovipositional profiles of four members of the Papilio glaucus group, P. glaucus , P. multicaudatus , P. canadensis , and P. rutulus. We used seven choice oviposition bioassays containing leaves from hosts in seven plant families utilized by members of the P. glaucus group. Specifically, we contrast the overall ovipositional profiles of these species and their acceptance of a host in a novel plant family ( Populus tremuloides : Salicaceae) and a host in a putatively ancestral host plant family ( Liriodendron tulipifera : Magnoliaceae). Significant differences were observed between the ovipositional profiles of P. glaucus and P. multicaudatus relative to each other and to P. canadensis and P. rutulus. In contrast, no significant differences were observed between the ovipositional profiles of P. canadensis and P. rutulus , which were also the only species that accepted P. tremuloides. Unlike the acceptance of P. tremuloides , the acceptance of L. tulipifera was present throughout the group despite the inability of the larvae of most species in the group to utilize this host. These results support the prediction of the "hierarchical threshold model" that ancestral host plants are likely to be retained in the ovipositional hierarchy while novel hosts should only be accepted by derived populations.     

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.     

Evidence of a sex-linked diapause response in Papilio glaucus subspecies and their hybrids

ABSTRACT. Pupal diapause in the eastern tiger swallowtail. Papilio glaucus L., is obligatory or facultative depending on subspecies. The southern subspecies, P. g. glaucus has a facultative diapause, induced by a larval exposure to short photoperiods. In the northern subspecies. P.g. canadensis , diapause is obligatory, occurring in each generation. F*** hybrids inherit the diapause response of their father or respond differently from either parent. The incidence of diapause in hybrids was associated with sex, indicating that the diapause response may be determined by an X-linked gene.     

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.     

Diversification of host use in two polyphagous butterflies: differences in oviposition specificity or host rank hierarchy?

Novel host usage may represent an initial step towards diversification or radiation onto novel hosts within an evolutionary lineage, particularly if a shift in host plant preference ranking takes place. Polyphagous stages of evolutionary lineages may represent transitional states in which novel host associations are more likely to develop, but may be more difficult to detect experimentally. The polyphagous sister species Papilio glaucus L. and Papilio canadensis (Lepidoptera: Papilionidae; these Papilio = Pterourus) are known to exhibit differences in host‐plant use, despite significant overlap in host‐use abilities, providing an opportunity to examine how host shifts in polyphagous species may occur and what the implications for future divergence may be. In particular, we were interested in (i) determining whether differences in oviposition behavior of these species were due to changes in specificity or shifts in host‐plant hierarchy, (ii) whether the varying preference for primary hosts also affected the preference for secondary hosts, and (iii) what the oviposition preferences of a new hybrid swarm population are. We examined more than 40 000 oviposition bouts from more than 400 P. glaucus, P. canadensis, and hybrid females placed in seven‐, three‐, or two‐choice assays. In each of the choice assays, leaves from plants in different plant families of varying suitability for P. glaucus and P. canadensis larvae were used. We found the primary difference between P. glaucus and P. canadensis to be limited to a Z‐linked shift in host rank hierarchy due to an acceptance of Populus tremuloides Michx. (Salicaceae) and reduced specificity for Liriodendron tulipifera L. (Magnoliaceae) in P. canadensis. In addition, we found the absence of the Z‐linked oviposition acceptance of P. tremuloides in a recently formed allochronically separated hybrid swarm population found in P. canadensis territory at the northern border of the P. glaucus and P. canadensis hybrid zone.     

POPULATION STRUCTURE AND HOST USE IN HYBRIDIZING SUBSPECIES OF PAPILIO GLAUCUS (LEPIDOPTERA: PAPILIONIDAE)

Two parapatric subspecies of the eastern tiger swallowtail butterfly, Papilio glaucus glaucus and P. g. canadensis, differ greatly in larval survival and growth on host plants in the Magnoliaceae, Salicaceae, and Betulaceae. In the first part of this study, butterflies were collected from 17 sites along a transect from Georgia to Quebec and used for allozyme electrophoresis. Two X- (or Z-)linked enzyme loci (Ldh and Pgd) showed allele frequency differences that delineated a hybrid zone between the subspecies in northern Pennsylvania and south-central New York. No significant linkage disequilibrium could be detected among allozyme loci within the hybrid zone samples. Genetic differentiation at Ldh and Pgd coincides with subspecies differences in diapause control and female mimicry, which are also sex-linked. Larval offspring of butterflies from 13 of the sites were then tested in the laboratory for survival and growth on Liriodendron tulipifera (Magnoliaceae), Populus deltoïdes (Salicaceae) and Betula lutea (Betulaceae). Steep clines in survival rates matched the position of the hybrid zone. Hybrid zone larvae showed intermediate survival rates and significant heterogeneity among families on all three plants, indicating presence of substantial genetic variation. The results suggest that differential host use by P. g. glaucus and P. g. canadensis is maintained primarily by independent clines in selection intensity for ability to use allopatric sets of host plants, coupled with restricted gene flow across the hybrid zone.     

Using hybrid and backcross larvae of Papilio canadensis and Papilio glaucus to detect induced phytochemical resistance in hybrid poplar trees experimentally defoliated by gypsy moths

Sub-plots of hybrid poplars were experimentally defoliated using 10 million gypsy moth larvae. Half of the defoliated (and undefoliated control) plots were fertilized to see if this would ameliorate the predicted induction of carbon-based phenolic defenses in the regrowth leaves. In order to bioassay the leaves of the four different treatments, we employed a continuum of genotypes (different hybrids and backcrosses of two different species of tiger swallowtail butterflies) with different abilities to detoxify these allelochemicals. Based on our previous studies with phytochemicals from the Salicaceae plant family, Papilio canadensis was likely to consume and process all Populus spp treatments, whereas P. glaucus predicted to either not consume or else quickly die on all Populus treatment leaves. Hybrid and backcross larvae of these two butterfly species are known to have intermediate levels of esterase detoxication enzymes and would therefore be likely to provide a continuum or at least varying degrees of sensitivity in bioassays for even the most subtle induction responses in the regrowth leaves. This presumption was supported in the feeding and growth studies conducted at different times post-defoliation during the 1997 growing season in Michigan.     

The strength of reproductive isolation in two hybridizing food-deceptive orchid species

Reproductive isolation is of fundamental importance for maintaining species boundaries in sympatry. In orchids, the wide variety of pollination systems and highly diverse floral traits have traditionally suggested a prominent role for pollinator isolation, and thus for prezygotic isolation, as an effective barrier to gene flow among species. Here, we examined the nature of reproductive isolation between Anacamptis morio and Anacamptis papilionacea, two sister species of Mediterranean food-deceptive orchids, in two natural hybrid zones. Comparative analyses of the two hybrid zones that are located on soils with volcanic origin and have different and well-dated ages consistently revealed that all hybrid individuals were morphologically and genetically intermediate between the parental species, but had strongly reduced fitness. Molecular analyses based on nuclear ITS1 and (amplified fragment length polymorphism) AFLP markers clearly showed that all examined hybrids were F1 hybrids, and that no introgression occurred between parental species. The maternally inherited plastid DNA markers indicated that hybridization between A. morio and A. papilionacea was bidirectional, as confirmed by the molecular analysis of seed families. The genetic architecture of the two hybrid zones suggests that the two parental species easily and frequently hybridize in sympatry as a consequence of partial pollinator overlap but that strong postzygotic barriers reduce hybrid fitness and prevent gene introgression. These results corroborate that chromosomal divergence is instrumental for reproductive isolation between these food-deceptive orchids and suggest that hybridization is of limited importance for their diversification.     

No association between mitochondrial DNA haplotypes and a female-limited mimicry phenotype in Papilio glaucus

Abstract Alternative alleles at a locus on the W chromosome of Papilio glaucus (causing dark or yellow wing colors, respectively) underlie a female-limited mimicry polymorphism thought to be maintained by balancing selection. In species with heterogametic females (i.e., the ZZ-male/ZW-female sex chromosome system), the mitochondrial DNA and the W chromosome are genetically linked because they are both maternally transmitted. We investigate the association of COI and COII mitochondrial DNA haplotypes with alternative W-linked phenotypes. Surprisingly, we find no congruence between mitochondrial DNA genealogies and inferred W-linked color alleles in P. glaucus. Using a maximum-likelihood phylogenetic approach, we reject the hypothesis of monophyly for darkmorph mitochondrial DNA lineages, even in the presence of putative low-frequency mimicry suppressor alleles or alternative melanizing factors. The most likely genealogical tree topologies assume more than one exchange event between mitochondrial DNA cytotype and the W-linked color morph. These results suggest that there is either paternal leakage of mitochondrial DNA or that more than two W-linked alleles underlie the alternative color morphs. Using data from an additional mitochondrial DNA locus, ND5, we show that pairwise linkage disequilibrium decays with physical distance between polymorphic sites. This finding suggests that genetic exchanges between maternal and paternal mitochondrial DNAs may have contributed to the lack of association we observe between phenotype and genotype.     

The molecular basis of melanism and mimicry in a swallowtail butterfly

Melanism in Lepidoptera, either industrial or in mimicry, is one of the most commonly cited examples of natural selection [1] [2]. Despite extensive studies of the frequency and maintenance of melanic genes in insect populations [1] [2], there has been little work on the underlying molecular mechanisms. Nowhere is butterfly melanism more striking than in the Eastern Tiger Swallowtail (Papilio glaucus) of North America [3] [4] [5]. In this species, females can be either yellow (wild type) or black (melanic). The melanic form is a Batesian mimic of the distasteful Pipevine Swallowtail (Battus philenor), which is also black in overall color. Melanism in P. glaucus is controlled by a single Y-linked (female) black gene [6]. Melanic females, therefore, always have melanic daughters. Black melanin replaces the background yellow in melanic females. Here, we show that the key enzyme involved is N-beta-alanyl-dopamine-synthase (BAS), which shunts dopamine from the melanin pathway into the production of the yellow color pigment papiliochrome and also provides products for cuticle sclerotization. In melanic females, this enzyme is suppressed, leading to abnormal melanization of a formerly yellow area, and wing scale maturation is also delayed in the same area. This raises the possibility that either reduced BAS activity itself is preventing scale sclerotization (maturation) or, in contrast, that the delay in scale maturation precludes expression of BAS at the correct stage. Together, these data show how changes in expression of a single gene product could result in multiple wing color phenotypes. The implications for the genetic control of mimicry in other Lepidoptera are discussed.     

The origin of a novel form of Senecio (Asteraceae) restricted to sand dunes in southern Sicily

The taxonomy of diploid Mediterranean Senecio sect. Senecio (Asteraceae) is complex, owing to a recent species radiation, high morphological plasticity and occasional interspecific hybridization. A study was conducted to resolve the origin of a novel form of Senecio restricted to sand dunes in southern Sicily, Italy. This has been described previously as morphologically intermediate to Senecio gallicus and Senecio glaucus ssp. coronopifolius, indicating a possible hybrid origin, or as a variant of Senecio leucanthemifolius. Plants of this form grown in a glasshouse were morphologically intermediate to S. glaucus and S. leucanthemifolius, but were also similar to some cultivated individuals of S. gallicus. No evidence for a hybrid origin was obtained from a survey of random amplified polymorphic DNA variation; instead the plants surveyed were most closely allied to Tunisian S. glaucus. They were also polymorphic for the same set of cpDNA haplotypes present in Tunisian S. glaucus. We conclude that the Sicilian Senecio is a variant form of North African S. glaucus ssp. coronopifolius, which most probably dispersed to sand dunes in southern Sicily in the relatively recent past. The presence of several cpDNA haplotypes in this material indicates that there have been multiple introductions of the species to Sicily.     

Cytogenetic analysis of a hybrid, Glyptotendipes pallens Mg. × Glyptotendipes glaucus Mg. (Diptera, Chironomidae): evolutionary considerations

The karyotype of experimentally obtained hybrids between the two closely related species Glyptotendipes pallens and Glyptotendipes glaucus is described. Hybridization was successful in one direction only ( G. pallens ♂ x G. glaucus ♀). The polytene chromosomes AB and EF of the hybrid show a more or less intimate pairing throughout their length. In the chromosomes CD in which an inversion occurs the characteristic loop is formed. The homologues of chromosome G are almost completely asynaptic. The localization of centromere heterochromatin was also studied. Centromere heterochromatin as well as intercalary heterochromatin could be observed in all chromosomes. By C banding analyses it could be shown that G. pallens has a telomeric chromosome G while in G. glaucus it is acrocentric. According to karyotype similarity it can be assumed that these two species have quite recently derived from a common ancestor since they still share much of their genomic organization. On the Black Sea coast (southeast part of Bulgaria) a natural hybridization zone between the sympatric species G. pallens and G. glaucus has been detected. The idea that hybridization between the two species might finally proceed to the formation of a new species by hybrid origin and introgression is discussed.     

Local Enhancement in Mud-Puddling Swallowtail Butterflies (Battus philenor and Papilio glaucus)

Male butterflies aggregate at moist soil to acquire nutrients, a phenomenon termed “mud-puddling.” We studied the attraction of free-flying Papilio glaucus and Battus philenor swallowtails to dead decoys of those two species at artificial puddles moistened with NaCl solution. Both species landed preferentially at puddles with a decoy present rather than at unbaited puddles, demonstrating very strong local enhancement, a form of social facilitation. Papilio glaucus were attracted only to intraspecific decoys, whereas Battus philenor exhibited both intraspecific and interspecific attraction. Circular discs cut from the hindwings of male Battus were highly attractive to male Battus but completely unattractive to Papilio glaucus. The visual cues attractive to males in their search for salts differ between these two swallowtail species for unexplained reasons.     

Unexpected ancestry of Populus seedlings from a hybrid zone implies a large role for postzygotic selection in the maintenance of species

In the context of potential interspecific gene flow, the integrity of species will be maintained by reproductive barriers that reduce genetic exchange, including traits associated with prezygotic isolation or poor performance of hybrids. Hybrid zones can be used to study the importance of different reproductive barriers, particularly when both parental species and hybrids occur in close spatial proximity. We investigated the importance of barriers to gene flow that act early vs. late in the life cycle of European Populus by quantifying the prevalence of homospecific and hybrid matings within a mosaic hybrid zone. We obtained genotypic data for 11 976 loci from progeny and their maternal parents and constructed a Bayesian model to estimate individual admixture proportions and hybrid classes for sampled trees and for the unsampled pollen parent. Matings that included one or two hybrid parents were common, resulting in admixture proportions of progeny that spanned the whole range of potential ancestries between the two parental species. This result contrasts strongly with the distribution of admixture proportions in adult trees, where intermediate hybrids and each of the parental species are separated into three discrete ancestry clusters. The existence of the full range of hybrids in seedlings is consistent with weak reproductive isolation early in the life cycle of Populus. Instead, a considerable amount of selection must take place between the seedling stage and maturity to remove many hybrid seedlings. Our results highlight that high hybridization rates and appreciable hybrid fitness do not necessarily conflict with the maintenance of species integrity.     

Discordant divergence times among Z-chromosome regions between two ecologically distinct swallowtail butterfly species

We investigate multilocus patterns of differentiation between parental populations of two swallowtail butterfly species that differ at a number of ecologically important sex-linked traits. Using a new coalescent-based approach, we show that there is significant heterogeneity in estimated divergence times among five Z-linked markers, rejecting a purely allopatric speciation model. We infer that the Z chromosome is a mosaic of regions that differ in the extent of historical gene flow, potentially due to isolating barriers that prevent the introgression of species-specific traits that result in hybrid incompatibilities. Surprisingly, a candidate region for a strong barrier to introgression, Ldh, does not show a significantly deeper divergence time than other markers on the Z chromosome. Our approach can be used to test alternative models of speciation and can potentially assign chronological order to the appearance of factors contributing to reproductive isolation between species.