Frequency dependent selection: I. Rare male phenomenon in D. subobscura dependent on the proportion of Amy genotypes and substrate composition

Analysis of the rare male mating advantage in D. subobscura, as a type of frequency dependent selection on maltose and starch media, was done by applying different statistical approaches (χ², cross-product ratio, variance and regression analysis). They reveal that mating occurs at random when proportions of prospective mates are equal, and that mating success of the males homozygous for Amy-locus genotypes (S/S and F/F) depends on their proportion. Regression analysis showed that the F/F males are sexually more active (have higher vigour) than S/S males. Rare male effect is one-sided and appears in F/F males that partake in more heterogamic matings. Comparison of the number of observed and expected homo- and heterogamic matings shows that homogamic matings are more frequent. Multifactorial analysis of variance shows that the number of matings are different for nine pairs of lines and four possible mating types (S_fS_m, S_fF_m, F_fS_m, F_fF_m). The rare male phenomenon is not dependent on different food composition, but is associated with variations in individual genotypes.     

Paternal‐specific S‐allele transmission in sweet cherry (Prunus avium L.): the potential for sexual selection

Homomorphic self‐incompatibility is a well‐studied example of a physiological process that is thought to increase population diversity and reduce the expression of inbreeding depression. Whereas theoretical models predict the presence of a large number of S‐haplotypes with equal frequencies at equilibrium, unequal allele frequencies have been repeatedly reported and attributed to sampling effects, population structure, demographic perturbation, sheltered deleterious mutations or selection pressure on linked genes. However, it is unclear to what extent unequal segregations are the results of gametophytic or sexual selection. Although these two forces are difficult to disentangle, testing S‐alleles in the offspring of controlled crosses provides an opportunity to separate these two phenomena. In this work, segregation and transmission of S‐alleles have been characterized in progenies of mixed donors and fully compatible pollinations under field conditions in Prunus avium. Seed set patterns and pollen performance have also been characterized. The results reveal paternal‐specific distorted transmission of S‐alleles in most of the crosses. Interestingly, S‐allele segregation within any given paternal or maternal S‐locus was random. Observations on pollen germination, pollen tube growth rate, pollen tube cohort size, seed set dynamics and transmission patterns strongly suggest post‐pollination, prezygotic sexual selection, with male–male competition as the most likely mechanism. According to these results, post‐pollination sexual selection takes precedence over frequency‐dependent selection in explaining unequal S‐haplotype frequencies.     

An attempt to measure selection coefficients affecting the alcohol dehydrogenase polymorphism in Drosophila melanogaster populations maintained on ethanol media

Eleven populations polymorphic for Adh F and S alleles were established from wild-caught D. melanogaster. Seven of the populations were founded from mass collections each of several hundred flies and the other four were isofemale lines. Subcultures from all eleven were maintained on one of four different types of medium—standard laboratory medium, standard media supplemented to 3% or 9% (v/v) ethanol, and simulated wine seepages. These subcultures were rescored for F and S gene frequencies after 10, 20 and 30 generations. Maximum-likelihood methods were then used to estimate selection coefficients among the Adh genotypes and analyses of deviance were carried out to test the coefficients against hypotheses of neutrality and various modes of selection. No significant selective changes in Adh frequencies were observed in the seven mass cultures on any of the four different media types. However, highly significant selective effects were detected in the four isofemale lines; these effects were homogeneous across the four lines and the four media types and the underlying fitness set was estimated as 1.00:1.04:1.09 for S/S:F/S:F/F.     

Density‐dependent natural selection in Drosophila: correlations between feeding rate,development time and viability

We have previously hypothesized that density‐dependent natural selection is responsible for a genetic polymorphism in crowded cultures of Drosophila. This genetic polymorphism entails two alternative phenotypes for dealing with crowded Drosophila larval cultures. The first phenotype is associated with rapid development, fast larval feeding rates but reduced absolute viability, especially in the presence of nitrogenous wastes like ammonia. The second phenotype has associated with it the opposite set of traits, slow development, slow feeding rates and higher viability. We suggested that these traits are associated due to genetic correlations and that an important selective agent in crowded larval cultures was high levels of ammonia. To test this hypothesis we have examined viability and larval feeding rates in populations kept at low larval densities but selected directly for (i) rapid egg‐to‐adult development, (ii) tolerance of ammonia in the larval environment and (iii) tolerance of urea in the larval environment. Consistent with our hypothesis we found that (i) larvae selected for rapid development exhibited increased feeding rates, and decreased viability in food laced with ammonia or urea relative to controls, and (ii) larvae selected to tolerate either ammonia or urea in their larval environment show reduced feeding rates but elevated survival in toxin‐laced food relative to controls. It would appear that development time and larval feeding rate are important characters for larvae adapting to crowded cultures. The correlated fitness effects of these characters provide important insights into the nature of density‐dependent natural selection.     

Adaptive significance of amylase polymorphism in Drosophila: XIV. Effect of substrates with different carbohydrate composition on some life-history traits of Drosophila subobscura

The Amy-locus polymorphism of Drosophila subobscura is used as a model-system for an experimental population genetic study of adaptive significance of alpha-amylase activity on substrates of different carbohydrate compositions. So far, fitness components have not commonly been included in ecological-genetic studies of alpha-amylase polymorphism in this species. In the present paper fitness components are analyzed in relation to different amylase activities in D. subobscura individuals homozygous for "slow" and "fast" Amy allele, associated with substrates of different carbohydrate compositions. The results indicate a significant effect of substrate carbohydrate composition on fitness components of the genotypes homozygous for S or F Amy allele in D. subobscura through their enzyme activity.     

Genome‐wide selection components analysis in a fish with male pregnancy

A major goal of evolutionary biology is to identify the genome‐level targets of natural and sexual selection. With the advent of next‐generation sequencing, whole‐genome selection components analysis provides a promising avenue in the search for loci affected by selection in nature. Here, we implement a genome‐wide selection components analysis in the sex role reversed Gulf pipefish, Syngnathus scovelli. Our approach involves a double‐digest restriction‐site associated DNA sequencing (ddRAD‐seq) technique, applied to adult females, nonpregnant males, pregnant males, and their offspring. An F_ST comparison of allele frequencies among these groups reveals 47 genomic regions putatively experiencing sexual selection, as well as 468 regions showing a signature of differential viability selection between males and females. A complementary likelihood ratio test identifies similar patterns in the data as the F_ST analysis. Sexual selection and viability selection both tend to favor the rare alleles in the population. Ultimately, we conclude that genome‐wide selection components analysis can be a useful tool to complement other approaches in the effort to pinpoint genome‐level targets of selection in the wild.     

High‐throughput genomics in sorghum: from whole‐genome resequencing to a SNP screening array

With its small, diploid and completely sequenced genome, sorghum (Sorghum bicolor L. Moench) is highly amenable to genomics‐based breeding approaches. Here, we describe the development and testing of a robust single‐nucleotide polymorphism (SNP) array platform that enables polymorphism screening for genome‐wide and trait‐linked polymorphisms in genetically diverse S. bicolor populations. Whole‐genome sequences with 6× to 12× coverage from five genetically diverse S. bicolor genotypes, including three sweet sorghums and two grain sorghums, were aligned to the sorghum reference genome. From over 1 million high‐quality SNPs, we selected 2124 Infinium Type II SNPs that were informative in all six source genomes, gave an optimal Assay Design Tool (ADT) score, had allele frequencies of 50% in the six genotypes and were evenly spaced throughout the S. bicolor genome. Furthermore, by phenotype‐based pool sequencing, we selected an additional 876 SNPs with a phenotypic association to early‐stage chilling tolerance, a key trait for European sorghum breeding. The 3000 attempted bead types were used to populate half of a dual‐species Illumina iSelect SNP array. The array was tested using 564 Sorghum spp. genotypes, including offspring from four unrelated recombinant inbred line (RIL) and F₂ populations and a genetic diversity collection. A high call rate of over 80% enabled validation of 2620 robust and polymorphic sorghum SNPs, underlining the efficiency of the array development scheme for whole‐genome SNP selection and screening, with diverse applications including genetic mapping, genome‐wide association studies and genomic selection.     

DOES MATE LIMITATION IN SELF‐INCOMPATIBLE SPECIES PROMOTE THE EVOLUTION OF SELFING? THE CASE OF LEAVENWORTHIA ALABAMICA

Genetic diversity at the S‐locus controlling self‐incompatibility (SI) is often high because of negative frequency‐dependent selection. In species with highly patchy spatial distributions, genetic drift can overwhelm balancing selection and cause stochastic loss of S‐alleles. Natural selection may favor the breakdown of SI in populations with few S‐alleles because low S‐allele diversity constrains the seed production of self‐incompatible plants. We estimated S‐allele diversity, effective population sizes, and migration rates in Leavenworthia alabamica, a self‐incompatible mustard species restricted to discrete habitat patches in rocky glades. Patterns of polymorphism were investigated at the S‐locus and 15 neutral microsatellites in three large and three small populations with 100‐fold variation in glade size. Populations on larger glades maintained more S‐alleles, but all populations were estimated to harbor at least 20 S‐alleles, and mate availabilities typically exceeded 0.80, which is consistent with little mate limitation in nature. Estimates of the effective size (N_e) in each population ranged from 600 to 1600, and estimated rates of migration (m) ranged from 3 × 10⁻⁴ to nearly 1 × 10⁻³. According to theoretical models, there is limited opportunity for genetic drift to reduce S‐allele diversity in populations with these attributes. Although pollinators or resources limit seed production in small glades, limited S‐allele diversity does not appear to be a factor promoting the incipient breakdown of SI in populations of this species that were studied.     

Adaptive significance of amylase polymorphism in <Emphasis Type="Italic">Drosophila</Emphasis>: Effect of substrates with different carbohydrate composition on some life-history traits of <Emphasis Type="Italic">Drosophila subobscura</Emphasis>

The Amy locus polymorphism of Drosophila subobscura is used as a model system for an experimental population genetic study of adaptive significance of α-amylase activity on substrates of different carbohydrate compositions. So far, fitness components have not commonly been included in ecological-genetic studies of α-amylase polymorphism in this species. In the present paper, fitness components are analyzed in relation to different amylase activities in D. subobscura individuals homozygous for the “slow” and the “fast” Amy allele, associated with substrates of different carbohydrate compositions. The results indicate a significant effect of substrate carbohydrate composition on fitness components of the genotypes homozygous for S or F Amy allele in D. subobscura through their enzyme activity. The text was submitted by the authors in English.     

Effect of Low‐ and High‐Calcium Dairy‐Based Diets on Macronutrient Oxidation in Humans

Objective: Higher calcium and dairy intakes may be associated with lower body weights, but a mechanism in humans has yet to be elucidated. We compared the effects of a dairy‐based high‐calcium diet and a low‐calcium diet on macronutrient oxidation. Research Methods and Procedures: Subjects (10 men and nine women) consumed a low‐dairy (LD, ～one serving per day, ～500 mg Ca²⁺/d) or high‐dairy (HD, ～three to four servings per day, ～1400 mg Ca²⁺/d) energy balance diet for 1 week. Each diet condition was performed twice. On the 7th day, subjects were studied in a room calorimeter under one of four study conditions, applied in a randomized crossover design. Within each diet condition, subjects were studied under conditions of energy balance and acute energy deficit. The deficit (?600 kcal/d) was induced only for the 24 hours that subjects resided in the room and was achieved by a combination of caloric restriction and exercise. Results: Under energy balance conditions, there was no effect of diet treatment on respiratory quotient or 24‐hour macronutrient oxidation. Under energy deficit conditions, 24‐hour fat oxidation was significantly increased on the HD diet (HD with deficit = 136 ± 13 g/d, LD with deficit = 106 ± 7 g/d, p = 0.02). Discussion: Consumption of a dairy‐based high‐calcium diet increased 24‐hour fat oxidation under conditions of acute energy deficit. We hypothesize that these effects are due to an increased fat oxidation during exercise.     

Estimations of linkage disequilibrium,effective population size and ROH‐based inbreeding coefficients in Spanish Churra sheep using imputed high‐density SNP genotypes

In this study, the availability of the Ovine HD SNP BeadChip (HD‐chip) and the development of an imputation strategy provided an opportunity to further investigate the extent of linkage disequilibrium (LD) at short distances in the genome of the Spanish Churra dairy sheep breed. A population of 1686 animals, including 16 rams and their half‐sib daughters, previously genotyped for the 50K‐chip, was imputed to the HD‐chip density based on a reference population of 335 individuals. After assessing the imputation accuracy for beagle v4.0 (0.922) and fimpute v2.2 (0.921) using a cross‐validation approach, the imputed HD‐chip genotypes obtained with beagle were used to update the estimates of LD and effective population size for the studied population. The imputed genotypes were also used to assess the degree of homozygosity by calculating runs of homozygosity and to obtain genomic‐based inbreeding coefficients. The updated LD estimations provided evidence that the extent of LD in Churra sheep is even shorter than that reported based on the 50K‐chip and is one of the shortest extents compared with other sheep breeds. Through different comparisons we have also assessed the impact of imputation on LD and effective population size estimates. The inbreeding coefficient, considering the total length of the run of homozygosity, showed an average estimate (0.0404) lower than the critical level. Overall, the improved accuracy of the updated LD estimates suggests that the HD‐chip, combined with an imputation strategy, offers a powerful tool that will increase the opportunities to identify genuine marker‐phenotype associations and to successfully implement genomic selection in Churra sheep.     

The effect of frequency‐dependent selection on resistance and tolerance to herbivory

Negative frequency‐dependent selection (FDS), where rare genotypes are favoured by selection, is commonly invoked as a mechanism explaining the maintenance of genetic variation in plant defences. However, empirical tests of FDS in plant–herbivore interactions are lacking. We evaluated whether the oviposition preference of the specialist herbivore Lema daturaphila is a mechanism through which this herbivore can exert FDS on its host plant Datura stramonium. The frequency of contrasting resistance–tolerance strategies was manipulated within experimental plots, and the plants were exposed to a similar initial density of their natural herbivore. Herbivore oviposition preference and final density, as well as plant damage and seed production, were estimated. Overall, we found that the high‐resistant–low‐tolerant genotypes produced four times more seeds when common than when rare, whereas the high‐tolerant–low‐resistant genotypes achieved twice its fitness when rare than when common. This pattern was the result of differential oviposition preferences. In addition, when the high‐resistant–low‐tolerant genotypes were common, there was a three‐fold decreased in herbivore final density which led to a decrease in damage level by 10%. Thus, in our experiment positive FDS seems to favour resistance over tolerance. We discuss how this result would change if the extent of herbivore local adaptation and damage modify the pattern of positive FDS acting on resistance and the optimal allocation to tolerance.     

Comparison of Fat Storage between Fischer 344 and Obesity‐Resistant Lou/C Rats Fed Different Diets**

Objective: We aimed to characterize further the Lou/C (LOU) and Fischer 344 (F344) rat strains for nutritional traits to validate their use as contrasting strains for molecular genetic studies. Research Methods and Procedures: Five batches of LOU and F344 rats were used to measure caloric intake, weight gain, and body composition when fed a chow diet, a self‐selection diet (together with the study of preferences for macronutrients), hypercaloric diets, and a chow diet in a cold environment. Results: Despite a higher caloric intake when fed a chow diet, LOU rats showed a lower weight gain, final body weight, and percentage of fat tissue, together with a higher percentage of carcass weight, than F344 rats. When fed a self‐selection diet, LOU males ingested less protein and more fat than F344 males, and the reverse was observed for females. In this condition, feed efficiency was reduced in LOU but increased in F344 rats compared with the chow diet. Diet‐induced obesity was observed in F344 rats but not in LOU rats fed hypercaloric diets. In a cold environment, both LOU and F344 rats displayed an increased percentage of brown adipose tissue compared with control groups, together with a higher caloric intake. Discussion: The study shows robust nutritional differences between the LOU rat, a lean strain with a low feed efficiency and resistant to diet‐induced obesity, and the contrasting F344 rat strain. It also shows the interest in these strains for studying the genetic components of resistance to obesity.     

Nonlinear frequency‐dependent selection promotes long‐term coexistence between bacteria species

Negative frequency‐dependent selection (NFDS) is an important mechanism for species coexistence and for the maintenance of genetic polymorphism. Long‐term coexistence nevertheless requires NFDS interactions to be resilient to further evolution of the interacting species or genotypes. For closely related genotypes, NFDS interactions have been shown to be preserved through successive rounds of evolution in coexisting lineages. On the contrary, the evolution of NFDS interactions between distantly related species has received less attention. Here, we tracked the co‐evolution of Escherichia coli and Citrobacter freundii that initially differ in their ecological characteristics. We showed that these two bacterial species engaged in an NFDS interaction particularly resilient to further evolution: despite a very strong asymmetric rate of adaptation, their coexistence was maintained owing to an NFDS pattern where fitness increases steeply as the frequency decreases towards zero. Using a model, we showed how and why such NFDS pattern can emerge. These findings provide a robust explanation for the long‐term maintenance of species at very low frequencies.     

Life‐history evolution under fluctuating density‐dependent selection and the adaptive alignment of pace‐of‐life syndromes

We present a novel perspective on life‐history evolution that combines recent theoretical advances in fluctuating density‐dependent selection with the notion of pace‐of‐life syndromes (POLSs) in behavioural ecology. These ideas posit phenotypic co‐variation in life‐history, physiological, morphological and behavioural traits as a continuum from the highly fecund, short‐lived, bold, aggressive and highly dispersive ‘fast’ types at one end of the POLS to the less fecund, long‐lived, cautious, shy, plastic and socially responsive ‘slow’ types at the other. We propose that such variation in life histories and the associated individual differences in behaviour can be explained through their eco‐evolutionary dynamics with population density – a single and ubiquitous selective factor that is present in all biological systems. Contrasting regimes of environmental stochasticity are expected to affect population density in time and space and create differing patterns of fluctuating density‐dependent selection, which generates variation in fast versus slow life histories within and among populations. We therefore predict that a major axis of phenotypic co‐variation in life‐history, physiological, morphological and behavioural traits (i.e. the POLS) should align with these stochastic fluctuations in the multivariate fitness landscape created by variation in density‐dependent selection. Phenotypic plasticity and/or genetic (co‐)variation oriented along this major POLS axis are thus expected to facilitate rapid and adaptively integrated changes in various aspects of life histories within and among populations and/or species. The fluctuating density‐dependent selection POLS framework presented here therefore provides a series of clear testable predictions, the investigation of which should further our fundamental understanding of life‐history evolution and thus our ability to predict natural population dynamics.     

SINGLE‐GENE SPECIATION WITH PLEIOTROPY: EFFECTS OF ALLELE DOMINANCE,POPULATION SIZE,AND DELAYED INHERITANCE

Single‐gene speciation is considered to be unlikely, but an excellent example is found in land snails, in which a gene for left‐right reversal has given rise to new species multiple times. This reversal might be facilitated by their small population sizes and maternal effect (i.e., “delayed inheritance,” in which an individual's phenotype is determined by the genotype of its mother). Recent evidence suggests that a pleiotropic effect of the speciation gene on antipredator survival may also promote speciation. Here we theoretically demonstrate that, without a pleiotropic effect, in small populations the fixation probability of a recessive mutant is higher than a dominant mutant, but they are identical for large populations and sufficiently weak selection. With a pleiotropic effect that increases mutant viability, a dominant mutant has a higher fixation probability if the strength of viability selection is sufficiently greater than that of reproductive incompatibility, whereas a recessive mutant has a higher fixation probability otherwise. Delayed inheritance increases the fixation probability of a mutant if viability selection is sufficiently weaker than reproductive incompatibility. Our results clarify the conflicting effects of viability selection and positive frequency‐dependent selection due to reproductive incompatibility and provide a new perspective to single‐gene speciation theory.     

Life‐history trade‐offs under different larval diets in Drosophila suzukii (Diptera: Drosophilidae)

Most larval drosophilids eat the microorganisms that develop in rotting fruit, a relatively protein‐rich resource. By contrast, the spotted‐wing Drosophila suzukii Matsumara (Diptera: Drosophilidae) uniquely develops in ripening fruit, a protein‐poor, carbohydrate‐rich resource. This shift in larval nutritional niche has led to D. suzukii being a significant agricultural pest in the U.S.A. and Europe. Although occupying a new niche may benefit a species by reducing competition, adaptation in host use may generate trade‐offs affecting fitness. To test the hypothesis that fitness trade‐offs will change with adaptation to novel larval diets, D. suzukii larval development on either a diet of a fresh, ripe blueberry (a natural host) or standard artificial Drosophila media (protein‐rich) is compared and the effect of diet on development time from egg to adult, adult body size and male wing spot area, and female fecundity is assessed. Larval development time differs, with larvae on the blueberry emerging as adults earlier than those on the artificial medium, although other fitness measures do not vary between the two diets. In addition, the faster development time on a blueberry does not trade off with body size as expected, although early fecundity is delayed in females that develop on blueberries. Thus, adaptation to a novel larval diet environment does not come at a cost to the ability to develop in protein‐rich resources.     

Evidence for complex selection on four‐fold degenerate sites in Drosophila melanogaster

We considered genome‐wide four‐fold degenerate sites from an African Drosophila melanogaster population and compared them to short introns. To include divergence and to polarize the data, we used its close relatives Drosophila simulans, Drosophila sechellia, Drosophila erecta and Drosophila yakuba as outgroups. In D. melanogaster, the GC content at four‐fold degenerate sites is higher than in short introns; compared to its relatives, more AT than GC is fixed. The former has been explained by codon usage bias (CUB) favouring GC; the latter by decreased intensity of directional selection or by increased mutation bias towards AT. With a biallelic equilibrium model, evidence for directional selection comes mostly from the GC‐rich ancestral base composition. Together with a slight mutation bias, it leads to an asymmetry of the unpolarized allele frequency spectrum, from which directional selection is inferred. Using a quasi‐equilibrium model and polarized spectra, however, only purifying and no directional selection is detected. Furthermore, polarized spectra are proportional to those of the presumably unselected short introns. As we have no evidence for a decrease in effective population size, relaxed CUB must be due to a reduction in the selection coefficient. Going beyond the biallelic model and considering all four bases, signs of directional selection are stronger. In contrast to short introns, complementary bases show strand specificity and allele frequency spectra depend on mutation directions. Hence, the traditional biallelic model to describe the evolution of four‐fold degenerate sites should be replaced by more complex models assuming only quasi‐equilibrium and accounting for all four bases.     

Short‐term hybridisation activates Tnt1 and Tto1 Copia retrotransposons in wild tuber‐bearing Solanum species

Interspecific hybridisation in tuber‐bearing species of Solanum is a common phenomenon and represents an important source of variability, crucial for adaptation and speciation of potato species. In this regard, the effects of interspecific hybridisation on retrotransposon families present in the genomes, and their consequent effects on generation of genetic variability in wild tuber‐bearing Solanum species, are poorly characterised. The aim of this study was to analyse the activity of retrotransposons in inter‐ and intraspecific hybrids between S. kurtzianum and S. microdontum, obtained by controlled crosses, and the effects on morphological, genetic and epigenetic variability. For genetic and epigenetic analysis, S‐SAP (sequence‐specific amplification polymorphism) and TMD (transposon methylation display) techniques were used, respectively, with specific primers for Tnt1 and Tto1 retrotransposon families (Order LTR, Superfamily Copia). The results indicate that at morphological level, interspecific hybrid genotypes differ from their parental species, whereas derived intraspecific hybrids do not. In both cases, we observed significant reductions in pollen grain viability, and a negative correlation with Tnt1 mobility. Both retrotransposons, Tto1 and Tnt1, were mobilised in the genotypes analysed, with mobility ranging from 0 to 7.8%. Furthermore, at the epigenetic level, demethylation was detected in the vicinity of Tnt1 and Tto1 in the hybrids compared with the parental genotypes. These patterns were positively correlated with the activity of the retrotransposons. The results suggest a possible mechanism through which hybridisation events generate genetic variability in tuber‐bearing species of Solanum through retrotranposon activation.     

Fitness of allozyme variants in Drosophila pseudoobscura III. Possible factors contributing to the maintenance of polymorphisms in nature

Experiments have been performed to investigate the mechanisms maintaining enzyme polymorphisms in natural populations. We have measured effects on fitness of genotypic variants at three loci, Est-5, Odh, and Mdh-2, in D. pseudoobscura. Significant differences exist among the genotypes in the rate of development from egg-to-adult; there is also indication of differences in larval survival. In a population segregating for allelic variants at all three loci, there is indication that segregation distortion at meiosis or some form of gametic selection might be involved. The relative fitnesses of alternative genotypes are reversed when either different fitness components are considered, or the genotypic frequencies are changed, or the larval density is increased. These fitness reversals may contribute to the maintenance of the polymorphisms, and may account for cyclical oscillations of allozyme frequencies observed in natural populations.Research supported by U.S. Public Health Service Research Fellowship (1F05 TWO 1991-01) to D.M. and by contract AT(04-3)34 with the U.S. Atomic Energy Commission. Adress reprint requests from Europe to D.M.; from elsewhere to F.J.A.