A Genetic Analysis of Habitat Selection in the Cactophilic Species, Drosophila mojavensis

Introduction

Katherine L. Lofdahl

Committee on Evolutionary Biology University of Chicago Chicago, Illinois 60637 USA

Past studies of habitat selection in insects have addressed various relationships among variation, heredity and natural selection; three basic factors for the process of evolution. Most research on habitat choice in insects concentrates on the selection of host plants as feeding and oviposition sites because these resources are demonstrably related to fitness (Whitham 1980). Indeed, the concept of host plant races even implies a degree of coevolution of an insect with such resources (Jaenike 1981). Demonstration that two populations or species currently using different host plant species also have different loci or alleles at a single locus that exerts con­trol over habitat choice is a frequent test for past coevolution with the respective host plants (Huettel and Bush 1972). These studies are helpful in comparing the outcome of evolution with theoretical models of population subdivision and their associated speciation patterns (Maynard Smith 1966, Bush 1969). To predict future evolutionary possibilities, however, an investigation of genetic polymorphism for habitat preference behavior within a single popula­tion is valuable. Establishing that individual genetic differences in host plant preferences exist allows the prediction of a response to any form of natural selection: directional, stabilizing or disruptive. Quantification of the amount of genetic variation in behavior, which is the trait's heritability when measured as a precentage of the phenotypic variance, reveals the rate of evolution of behavior when information on the intensity of natural selection is available also (Falconer 1981). The aims of this study are: (1) to learn whether a monophagous insect population has genetic variation for acceptance of an unfamiliar host plant species; and (2) to quantify the amount of such genetic variation in behavior that is available to produce a response to natural selection in future generations.

A search for genetic variation can usefully begin with a species that shows geographic variation in the relevant behavioral phenotype. An ecologically well known insect that exhibits geographic variation in host plant utilization is the cactus breeding species, Drosophila mojavensis Patterson and Crow. Most populations use only one or two host cactus species, but there is geographic replacement in the species of cacti used (Fellows and Heed 1972). Thus, D. mojavensis uses five to six cactus species as breeding sites when all populations are considered together. Within the center of its distribution, in the Sonoran Desert of North America, the Arizona and Sonora (Mexico) populations use organpipe cactus, Stenocereus thurberi (Engelm.) Buxbaum, with a possible seasonal shift to saguaro cactus, Carnegiea gigantea (Engelm.) Br. & R. In Baja California, D. mojavensis breeds almost exclusively in

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154 Katherine L. Lofdahl

agria cactus, Stenocereus gummosus (Engelm). Britt. & Rose, despite the presence of organ­pipe cactus there. A peripheral population in the Mojave Desert uses barrel cactus, Ferocactus acanthodes (Lemairz) Br. & R. A second isolated population, located on Santa Catalina Island, California, breeds in the only available cactus (genus Opuntia). The present research asks whether this monophagous Opuntia-breeding population possesses selectable genetic variation for use of another cactus, agria, which is highly preferred by D. mojavensis wherever the insect and agria occur together.

Experimental studies of habitat selection require a definition of this behavior in terms relevant to the ecology of the species investigated. In D. mojavensis, the behavior is designated correctly as habitat rather than as host plant selection. Drosophila mojavensis breeds only in cactus necroses in which the actual resource is the microbial community of bacteria and cactophilic yeasts that serve as food for both adults and larvae. The fresh tissue of the host cactus is not itself the direct source of most nutrients for D. mojavensis. These bacteria and yeasts also provide chemical cues that can attract D. mojavensis to the cactus necroses (Fogleman 1982). Each cactus species may produce a characteristic array of such stimuli (Vacek 1979). Drosophila mojavensis can therefore potentially discriminate among host cacti on this basis. Both because oviposition behavior is more easily quantified for an individual than is chemotaxis and because it is more closely tied to research on fitnesses of progeny reared on different cactus species (Mangan 1978), egg-laying behavior is the focus of the present assay for genetic variation in habitat selection.

The experiment tested the oviposition preference of an Opuntia-breeding population of D. mojavensis on a simulated agria cactus necrosis, representing a cactus habitat that the population never experiences in nature (Heed 1982). The method used allows the estimation of genetic variance for oviposition acceptance measured in two complementary ways: (1) in the initial acceptance of agria cactus as an oviposition substrate and; (2) as the number of eggs laid on agria given that initial acceptance has occurred. The results demonstrate that signifi­cant genetic variance exists for habitat selection measured in either way. The amount of genetic variation that is available for response to natural selection is, in both cases, equal in magnitude to 10-20% of the phenotypic variance in the behavior. The biological meaning of these estimates concerns the genetic consequences of coevolution of the Santa Catalina Island population with its natural Opuntia host. Regardless of the nature of any genetic changes that may have occurred in this D. mojavensis population after it colonized Opuntia, genetic varia­tion for acceptance of a cactus species used by other D. mojavensis populations is still present. The monophagous population studied here is therefore not genetically restricted to oviposi­tion on its usual host cactus.

Methods and Materials

Establishment of the Laboratory Stock

The population sample of D. mojavensis used was University of Arizona stock no. A826, which was collected by William B. Heed in November, 1981. This stock was about six genera­tions removed from nature when heritabilities were estimated. Originally, this stock was taken as two separate collections from Santa Catalina Island, California. Each sample was reared from one of the following substrates: Opuntia "demissa" cladodes (pads) (producing 28 eclosed adult D. mojavensis) or O. "demissa" fruits (969 adults eclosing from 20-30 fruits). The sole host cactus on Santa Catalina Island is Opuntia "demissa" Griffiths, which is thought to be a hybrid swarm. The introduced mission cactus, Opuntia ficus-indica (L.) Miller, has apparently been heavily introgressed by a smaller native Opuntia species to form O. "demissa" (Thorne 1967). The population of D. mojavensis on Santa Catalina Island is in-

Habitat Selection in Drosophila mojavensis 155

ferred to be a recent colonist because its host cactus, o. ficus-indica, is recently introduced, and D. mOjavensis is not known to breed in the smaller Opuntia species (Heed 1982).

These two samples were maintained on standard banana Drosophila food inoculated with live baker's yeast (Saccharomyces cerevisiae). In early January, 1982, these two stocks were mixed, using about 100 flies from each stock, to form a new stock designated A826 M. The females of this pooled stock were allowed to lay eggs. When these eggs produced adults, males and virgin females were collected to be used as parents in the heritability experiment. Tran­sient linkage disequilibrium, arising from mixing the two collections, should not seriously bias the estimates of genetic variance made here (Lewontin 1974).

Behavioral Assay Procedure

The fresh agria cactus used for the oviposition medium was collected in Baja California near San Ignacio in January, 1982. The sample comprised two to three arms of this cactus. All tissue was removed from the woody core, coarsely chopped and then autoclaved. This was then inoculated with Erwinia carnegieana (strain no. 1-12, Department of Plant Pathology, University of Arizona), which is the saguaro cactus necrosis bacterium. This bacterium also occurs in agria rots in nature (Vacek 1979). The cactus tissue was then placed in a 30°C in­cubator for two weeks. At this point, the cactus was a bright yellow color that characterizes a natural agria rot when it has high concentrations of chemicals suitable for attracting Drosophila (Vacek 1979). By behavioral assay, this medium also elicited oviposition from most females. The cactus medium was then put through a fine (2 x 2 mm mesh) sieve to stan­dardize the texture of the medium. The same batch of agria was used to test all females. The medium was kept refrigerated at 10 °C except when aliquots were taken for the day's experi­ment. To make the eggs more easily visible, 5 to 10 drops of green food coloring (McCormick & Co.) were added to each liter of cactus homogenate. This dye has no effect on oviposition behavior in D. mojavensis (W. B. Heed, pers. comm.). Scoring is improved, since the eggs ab­sorb the dye and become blue-green, contrasting with the yellow-green medium.

The test apparatus was a pair of Microtest III plates (Falcon Plastics). One plate of each pair had each of the 96 wells filled level with the surface with the cactus medium. Each well in the plate will hold approximately 0.6 ml of liquid. This plate with the medium was refrigerated until 6 to 8 h prior to the test period when it was placed at room temperature. The second Microtest plate of each pair was left empty as a chamber for the flies.

The test regime began when single females were aspirated without anaesthesia into empty individual vials to which an aged male was added. These females were then starved for 6 h without water to increase their response to cactus stimuli. It is important to note that D. mo­javensis is not induced to oviposit in the absence of cactus stimuli merely by starvation, nor are the females rendered nondiscriminating in oviposition behavior by being held as virgins for several weeks (Lofdahl, unpublished). During these 6 h, each pair of flies was lightly anaesthetized with CO2 and transferred to a well in the empty Microtest plate. This plate was then covered with microscope slides that were secured with rubber bands. At the end of the starvation period, the entire Microtest plate with the flies was put under CO2 anaesthesia for several minutes, the slides removed, and the Microtest plate with the cactus medium inverted over it. The paired Microtest plates were held together with rubber bands. Within one min, all pairs had awakened. After 15 min, the test apparatus was inverted, placing the cactus medium on the bottom. The apparatus was then placed in the dark at room temperature (21-23 0c) for 18 h. This method excludes visual cues, so the test is for genetic variation on chemical and tac­tile cues. At the end of the test period, the paired Microtest plates were put in the CO2

chamber, the flies tapped into the empty Microtest plate, and then all flies were discarded. The

156 Katherine L. Lofdahl

oviposition medium plate was covered with plastic film and refrigerated until the eggs were counted. Those cases in which the female was trapped in the medium were not scored.

Genetic Design and Statistical Analysis

The experimental design was a sire-dam breeding analysis as is outlined by Falconer (1981) for the study of polygenic traits. This method relies on the establishment of either full­or half-sibling families; the degree of resemblance in the scores for the trait among and within families is then used to arrive at an estimate of genetic variance in relation to the total (phenotypic) variance in the trait. An analysis of variance is used to do this. The present study employed a nested ANOV A because full-and half-sibling families were both created in the breeding design. Because the parents used are considered a sample of a natural population, in­terest is in knowing the amount of genetic variance present in the population rather than in estimating the genotypes of the parents. Since population parameters (the heritabilities) are estimated, the ANOV A is a Model II or variance components model. In the nested ANOV A, the among-sires component of variance refers to the degree of resemblance of members of half-sibling families. Likewise, the among-dams component of variance measures the similari­ty of members of full-sibling families in relation to the total variance in the behavior.

Each of 38 males was randomly mated to 42 different females. Allowing for failed matings and nonovipositing females, this gave approximately 1100 full-sibling families. The female parents were each placed in an individual I-dram vial of banana food that had been in­oculated with live baker's yeast. All members of a full-sibling family shared the same larval environment. The progeny for testing in the heritability experiment were collected from the rearing vials as virgin females. Females from each full-sibling family were kept together in a vial of banana food until tested. All males collected were pooled into larger groups and aged for 10 days to allow them to become sexually mature. The females tested were of various ages, but all were approximately 2-4 weeks past eclosion. For the first test series, one female was chosen at random from each full-sibling family and allowed to oviposit. The second replicate took one female from each of the remaining families. This process was continued until all the females were tested. Approximately 300-400 progeny were tested per day.

Data from the sire-dam analysis were examined in two ways: as a metric trait and as a threshold trait. Since the number of eggs laid was recorded for each female, the heritability can be calculated for these continuously distributed data. The percentage of females ovipositing, however, depends on how long the cactus medium has been fermented as this determines the concentrations of volatile oviposition cues such as ethanol. In the present ex­periment, only 670/0 of all females tested laid eggs. The NESTED program for the analysis of variance of hierarchic designs from the Statistical Analysis System (Helwig and Council 1979) was therefore used to estimate the variance components excluding cases where the female fail­ed to lay any eggs. Formulae for calculating the heritability from a sire-dam breeding analysis, given by Falconer (1981), were used. The among-sires component of variance was preferred to the among-dams component as a means of estimating heritability, for, unlike estimates from the dam component, the estimate is not biased with dominance effects and the influences of a common environment on the progeny. The heritability estimated from the sire component thus gives an accurate value for the amount of genetic variance in the number of eggs laid on agria that, in combination with knowledge of the intensity of selection, determines the rate of response to natural or artificial selection (Falconer 1981).

The data also gave the percentage of each sire's progeny ovipositing on agria. The trait can therefore be treated as a threshold character with the female's initial acceptance of the oviposition medium occurring if she is beyond a physiological threshold for egg laying on agria. The individual females are then scored as 1 (one or more eggs laid on agria) or as 0 (no

Habitat Selection in Drosophila mojavensis 157

eggs laid). This gives a 2 x N Chi-square table where N = number of sires and there are two response categories (oviposition vs. no oviposition). An estimate of the heritability of this threshold trait, initial acceptance of agria for oviposition, can be made from the sample Chi­square value that is used to test for heterogeneity among sire percentages (Robertson and Lerner 1949).

Results

Genetic Variation jor Initial Acceptance oj Agria Cactus

The formula for estimating the amount of selectable genetic variance for the threshold trait, initial acceptance of agria for oviposition, is given by Robertson and Lerner (1949). The percentage of each sire's female progeny ovipositing on agria is given in Table l. A Chi-square test for homogeneity of these percentages reveals that different genotypes (sires) have dif­ferent genetic predispositions to oviposit on agria (p <0.001). Although males do not express oviposition behavior, as in most sex-limited traits, they do transmit genes for initial accep­tance of agria to their daughters. A heritability estimate for the initial acceptance of agria is obtained from the sample Chi-square value. Robertson and Lerner (1949) suggested that the arc-sin transformation of these percentages can be dispensed with when the amount of genetic variance is small, as is the case here. The untransformed percentages were therefore used. The Chi-square test is robust to small cell sizes in a 2 x Ntable (Lewontin and Felsenstein 1965), so no sire's data were excluded on this basis. The Chi-square values are virtually identical whether the cells with fewer than five females ovipositing are exluded or not. Using Robertson and Lerner's (1949) formula, this gives a heritability estimate of h2 = 0.11. Although the same authors give a formula for the standard error of heritability estimates for threshold traits, this was not applied here because it relies on equal cell sizes for the estimate. The main conclusion is that significant genetic variation, equal in magnitude to 11070 of the phenotypic variance, ex­ists for the initial acceptance of agria cactus as an oviposition substrate.

The problem with estimating the heritability of a threshold trait on the observed phenotypic (binomial) scale, as given above, is that the magnitude of the heritability depends on the incidence of the trait (in this case, the mean percentage of females ovipositing). The in­cidence in the present study was 67 %. To avoid this difficulty, Falconer (1981) gives a transformation to an estimate of heritability on an underlying physiological scale where gene action is additive and the tendencies for initial acceptance of agria are normally distributed. When this is applied to the estimate of h2 on the binomial scale reached above, this gives h2

(underlying scale) = 0.18. Measured on either the phenotypic or the underlying physiological scale, the Opuntia-breeding population of D. mojavensis has genetic variation for initial ac­ceptance of agria cactus for oviposition. Natural selection can act on this genetic variation to increase or decrease the percentage of females in the population using agria for egg laying.

Genetic Variation jor Number oj Eggs Laid on Agria Cactus

Before performing the analysis of variance, the data were square-root transformed. This is appropriate because count data are often Poisson distributed, and this transformation helps remove the dependence of the variance on the mean (Sokal and Rohlf 1981). This data transformation also significantly reduced the degree of kurtosis in comparison with the raw or logarithm-transformed scores, which is important in drawing valid inferences from variance components models (Scheffe 1959).

The F-test for significance of the sire component of variance, i.e., for genetic variance in the number of eggs laid on agria, can be performed on the nested ANOYA (Table 2) in several forms. The first is the simple test assuming equal cell sizes, which gives a highly significant

158 Katherine L. Lofdahl

Table l. Oviposition of Drosophila mojavensis on agria cactus (Stenocereus gummosus): analysis of oviposition substrate acceptance as a threshold trait.

Male number

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Total number of females tested

91 52

113 94 38 39 77 32 29 75 66 24 52 72 24 43 33 49 33 31 44 64 23 37 65 57 61 45 39 20

2 9

67 54 94 40 23 29

Percentage females ovipositing

0.82 0.65 0.71 0.52 0.58 0.62 0.77 0.66 0.62 0.64 0.59 0.67 0.81 0.65 0.79 0.65 0.76 0.88 0.52 0.55 0.77 0.66 0.70 0.57 0.68 0.72 0.77 0.76 0.64 0.65 1.00 0.67 0.76 0.74 0.46 0.68 0.61 0.72

Chi-square test for homogeneity of binomial samples:

;'37 (.001) = 69.4

Habitat Selection in Drosophila mojavensis 159

Table 2. Nested analysis of variance using square-root transformed oviposition scores of Drosophila mojavensis on agria cactus (Stenocereus gummosus).

Source of Mean Variance Percent of variation D.F. squares component total variance

Sires 36 14.89 0.23 3.4

Dames (within sires) 539 6.88 0.25 3.6

Error 665 6.36 6.36 93.0

Total 1240 6.83 6.84 100.0

F-test for significance of sire component of variance using Satterthwait's approximation: F(36,237) = 2.09 P < 0.005

F-test for significance of dam component of variance: F(S39.665) = 1.08 n.s.

result (p < 0.005). In the case of highly unequal cell sizes, as in the present experiment, an F-test employing Satterthwaite's approximation is preferred. Although one of the conditions suggested for the application of this approximation is violated, the result is merely to give a test that is overly conservative (Sokal and Rohlf 1981). The fact that the F-test with Satter­thwaite's approximation still gives a very high significance level (p < 0.005) is strong evidence for the presence of additive (selectable) genetic variance in the number of eggs laid by D. mo­javensis on agria cactus.

The analysis of variance on the sire component is quite significant while the dam compo­nent of variance is just below the p = significance level. The heritability as estimated from the sire component is h2 ± S.E. = 0.136 ± 0.062 on the square-root scale (Falconer 1981, Turner and Young 1969). If an estimate of heritability were to be made on the raw data, the value would be almost identical (h2 ± S.E. = 0.127 ± 0.062). Thus, genetic variance, equal in amount of 140/0 of the phenotypic variance, is present and available for response to either natural or artificial selection on the number of eggs laid on agria cactus.

Discussion

Before relating the present estimates of genetic variance to the evolutionary potential of the D. mojavensis population studied, it must be demonstrated that these significant results are not a laboratory artifact. The acceptance of agria cactus as an oviposition substrate is con­sistent with studies of chemotaxis in wild D. mojavensis populations. The species is most at­tracted to agria baits in the field, even in areas where this cactus is wholly absent (Fellows and Heed 1972). Agria preference itself is therefore not laboratory-generated, but rather characterizes a number of D. mojavensis populations (Mangan 1978).

Because the amount of genetic variation, i.e., the heritability, partially determines the rate of response to natural selection, it is valuable to know if the magnitudes of the heritabilities are comparable in the laboratory and in nature. Certainly, the population sample

160 Katherine L. Lofdahl

assayed in the oviposition tests was only a few generations removed from nature. Yet this is not sufficient to infer a lack of change in the genetic parameters of the population. Natural selection in the laboratory can, for example, increase the tendency of Drosophila melanogaster females to lay eggs on a sucrose medium over a period of only a few generations (Mazing 1946). To the extent that continued selection exhausts the genetic variance (Bulmer 1971), the percentage of genetic variance in relation to the phenotypic variance can decrease during laboratory maintenance of a stock. Natural selection based on differential fitnesses in the laboratory environment can alter both the oviposition phenotype and in some cases also the amount of genetic variation present for response to selection in future generations. The amount of genetic variation for oviposition on agria in nature may be greater or less than that estimated here, but the presence of genes for habitat acceptance is not put in doubt. The fact that all the genes influencing agria acceptance are not fixed at frequencies of 100070, but that individual genetic differences exist for this behavior, is also a laboratory result that may well hold true in nature. Natural selection acting in the Santa Catalina Island population could therefore increase the preference of D. mojavensis for agria if this cactus were introduced and fitnesses of offspring were higher on agria than on Opuntia cactus.

A second relevant issue in evaluating these results on oviposition behavior against empirical studies on other species and models of habitat selection, is whether the genetic differences measured are in fecundity or in degree of agria acceptance. Fecundity is here defin­ed as the number of mature eggs present in a virgin female. Indirect evidence suggests that the present experiment has employed a valid measure of agria acceptance. Thorax length, an in­dex of body size, is known to have a high phenotypic correlation with ovariole number (Mangan 1978), which is itself highly correlated with the number of mature eggs present in virgin females (David 1970). Therefore, thorax length is a good index of fecundity in Drosophila. Since the number of eggs laid on agria gave a nonsignificant regression on thorax length (p >0.83) for the first 150 females tested in the present experiment, acceptance of agria necroses is being measured. The number of eggs laid on agria is at least partially independent of the number of eggs stored by virgin females. The heritability of number of eggs laid on agria can be regarded as a true measure of genetic variation in cactus acceptance.

Often it is useful to ask how a behavior in a temporal sequence, such as that involved in courtship or prey-catching, evolves in relation to others in the sequence. Then one needs to know the amount of genetic variation present for that element in the series given that all previous elements have occurred. For example, D. mojavensis females may not lay eggs readily unless they have mated (Markow 1982). To determine the degree of expression of oviposition acceptance in response to host plant traits, it is reasonable to discard those females not laying eggs. This is merely measuring heritability in a more defined environment; an en­vironment in which causes not relevant to preference, such as whether the female has mated, are removed. The opposite tactic is to measure genetic variation for the initial acceptance of agria as an oviposition substrate to estimate the penetrance of oviposition on agria. Knowing that heritability values for both the penetrance and expressivity of oviposition on agria are statistically significant, demonstrates additive genetic (Le., selectable) variance for the initial decision and for the degree of acceptance in the agria stimulus environment. This dual knowledge gives a clear impression of the ways in which natural selection can change genetically based habitat selection.

Establishing the existence of genes for oviposition on agria reveals nothing about the ontogeny of this behavior. Perhaps variation in oviposition acceptance may be due wholly or in part to genetic variation among individuals in tendency to undergo habituation or associative olfactory conditioning to chemical cues in the larval or adult environments (Jaenike 1982). Alternatively, these preferences may be due to congenital genetic variation in

Habitat Selection in Drosophila mojavensis 161

oviposition responses. The design of the present experiment cannot determine whether the genetic variances for the initial acceptance and for the number of eggs laid refer to genes for learning, for congenital preferences or both. The need to consider genetic variation in learning abilities in studies of insects is demonstrated by recent successful artificial selection for classical conditioning in the black blow fly, Phormia regina Meigen (McGuire and Hirsch 1977). Fuyama (1976, 1978) documented genetic variation for congenital olfactory preferences in Drosophila. Further research is needed to estimate the relative influences of genes for learn­ing and for congenital preferences on the oviposition behavior of D. mojavensis. The knowledge of how cactus preferences are acquired in ontogeny is critical because learned preferences and congenital predispositions can have very different implications for the extent of host plant restriction.

The evidence for genetic variation in host acceptance in D. mojavensis has a fundamental bearing on allopatric host colonization in this species. Since the Santa Catalina Island popula­tion represents a recent colonization, it is accurate to claim that D. mojavensis has extended its degree of polyphagy. This is similar to the situation in Drosophila grimshawi Oldenberg to the extent that both colonizing populations added a new host plant or plants to the species' list of habitats (Carson and Ohta 1981). The genetic prerequisites for Opuntia colonization by D. mojavensis are unknown. Perhaps they involve physiological adaptations to the new host or genetic predispositions to feed and oviposit on Opuntia. Yet such hypothesized genetic predispositions do not require the loss of genetic variation for use of an earlier host cactus species. Addition of a new host cactus need not involve the interchange of genes for agria use and genes for Opuntia use. The same genes creating a predisposition to use agria also may pro­duce a tendency to successfully colonize Opuntia due to pleiotropic genetic effects or to the ex­istence of common oviposition cues in the two cactus species. The present analysis of the genetics of allopatric host colonization allows a prediction: if the agria- and Opuntia-breeding populations of D. mojavensis are brought in contact, absolute niche separation in breeding sites is not likely to be present, at least in the early stages. Colonization of a new host plant species has not promoted genetic differentiation in cactus use of the sort that can lead to im­mediate, total population subdivision during secondary contact.

Acknowledgments I am extremely grateful to Dr. William B. Heed who kindly provided me with D. mo­

javensis stocks and laboratory facilities during my research visit at the University of Arizona. Drs. W. B. Heed and James C. Fogleman also provided many helpful discussions on the evolutionary ecology of Drosophila. I also thank Tom Orum of the Department of Plant Pathology, University of Arizona, for the culture of Erwinia carnegieana. The manuscript benefited greatly from comments by S. Arnold, M. Huettel, R. Lande, M. Rauscher, M. Rose and W. T. Starmer. This research was supported by a Nierman Research Award from the University of Chicago.

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