Multi-locus genomic signatures of local adaptation to snow across the landscape in California populations of a willow leaf beetle.

Organisms living in mountains contend with extreme climatic conditions, including short growing seasons and long winters with extensive snow cover. Anthropogenic climate change is driving unprecedented, rapid warming of montane regions across the globe, resulting in reduced winter snowpack. Loss of snow as a thermal buffer may have serious consequences for animals overwintering in soil, yet little is known about how variability in snowpack acts as a selective agent in montane ecosystems. Here, we examine genomic variation in California populations of the leaf beetle Chrysomela aeneicollis, an emerging natural model system for understanding how organisms respond to climate change. We used a genotype-environment association approach to identify genomic signatures of local adaptation to microclimate in populations from three montane regions with variable snowpack and a coastal region with no snow. We found that both winter-associated environmental variation and geographical distance contribute to overall genomic variation across the landscape. We identified non-synonymous variation in novel candidate loci associated with cytoskeletal function, ion transport and membrane stability, cellular processes associated with cold tolerance in other insects. These findings provide intriguing evidence that variation in snowpack imposes selective gradients in montane ecosystems.

A chromosome-scale genome assembly and evaluation of mtDNA variation in the willow leaf beetle Chrysomela aeneicollis.

The leaf beetle Chrysomela aeneicollis has a broad geographic range across Western North America but is restricted to cool habitats at high elevations along the west coast. Central California populations occur only at high altitudes (2,700-3,500 m) where they are limited by reduced oxygen supply and recent drought conditions that are associated with climate change. Here, we report a chromosome-scale genome assembly alongside a complete mitochondrial genome and characterize differences among mitochondrial genomes along a latitudinal gradient over which beetles show substantial population structure and adaptation to fluctuating temperatures. Our scaffolded genome assembly consists of 21 linkage groups; one of which we identified as the X chromosome based on female/male whole genome sequencing coverage and orthology with Tribolium castaneum. We identified repetitive sequences in the genome and found them to be broadly distributed across all linkage groups. Using a reference transcriptome, we annotated a total of 12,586 protein-coding genes. We also describe differences in putative secondary structures of mitochondrial RNA molecules, which may generate functional differences important in adaptation to harsh abiotic conditions. We document substitutions at mitochondrial tRNA molecules and substitutions and insertions in the 16S rRNA region that could affect intermolecular interactions with products from the nuclear genome. This first chromosome-level reference genome will enable genomic research in this important model organism for understanding the biological impacts of climate change on montane insects.

Transcriptomic evidence indicates that montane leaf beetles prioritize digestion and reproduction in a sex-specific manner during emergence from dormancy.

During winter, many organisms conserve resources by entering dormancy, suppressing metabolism and biosynthesis. The transition out of winter dormancy to summer activity requires a quick reversal of this suppression, in order to exploit now-favorable environmental conditions. To date, mechanisms by which winter climate variation affects this transition remains unelucidated. Here we experimentally manipulated snow cover for naturally overwintering montane leaf beetles (Chrysomela aeneicollis), and profiled changes in gene expression during the transition out of dormancy in spring. Upon emergence, beetles up-regulate transcripts associated with digestion and nutrient acquisition and down regulate those associated with lipid metabolism, suggesting a shift away from utilizing stored lipid and towards digestion of carbohydrate-rich host plant tissue. Development of digestive capacity is followed by up-regulation of transcripts associated with reproduction; a transition that occurs earlier in females than males. Snow manipulation strongly affected the ground thermal regime and correspondingly gene expression profiles, with beetles showing a delayed up-regulation of reproduction in the dry compared to snowy plots. This suggests that winter conditions can alter the timing and prioritization of processes during emergence from dormancy, potentially magnifying the effects of declining snow cover in the Sierra's and other snowy mountains.

Snow modulates winter energy use and cold exposure across an elevation gradient in a montane ectotherm.

Snow insulates the soil from air temperature, decreasing winter cold stress and altering energy use for organisms that overwinter in the soil. As climate change alters snowpack and air temperatures, it is critical to account for the role of snow in modulating vulnerability to winter climate change. Along elevational gradients in snowy mountains, snow cover increases but air temperature decreases, and it is unknown how these opposing gradients impact performance and fitness of organisms overwintering in the soil. We developed experimentally validated ecophysiological models of cold and energy stress over the past decade for the montane leaf beetle Chrysomela aeneicollis, along five replicated elevational transects in the Sierra Nevada mountains in California. Cold stress peaks at mid-elevations, while high elevations are buffered by persistent snow cover, even in dry years. While protective against cold, snow increases energy stress for overwintering beetles, particularly at low elevations, potentially leading to mortality or energetic tradeoffs. Declining snowpack will predominantly impact mid-elevation populations by increasing cold exposure, while high elevation habitats may provide refugia as drier winters become more common.

Mitonuclear mismatch alters performance and reproductive success in naturally introgressed populations of a montane leaf beetle.

Coordination between nuclear and mitochondrial genomes is critical to metabolic processes underlying animals' ability to adapt to local environments, yet consequences of mitonuclear interactions have rarely been investigated in populations where individuals with divergent mitochondrial and nuclear genomes naturally interbreed. Genetic variation in the leaf beetle Chrysomela aeneicollis was assessed along a latitudinal thermal gradient in California's Sierra Nevada. Variation at mitochondrial cytochrome oxidase II (COII) and the nuclear gene phosphoglucose isomerase (PGI) shows concordance and was significantly greater along a 65 km transect than 10 other loci. STRUCTURE analyses using neutral loci identified a southern and northern subpopulation, which interbreed in the central drainage Bishop Creek. COII and PGI were used as indicators of mitochondrial and nuclear genetic variation in field and laboratory experiments conducted on beetles from this admixed population. Fecundity, larval development rate, running speed and male mating frequency were higher for beetles with geographically "matched" than "mismatched" mitonuclear genotypes. Effects of mitonuclear mismatch were largest for individuals with northern nuclear genotypes possessing southern mitochondria and were most pronounced after heat treatment or at high elevation. These findings suggest that mitonuclear incompatibility diminishes performance and reproductive success in nature, effects that could intensify at environmental extremes.

Effects of individual, community, and landscape drivers on the dynamics of a wildland forest epidemic.

The challenges posed by observing host-pathogen-environment interactions across large geographic extents and over meaningful time scales limit our ability to understand and manage wildland epidemics. We conducted a landscape-scale, longitudinal study designed to analyze the dynamics of sudden oak death (an emerging forest disease caused by Phytophthora ramorum) across hierarchical levels of ecological interactions, from individual hosts up to the community and across the broader landscape. From 2004 to 2011, we annually assessed disease status of 732 coast live oak, 271 black oak, and 122 canyon live oak trees in 202 plots across a 275-km2 landscape in central California. The number of infected oak stems steadily increased during the eight-year study period. A survival analysis modeling framework was used to examine which level of ecological heterogeneity best predicted infection risk of susceptible oak species, considering variability at the level of individuals (species identity, stem size), the community (host density, inoculum load, and species richness), and the landscape (seasonal climate variability, habitat connectivity, and topographic gradients). After accounting for unobserved risk shared among oaks in the same plot, survival models incorporating heterogeneity across all three levels better predicted oak infection than did models focusing on only one level. We show that larger oak trees (especially coast live oak) were more susceptible, and that interannual variability in inoculum production by the highly infectious reservoir host, California bay laurel, more strongly influenced disease risk than simply the density of this important host. Concurrently, warmer and wetter rainy-season conditions in consecutive years intensified infection risk, presumably by creating a longer period of inoculum build-up and increased probability of pathogen spillover from bay laurel to oaks. Despite the presence of many alternate host species, we found evidence of pathogen dilution, where less competent hosts in species-rich communities reduce pathogen transmission and overall risk of oak infection. These results identify key parameters driving the dynamics of emerging infectious disease in California woodlands, while demonstrating how multiple levels of ecological heterogeneity jointly determine epidemic trajectories in wildland settings.

Host Phenology and Leaf Effects on Susceptibility of California Bay Laurel to Phytophthora ramorum.

Spread of the plant pathogen Phytophthora ramorum, causal agent of the forest disease sudden oak death, is driven by a few competent hosts that support spore production from foliar lesions. The relationship between traits of a principal foliar host, California bay laurel (Umbellularia californica), and susceptibility to P. ramorum infection were investigated with multiple P. ramorum isolates and leaves collected from multiple trees in leaf-droplet assays. We examined whether susceptibility varies with season, leaf age, or inoculum position. Bay laurel susceptibility was highest during spring and summer and lowest in winter. Older leaves (>1 year) were more susceptible than younger ones (8 to 11 months). Susceptibility was greater at leaf tips and edges than the middle of the leaf. Leaf surfaces wiped with 70% ethanol were more susceptible to P. ramorum infection than untreated leaf surfaces. Our results indicate that seasonal changes in susceptibility of U. californica significantly influence P. ramorum infection levels. Thus, in addition to environmental variables such as temperature and moisture, variability in host plant susceptibility contributes to disease establishment of P. ramorum.

Cold tolerance of the montane Sierra leaf beetle, Chrysomela aeneicollis.

Small ectothermic animals living at high altitude in temperate latitudes are vulnerable to lethal cold throughout the year. Here we investigated the cold tolerance of the leaf beetle Chrysomela aeneicollis living at high elevation in California's Sierra Nevada mountains. These insects spend over half their life cycle overwintering, and may therefore be vulnerable to winter cold, and prior studies have demonstrated that survival is reduced by exposure to summertime cold. We identify overwintering microhabitat of this insect, describe cold tolerance strategies in all life stages, and use microclimate data to determine the importance of snow cover and microhabitat buffering for overwinter survival. Cold tolerance varies among life history stages and is typically correlated with microhabitat temperature: cold hardiness is lowest in chill-susceptible larvae, and highest in freeze-tolerant adults. Hemolymph osmolality is higher in quiescent (overwintering) than summer adults, primarily, but not exclusively, due to elevated hemolymph glycerol. In nature, adult beetles overwinter primarily in leaf litter and suffer high mortality if early, unseasonable cold prevents them from entering this refuge. These data suggest that cold tolerance is tightly linked to life stage. Thus, population persistence of montane insects may become problematic as climate becomes more unpredictable and climate change uncouples the phenology of cold tolerance and development from the timing of extreme cold events.

Inferring the past and present connectivity across the range of a North American leaf beetle: combining ecological niche modeling and a geographically explicit model of coalescence.

The leaf beetle Chrysomela aeneicollis occurs across Western North America, either at high elevation or in small, isolated populations along the coast, and thus has a highly fragmented distribution. DNA sequence data (three loci) were collected from five regions across the species range. Population connectivity was examined using traditional ecological niche modeling, which suggested that gene flow could occur among regions now and in the past. We developed geographically explicit coalescence models of sequence evolution that incorporated a two-dimensional representation of the hypothesized ranges suggested by the niche-modeling estimates. We simulated sequence data according to these models and compared them to observed sequences to identify most probable scenarios regarding the migration history of C. aeneicollis. Our results disagreed with initial niche-modeling estimates by clearly rejecting recent connectivity among regions, and were instead most consistent with a long period of range fragmentation, extending well beyond the last glacial maximum. This application of geographically explicit models of coalescence has highlighted some limitations of the use of climatic variables for predicting the present and past range of a species and has explained aspects of the Pleistocene evolutionary history of a cold-adapted organism in Western North America.

Effects of temperature variation on male behavior and mating success in a montane beetle.

Locomotion and mating ability are crucial for male reproductive success yet are energetically costly and susceptible to physiological stress. In the Sierra willow beetle Chrysomela aeneicollis, male mating success depends on locating and mating with as many females as possible. Variation at the glycolytic enzyme locus phosphoglucose isomerase (Pgi) is concordant with a latitudinal temperature gradient in these populations, with Pgi-1 frequent in the cooler north, Pgi-4 in the warmer south, and alleles 1 and 4 in relatively equal frequency in areas intermediate in geography and climate. Beetles experience elevated air temperatures during a mating season that causes differential physiological stress among Pgi genotypes, and running speeds of individuals homozygous for Pgi-4 are more tolerant of repeated thermal stress than individuals possessing Pgi-1. Here the importance of running behavior for male mating activity was examined, and differential effects of thermal stress among Pgi genotypes on male mating activity were measured. In nature, males run more than females, and nearly half of males mate or fight for a mate after running. In the laboratory, mating activity was positively correlated with running speed, and repeated mating did not reduce running speed or subsequent mating activity. Males homozygous for Pgi-4 mated longer and more frequently after heat treatment than 1-1 and 1-4 males. All heat-treated males had lower mating frequencies and higher heat shock protein expression than control males; however, mating frequency of recovering 4-4 males increased throughout mating trials, while treated 1-1 and 1-4 males remained low. These results suggest that effects of stress on mating activity differ between Pgi genotypes, implying a critical role for energy metabolism in organisms' response to stressful temperatures.

Effects of temperature on physiology and reproductive success of a montane leaf beetle: implications for persistence of native populations enduring climate change.

Understanding how climate change impacts natural systems requires investigations of the effects of environmental variation on vulnerable species and documentation of how populations respond to change. The willow beetle Chrysomela aeneicollis is ideal for such studies. It lives in California's Sierra Nevada on the southern edge of its worldwide range. Beetles experience elevated air temperatures during summertime egg laying and larval development. Exposure to these temperatures causes physiological stress, which may reduce reproductive success and endanger populations. The glycolytic enzyme phosphoglucose isomerase (PGI) is a marker of temperature adaptation in C. aeneicollis. PGI allele frequency varies across a latitudinal gradient: allele 1 is common in Rock Creek (RC), which is cooler and to the north, and allele 4 is common in Big Pine Creek (BPC), which is warmer and to the south. In populations that are intermediate in geography and climate (e.g., Bishop Creek [BC]), PGI-4 frequency increases from north to south such that alleles 1 and 4 are in relatively equal frequency in southern BC. Over the past decade, Sierra Nevada beetle populations have colonized high elevations and have become extinct at lower elevations where they were once common. In BC, the magnitude of PGI allele frequency fluctuations among life-history stages is related to maximal air temperature, with the frequency of PGI-4 increasing after the hottest part of summer. To identify mechanisms that may cause shifts in PGI allele frequency, we measured metabolic rate and fecundity for beetles collected at BC. Metabolic rate of males and females was measured at 20 degrees and 36 degrees C using flow-through respirometry. To measure laboratory fecundity, mating pairs were acclimated for 4 h each afternoon at a control temperature (20 degrees C) or at mildly elevated temperatures (26 degrees or 32 degrees C) and number of eggs laid was counted daily for 24 d, after which tissue levels of 70-kD heat shock proteins (Hsp70) were determined. Previous studies had demonstrated differences in Hsp70 expression among PGI genotypes at these temperatures. To measure field fecundity, mating pairs from BC were transplanted to similar elevations in BPC, BC, and RC and were monitored in situ for 24 d. Metabolic rate was higher for PGI 4-4 genotypes than for PGI 1-4 or PGI 1-1 individuals at 36 degrees C but not at 20 degrees C. In contrast, laboratory fecundity was greatest for females possessing PGI-1, independent of acclimation temperature. At the end of the laboratory fecundity experiment, Hsp70 expression was positively related to fecundity, suggesting minimal reproductive cost of upregulation of heat shock proteins in response to mild heat stress. In the field, fecundity was highest for PGI 1-1 and PGI 1-4 individuals in RC and PGI 4-4 individuals in BPC and was similar for all genotypes in BC. Thus, fecundity in nature was greatest for the genotypes that were most common in each area. Taken together, data reported here suggest that hot, dry summers in the Sierra Nevada may result in an increase in frequency of the PGI-4 allele and shifts to higher elevations for C. aeneicollis populations.

Influence of land-cover change on the spread of an invasive forest pathogen.

Human-caused changes in land use and land cover have dramatically altered ecosystems worldwide and may facilitate the spread of infectious diseases. To address this issue, we examined the influence of land-cover changes between 1942 and 2000 on the establishment of an invasive pathogen, Phytophthora ramorum, which causes the forest disease known as Sudden Oak Death. We assessed effects of land-cover change, forest structure, and understory microclimate on measures of inoculum load and disease prevalence in 102 15 x 15 m plots within a 275-km2 region in northern California. Within a 150 m radius area around each plot, we mapped types of land cover (oak woodland, chaparral, grassland, vineyard, and development) in 1942 and 2000 using detailed aerial photos. During this 58-year period, oak woodlands significantly increased in area by 25%, while grassland and chaparral decreased by 34% and 51%, respectively. Analysis of covariance revealed that vegetation type in 1942 and woodland expansion were significant predictors of pathogen inoculum load in bay laurel (Umbellularia californica), the primary inoculum-producing host for P. ramorum in mixed evergreen forests. Path analysis showed that woodland expansion resulted in larger forests with higher densities of the primary host trees (U. californica, Quercus agrifolia, Q. kelloggii) and cooler understory temperatures. Together, the positive effects of woodland size and negative effects of understory temperature explained significant variation in inoculum load and disease prevalence in bay laurel; host stem density had additional positive effects on inoculum load. We conclude that enlargement of woodlands and closure of canopy gaps, likely due largely to years of fire suppression, facilitated establishment of P. ramorum by increasing the area occupied by inoculum-production foliar hosts and enhancing forest microclimate conditions. Epidemiological studies that incorporate land-use change are rare but may increase understanding of disease dynamics and improve our ability to manage invasive forest pathogens.

Predator diversity and identity drive interaction strength and trophic cascades in a food web.

Declining predator diversity may drastically affect the biomass and productivity of herbivores and plants. Understanding how changes in predator diversity can propagate through food webs to alter ecosystem function is one of the most challenging ecological research topics today. We studied the effects of predator removal in a simple natural food web in the Sierra Nevada mountains of California (USA). By excluding the predators of the third trophic level of a food web in a full-factorial design, we monitored cascading effects of varying predator diversity and composition on the herbivorous beetle Chrysomela aeneicollis and the willow Salix orestera, which compose the first and second trophic levels of the food web. Decreasing predator diversity increased herbivore biomass and survivorship, and consequently increased the amount of plant biomass consumed via a trophic cascade. Despite this simple linear mean effect of diversity on the strength of the trophic cascade, we found additivity, compensation, and interference in the effects of multiple predators on herbivores and plants. Herbivore survivorship and predator-prey interaction strengths varied with predator diversity, predator identity, and the identity of coexisting predators. Additive effects of predators on herbivores and plants may have been driven by temporal niche separation, whereas compensatory effects and interference occurred among predators with a similar phenology. Together, these results suggest that while the general trends of diversity effects may appear linear and additive, other information about species identity was required to predict the effects of removing individual predators. In a community that is not temporally well-mixed, predator traits such as phenology may help predict impacts of species loss on other species. Information about predator natural history and food web structure may help explain variation in predator diversity effects on trophic cascades and ecosystem function.

Susceptibility to Phytophthora ramorum in a key infectious host: landscape variation in host genotype, host phenotype, and environmental factors.

Sudden oak death is an emerging forest disease caused by the invasive pathogen Phytophthora ramorum. Genetic and environmental factors affecting susceptibility to P. ramorum in the key inoculum-producing host tree Umbellularia californica (bay laurel) were examined across a heterogeneous landscape in California, USA. Laboratory susceptibility trials were conducted on detached leaves and assessed field disease levels for 97 host trees from 12 225-m(2) plots. Genotype and phenotype characteristics were assessed for each tree. Effects of plot-level environmental conditions (understory microclimate, amount of solar radiation and topographic moisture potential) on disease expression were also evaluated. Susceptibility varied significantly among U. californica trees, with a fivefold difference in leaf lesion size. Lesion size was positively related to leaf area, but not to other phenotypic traits or to field disease level. Genetic diversity was structured at three spatial scales, but primarily among individuals within plots. Lesion size was significantly related to amplified fragment length polymorphism (AFLP) markers, but local environment explained most variation in field disease level. Thus, substantial genetic variation in susceptibility to P. ramorum occurs in its principal foliar host U. californica, but local environment mediates expression of susceptibility in nature.

The role of stress proteins in responses of a montane willow leaf beetle to environmental temperature variation.

The heat shock response is a critical mechanism by which organisms buffer effects of variable and unpredictable environmental temperatures. Upregulation of heat shock proteins (Hsps) increases survival after exposure to stressful conditions in nature, although benefits of Hsp expression are often balanced by costs to growth and reproductive success. Hsp-assisted folding of variant polypeptides may prevent development of unfit phenotypes; thus, some differences in Hsp expression among natural populations of ectotherms may be due to interactions between enzyme variants (allozymes) and Hsps. In the Sierra willow leaf beetle Chrysomela aeneicollis, which lives in highly variable thermal habitats at the southern edge of their range in the Eastern Sierra Nevada, California, allele frequencies at the enzyme locus phosphoglucose isomerase (PGI) vary across a climatic latitudinal gradient. PGI allozymes differ in kinetic properties,and expression of a 70 kDa Hsp differs between populations, along elevation gradients,and among PGI genotypes. Differences in Hsp70 expression among PGI genotypes correspond to differences in thermal tolerance and traits important for reproductive success, such as running speed, survival and fecundity. Thus, differential Hsp expression among genotypes may allow functionally important genetic variation to persist, allowing populations to respond effectively to environmental change.

Phosphoglucose isomerase genotype affects running speed and heat shock protein expression after exposure to extreme temperatures in a montane willow beetle.

Eastern Sierra Nevada populations of the willow beetle Chrysomela aeneicollis commonly experience stressfully high and low environmental temperatures that may influence survival and reproduction. Allele frequencies at the enzyme locus phosphoglucose isomerase (PGI) vary across a climatic latitudinal gradient in these populations, with PGI allele 1 being most common in cooler regions and PGI allele 4 in warmer ones. PGI genotypes differ in heat and cold tolerance and in expression of a 70 kDa heat shock protein. Here we examine genetic, behavioral and environmental factors affecting a performance character, running speed, for willow beetles, and assess effects of consecutive cold and heat exposure on running speed and expression of Hsp70 in the laboratory. In nature, running speed depends on air temperature and is higher for males than females. Mating beetles ran faster than single beetles, and differences among PGI genotypes in male running speed depended on the presence of females. In the laboratory, exposure to cold reduced subsequent running speed, but the amount of this reduction depended on PGI genotype and previous thermal history. Effects of exposure to heat also depended on life history stage and PGI genotype. Adults possessing allele 1 ran fastest after a single exposure to stressful temperature, whereas those possessing allele 4 ran faster after repeated exposure. Larvae possessing allele 4 ran fastest after a single stressful exposure, but running speed generally declined after a second exposure to stressful temperature. The ranking of PGI genotypes after the second exposure depended on whether a larva had been exposed to cold or heat. Effects of temperature on Hsp70 expression also varied among PGI genotypes and depended on type of exposure, especially for adults (single heat exposure, two cold exposures: PGI 1-1>1-4>4-4; other multiple extreme exposures: 4-4>1-4>1-1). There was no consistent association between alleles at other polymorphic enzyme loci and running speed or Hsp70 expression. These data suggest that variation at PGI is associated with considerable plasticity in running speed. Differences in Hsp70 expression among PGI genotypes suggest that the heat-shock response may buffer differences in thermal tolerance and performance among genotypes and help maintain the PGI polymorphism in a thermally variable environment.

Allele frequency shifts in response to climate change and physiological consequences of allozyme variation in a montane insect.

Rapid changes in climate may impose strong selective pressures on organisms. Evolutionary responses to climate change have been observed in natural populations, yet no example has been documented for a metabolic enzyme locus. Furthermore, few studies have linked physiological responses to stress with allozyme genotypic variation. We quantified changes in allele frequency between 1988 and 1996 at three allozyme loci (isocitrate dehydrogenase, Idh; phosphoglucose isomerase, Pgi; and phosphoglucomutase, Pgm) for the leaf beetle Chrysomela aeneicollis in the Bishop Creek region of the Sierra Nevada of California (2900-3300 m). Beetles often experience high daytime (> 32 degrees C) and extremely low nighttime (< -5 degrees C) temperatures during summer. Bishop Creek weather station data indicated that conditions were unusually dry before 1988, and that conditions were cool and wet during the years preceding the 1996 collection. We found directional changes in allele frequency at Pgi (11% increase in the Pgi-1 allele), but not at Idh or Pgm. We also found that physiological response to thermal extremes depended on Pgi genotype. Pgi 1-1 individuals induced expression of a 70-kD heat shock protein (HSP) at lower temperatures than 1-4 or 4-4 individuals, and 1-1 individuals expressed higher levels of HSP70 after laboratory exposure to temperatures routinely experienced in nature. Survival after nighttime laboratory exposure to subzero temperatures depended on gender, previous exposure to cold, and Pgi genotype. Females expressed higher levels of HSP70 than males after exposure to heat, and recovery by female Pgi 1-1 homozygotes after exposure to cold (-5 degrees C) was significantly better than 1-4 or 4-4 genotypes. These data suggest that the cooler climate of the mid-1990s may have caused an increase in frequency of the Pgi-1 allele, due to a more robust physiological response to cold by Pgi 1-1 and 1-4 genotypes.

THE EVOLUTION OF HOST-PLANT USE AND SEQUESTRATION IN THE LEAF BEETLE GENUS PHRATORA (COLEOPTERA: CHRYSOMELIDAE).

Leaf beetles in the genus Phratora differ in host plant use and in the chemical composition of their larval defensive secretion. Most species specialize on either poplars or willows (family Salicaceae), but two species feed on birch (family Betulaceae). Phratora vitellinae utilizes salicylates from the host plant to produce its larval secretion, which contains salicylaldehyde, while other Phratora species produce an autogenous secretion. To reconstruct the evolutionary history of host plant use and the larval secretion chemistry in this genus, we sequenced 1383 base pairs of the mt cytochrome oxidase I gene for six European and one North American Phratora species and three outgroup taxa. Bootstrap values of the complete nucleotide sequence were 99-100% for six of eight nodes in the maximum parsimony tree. They were 71% and 77% for the two other nodes. The maximum parsimony tree and the maximum likelihood tree based on nucleotide sequence showed the same relationships as a maximum parsimony tree based on the amino acid sequence. Beetle phylogeny overlapped broadly with host plant taxonomy and chemistry, and it revealed historical constraints influencing host plant use. However, there was one host shift from the willow family (Salicaceae) to the birch family (Betulaceae). The use of host plant phenol glycosides for the larval defensive secretion evolved along the lineage that led to P. vitellinae. Phratora vitellinae feeds on the taxonomically widest range of host plants, which are characterized by moderate to high levels of salicylates. The results support the hypothesis that the use of salicylates for the larval secretion evolved twice independently in chrysomeline leaf beetles.

Predator protection versus rapid growth in a montane leaf beetle.

Adults and larvae of Chrysomela aenicollis (Coleoptera: Chrysomelidae) feed on foliage of Salix species (Salicaceae) between 2,400-3,400 m above sea level in the eastcentral Sierra Nevada mountains of California. We predicted that (1) cold climatic conditions would be a more frequent source of mortality at higher elevations, (2) mildweather agents of mortality such as predation should be more severe at lower elevations, and (3) populations of C. aenicollis would be adapted to the local selective regime at each elevation. We tested these predictions in 1984 and 1985 by transferring over 6,000 eggs and larvae within and between two sites at 2,810 and 3,240 m elevation above sea level. During mild summer weather at both sites, survivorship on Salix branches isolated by a barrier of sticky resin was similar to that on control branches, and we concluded that aerial predators were the primary cause of mortality. At least one major predator, a solitary wasp (Symmorphus sp., Hymenoptera: Eumenidae), was specifically associated with C. aenicollis at the lower site, where beetle mortality was highest. At both sites in 1984 and 1985, larvae originating from the lower site remained in aggregations and survived more frequently than larvae from the upper site, suggesting that they are better defended against predators. During a storm with cold weather late in the 1984 season, larvae and pupae died more frequently at the upper site, and there was a marginally significant trend (P<0.1) for the lower site individuals to die more frequently than upper site larvae during the cold storm. Upper site larvae grew approximately 10% faster than lower site larvae at the lower site and under controlled conditions in the laboratory. These findings indicate that upper and lower site populations were adapted to the local selective regime, which suggest how populations of montane phytopagous insects may adapt to changing elevations.