Reciprocal latitudinal clines in oviposition behavior ofPapilio glaucus andP. canadensis across the Great Lakes hybrid zone: possible sex-linkage of oviposition preferences

Summary The eastern tiger swallowtail butterfly,Papilio glaucus, is the most polyphagous of all Papilionidae species. While diverse larval detoxication abilities are known for bothPapilio glaucus and the closely relatedP. canadensis, the factors affecting oviposition preferences in adult females are unknown. These congeneric species were studied to determine the extent of oviposition mistakes on toxic plants. We were also interested in comparing the geographic patterns of variation in oviposition preferences and the genetic basis of these differences. We conducted oviposition three-choice studies with the Canadian tiger swallowtail butterfly,Papilio canadensis, and the Eastern tiger swallowtail,Papilio glaucus, giving them the choice of (1) tuliptree,Liriodendron tulipifera, which is toxic to virtually all populations (P. canadensis) north of the Great Lakes Region of North America, (2) quaking aspen,Populus tremuloides, which is toxic to essentially all populations (P. glaucus) south of the Great Lakes, and (3) black cherry,Prunus serotina, which is an excellent foodplant for all members of thePapilio glaucus group, but which does not occur at latitudinal extremes of North America (in Alaska and most of Canada or the southern half of Florida). Handpaired interspecific hybrids were tested under the same experimental design to evaluate the possibility of sexlinked oviposition behavior. There was considerable variability in the choice of plants by individual butterflies, but a general trend suggesting that the females of each species had a lower preference for the plant toxic to their larvae. More than 6000 oviposition bouts were counted from 37 differentp. canadensis and 54p. glaucus females along a latitudinal transect of approximately 5000 km from Alaska south through the Great Lakes hybrid zone region to southern Florida. While not exceptionally high anywhere, the preference for aspen (Salicaceae) declined precipitously in central Michigan (45° N latitude) and remained very low (5–12%) in all locations southward to Florida, whereas we observed a reciprocal trend in preference for tuliptree (Magnoliaceae) which was greatest in Florida (87% of all eggs) and steadily declined northward across the Great Lakes region. Cherry was selected in these 3-choice tests at a relatively consistent and low frequency at all latitudes. Fixed allele differences in sex-linked (LDH and PGD) and autosomal (HK) electromorphs are known forP. glaucus andP. canadensis. Our electrophoretic data suggest that the preference of an individual female for aspen is not simply a characteristic of the northern species (P. canadensis) but can occur inP. glaucus females. The reciprocal situation is also evident in Northern Michigan and Wisconsin females (scored electrophoretically and morphologically asP. canadensis) which sometimes exhibit a clear preference for the toxic tuliptree. In fact, Alaskan populations ofP. canadensis chose tuliptree for about 52% of their eggs, even though none of their offspring has ever survived on this plant species in laboratory studies. We conclude that even with distinctive latitudinal trends, a considerable amount of local variation in relative oviposition preference exists among individuals of these polyphagous species. BothP. glaucus andP. canadensis will lay eggs on toxic plants. It appears that factors selecting against oviposition on toxic tuliptrees have been minimal (relative to other factors) in Alaska and somewhat stronger in the Great Lakes hybrid zone. It is in this zone of contact with tuliptree where selection against theP. canadensis populations ovipositing on tuliptree may be strong due to high larval mortality when such natural mistakes are made. We do not know whether behavioral preference changes evolutionarily preceded or followed the development of specific physiological detoxication abilities for tuliptree or quaking aspen. However, for bothP. canadensis andP. glaucus the occurrence of oviposition mistakes on toxic plants by adults extends geographically well beyond the larval detoxication abilities of their offspring. Hybrid female offspring of pairings with Michigancanadensis females andglaucus males show distinct preferences for tuliptree, suggesting that oviposition may be controlled by a factor (or factors) on the sex chromosome. Unfortunately we were unable to obtain reciprocal hybrids to evaluate the possibility of sex-linked aspen preference.     

Biochemical detoxication: mechanism of differential tiger swallowtail tolerance to phenolic glycosides

Summary Phenolic glycosides, commonly occurring allelochemicals in the plant family Salicaceae, are differentially toxic to subspecies of the eastern tiger swallowtail and responsible for striking differences in the abilities of Papilio glaucus canadensis and P.g. glaucus to utilize the Salicaceae as food plants. This research was designed to test the hypothesis that particularly high esterase activity confers resistance to phenolic glycosides in P.g. canadensis. I conducted larval survival trials in which the phenolic glycosides salicortin and tremulacin were administered with and without inhibitors of the major detoxication enzymes. Results for P.g. canadensis showed that when esterases were inhibited, toxicity of the phenolic glycosides increased greatly. None of the inhibitors significantly increased toxicity of the compounds to P.g. glaucus. I also conducted in vitro assays of the major detoxication enzymes (polysubstrate monooxygenases, esterases, and glutathione transferases) in larval midguts. Soluble esterase activity was 3-fold higher in P.g. canadensis than in P.g. glaucus. Moreover, esterase activity was inducible by prior consumption of phenolic glycosides in P.g. canadensis but not in P.g. glaucus. Glutathione transferases may also be involved in the terminal metabolism of phenolic glycosides. This is the first verified case of detoxication of an allelochemical by esterase enzymes in herbivores. The biochemical adaptation has played an important role in the evolution of food plant preferences in P. glaucus subspecies.     

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.     

Hybridization leads to host-use divergence in a polyphagous butterfly sibling species pair

Climate warming has lead to increased genetic introgression across a narrow hybrid zone separating the eastern and Canadian tiger swallowtails (Papilio glaucus and Papilio canadensis). This situation has led to the formation of an allochronically separated hybrid population with a delayed emerging phenotype or “late flight”. Here, we assess how the recombination of the parental genomes that lead to this phenotype may have facilitated another major ecological shift, host-use divergence. We first contrast the ovipositional profiles of the late flight population to that of the parental species P. glaucus and P. canadensis. Subsequently we contrast the larval survival and growth of the late flight, a P. canadensis and a P. glaucus population, and a population from the northern edge of the hybrid zone on five hosts. Our results indicate that the ovipositional preference of this hybrid swarm is identical to that of the introgressing parental species, P. glaucus. Due to the absence of the preferred hosts of P. glaucus (Liriodendron tulipifera L. and Ptelea trifoliata L.) where the late flight occurs, this ovipositional pattern implies a functional specialization onto a secondary host of both parental species, Fraxinus americana L. In contrast, the larval host-use abilities represent a mixture of P. glaucus and P. canadensis, indicating divergence in larval host-use abilities has not taken place. However, high genetic variability (genetic coefficient of variation) is present for growth on F. americana in the late flight hybrid swarm and tradeoffs for larval performance on the preferred hosts of the parental species are evident; indicating a strong potential for future specialization in larval host-use abilities. This current scenario represents an instance where a shift in a major ecological trait, host use, is likely occurring as a byproduct of a shift in an unrelated trait (delayed emergence) leading to partial reproductive isolation.     

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     

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

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

Differential responses of tiger swallowtail subspecies to secondary metabolites from tulip tree and quaking aspen

Summary Two subspecies of the eastern tiger swallowtail butterfly, Papilio glaucus, exhibit reciprocal inabilities to survive and grow on each other's preferred foodplant. P. g. canadensis R. & J. performs well on quaking aspen (Populus tremuloides Michx.) but not on tulip tree (Liriodendron tulipifera L.); P. g. glaucus L. performs well on tulip tree but not on quaking aspen. This study was designed to test the hypothesis that secondary metabolites in tulip tree and quaking aspen are responsible for these differential utilization abilities. We extracted and fractionated leaf constituents into different chemical classes, applied them to a mutually acceptable diet (black cherry, Prunus serotina, leaves), and bioassayed them against neonate larvae (survival) and penultimate instar larvae (survival, growth, digestibility and conversion efficiencies). For each plant species, one fraction in particular showed activity against the unadapted subspecies. One tulip tree fraction dramatically reduced survival of P. g. canadensis neonates, and reduced consumption rates, growth rates, and ECI's of fourth instar larvae. The tulip tree constituents most likely responsible for these effects are sesquiterpene lactones. One quaking aspen fraction greatly lowered survival of P. g. glaucus neonates, and decreased survival, consumption rates, growth rates and ECD's of fourth instar larvae. The compounds responsible for these results are probably simple phenols or phenolic glycosides. Surprisingly, P. g. glaucus and P. g. canadensis showed slightly poorer performance on the active tulip tree and quaking aspen fractions, respectively, indicating that even adapted insects incur a metabolic cost in the processing of their host's phytochemicals.     

Salicaceae detoxification abilities in Florida tiger swallowtail butterflies (Papilio glaucus maynardi Gauthier): Novel ability or Pleistocene holdover?

Abstract Florida populations of the eastern tiger swallowtail butterfly, Papilio glaucus L., have unique morphological features and ecological adaptations that have contributed to their subspecies status (P. g. maynardi Gauthier). We describe geographically unique abilities for detoxification of Carolina willow, Salix caroliniana Michx. (Salicaceae), for several Florida populations of P. g. maynardi. Of all the approximately 570 worldwide species of the Papilionidae, such Salicaceae detoxification abilities exist only in the allopatric North American western and northernmost species (P. rutulus Lucas, P. eurymedon Lucas and P. canadensis Rothschild & Jordan). Females of P. glaucus collected from populations in southeastern USA were examined for oviposition preference in 5‐choice assays, and displayed a low preference for Salicaceae (<5%), but larvae from Florida populations exhibited a high survival (>60%) on these plants. Detoxification abilities have previously shown to be autosomally inherited, and can be transferred via natural or hand‐paired interspecific hybrid introgression. However, these Florida populations are at least 700–1 500 km from the nearest hybrids or the hybrid species, P. appalachiensis Pavulaan & Wright, which possess these detoxification abilities. In any case, the Z ( = X)‐linked oviposition preferences for Salicaceae are lacking in these Florida populations, illustrating genetic independence of oviposition preference determination and larval survival/performance abilities. The origins of detoxification abilities are unlikely to be due to recent climate‐driven introgression, and may represent ancestral trait carry‐overs from interglacial refugium populations of the Pleistocene epoch.     

Origins of the regional feeding abilities in the tiger swallowtail butterfly: ecological monophagy and the Papilio glaucus australis subspecies in Florida

Summary The Highlands County, Florida populations of the eastern tiger swallowtail butterfly, Papilio glaucus L., represent a putative subspecies, P. g. australis Maynard. The only foodplant ever reported in southern half of Florida is sweetbay, Magnolia virginiana L. (Magnoliaceae). In fact, there are apparently no other reported foodplants for the polyphagous tiger swallowtail species which exist in southern Florida. These ecologically monophagous P. g. australis populations were studied in order to determine whether or not significant behavioral and biochemical adaptations in the larvae are detectable as a result of the ecological specialization on sweetbay. These results suggest an emphatic yes. On sweetbay, neonate larval survival of these Florida populations is the best of any other population tested from across North America. In addition, penultimate instar larval growth rates are significantly faster on sweet bay than any other foodplant tested. Furthermore, these biochemical adaptations to sweetbay by P. g. australis in Florida are accompanied by a significantly poorer neonate survival and growth performance on northern foodplants (most notably the Salicaceae and Betulaceae). Reciprocal inabilities of the northern subspecies, P. g. canadensis, and the western P. rutulus to utilize sweetbay are described, as well as the reciprocal inabilities of two congeneric and sympatric (Florida) species, Papilio palamedes and Papilio troilus, that have specialized on another bay (redbay, Persea borbonia; of the Lauraceae). It remains uncertain to what degree such apparent negative genetic correlations in foodplant adaptations represent a driving force in the formation of host races or species, especially since so little is known about oviposition preferences of Papilio glaucus across North America. In Florida we have an apparently strict ecological monophagy coupled with a degree of reproductive isolation that has resulted in its tentative classification as a subspecies, P. g. australis. The role of foodplants in the speciation process can be significant even for P. glaucus, the most polyphagous of all 563 species of world Papilionidae. It remains uncertain whether the significant differences in detoxication and biochemical processing abilities observed in this study were evolved in parapatry or allopatry. Disjunct populations of P. alexiares in Mexico retain some ability to detoxify sweetbay, as do some individuals in most P. glaucus glaucus populations tested from ten different states north of Florida.     

Assessing ecological and physiological costs of melanism in North American Papilio glaucus females: two decades of dark morph frequency declines

Polymorphisms for melanic form of insects may provide various selective advantages. However, melanic alleles may have significant/subtle pleiotrophic “costs.” Several potential pleiotrophic effects of the W (=Y)‐linked melanism gene in Papilio glaucus L. (Lepidoptera) showed no costs for melanic versus yellow in adult size, oviposition preferences, fecundity, egg viability, larval survival/growth rates, cold stress tolerance, or postdiapause emergence times. Sexual selection (males choosing yellow rather than mimetic dark females) had been suggested to provide a balanced polymorphism in P. glaucus, but spermatophore counts in wild females and direct field tethering studies of size‐matched pairs of virgin females (dark and yellow), show that male preferences are random or frequency‐dependent from Florida to Michigan, providing no yellow counter‐advantages. Recent frequency declines of dark (melanic/mimetic) females in P. glaucus populations are shown in several major populations from Florida (27.3°N latitude) to Ohio (38.5° N). Summer temperatures have increased significantly at all these locations during this time (1999–2018), but whether dark morphs may be more vulnerable (in any stage) to such climate warming remains to be determined. Additional potential reasons for the frequency declines in mimetic females are discussed: (i) genetic introgression of Z‐linked melanism suppressor genes from P. canadensis (R & J) and the hybrid species, P. appalachiensis (Pavulaan & Wright), (ii) differential developmental incompatibilities, or Haldane effects, known to occur in hybrids, (iii) selection against intermediately melanic (“dusty”) females (with the W‐linked melanic gene, b+) which higher temperatures can cause.     

Larval developmental rates of three putative subspecies of tiger swallowtail butterflies,Papilio glaucus,and their hybrids in relation to temperature

Summary The eastern tiger swallowtail butterfly, Papilio glaucus L. has been presumed to be comprised of three parapatric subspecies (P. g. canadensis R & J, across Canada and the northern U.S.; P. g. glaucus L., in the eastern half of the U.S.; and P. g. australis Maynard, in Florida). Populations from 27° north latitude in Florida to 50° north latitude in Manitoba, Canada and from 75° west longitude in Pennsylvania to 96° west longitide in Canada were tested by laboratory larval feeding studies at various constant temperatures ranging from 16°–28° C. These data indicate that populations of Papilio glaucus show generally similar developmental responses to temperature, regardless of their geographic origin. Limited differences do exist between some populations studied, involving such traits as upper threshold temperature and differences between individual developmental rate curves. No consistent differences were noted between the three putative subspecies in their developmental response under these experimental conditions. In Wisconsin, the twenty year average of total season degree day accumulations above a base temperature (developmental threshold) of 10° C delineates a relatively narrow geographical region across the center of the state for which the bivoltine potential reaches its northernmost limits. This zone also closely corresponds to the southernmost limit of the P. g. canadensis subspecies (generally believed to be obligately univoltine).     

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.     

Differential toxicity of a phenolic glycoside from quaking aspen to Papilio glaucus butterfly subspecies,hybrids and backcrosses

Summary Papilio glaucus subspecies, hybrids and backcrosses exhibit greatly different abilities to use quaking aspen (Populus tremuloides) and other members of the Salicaceae as host plants. This study was conducted to test the hypotheses that phenolic glycosides account for the differences in larval performance, and that differential performance is correlated with differential larval esterase activities. To test the hypotheses we conducted first instar survival trials and fourth (penultimate) instar feeding trials with tremulacin, a phenolic glycoside. We also conducted assays of -glucosidase, esterase, and glutathione transferase activities, using midgut enzyme preparations from fifth instars. First instar survival on the tremulacin treated diet generally improved with a higher proportion of Papilio glaucus canadensis genes in the genotype, although survival in one backcross treatment was surprisingly low. Penultimate instars of P.g. glaucus and P.g. australis fed tremulacin treated black cherry leaves experienced a severe reduction in growth rate relative to larvae fed control leaves. This seriously suppressed growth was partially due to reduced consumption rates and reduced conversion efficiencies, however, approximate digestibility was not affected. In contrast, P. g. canadensis and hybrids showed no differences in growth rates between tremulacin treated and control leaves. Reciprocal backcrosses of hybrids with P. g. glaucus resulted in slightly suppressed growth on treated versus control leaves. The results suggest that after a certain threshold, increased proportions of P. g. glaucus genes resulted in poorer growth performance with tremulacin in the diet. Soluble esterase activities generally increased with the proportion of Papilio glaucus canadensis genes in the genotype, and paralleled overall trends in larval survival and feeding performance. We conclude that phenolic glycosides such as tremulacin are responsible for differential performance of Papilio glaucus subspecies, hybrids and backcrosses fed plants in the Salicaceae, and that detoxification of phenolic glycosides by midgut esterase explains why some Papilio glaucus genotypes can effectively utilize these plants.     

Ecological speciation without host plant specialization; possible origins of a recently described cryptic Papilio species

North American Papilio canadensis and P. glaucus (Lepidoptera: Papilionidae, these Papilio = Pterourus) have previously been described as having allopatric distributions separated by a narrow hybrid zone running from Minnesota to southern New England, and southward in the Appalachian Mountains (possibly to northern Georgia). Recent patterns of hybridization and introgression suggest a more complex interaction between the two, possibly even resulting in the formation of a new species (Pterourus appalachiensis Pavulaan & Wright, 2002). Recently, extensive northward interspecific introgression of P. glaucus‐diagnostic traits has been observed in the hybrid zone. These include wing bands and other color patterns, the ability to feed on tulip tree leaves, and Hk‐100 allozymes; all are autosomally encoded. However, there has been little northward introgression of certain other P. glaucus traits (such as facultative diapause and bivoltinism, and Ldh‐100 allozymes, both X‐linked; and the Y‐linked melanic mimicry gene in females). Interspecific recombination of the X‐chromosome has evidently occurred, as shown by discordant patterns of X‐linked markers. The P. glaucus X‐linked Pgd‐100 and Pgd‐50 alleles have introgressed 200–400 km north of the historical hybrid zone, yet the P. glaucus X‐linked Ldh‐100 allele has not. The allele frequency shift for both genes is more closely related to the ‘thermal landscape’ (i.e., accumulated degree‐days above a developmental base threshold of 50 °F (=10 °C)) than to latitude. Delayed post‐diapause eclosion of cohorts within the hybrid zone, e.g., the New York/Vermont border area, has produced a natural ‘false‐second generation’ flight (a hybrid swarm of synchronous males and females, where 2300–2700 °F degree‐days have accumulated each year since 1998) that is reproductively isolated from flights of both parental species. Moreover, the newly described P. appalachiensis exhibits a unique combination of traits. These include obligate diapause, a univoltine habit, and the Ldh‐80 or Ldh‐40 alleles (as for P. canadensis), the Pgd‐100 or Pgd‐50 alleles (as for P. glaucus), and a delayed ‘false‐second generation’ reproductive flight period (as observed in the hybrid zone). Since 2001, a rare allele or ‘hybrizyme’ (Ldh‐20) has appeared in this false second generation at high frequencies (40–50%). We hypothesize that strong selection against the facultative diapause (od‐)trait (and the linked Ldh‐100 allele) in regions with 2800 °F degree‐days or less, and divergent selection in favor of Pgd‐100 (or a closely linked trait) combined with allochronic reproductive isolation, has resulted in recombinational, parapatric, hybrid speciation. There is no evidence at present that host‐plant shifts or changes in sex pheromones have driven this process, in contrast to many other speciation events in the Lepidoptera.     

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.     

Cover Caption

Cover: North American Papilio glauscus females nectaring upon bull thistles. The dark (mimetic) wing pattern in females is regulated by both a Z (=X) linked enabler and a W (=Y) linked dark melanism factor which introgress independently across an interspecific hybrid zone with the sister species P. canadensis further north. Across this changing thermal landscape, recombinant homoploid hybrids reflect differential geographic movement for several additional Z‐linked, as well as important autosomal, traits. See page 121. Image: J.M. Scriber     

The role of latitudinal,genetic and temperature variation in the induction of diapause of Papilio glaucus (Lepidoptera: Papilionidae)

A key adaptation in insects for dealing with variable environmental conditions is the ability to diapause. The tiger swallowtail butterflies, Papilio glaucus and P. canadensis are ideal species to explore the genetic causes and population genetic consequences of diapause because divergence in this trait is believed to be a salient factor in maintaining a hybrid zone between these species. Yet little is known about the factors that influence diapause induction in this system. Here we explored how spatial (latitudinal), environmental (temperature) and genetic (hybridization) factors affect diapause induction in this system. Specifically, a series of growth chamber experiments using wild caught individuals from across the eastern United States were performed to: (1) evaluate how critical photoperiod varies with latitude, (2) isolate the stage in which induction occurs, (3) test whether changes in temperature affected rates of diapause induction, and (4) explore how the incidence of diapause is affected in hybrid offspring. We find that induction occurs in the larval stage, is not sensitive to a relatively broad range of temperatures, appears to have a complex genetic basis (i.e., is not simply a dominant trait following a Mendelian inheritance pattern) and that the critical photoperiod increases by 0.4 h with each increasing degree in latitude. This work deepens our understanding of how spatial, environmental and genetic variation influences a key seasonal adaptation (diapause induction) in a well‐developed ecological model system and will make possible future studies that explore how climatic variation affects the population dynamics and genetics of this system.     

Interactions between Papilionidae and ancient Australian Angiosperms: evolutionary specialization or ecological monophagy?

The evolution of host range for insect herbivores involves many behavioral, physiological, and biochemical adaptations that often lead to locally specialized populations or species. Such specialization may be constrained by ecological factors (e.g., local host availability) or by evolutionary factors (e.g., phylogenetic divergence in behavioral, physiological, or biochemical detoxification enzymes; and potential inabilities to return to ancestral hosts). While insect adaptations to new hosts can be rapid, ancient detoxification systems may persist in some lineages of swallowtail butterflies (Papilionidae) for millions of years. Here, we test various species of specialized species/populations of Papilionidae (Lepidoptera) from North America and from Australia on an array of Australian host plant families in order to determine whether the current feeding constraints reflect loss of capabilities to recognize and use hosts other than their current (local) favorites. We selected two species of Lauraceae specialists (Papilio troilus L. and Papilio palamedes Drury) from North America and one locally specialized population of Papilio glaucus L. that only uses one plant species in the Magnoliaceae in Florida. We also examined three species/populations of Australian swallowtails for comparison, including the Monimiaceae‐specialized Graphium macleayanum moggana L. E. Couchman, the Rutaceae‐specialized Papilio aegeus Donovan, and the Annonaceae‐specialized Graphium eurypylus L. Our aim was to determine whether neonate larvae of these six specialists could survive on any plants other than their currently favored species. While the Lauraceae specialists could use nothing else and were thus evolutionarily constrained, the Magnoliaceae‐, Rutaceae‐, and Monimiaceae specialists all had common abilities to accept, feed, and grow on plants in the Lauraceae, Monimiaceae, Magnoliaceae, and Rutaceae families. Even the Annonaceae specialist was discovered using Magnoliaceae in the field, suggesting existence here also of both flexiblity in preferences and detoxification abilities and ‘ecological monophagy’.     

The effects of sequentially switching foodplants upon biomass and nitrogen utilization by polyphagous and stenophagous Papilio larvae

The eastern swallowtail butterfly, Papilio glaucus, is often classified as a polyphagous species but growth of individuals is hindered rather than aided by feeding on a sequence of plants in no-choice situations. The spicebush swallowtail, Papilio troilus, is a close relative of P. glaucus. P. troilus has restricted its feeding primarily to the Lauraceae and the efficiency of P. troilus larvae in utilizing foodplants in this family is 2 x – 3 x that of the generalized P. glaucus larvae. Again, however, a sequence of acceptable foods reduces rather than favors larval growth rate.

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Résumé Les taux de consommation, les rendements de transformation, les bilans de biomasse et d'azote, et la croissance de chenilles herbivores de Papilio glaucus et Papilio troilus ont été déterminés sur les plantes-hôtes naturelles dans des conditions écologiques contrôlées. Les effets d'une alternance périodique des plantes-hôtes tous les 2 jours ont été déterminés dans des expériences supplémentaires. Sur Sassafras (Lauraceae), Papilio troilus, plus spécialiste, croît plus vite et avec un meilleur rendement que Papilio glaucus, espèce généraliste. La croissance des 2 espèces de Papilio est supprimée quand on les oblige à changes d'aliments. Les gains en poids des larves et les poids finaux des chrysalides dans des séries alternantes sont intermédiaires, atteignant les moyennes observées entre les meilleures et pires alimentations avec une espèce végétale unique.Bien que l'on puisse conclure que l'alternance des plantes consommées réduit généralement la consommation et la conversion de la biomasse végétale des plantes-hôtes, Papilio glaucus est capable de survivre sur Lindera benzoin (Lauraceae) alternant avec cerisier et frêne, alors que toutes les chenilles meurent sur L. benzoin seul. D'autres faits montrent que cette Lauraceae ne peut être qu'occasionnellement acceptable comme plante-hôte naturelle par Papilio glaucus, tandis qu'elle est essentielle pour les populations plus spécialisées de P. troilus.Ceci suggère que toute classification en généraliste, ou polyphage, doit être accompagnée d'une discussion se référant à l'espèce, la population ou aux larves elles-mêmes. Des informations quantitatives sur des plantes supplémentaires seraient utiles pour distinguer entre l'aptitude des espèces et celle des individus à consommer des plantes-hôtes fournies alternativement ou simultanément.

     

Differential effects of short term winter thermal stress on diapausing tiger swallowtail butterflies (Papilio spp.)

Abstract It is generally thought that insects inhabiting lower latitudes are more severely impacted by changes in their thermal environment than are high latitude species. This is attributed to the wider range of temperatures to which high‐latitude species are exposed. By contrast, low‐latitude species have typically evolved in more thermally stable environments with a narrower range of temperature variation. However, deviation from this pattern can occur and here we report that under variable winter conditions a higher latitude species may be more sensitive to thermal variation than its lower latitude sister species. Using split broods, we examined the survival and adult emergence success of diapausing pupae of Papilio canadensis and P. glaucus, as well as a unique, recombinant hybrid population (“late‐flight”) to short periods of mid‐winter cold and heat stress. Our results indicate that the higher latitude, univoltine populations (P. canadensis and late‐flights) exhibit lower pupal survival than the lower latitude, facultative diapauser (P. glaucus) for all mid‐winter thermal stress treatments, both high and low. Size differences alone do not appear to account for the observed differences in survival or metabolic costs in these three phenotypes, as late‐flight individuals are similar in size to P. glaucus. We attribute the observed differences in survival and weight loss to potential metabolic differences and variation in the intensity of diapause, in addition to divergent adaptation to winter precipitation levels (e.g. snow cover) and the influences this may have on microhabitat temperature moderation.