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.

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.

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.

Local distribution of the lycaenid butterfly, Jalmenus evagoras, in response to host ants and plants.

The caterpillars of Jalmenus evagoras are tended by ants as they feed upon Acacia trees. In the area of Brisbane, Australia, J. evagoras require ants of the Iridomyrmex anceps species group; predation and parasitism are so intense that larvae and pupae deprived of attendant ants cannot survive (Pierce 1983). We investigated the efficiency with which J. evagoras locate and exploit the "host ant" resource by sampling 737 quadrats in 30 sampling grids and six study sites containing appropriate host plants; ants were collected at baits located in the center of each quadrat. J. evagoras was found in all habitats where I. anceps cooccurred with host Acacia. Nine of the ten sampling grids which had three or more I. anceps/Acacia "host" quadrats also had colonies of J. evagoras present (or immediately adjacent), including sites as far as 35 km apart. Of 19 sampling grids on which "host" quadrats were rare (i.e., less than three quadrats), none had J. evagoras (P<0.001). Within sample grids, I. anceps was distributed indepedently from Acacia trees, suggesting that they are not dependent for their survival on either Acacia or on J. evagoras. Within montane pasture habitats, I. anceps and at least one other ground-dwelling Iridomyrmex species were distributed in mutually exclusive "ant mosaic" territories which were stable during a one month period. I. anceps did not colonize or tend pupae of J. evagoras experimentally placed in adjacent territories of a different, nontending species of Iridomyrmex, demonstrating the integrity of territory boundaries. Sampling of ants in Acacia trees revealed that, in the absence of J. evagoras, Iridomyrmex workers are not common above ground level, and that their numbers decline in larger trees (P=0.02). In I. anceps territories, eight of nine J. evagoras pupae placed in trees over 3.0 m tall were not found after 24 h whereas all ten controls placed in low trees were found and tended (P=0.00012). This may explain why J. evagoras tends to oviposit in trees less than 2.0 m tall. An alternative hypothesis, that smaller trees have higher content of total nitrogen, and are threfore more nutritious, was not supported. We conclude that the local distribution and host tree selection by J. evagoras is dependent upon the distribution, patchiness, and foraging behavior of the host ant, I. anceps, and its spatial overlap with a number of species of host Acacia.

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.

Are chemical barriers necessary for evolution of butterfly-plant associations?

The association between heliconiine butterflies and Passion flower vines is composed of three or more subassociations, in which each Heliconius species group feeds on a different Passiflora subgenus. The relationships are consistent with the adaptive zone hypothesis of Ehrlich and Raven, which would suggest that (1) species of the subgenus Plectostemma proliferated as a result of chemical barriers to herbivory, which created a herbivore-free adaptive zone in which speciation and diversification took place, and (2) species of the H. erato-charitonia group overcame these barriers and entered a competitor-free adaptive zone, in which they proliferated and speciated with those plants as hosts. The hypothesis that plant secondary chemicals were responsible for creating such barriers to herbivory was tested using heliconiine species as bioassays, in which reduced growth rates indicated presence of chemical barriers to feeding. Contrary to expectation, plants of the subgenus Plectostemma showed little or no chemical defense against any species of heliconiine caterpillar. In contrast many plants of the "primitive" subgenus Granadilla possessed significant chemical barriers against herbivory by heliconiine larvae, excepting those species in the H. numata-melpomene species group. I concluded that chemical barriers to feeding were not responsible for proliferation and diversification in the subgenus Plectostemma, nor did chemicals create a competitor-free "adaptive zone" in which the H. erato-charitonia species-group could proliferate and speciate. Chemical barriers may have been important in the evolution of the subgenus Granadilla-heliconiine association. I suggest that plant allelochemics are only one of many possible barriers to herbivory which can help create "adaptive zones" for plants and their herbivores, and that the patterns of butterfly foodplant specialization discussed by Ehrlich and Raven (1964) are not necessarily the result of biochemical adaptation and counteradaptation.

Patterns of pollen exploitation by Heliconius butterflies.

We have studied pollen feeding habits of ten species of Heliconius butterflies in six major study areas in Trinidad and Costa Rica, and examined inter-species differences in pollen utilization under greenhouse conditions. We found: 1. Females collected significantly more pollen than males. 2. Older individuals generally collected significantly more pollen than younger individuals. 3. Amount of pollen collected may vary significantly through time at a single site, apparently because of changing weather conditions. 4. Amount of pollen collected may vary significantly between species at a single site at a given time, apparently because of differences in per capita resource availability to species using different habitats. 5. Different species had significantly differing abilities to exploit small grained or large grained pollens, which resulted in significant differences in mean pollen collected at different sites by the same species. 6. Species exploiting both large and small grained pollens showed significant differences through time in the ratio of large to small grained pollens utilized.