Variation In Leaf Chemistry Research Articles

The chemical biogeography of a widespread aromatic plant species shows both spatial and temporal variation.

Plants produce a wide variety of secondary metabolites, but intraspecific variation in space and time can alter the ecological interactions these compounds mediate. The aim of this work was to document the spatial and temporal chemical biogeography of Monarda fistulosa. I collected leaves from 1587 M. fistulosa individuals from 86 populations from Colorado to Manitoba, extracted and analyzed their terpenes with gas chromatography, mapped monoterpene chemotypes, and analyzed chemical variation with principal component analysis. I also measured the amounts of terpenes in different plant tissues to examine intraplant variation and monitored leaf terpene chemistry over a single growing season to examine temporal patterns. Finally, I extracted terpenes from herbarium samples up to 125 years old and compared the chemotypes with recent samples from the same sites. M. fistulosa populations consisted mostly of thymol (T) and carvacrol (C) chemotypes, but geraniol (G) and (R)‐(−)‐linalool (L), a chemotype new to this species, were also present. A principal component analysis showed that most of the chemical variation across populations was due to the amounts of the dominant terpene in plants. Intraplant tissue chemistry revealed that leaves mostly had the greatest amounts of terpenes, followed by floral structures, stems, and roots. Short‐term temporal variation in leaf chemistry of T and C plants over a growing season showed that plants produced the highest levels of biosynthetic precursors early in the season and their dominant monoterpenes peaked in mid‐summer. Plant chemotype was discernable in the oldest herbarium samples, and 15 of 18 historic samples matched the majority chemotype currently at the site, indicating that population chemotype ratios may remain stable over longer time scales. Overall, the results show that plant species' secondary chemistry can vary both spatially and temporally, which may alter the biological interactions that these compounds mediate over space and time.

Genotypic richness predicts phenotypic variation in an endangered clonal plant.

Declines in genetic diversity within a species can affect the stability and functioning of populations. The conservation of genetic diversity is thus a priority, especially for threatened or endangered species. The importance of genetic variation, however, is dependent on the degree to which it translates into phenotypic variation for traits that affect individual performance and ecological processes. This is especially important for predominantly clonal species, as no single clone is likely to maximise all aspects of performance. Here we show that intraspecific genotypic diversity as measured using microsatellites is a strong predictor of phenotypic variation in morphological traits and shoot productivity of the threatened, predominantly clonal seagrass Posidonia australis, on the east coast of Australia. Biomass and surface area variation was most strongly predicted by genotypic richness, while variation in leaf chemistry (phenolics and nitrogen) was unrelated to genotypic richness. Genotypic richness did not predict tissue loss to herbivores or epiphyte load, however we did find that increased herbivore damage was positively correlated with allelic richness. Although there was no clear relationship between higher primary productivity and genotypic richness, variation in shoot productivity within a meadow was significantly greater in more genotypically diverse meadows. The proportion of phenotypic variation explained by environmental conditions varied among different genotypes, and there was generally no variation in phenotypic traits among genotypes present in the same meadows. Our results show that genotypic richness as measured through the use of presumably neutral DNA markers does covary with phenotypic variation in functionally relevant traits such as leaf morphology and shoot productivity. The remarkably long lifespan of individual Posidonia plants suggests that plasticity within genotypes has played an important role in the longevity of the species. However, the strong link between genotypic and phenotypic variation suggests that a range of genotypes is still the best case scenario for adaptation to and recovery from predicted environmental change.

Transcriptome sequencing of two phenotypic mosaic Eucalyptus trees reveals large scale transcriptome re-modelling.

Phenotypic mosaic trees offer an ideal system for studying differential gene expression. We have investigated two mosaic eucalypt trees from two closely related species (Eucalyptus melliodora and E. sideroxylon), which each support two types of leaves: one part of the canopy is resistant to insect herbivory and the remaining leaves are susceptible. Driving this ecological distinction are differences in plant secondary metabolites. We used these phenotypic mosaics to investigate genome wide patterns of foliar gene expression with the aim of identifying patterns of differential gene expression and the somatic mutation(s) that lead to this phenotypic mosaicism. We sequenced the mRNA pool from leaves of the resistant and susceptible ecotypes from both mosaic eucalypts using the Illumina HiSeq 2000 platform. We found large differences in pathway regulation and gene expression between the ecotypes of each mosaic. The expression of the genes in the MVA and MEP pathways is reflected by variation in leaf chemistry, however this is not the case for the terpene synthases. Apart from the terpene biosynthetic pathway, there are several other metabolic pathways that are differentially regulated between the two ecotypes, suggesting there is much more phenotypic diversity than has been described. Despite the close relationship between the two species, they show large differences in the global patterns of gene and pathway regulation.

Recognizing cross‐ecosystem responses to changing temperatures: soil warming impacts pelagic food webs

The energy and materials that move across ecosystem boundaries influence food web structure and key ecosystem functions. Despite the acknowledged importance of such ecological subsidies, surprisingly little information is available regarding the role of environmental temperature in influencing subsidy quality and the response of the recipient ecosystem. We evaluated the impacts of temperature‐mediated changes in leaves from deciduous trees, an important subsidy from terrestrial to freshwater ecosystems, on both the producer‐based and detritivore‐based components of a pelagic pond food web in a field mesocosm experiment. We hypothesized that variation in leaf chemistry driven by increased soil temperature would alter both the quality of leaf subsidies and the pond response. We collected red maple Acer rubrum leaves from heated and ambient temperature plots from the long‐term soil warming experiment at the Harvard Experimental Forest and added them to 167‐l field mesocosms containing established plankton communities, creating ‘no leaf’, ‘ambient leaf’ and ‘heated leaf’ treatments during autumn 2012. We then monitored physical, chemical, and biological responses to treatments until the mesocosms froze six weeks later. Experimental soil warming altered the chemical composition of deciduous leaves, the physical and chemical environment of the aquatic ecosystems to which leaves were added, and the pelagic pond food webs as measured by community composition. Compared to leaves from ambient‐temperature soils, leaves from warmed soils initially resulted in lower water column phosphorus and dissolved organic carbon, reducing bacterial densities. However, the diminished carbon and phosphorus resulting from soil warming also increased light availability that ultimately stimulated cladoceran zooplankton relative to ambient‐temperature leaves. Our results suggest that changes in temperature can alter ecological subsidies in unanticipated ways, and suggest that accurately predicting the potential consequences of climate change will require conducting research across ecosystem boundaries.

Chemical composition of vegetation along urbanisation gradients in two European cities

Accumulation of particulate matter (PM) and metals on leaves of three deciduous woody species was studied along urbanisation gradients in Stavanger and Warsaw. Differences between rural and urban sites explained most of the observed variation in leaf chemistry, followed by differences between regions. Highest leaf accumulation of elements was found in Warsaw, but also composition of elements differed between the cities. Overall, species showed similar patterns of element accumulation, but differed in accumulation of specific elements. These differences could in part be explained by differences in epicuticular waxes and PM accumulation. Expected source of elements and their chemical characteristics did not explain the observed accumulation patterns. A better differentiation between elements taken up from soil and air would be required for his. Species specific accumulation of elements has to be taken into consideration using leaf samples for biomonitoring.

Phenological variation in leaf chemistry of Nothofagus macrocarpa regarding Ormiscodes sp. growth and survival

La fenologia de la planta hospedera puede afectar a insectos defoliadores a traves de cambios en rasgos fisico-quimicos de las hojas. Roble de Santiago (Nothofagus macrocarpa) forma bosques relictos al extremo norte de la distribucion del genero en America, constituyendose en un ecosistema unico. Ciclos poblacionales eruptivos de especies del genero Ormiscodes (Lepidoptera: Saturniidae) tienen el potencial de defoliar vastas areas de Nothofagus spp., pero los mecanismos reguladores de su dinamica poblacional han sido poco estudiados. Se estudio una especie univoltina del genero Ormiscodes que consume follaje de N. macrocarpa desde mediados de octubre a noviembre. Se evaluo la sobrevivencia y crecimiento larval al momento de su emergencia natural y al retrasarla 21, 30, y 79 dias. Se cuantifico la concentracion foliar de nitrogeno total y fenoles totales. Como resultado, se determino que la sobrevivencia y crecimiento larval decrecieron a medida que su emergencia se retraso. El crecimiento larval fue mas sensible a cambios en la calidad del follaje en comparacion con su sobrevivencia. La disminucion del crecimiento y sobrevivencia de los insectos se correlaciono significativamente con la concentracion de nitrogeno foliar, pero no con la concentracion de fenoles totales. Estos resultados indican que el momento de emergencia de las larvas, y la concentracion foliar de nitrogeno total son importantes factores que podrian tener un rol en la dinamica poblacional de este insecto

A chemical perspective on the evolution of variation in Eucalyptus globulus

It is becoming increasingly easy to generate genotypic data but much harder to gather an equivalent amount of phenotypic information, particularly for chemical traits. In this study of Eucalyptus globulus ssp. globulus, we measured about 60 chemical leaf traits of trees growing in a common garden to address the following questions: (1) how much variation is there between geographic regions, populations within regions and within populations? (2) How do chemical traits vary over the species’ geographic range? (3) If so, does E. globulus ssp. globulus exhibit distinct chemotypes – plants that are morphologically similar but which differ chemically? (4) Are the affinities between E. globulus ssp. globulus and closely related subspecies apparent in the chemical variation? Variation among trees within populations contributed most variation in leaf chemistry followed by variation between geographic regions. For many traits, variation among populations within proposed races and variation among proposed races within geographic regions explained little of the total variation. There was a cline in the concentration of secondary chemicals with the lowest concentrations in Tasmanian populations and the highest in those from eastern Victoria, with intermediate concentrations in populations from Bass Strait Islands. We identified three chemotypes, characterised by specific terpenes and formylated phloroglucinol compounds. The frequency of occurrence of these chemotypes showed a geographic pattern also, with “chemotype 1” predominating in Tasmania, while “chemotypes 2 and 3” occurred at highest frequency in eastern Victoria. We suggest that the chemotypes reflect introgression between E. globulus ssp. globulus and the three closely related subspecies – E. globulus ssp. bicostata, E. globulus ssp. maidenii and E. globulus ssp. pseudoglobulus. Although the formation of land-bridges with fluctuating sea levels has no doubt shaped the evolutionary history of all four subspecies, we propose that the migratory swift parrot (Lathamus discolor), an important pollinator and a species closely associated with E. globulus, has helped shape the evolution of the four tree subspecies.

Foliar nitrogen and phosphorus resorption and decomposition in the nitrogen-fixing tree Lysiloma microphyllum in primary and secondary seasonally tropical dry forests in Mexico

The tree Lysiloma microphyllum (Fabaceae) dominates in the seasonally tropical dry forests of central Mexico. In this study foliar N and P concentrations (on leaf mass basis), foliar N and P resorption efficiency and proficiency, as well as the decomposition of senescent leaves of L. microphyllum were studied in primary and in regenerating, secondary seasonally tropical dry forests. Our study included an area of early successional forest (10 y old), with phosphorus-poor soils and comparatively abundant nitrogen, an area of late-successional forest (∼60 y old), in which soil P and N were comparatively abundant, and an area of primary forest, in which soil P was comparatively abundant and N was less abundant than in the secondary counterparts. N and P concentrations in mature leaves varied across forests, reflecting soil nutrient availability. Nitrogen concentration in senescent leaves did not change among sites, which led to very different patterns of N resorption. In contrast, P concentration in senescent leaves was lower in the early than in late-successional and primary forests, which resulted in similar patterns of resorption. Leaf decomposition increased from 70% mass loss in the first year in the early successional to ∼80% in the same period in late-successional and primary forests. The element loss during decomposition change across forests in the following order: for N, early successional = late-successional > primary forest, and for P, primary forest > late-successional > early successional forest. Overall, the pattern of variation in leaf chemistry and nutrient release on the forest floor among sites is consistent with soil nutrient availability along this sequence, while decomposition rate may be related with the P concentration in senescent leaves.

Leaf ontogeny influences leaf phenolics and the efficacy of genetically expressed Bacillus thuringiensis cry1A(a) d-endotoxin in hybrid poplar against gypsy moth.

We tested the hypothesis that ontogenetic variation in leaf chemistry could affect the efficacy of genetically expressed Bacillus thuringiensis cry1A(a) d-endotoxin, and thus provide spatial variation in (1) foliage protection and (2) selective pressures that could delay the resistance of folivores. Our model consisted of clonal hybrid Populus plants (NC5339). Consumption of foliage and relative growth rates of gypsy moth, Lymantria dispar (L.) increased, and phenolic glycoside concentrations decreased, as leaves from transformed plants containing the cry1A(a) d-endotoxin and nontransformed plants matured from leaf plastochron index (LPI) 1-6. Feeding and growth rates were negatively correlated with phenolic glycosides in both transformed and nontransformed foliage. The presence of the B. thuringiensis d-endotoxin was at most, additive to the effect of the phenolic glycosides. Feeding and growth rates were positively correlated with condensed tannins in transformed foliage, but there was no relationship with condensed tannins in nontransformed foliage. The results indicate that the presence of foliar allelochemicals of poplar can enhance the effectiveness of genetically expressed B. thuringiensis d-endotoxin against gypsy moth larvae. However, the spatial variation in gypsy moth performance in response to the combination of foliar allelochemicals and d-endotoxin was not greater than the effect of ontogenetic variation in foliar allelochemicals alone. These results suggest that for this important pest, foliage protection may be obtained without genetically engineered defenses, and instead, by relying on ontogenetic and clonal variation in allelochemicals. The benefits of combining novel resistance mechanisms with natural ones will depend upon the specific folivore's adaptation to natural resistance mechanisms, such as allelochemicals. Moreover, some of the greatest benefits from transgenic resistance may arise from the need to protect trees from multiple pests, some of which may not be deterred by, or may even prefer, allelochemicals that confer protection from a few species.

Nursery conditions affect seedling chemistry, morphology and herbivore preferences for Eucalyptus nitens

We examined intraspecific variation in leaf chemistry and seedling morphology of Eucalyptus nitens grown under three different nursery conditions, and compared these with preferences of two species of marsupial herbivore: red-bellied pademelons, Thylogale billardierii, and common brushtail possums, Trichosurus vulpecula. Chemical and morphological variation with nursery treatment reflected variation in light and nitrogen limitation to seedlings. This variation is discussed in terms of the carbon-nutrient balance hypothesis. The most resource-limited seedlings had the lowest biomass and nitrogen level, but highest specific leaf area, lignin:leaf area ratio, gallotannin:nitrogen ratio and sideroxylonal levels. Both pademelons and possums preferred these resource-limited seedlings least. Possums showed greater discrimination than pademelons for the two other nursery treatments, indicating differences in the two species’ responses to seedling characteristics. Results show that environmental variation in nursery growing conditions has potential in reducing palatability to seedlings, and hence browsing damage, in plantation forestry.

Effect of nitrogen and water treatment on leaf chemistry in horsenettle (Solanum carolinense), and relationship to herbivory by flea beetles (Epitrix spp.) and tobacco hornworm (Manduca sexta).

We studied the interaction between plants (horsenettle; Solanum carolinense) and herbivorous insects (flea beetles; Epitrix spp., and tobacco hornworm; Manduca sexta) by focusing on three questions: (1) Does variation in nitrogen availability affect leaf chemistry as predicted by the carbon-nutrient balance (CNB) hypothesis? (2) Does variation in plant treatment and leaf chemistry affect insect feeding? (3) Is there an interaction between the insect herbivores that is mediated by variation in leaf chemistry? For three successive years (1998-2001), we grew a set of clones of 10 maternal plants under two nitrogen treatments and two water treatments. For each plant in the summer of 2000, we assayed herbivory by hornworms in both indoor (detached leaf) and outdoor (attached leaf) assays, as well as ambient flea beetle damage. Estimates of leaf material consumed were made via analysis of digitized leaf images. We also assayed leaves for total protein, phenolic, and glycoalkaloid content, and for trypsin inhibitor, polyphenol oxidase, and peroxidase activity. Despite strong effects of nitrogen treatment on growth and reproduction, only total protein responded as predicted by CNB. Leaf phenolic levels were increased by nitrogen treatment, polyphenol oxidase activity was decreased, and other leaf parameters were unaffected. Neither hornworm nor flea beetle herbivory could be related to plant treatment or genotype or to variation in any of the six leaf chemical parameters. A negative relationship between flea beetle and hornworm herbivory was found, but was not apparently mediated by any of the measured leaf chemicals. Because leaf resistance was maintained in low nitrogen plants at the apparent expense of growth and reproduction, our results support the concept of a fitness cost of defense, as predicted by the optimal defense hypothesis.

The effects of leaf quality on herbivore performance and attack from natural enemies.

A diverse array of sublethal plant secondary compounds are commonly found in the foliage of temperate deciduous trees. These traits are thought to defend a plant in two principal ways, either directly by reducing insect oviposition, feeding, or biomass gain, or indirectly, through digestive inhibition. Such inhibition is hypothesized to slow the rate of herbivore development, thereby increasing their susceptibility to natural enemies (the slow-growth-high-mortality hypothesis). To clarify the defensive role of these compounds, field experiments were conducted to examine the relationships among oak leaf quality, herbivore family, and three herbivore performance measures: survivorship, development time, and pupal mass, for a bivoltine leaf-tying caterpillar, Psilocorsis quercicella (Lepidoptera: Oecophoridae). Two experiments, one for each generation of the insect, were conducted to examine the effects of intraspecific variation in leaf chemistry of its host, white oak trees (Quercus alba). In each experiment, full-sib neonate larvae were placed in experimental leaf ties on high- versus low-quality trees and allowed to feed for 2weeks under field conditions. To determine the effect of the third trophic level, a portion of each family in each leaf-quality treatment was bagged to prevent attack from natural enemies. This treatment also allowed us to test a prediction of the slow-growth-high-mortality hypothesis, i.e., that development time, as measured for full sibs in the bagged treatment, should be positively correlated with mortality of their full sibs exposed to natural enemies. Low leaf quality significantly reduced survivorship of the caterpillars in the first generation but not the second. The third trophic level decreased survivorship in both generations. Larval development time was not affected by leaf quality in either generation, but varied significantly among insect families in both generations. In turn, larvae from slower-developing families did not suffer increased predation and parasitism, as predicted by the slow-growth-high mortality hypothesis. In contrast to development time, pupal mass showed a greater response to intraspecific variation in leaf quality, although the effect was only significant in generation1. Concentrations of both total phenolics and hydrolyzable tannins in Q. alba foliage appear to be important negative predictors of pupal mass in P. quercicella. In marked contrast to development time, no main family effect was found for pupal mass in either experiment; however, significant family×environment interactions were found for the effects of the bagging treatment (generation1) and the leaf-quality treatment (generation2). Overall, the first trophic level had a greater influence on pupal mass (a fecundity correlate), while larval development time was determined more by the insect's family (genotype+maternal environment). The third trophic level was a consistently strong source of mortality in both experiments, but as a whole did not respond to familial differences in development time. Thus, from the perspective of P. quercicella, plant quality appears to serve as a defense more through its direct effect on herbivore survivorship and fecundity than through an indirect effect on predation via changes in development time.

An analysis of the pattern of feeding in the Costa Rican weevil, Exopthalmus jekelianus, on the tropical tree, Cedrela odorata

AbstractFeeding patterns were recorded and analysed for adult female weevils, Exopthalmus jekelianus (White) (Coleoptera: Curculionidae), feeding on Central American mahogany, Cedrela odorata L., in the field in Costa Rica. The study forms part of an investigation into the relationship between feeding patterns and the fine‐scale variation in leaf chemistry occurring within the host plant. The weevils’ feeding patterns were the simplest in temporal structure of any reported to date for an insect herbivore. Weevils spent an average of only 3% of their time feeding during the 10‐h observation periods. Meals lasted an average of 2.8 min and occurred at a mean intermeal interval of 84 min. The feeding patterns gave the appearance of a short‐term rhythm underlying the onset of feeding (as has been found in locusts and caterpillars), although there were insufficient meals taken by individuals over the 10‐h period to test this suggestion. Meals were notable in apparently lacking intrameal pauses and also commencing without preliminary sampling behaviours, such as palpating or biting. Whether the combination of short, infrequent meals, ingested without pauses and not preceded by sampling behaviour, represents an adaptation reducing apparency to natural enemies, or else simply reflects low nutritional needs, is discussed. Correlations between meal durations and following and preceding intermeal intervals suggested that variation in intermeal intervals stemmed largely from variation in meal duration, not vice versa, with variation in meal duration resulting from an external influence such as leaf nutritional and/or allelo‐chemistry. The latter suggestion is currently being tested.

Seasonal variation in leaf chemistry of the coast live oak Quercus agrifolia and implications for the California oak moth Phryganidia californica.

The perennial foliage of the California coast live oak (Quercus agrifolia Nee) permits herbivores to feed on this oak species throughout the year. Patterns of herbivory for a two-year period on Q. agrifolia were observed in relation to seasonal and age-related changes in the nutritional and defensive characteristics of leaves. Nitrogen and phosphorus contents were higher in new leaves compared to mature foliage. Structural compounds (e.g., cellulose) in leaves rapidly increased with age. Concentrations of tatal phenolics (Folin-Denis) and astringency were higher in new foliage, and concentrations of condensed tannins gradually increased as the leaves matured. Peaks of herbivore damage were observed in June and in September-October, and were caused by outbreaks of the California oak moth (Phryganidia californica). P. californica, a bivoltine oak specialist, exhibited feeding preferences in June for old leaves over emerging leaves, and showed no preferences for leaf classes in September. These results suggest that P. californica is adapted to survive on nutritionally poor foliage and to circumvent "quantitative defenses" such as condensed tannins.

Influence on Larval Growth of the Eastern Black Swallowtail Butterfly Papilio polyxenes (Lepidoptera: Papilionidae) of Seasonal Changes in Nutritional Parameters of Umbelliferae Species

Larvae of the eastern black swallowtail butterfly, Papilio polyxenes asterias (Stoll) were fed a variety of plants of the family Umbelliferae and were analyzed for larval growth, feeding efficiencies and nitrogen budgets as a function of food plant age and nutrient content. Several food plants (Hydrocotyle americana, Sanicula gregaria, Osmorhiza claytoni, Osmorhiza longistylis and Torilis japonica of the Umbelliferae; and Xanthoxylum americanum of the Rutaceae) proved to be unacceptable to the larvae in no-choice situations for a variety of reasons. Among these, we found escape in space, escape in time, and a newly discovered physical trichome defense of Torilisjaponica to be involved. The food plants suceessfully fed upon (Daucus carota, Pastinaca sativa, Conium maculatum, Cicuta bulbifera, Angelica atropurpurea, Thaspium trifoliatum, Cryptotaenia canadensis, Chaerophyllum procumbens, and Heracleum maximum) showed significant seasonal differences in their nutritional value as food for Papilio polyxenes larvae. An analysis of both the nutrient composition of the plant and the insect utilization indices upon each food plant showed no consistent seasonal trends in plant quality. Instead, the leaves of each plant species varied in their nutritional value (nitrogen and water contents) according to its own particular phenology. Larvae fed on plants which had low leafwater contents (less than 759%) were unable to increase their assimilation or nitrogen utilization efficiencies enough to maximize growth rates. Larvae on certain low-water plants achieved higher consumption rates, but this could not adequately compensate for the low efficiency of assimilation and utilization efficiency of plant biomass and nitrogen, so growth rates remained low. INTRODUCTION Phylogenetic constraints upon the adaptations of herbivores to their food plants remain poorly understood (Diehl and Bush, 1984; Futuyma and Peterson, 1985). Local specialization upon preferred food plants appears to be the rule rather than the exception in Lepidoptera (see reviews in Wiklund, 1975; Fox and Morrow, 1981; Scriber, 1983, 1986a, 1986b; Scriber et al., 1986). The North American Umbellifer-feeding swallowtail butterflies are no exception, and local oviposition favorites are known to exist (Scudder, 1889; Heitzman, 1973; Emmel and Shields, 1978; Scriber and Finke, 1978; Blau, 1980; Shapiro and Masuda, 1980; Sims, 1980a, 1980b; Wiklund, 1981; Goeden and Ricker, 1982; Jackson, 1982; Opler and Krizek, 1984; Feeny et al., 1985). Leaf nutritional quality can be a driving force in the growth rate of insects, and seasonal variation in leaf chemistry can have dramatic effects upon insect preference/ performance (Feeny, 1970; Scriber, 1977; Scriber and Slansky, 1981). This study was designed to delineate the suitability of the species of Umbelliferae over the course of the growing season in Greene County, Ohio, to the umbellifer-feeding specialist, Papilio polyxenes. Specifically, we determined the effects of seasonal variation in leaf quality upon the consumption rates and efficiencies of leaf tissue processing for larval growth 'Current address: 1028 Labonne Parkway, Valley Park, Missouri 63088. 2Current address: Department of Entomology, Michigan State University, East Lansing 48824. 3Send reprint requests to J. M. Scriber.