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.     

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).     

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.     

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.     

MAINTENANCE OF ECOLOGICALLY SIGNIFICANT GENETIC VARIATION IN THE TIGER SWALLOWTAIL BUTTERFLY THROUGH DIFFERENTIAL SELECTION AND GENE FLOW

Differential selection in a heterogeneous environment is thought to promote the maintenance of ecologically significant genetic variation. Variation is maintained when selection is counterbalanced by the homogenizing effects of gene flow and random mating. In this study, we examine the relative importance of differential selection and gene flow in maintaining genetic variation in Papilio glaucus. Differential selection on traits contributing to successful use of host plants (oviposition preference and larval performance) was assessed by comparing the responses of southern Ohio, north central Georgia, and southern Florida populations of P. glaucus to three hosts: Liriodendron tulipifera, Magnolia virginiana, and Prunus serotina. Gene flow among populations was estimated using allozyme frequencies from nine polymorphic loci. Significant genetic differentiation was observed among populations for both oviposition preference and larval performance. This differentiation was interpreted to be the result of selection acting on Florida P. glaucus for enhanced use of Magnolia, the prevalent host in Florida. In contrast, no evidence of population differentiation was revealed by allozyme frequencies. F_ST-values were very small and Nm, an estimate of the relative strengths of gene flow and genetic drift, was large, indicating that genetic exchange among P. glaucus populations is relatively unrestricted. The contrasting patterns of spatial differentiation for host-use traits and lack of differentiation for electrophoretically detectable variation implies that differential selection among populations will be counterbalanced by gene flow, thereby maintaining genetic variation for host-use traits.     

Isozyme variation and correspondence with unusual infrared reflectance patterns inPhragmites australis (Poaceae)

Isozyme variation was investigated in populations ofPhragmites australis (Poaceae) which have recently invaded and taken over marsh habitat of the Mississippi River delta. Infrared aerial photographs in the Garden Island Bay subdelta of the extensiveP. australis populations reveal distinct, clone-like circular patches within a predominant background. Preliminary evidence indicates that the infrared color differences represent distinct morphological types. However, there are no obvious environmental factors that could account for the peculiar patterns.P. australis collections were taken from five separate and distinct patches and adjacent background. Only two electrophoretic phenotypes were found: one from patches and one from the background. In comparing the two, 20% of the 40 loci scored are fixed for alternate alleles. These results indicate a clear correspondence of infrared reflectance with electrophoretic phenotype. In addition, the genetic uniformity as evidenced by the discovery of only two electrophoretic phenotypes supports the contention that the recent spread ofP. australis throughout the Mississippi River delta has been primarily, if not exclusively, a result of vegetative propagation.     

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     

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.

     

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.     

GENETICS OF MIMICRY IN THE TIGER SWALLOWTAIL BUTTERFLIES,PAPILIO GLAUCUS AND P. CANADENSIS (LEPIDOPTERA: PAPILIONIDAE)

The tiger swallowtail butterfly, Papilio glaucus, exhibits a female-limited polymorphism for Batesian mimicry; the Canadian tiger swallowtail, Papilio canadensis, lacks the mimetic (dark) form entirely. The species hybridize to a limited extent where their ranges overlap. Field collections and censuses indicate that mimetic females occur throughout the range of P. glaucus but at lowest frequencies in populations at the latitudinal edges of its geographic range such as the southernmost part of Florida and along the entire northern edge of its distribution from Massachusetts to Minnesota. Frequencies of mimetic females have remained relatively stable over time. Inheritance of the mimetic form is controlled primarily by two interacting sex-linked loci. The typical matrilineal pattern of inheritance in P. glaucus can be explained by polymorphism at a Y-linked locus, b. Analysis of P. glaucus × P. canadensis crosses has also revealed an X-linked locus, s, which controls the expression of the mimetic phenotype. The P. canadensis allele, s^can, suppresses the mimetic phenotype in hybrid and backcross females. Results from more than 12 yr of rearing tiger swallowtails, including interspecies hybrids, indicate that the absence of mimetic P. canadensis females is due to both a high frequency of the “suppressing” allele s^can and low frequency of the black-pigment-determining b + allele. The frequency of s^can (or other suppressing alleles of s) in P. glaucus populations outside the hybrid zone is low. Some males heterozygous at the s locus and some suppressed mimetic females occur within the hybrid zone. A simple genetic model predicts the frequency of daughters that differ in phenotype from their mothers.     

Basal oxygen consumption,ventilatory frequency,and heart rate during the protracted spawning run of the Southern Hemisphere lamprey Geotria australis

Summary Basal oxygen consumption, ventilatory frequency, and heart rate were recorded at four different times during the unusually protracted 15–16-month spawning run of the Southern Hemisphere lamprey Geotria australis. At 15°C, the mean basal oxygen consumption of G. australis caught immediately after they had left the sea and embarked on the spawning run (45 l · g^-1 · h^-1) was less than in young adults about to commence their marine feeding phase (64 l · g^-1 · h^-1), but greater than in large ammocoetes (26.5 l · g^-1 · h^-1). Basal oxygen consumption fell progressively during the spawning-run of to 33 l · g^-1 · h^-1 after 5 months and 25 l · g^-1 · h^-1 after 10 months, before rising to 35 l · g^-1 · h^-1 after 15 months when the animals were approaching sexual maturity. The downwards trend in basal oxygen consumption contrasts with that recorded during the spawning run of Lampetra fluviatilis. Furthermore, these values for spawning-run of G. australis are far lower than those measured at any time during the upstream migration of L. fluviatilis or during the parasitic phase of landlocked Petromyzon marinus. A low and declining metabolic rate during much of the spawning run of G. australis would facilitate the conservation of energy reserves during this very long non-feeding period. Trends shown by ventilatory frequency and heart rate essentially parallel those of basal oxygen consumption. The Q₁₀s for basal oxygen consumption, ventilatory frequency and heart rate over the temperature range 5–25°C were 1.6, 1.6, and 1.7, respectively. The trends shown by basal oxygen consumption during metamorphosis and the upstream migration did not parallel those exhibited by circulating thyroid hormones.     

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.     

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.     

Genetic differentiation in carbon isotope discrimination and gas exchange in Pseudotsuga menziesii

Patterns of genetic variation in gas-exchange physiology were analyzed in a 15-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) plantation that contains 25 populations grown from seed collected from across the natural distribution of the species. Seed was collected from 33°30 to 53°12 north latitude and from 170 m to 2930 m above sea level, and from the coastal and interior (Rocky Mountain) varieties of the species. Carbon isotope discrimination () ranged from 19.70() to 22.43() and was closely related to geographic location of the seed source. The coastal variety (20.50 (SE=0.21)) was not significantly different from the interior variety (20.91 (0.15)). Instead, most variation was found within the interior variety; populations from the southern Rockies had the highest discrimination (21.53 (0.20)) (lowest water-use efficiency). Carbon isotope discrimination (), stomatal conductance to water vapor (g), the ratio of intercellular to ambient CO₂ concentration (c_i/c_a), and intrinsic water-use efficiency (A/g) were all correlated with altitude of origin (r=0.76, 0.73, 0.74, and –0.63 respectively); all were statistically significant at the 0.01 level. The same variables were correlated with both height and diameter at age 15 (all at P0.0005). Observed patterns in the common garden did not conform to our expectation of higher WUE, measured by both A/g and , in trees from the drier habitats of the interior, nor did they agree with published in situ observations of decreasing g and with altitude. The genetic effect opposes the altitudinal one, leading to some degree of homeostasis in physiological characteri tics in situ.     

Relationships of herbivore feeding and plant flavonoids (Coleoptera: Chrysomelidae and Anacardiaceae: Rhus)

Summary Eighteen leaf flavonoid compounds were isolated from several populations of Rhus tripartita from xeric habitats in Israel, ornamental Schinus terebinthifolius from Israel, and three species of Rhus (vulgaris, natalensis, tenuinervis) from mesic habitats in Kenya. Foodplant preference testing of Rhus-feeding Leaf Beetles (Blepharida sacra from Israel and B. marginalis and B. conradsi from Kenya) correlated well with the flavonoid composition of the different foodplants. Blepharida sacra and B. marginalis foodplant preferences demonstrated an herbivore sibling relationship but there is evidence, including foodplant ecology and distribution, that they are separate species. The herbivorefoodplant coevolution of the xeric B. sacra-R. tripartita is distinct from that of the mesic B. marginalis-R. vulgaris/natalensis, however, this study also indicates possible ancestral relationships between the herbivore species as well as between the plant species.     

Growth,acetylene reduction activity and localization of nitrogenase in relation to vesicle formation in Frankia strains Cc1.17 and Cp1.2

A comparative study was conducted on the effect of NH₄Cl on growth, vesicle formation and formation of nitrogenase of Frankia strains Cc1.17 and Cp1.2, derived from root nodules of Colletia cruciata and Comptonia peregrina, respectively. On a medium without combined nitrogen (P-N), both strains formed spherical cells, called vesicles, like many other Frankia strains. Data are presented on the number of vesicles per mg protein, after cultivation in media with sodium propionate as C-source without combined nitrogen (P-N) or with 0.2 g NH₄Cl/l (P+N). Strain Cp1.2 as may other Frankia strains, showed on P+N medium a very strong reduction of vesicle formation of 99% relative to the number of vesicles formed on P-N medium, after 11 days growth. However, in strain Cc11.17 this reduction was only 70%. The occurence of relatively large numbers of vesicles in P+N media has not yet been reported for other Frankia strains. No acetylene reduction activity was found in NH ₄ ⁺ -grown cells. The regulation of induction of nitrogenase in Frankia by NH₄Cl was tested by immuno-gelectrophoresis using antisera against nitrogenase of Rhizobium leguminosarum PRE. The component I of the enzyme showed crossreactivity while the component II had only a weak crossreaction. The experiments indicated that no nitrogenase was detectable in the NH ₄ ⁺ -grown cells. For the localization of nitrogenase, relative amounts of the enzyme were compared in whole cells and vesicle-enriched fractions. Western blots showed a significant enrichment of nitrogenase in the vesicle fractions, which indicated that most of the nitrogenase was localized in the vesicle.     

Reconstitution of cytoplasmic streaming inCharaceae

Summary The active sites of actin of oneCharaceae species were found to interact with the endoplasmic factor from a different species. Protoplasm was suqueezed out of cells ofChara australis with vacuoles that had been perfused beforehand with a medium containing EGTA and Mg · ATP. Centrifugation of this protoplasmic mixture divided it into the supernatant composed of endoplasmic granules and the precipitate composed of chloroplasts and nuclei. When the endoplasmic granular aggregates were introduced into a tonoplast-freeNitella axilliformis cell treated with NEM to inactivate the endoplasmic factor, they became attached to theNitella gel and streamed longitudinally with the polarity. Treatment of the endoplasmic granules with the strong Mg²⁺chelator CyDTA (1,2-cyclohexane diamineN, N-tetraacetic acid) irreversibly inhibited reconstitution of the cytoplasmic streaming.Abbreviations APW artificial pond water - ATP adenosine-5-triphosphoric acid - CyDTA cyclohexanediamine-N,N-tetraacetic acid - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N-tetraacetic acid - HMM heavy meromyosin - NEM N-ethylmaleimide - PEP phosphoenolypyruvate - PIPES piperazine-N,N-bis-(2-ethane-sulfonic acid) - PK pyruvate kinase - PMSF phenylmethylsulfonylfluoride     

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat

A -amylase (EC 3.2.1.2) was identified in the outer pericarp (P) of developing seeds of wheat (Triticum aestivum L.) and compared with the well known -amylase which is synthesized during seed development in the starchy endosperm (E). The enzyme P already exists in the tissues before anthesis and vanishes at the time when E starts to accumulate. The isoelectric-focusing patterns of P and E are very similar. The relative molecular weight (M_r) of P is slightly higher than that of E (66 and 64.5 kDa, respectively). Both P and E exhibit common epitopes in addition to epitopes specific for each of them. The two enzymes were identified in small amounts in the green tissues of the developing seeds (inner pericarp and testa). No antigenic difference was detected between P and the -amylases of roots and leaves.Abbreviations P pericarp -amylase - E endosperm -amylase - IS1 anti--amylase immune serum - IS2 anti- and anti- amylase immune serum - IS3 anti- amylase immune serum - IEF isoelectric focusing - IgG immunoglobulin G The authors thank Dr. P. Ziegler (Universität Bayreuth, FRG) for stimulating discussion and for useful suggestions during the writing of the text. The authors thank Miss C. Mayer for her skillful technical assistance.     

Changes in phosphorus concentrations and pH in the rhizosphere of some agroforestry and crop species

The aim of this work was to assess whether agroforestry species have the ability to acquire P from pools unavailable to maize. Tithonia diversifolia(Hemsley) A. Gray, Tephrosia vogelii Hook f., Zea mays and Lupinus albusL. were grown in rhizopots and pH change and depletion of inorganic and organic P pools measured in the rhizosphere. Plants were harvested at the same growth stage, after 56 days for maize and white lupin and 70 days for tithonia and tephrosia, and the rhizosphere sampled. The rhizosphere was acidified by tithonia (pH change –0.3 units to pH 4.8) and lupins (–0.2 units to 4.9), alkalinised by tephrosia (+0.4 units to pH 5.4), and remained unchanged with maize growth. Concurrent with acidification in the rhizosphere of tithonia there was a decline in resin-P (0.8 g P g^–1). However, there was also a decline in NaOH extractable inorganic P (NaOH-P_i) (5.6 g P g^–1 at the root surface) and organic P pools (NaOH-P_o) (15.4 g P g^–1 at 1.5 mm from the root), which would not be expected without specific P acquisition mechanisms. Alkalinisation of tephrosia rhizosphere was accompanied by changes in all measured pools, although the large depletion of organic P (21.6 g P g^–1 at 5 mm from the root) suggests that mineralisation, as well as desorption of organic P, was stimulated. The size of changes of both pH and P pools varied with distance away from the rhizoplane. Decline of more recalcitrant P pools with the growth of the agroforestry species contrasted with the effect of maize growth, which was negligible on resin-P and NaOH-P_i, but led to an accumulation of P as NaOH-P_o (14.2 g P g^–1 at 5 mm from the root). Overall the depletion of recalcitrant P pools, particularly P_o, suggests that the growth of tithonia and tephrosia enhance desorption and dissolution of P, while also enhancing organic P mineralisation. Both species appear to have potential for agroforestry technologies designed to enhance the availability of P to crops, at least in the short term.