Climate-mediated hybrid zone movement revealed with genomics, museum collection, and simulation modeling.

Climate-mediated changes in hybridization will dramatically alter the genetic diversity, adaptive capacity, and evolutionary trajectory of interbreeding species. Our ability to predict the consequences of such changes will be key to future conservation and management decisions. Here we tested through simulations how recent warming (over the course of a 32-y period) is affecting the geographic extent of a climate-mediated developmental threshold implicated in maintaining a butterfly hybrid zone (Papilio glaucus and Papilio canadensis; Lepidoptera: Papilionidae). These simulations predict a 68-km shift of this hybrid zone. To empirically test this prediction, we assessed genetic and phenotypic changes using contemporary and museum collections and document a 40-km northward shift of this hybrid zone. Interactions between the two species appear relatively unchanged during hybrid zone movement. We found no change in the frequency of hybridization, and regions of the genome that experience little to no introgression moved largely in concert with the shifting hybrid zone. Model predictions based on climate scenarios predict this hybrid zone will continue to move northward, but with substantial spatial heterogeneity in the velocity (55-144 km/1 °C), shape, and contiguity of movement. Our findings suggest that the presence of nonclimatic barriers (e.g., genetic incompatibilities) and/or nonlinear responses to climatic gradients may preserve species boundaries as the species shift. Further, we show that variation in the geography of hybrid zone movement could result in evolutionary responses that differ for geographically distinct populations spanning hybrid zones, and thus have implications for the conservation and management of genetic diversity.

Patterns of divergence across the geographic and genomic landscape of a butterfly hybrid zone associated with a climatic gradient.

Hybrid zones are a valuable tool for studying the process of speciation and for identifying the genomic regions undergoing divergence and the ecological (extrinsic) and nonecological (intrinsic) factors involved. Here, we explored the genomic and geographic landscape of divergence in a hybrid zone between Papilio glaucus and Papilio canadensis. Using a genome scan of 28,417 ddRAD SNPs, we identified genomic regions under possible selection and examined their distribution in the context of previously identified candidate genes for ecological adaptations. We showed that differentiation was genomewide, including multiple candidate genes for ecological adaptations, particularly those involved in seasonal adaptation and host plant detoxification. The Z chromosome and four autosomes showed a disproportionate amount of differentiation, suggesting genes on these chromosomes play a potential role in reproductive isolation. Cline analyses of significantly differentiated genomic SNPs, and of species-diagnostic genetic markers, showed a high degree of geographic coincidence (81%) and concordance (80%) and were associated with the geographic distribution of a climate-mediated developmental threshold (length of the growing season). A relatively large proportion (1.3%) of the outliers for divergent selection were not associated with candidate genes for ecological adaptations and may reflect the presence of previously unrecognized intrinsic barriers between these species. These results suggest that exogenous (climate-mediated) and endogenous (unknown) clines may have become coupled and act together to reinforce reproductive isolation. This approach of assessing divergence across both the genomic and geographic landscape can provide insight about the interplay between the genetic architecture of reproductive isolation and endogenous and exogenous selection.

Sex chromosome mosaicism and hybrid speciation among tiger swallowtail butterflies.

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

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.

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.

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.

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

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 dark-morph 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.

Sequential diets, metabolic costs, and growth of Spodoptera eridania (Lepidoptera: Noctuidae) feeding upon dill, lima bean, and cabbage.

This study illustrates the diversity of feeding responses of individually polyphagous southern armyworms, Spodoptera eridania, to plants with differing allelochemics. In spite of the near optimal leaf water and nitrogen contents of the young foliage, it is apparent that vastly different larval growth performance results from dill, lima bean, and cabbage. Cabbage is the poorest food (as measured by larval growth rates and metabolic costs of processing the plant biomass). Unlike the case with certain other plant species or cultivars that are costly to process, with cabbage, S. eridania does not compensate for low efficiencies (E.C.D.'s) with increased consumption rates (R.C.R.'s). Biochemical or physiological reasons for this inability are unknown.A sequence of foods (changed each 18-24 h) apparently did not add sufficient stress upon the MFO system to be detected in the respiratory expenditures of S. eridania larvae, in spite of the fact that dill is known to contain insecticidal and synergistic chemicals (Lichtenstein et al. 1974). The larval growth performances and metabolic expenditures in these sequences were intermediate between the best food (dill) and the worse (cabbage). Significant differences were observed however between the sequential switching sequences, perhaps indicating that particular periods during the instar are especially more sensitive to certain allelochemics. Actual respiratory costs of the lima bean-cabbage-dill (i.e. B-C-D) sequence were 40-50% higher than observed for the other two sequences and more than 50% higher than the theoretical metabolic costs based on the proportions actually eaten and known costs associated with each food.This study and a related one (Scriber 1981a) illustrate how consumption rates, feeding efficiences, and larval growth of Spodoptera eridania are not species, population, or even individual characteristics, (cf. Fox and Morrow 1981), but instead depend largely upon variations in plant allelochemics and plant nutritional quality (Wolfson 1978; Scriber, 1981 b; Scriber and Slansky 1981). More significantly they illustrate that the food consumed in earlier instars (Scriber 1981 a) as well as the food consumed earlier in an instar can be a major influence upon the observed armyworm growth performances under a given set of environmental conditions.

Limiting effects of low leaf-water content on the nitrogen utilization, energy budget, and larval growth ofHyalophora cecropia (Lepidoptera: Saturniidae).

Hyalophora cecropia larvae were reared on leaves of wild cherry,Prunus serotina, which contained variable amounts of leaf water but otherwise did not differ in fiber, total nitrogen, and caloric content. Larvae which were fed leaves low in leaf water grew more slowly and were less efficient at utilizing plant biomass, energy, and nitrogen than those larvae fed leaves which were fully supplemented with water.Experiments were performed using excised leaves under different regimes of relative humidity and leaf water supplementation in climatic control chambers maintained at identical temperatures and photoperiod. Foodplant biomass utilization efficiencies were severely reduced by decreasing amounts of leaf water. Growth rates were halved and the efficiency of conversion of assimilated dry matter into larval biomass was reduced from 82% in the treatment with fully supplemented leaves to 34% in the driest treatment. The nitrogen utilization efficiency (N.U.E.) was reduced from 75-80% to 48%, and the relative accumulation rate of nitrogen (N.A.R.) was suppressed nearly 2-fold for larvae on low-water leaves. Relative maintenance costs (calories expended in respiration/mg tissue/day) of larvae were nearly five times higher on dry leaves than on fully supplemented leaves. Larvae on leaves which were low in water content were themselves more desiccated, and metabolized greater portions of assimilated energy, perhaps in an attempt to supplement body water with metabolic water derived from respiration.The larval rates of consumption of biomass, energy, and nitrogen were the same for all treatments, indicating that leaf water affected larval growth primarily by restricting the efficiency of utilizing these nutrients. Where water was limiting (as in tree leaves), an increased consumption rate did not appear to be a successful means of increasing growth rates. There were daily and seasonal differences in leaf water content between different trees of the same species. Although absolute differences in leaf water exist between different trees and between young and old (fully expanded) leaves of a single tree, these differences are proportional and parallel each other through daily and seasonal cycles.In spite of evolutionary adaptations of herbivores to acquire adequate water and avoid desiccation, the leaf water content naturally encountered by cecropia larvae on cherry leaves may limit their growth, especially if the R.H. is low.

Effects of hydrolyzable and condensed tannin on growth and development of two species of polyphagous lepidoptera: Spodoptera eridania and Callosamia promethea.

The effects of tannins on survival, growth, and digestion were compared in two polyphagous species of Lepidoptera (one, the southern armyworm, a forb-feeder; and the other, the promethea silkmoth, a tree-feeder). Two different types of tannins (hydrolyzable and condensed) were incorporated into artificial basal diets in order to determine whether or not differential survival and growth would result between the forb feeder, which normally does not encounter tannins in its natural diet, and the tree-feeder, whose host species include many tanniniferous plants from several different families.Neonate larvae of the forb-feeding armyworms exhibited significantly suppressed 10-day growth rates at all tannin concentrations tested (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0% of wet weight) for both the hydrolyzable and the condensed tannin compared to the control diet, however no dose-effect was detectable. In contrast, there were no detectable differences in neonate survival or growth through the first 10 days for the tree-feeding promethea silkmoth larvae fed diets with either tannic acid or quebracho tree condensed tannin.In order to determine the physiological mechanisms of action of these tannins against armyworms, we conducted detailed physiological bioassays of biomass and nitrogen utilization by penultimate instar larvae. Standard gravimetric feeding studies with both tannic acid and the quebracho tree condensed tannin demonstrated that reduced relative growth rates (RGR's) of Spodoptera eridania Cram. were due to the suppressed relative consumption rates (RCR's) and decreased conversion efficiencies (ECD's) rather than due to digestibility-reduction (as reflected by approximate digestibility, AD). As with the neonate larval growth rate suppression, there were no detectable dose responses at the different concentrations of tannic acid (0.25, 0.50, 0.75, 1.00, 2.50, and 5.0 percent) and condensed tannins from quebracho (0.25, 0.50, 0.75, 1.0, and 2.5 percent) in our penultimate instar studies.

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

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).

Lack of physiological improvement in performance of Callosamia promethea larvae on local host plant favorites.

As a species, the promethea silkmoth, Callosamia promethea (Saturniidae: Lepidoptera) exhibits a wide host range on 6-10 families of plants, although specific populations are known to have local foodplant favorites. We tested the hypothesis that larvae from a particular host plant lineage would show physiological adaptations to this host compared with larvae from other host plant lineages. We found no evidence that larval survival and growth was any better for larvae fed the natural plant of the parental population than for larvae from other host lineages. These natural host lineages include: black cherry (Prunus serotina Ehrh.), tuliptree (Liriodendron tulipifera L.), sassafras (Sassafras albidum (Nutt.) Nees) and spicebush (Lindera benzoin (L.) Blume). The only apparent manifestation of physiological specialization was the inability of tuliptree lineages of C. promethea to survive on paper birch (Betula papyrifera Marsh), although this may reflect the geographical pattern of adaptation to birch, rather than a negative correlation with adaptation to tuliptree. These results suggest that for C. promethea larvae, growth performance and survival is primarily influenced by plant nutritional quality, rather than physiological adaptations to the locally preferred host plant.

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

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.

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

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 ß-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.

Adaptations to "Thermal Time" Constraints in Papilio: Latitudinal and Local Size Clines Differ in Response to Regional Climate Change.

Adaptations to "thermal time" (=Degree-day) constraints on developmental rates and voltinism for North American tiger swallowtail butterflies involve most life stages, and at higher latitudes include: smaller pupae/adults; larger eggs; oviposition on most nutritious larval host plants; earlier spring adult emergences; faster larval growth and shorter molting durations at lower temperatures. Here we report on forewing sizes through 30 years for both the northern univoltine P. canadensis (with obligate diapause) from the Great Lakes historical hybrid zone northward to central Alaska (65° N latitude), and the multivoltine, P. glaucus from this hybrid zone southward to central Florida (27° N latitude). Despite recent climate warming, no increases in mean forewing lengths of P. glaucus were observed at any major collection location (FL to MI) from the 1980s to 2013 across this long latitudinal transect (which reflects the "converse of Bergmann's size Rule", with smaller females at higher latitudes). Unlike lower latitudes, the Alaska, Ontonogon, and Chippewa/Mackinac locations (for P. canadensis) showed no significant increases in D-day accumulations, which could explain lack of size change in these northernmost locations. As a result of 3-4 decades of empirical data from major collection sites across these latitudinal clines of North America, a general "voltinism/size/D-day" model is presented, which more closely predicts female size based on D-day accumulations, than does latitude. However, local "climatic cold pockets" in northern Michigan and Wisconsin historically appeared to exert especially strong size constraints on female forewing lengths, but forewing lengths quickly increased with local summer warming during the recent decade, especially near the warming edges of the cold pockets. Results of fine-scale analyses of these "cold pockets" are in contrast to non-significant changes for other Papilio populations seen across the latitudinal transect for P. glaucus and P. canadensis in general, highlighting the importance of scale in adaptations to climate change. Furthermore, we also show that rapid size increases in cold pocket P. canadensis females with recent summer warming are more likely to result from phenotypic plasticity than genotypic introgression from P. glaucus, which does increase size in late-flight hybrids and P. appalachiensis.

INTRASEXUAL SELECTION CONSTRAINS THE EVOLUTION OF THE DORSAL COLOR PATTERN OF MALE BLACK SWALLOWTAIL BUTTERFLIES, PAPILIO POLYXENES.

Males of the eastern black swallowtail (Papilio polyxenes asterius Stoll) with typical coloration were more successful in intrasexual competition for mating territories than were males altered to have female-like mimetic coloration. Sibling males were matched for wingspan and emergence date and released as pairs, one with its pattern altered and one a control that was marked but with unaltered appearance. Significantly fewer altered males were resighted one or more days after release compared with control males (33% vs. 76%, 1990; 46% vs. 83%, 1993). Altered males were less able to establish and maintain themselves in preferred territories. The inability of released, altered males to establish a territory appears related to significantly longer male-male encounters. Encounters involving at least one participant with altered appearance averaged 66 s compared with 24 s if neither male was altered. However, altering the coloration of P. polyxenes males that already had established themselves in a territory had little effect. After courtships of similar duration (≈ 40 s), released virgin females were equally likely to mate with either altered or control males. This suggests that male-male intrasexual selection is of greater importance than female mate choice in maintaining a non-mimetic dorsal coloration in male P. polyxenes.

Competition between the gypsy moth, Lymantria dispar, and the northern tiger swallowtail, Papilio canadensis: interactions mediated by host plant chemistry, pathogens, and parasitoids.

The gypsy moth, Lymantria dispar, and the northern tiger swallowtail, Papilio canadensis, overlap geographically as well as in their host ranges. Adult female swallowtails are incapable of distinguishing between damaged and undamaged leaves, and the opportunities for competition between these two species are numerous. We designed field and laboratory experiments to look for evidence of indirect competition between P. canadensis and L. dispar larvae. Swallowtail caterpillars were reared in the laboratory on leaves from gypsy-moth-defoliated and undefoliated trees to explore host-plant effects. We tested for pathogen-mediated interactions by rearing swallowtail larvae on both sterilized and unsterilized leaves from defoliated and undefoliated sources. In addition, we measured the effects of known gypsy moth pathogens, as well as gypsy moth body fluids, on the growth and survival of swallowtail larvae. Field experiments were designed to detect the presence of parasitoid-mediated competition, as well: we recorded parasitism of swallowtail caterpillars placed in the field either where there were no gypsy moth larvae present, or where we had artificially created dense gypsy moth populations. We found evidence that swallowtails were negatively affected by gypsy moths in several ways: defoliation by gypsy moths depressed swallowtail growth rate and survival, whether leaves were sterilized or not; sterilization significantly reduced the effect of defoliation, and gypsy moth body fluids proved lethal; and swallowtail caterpillars suffered significantly increased rates of parasitism when they were placed in the field near gypsy moth infestations.

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, scan , 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 scan and low frequency of the black-pigment-determining b + allele. The frequency of scan (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.