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1 School of Biological Sciences, 348 Manter Hall, University of Nebraska, Lincoln, Nebraska 68588-0118 USA
Received for publication July 10, 1998. Accepted for publication May 18, 1999.
ABSTRACT
At Arapaho Prairie, in the sandhills of western Nebraska, the dioecious annual Croton texensis (Euphorbiaceae) exhibits biased sex ratios. Moreover, the direction of bias changes from year to year: in 1994 the study population was significantly female biased, in 1995 and 1996 it was significantly male biased, and in 1997 and 1998 the sex ratio did not differ from 1 : 1. Such variation in the observed sex ratio in plants is frequently attributed to environmental sex determination (ESD), which is favored by natural selection if the rate of fitness gain across an environmental gradient is greater for one sex than the other. We performed experiments to determine: (1) whether variation in the sex ratio is correlated with environmental conditions, as would be expected if ESD is operating, and (2) whether ESD, if present, would be favored by natural selection. In a common garden experiment in which water and fertilizer were manipulated the sex ratio was marginally male biased in treatments in which water was added, but not different from 1 : 1 in other treatments. In field plots into which seeds were planted none of several soil characteristics, nor overall plot quality for C. texensis (measured as average plant biomass) were correlated with plot sex ratio. However, plots in which a large number of planted seeds emerged tended to be female biased. These results provide very weak evidence for sex ratio bias across an environmental gradient, and thus provide little evidence for ESD. Moreover, sex-by-environment interactions for fitness, which are required for the evolution of ESD, were absent for all measured variables. Thus, ESD does not appear to be favored by natural selection in this population. Instead, these biases may have been caused by differences between the sexes in germination and/or early mortality.
Key Words: Croton texensis • environmental sex determination • ESD • Euphorbiaceae • sex ratio
In populations of sexually reproducing organisms, negative frequency-dependent selection generally acts to maintain a sex ratio that reflects equal investment in males and females (Fisher, 1930
). There are exceptions to this expectation, for example, when local mate competition (Hamilton, 1967
) or local resource competition (Clark, 1978
) is present. Another exception is predicted by sex allocation theory when the environment is heterogeneous and sex is environmentally determined (Freeman, Harper, and Charnov, 1980
; Charnov et al., 1981
; Charnov, 1982
). In the case of environmental sex determination (ESD), the sex ratio is expected to be 1 : 1 in the population as a whole, but locally biased, from predominantly male to predominantly female, along an environmental gradient (Bierzychudek and Eckhart, 1988
).
Charnov and others (Freeman, Harper, and Charnov, 1980
; Charnov et al., 1981
; Charnov, 1982
) argue that ESD will be favored by natural selection when the rate of fitness gain across an environmental gradient differs between males and females. Because reproduction is generally more costly for females, it is usually argued that female fitness will increase more rapidly as environmental quality improves (Freeman, Klikoff, and Harper, 1976
; Lloyd and Webb, 1977
; Freeman, Harper, and Charnov, 1980
; but see Bawa and Opler, 1977
), and thus, that the sex ratio will be female biased in high-quality environments. Charnov and Bull (1977)
suggest that ESD should be most strongly favored by natural selection when (1) the environment that an organism enters has a large impact on its lifetime fitness, (2) the environment is patchy, with some patches conferring more of an advantage to one sex than the other, and (3) the parent and offspring have little control over which patch type the offspring will enter. These conditions are especially likely to hold true for sessile organisms with limited dispersal mechanisms, a condition that includes most plants. Thus, if ESD is operating, one would expect to see locally biased sex ratios correlated with an environmental gradient. Moreover, a comparison of the relative fitness of males and females in each environment should show a sex-by-environment interaction. In particular, relative fitness of each sex should be greater in the environment in which it is most common.
Empirical tests have supported this sex allocation model for maternal control of the sex ratio in parasitic wasps (Charnov et al., 1981
), as well as for sex change in sequential hermaphrodites, such as jack-in-the-pulpit (Policansky, 1981
) and pandalid shrimp (Charnov, 1979
), and were suggestive in an orchid (Zimmerman, 1991
). However, there are few empirical tests of the ESD version of the model, which predicts nonlabile sex choice for a predominantly dioecious species. A series of experiments with Spinacia oleracea has shown that ESD can occur in this species (Onyekwelu and Harper, 1979
; Freeman and Vitale, 1985
) and that there is some evidence for differences in relative fitness gain of the sexes across an environmental gradient (Vitale and Freeman, 1986
). However, the interpretation of data from these studies is complicated by the high frequency of monoecious individuals because it is not clear how allocation to male vs. female function is distributed in these cosexual plants.
The objective of this study was to determine whether biased sex ratios observed in a population of the dioecious annual plant Croton texensis are consistent with the operation of environmental sex determination. Here we report that the sex ratio in our experimental population of C. texensis varies among years from significantly female biased to significantly male biased, and address the following questions: (1) do sex ratios in C. texensis vary as a function of the environment, as expected if ESD is operating? and (2) does C. texensis show the sex-by-environment interaction for fitness expected for ESD to be favored by natural selection?
MATERIALS AND METHODS
Experimental organism and field site
Croton texensis (Kl.) Muell. Arg. (Euphorbiaceae) is a dioecious annual herb of sandy soils. It is infrequent to common throughout its range in the Great Plains and is somewhat of a disturbance specialist (Great Plains Flora Association, 1986
). Staminate (male) plants have numerous small cream-colored flowers in racemes of several dozen flowers, which open sequentially. Male flowers are visited by a wide variety of insects, which collect pollen and nectar, and females are presumably insect pollinated. Pistillate (female) flowers are in smaller racemes, of one to four flowers each. Female flowers are apetalous, green, and apparently do not produce nectar (K. Keeler, University of Nebraska, personal communication). There is a measurable tendency toward sexual dimorphism such that male plants are more slender, with longer internodes and narrower leaves (Ferguson, 1901
; Decker, 1997
), but plants cannot be reliably sexed by these characters.
At our study site C. texensis germinates in late April and early May. Early growth is very slow and seedlings do not emerge from the soil until ~2 wk following germination (K. Decker, personal observation). Postgermination, pre-emergence mortality can be as high as 40% (Pilson and Decker, unpublished data). However, once plants reach the four-true-leaf stage mortality is very low (1–2%; Pilson and Decker, unpublished data; and see Results). Croton texensis flowers from July to September, and plants senesce by late September (Keeler, Harrison, and Vescio, 1980
; K. Decker, personal observation).
The field site for this study, Arapaho Prairie, is a 526-ha (two section) tract of sandhills prairie in Arthur County in western Nebraska. The area is owned by The Nature Conservancy and managed as a research preserve by the University of Nebraska, and has been protected from domestic grazing since 1977. Croton texensis is common at the study site, especially in areas disturbed by pocket gophers. Arapaho Prairie includes two interdunal valleys as well as parts of several dune slopes and ridges with varying exposures; maximum elevation differences are ~70 m (Keeler, Harrison, and Vescio, 1980
). Previous workers have described three habitat types (valley, slope, and ridge) characterized by edaphic features and associated vegetation assemblages (Barnes, Harrison, and Heinisch, 1984
). Valley-to-ridge gradients in moisture availability and soil nitrogen have also been detected (Alward and Joern, 1993
).
Field surveys
Population sex ratios were surveyed from 1994 to 1998 near the end of the flowering season. In September, when the surveys were performed, >99% of all plants have flowered but not senesced, allowing an accurate estimate of the population sex ratio. A grid system of permanent markers at 100-m intervals has been established on Arapaho Prairie. In each year, we surveyed all grid points on the western section, counting and sexing C. texensis individuals in 100-m2 circular quadrats at each of 240 markers. The habitat type (ridge, slope, or valley) at each grid point was also recorded. Sex ratios were compared to a 1 : 1 ratio using a log-likelihood ratio goodness-of-fit test (G test; Sokol and Rohlf, 1981
). The hypothesis of no difference in sex ratio between habitat types was tested by maximum likelihood ANOVA for categorical data (CATMOD procedure; SAS, 1990
).
Soil temperature and potential evapotranspiration are measures of environmental stress, and we hypothesized that yearly variation in these climate variables could affect C. texensis sex ratio or population size. For this reason we obtained daily weather data from the High Plains Climate Center (University of Nebraska), which maintains an automated weather station near the center of the west section of Arapaho Prairie. We calculated the mean monthly soil temperature and monthly total potential evapotranspiration (daily values calculated by the center using the methods of Hubbard, 1992
) for the months of April, May, and June 1994–1998. We then calculated Pearson product-moment correlation coefficients between these weather data and sex ratio and population size in the years 1994–1998 (CORR procedure; SAS, 1990
).
Environmental sex determination, common garden experiment
In order to obtain an estimate of the potential effect of environmental variation on sex ratio, we performed a common garden experiment at the University of Nebraska's Cedar Point Biological Station (~47 km south of Arapaho Prairie). Because soil moisture availability and nitrogen gradients are known to be present at Arapaho Prairie (Alward and Joern, 1993
), we used combinations of water and fertilizer to create variable environmental conditions. The experiment was set up as a randomized complete block design with four blocks of 500, for a total of 2000 plants. Treatment design was a 2 x 2 factorial arrangement of supplemental and ambient amounts of water and 1 : 1 : 1 N-P-K controlled release fertilizer (Osmocote).
The seeds used in this experiment were collected from Arapaho Prairie in fall of 1995, cleaned, bulked, buried in small lots at the collection site for overwintering, and rebulked before use. Seeds were germinated under uniform conditions, then planted individually in ~1-l Plastic beverage cups filled with well-mixed soil from Arapaho Prairie. Seeds with emerged radicals were planted at a depth of 5–7 cm and watered in. Because all seeds did not germinate at the same time, the experiment was blocked by planting date. On 30 April 1996, 500 seedlings were planted in each of the first two blocks, and, on 7–8 May 1996, 500 and 488 seedlings were planted in the third and fourth blocks, respectively. Posttreatment survival rates and sex ratios did not differ significantly among blocks; therefore they are not treated separately in the analyses presented below. Cups were arranged outdoors on cleared ground and later placed on top of large fiberboard sheets to prevent roots from growing into the soil beneath the cups.
Treatments were begun when ~50% of the seedlings in a block had emerged and before any seedling had reached the four-leaf stage. Fertilizer treatments were applied on 29 May 1996 for the first block and on 7 June 1996 for the remaining three blocks. Watering treatments were begun on 10 June for all blocks.
Some seedlings damaged in early, pretreatment hailstorms were replaced with spares growing in flats. Not all damaged seedlings could be replaced, so replacements were made in such a way as to ensure nearly equal numbers of each treatment in each block. All replacements were done prior to the initiation of treatments.
Fertilizer was applied one time in the form of pellets (~0.30 g/cup). Unfertilized cups received no fertilizer of any kind. Supplemental water was applied to individual cups in the watered treatment by a spray nozzle turned on for a constant number of seconds. Amounts delivered were checked using a calibrated empty cup. Over the course of the experiment, cups in the watered treatment received approximately three times as much water as unwatered cups. However, because the experiment was exposed to natural precipitation as well as to intentional watering, these relative amounts could not be precisely maintained over the duration of the experiment. During a few extended rainy periods and thunderstorms, all treatments received excess water and no supplemental watering was done. Cups were weeded frequently for most of the duration of the experiment, until nearly all plants had flowered.
The experiment was censused weekly to determine survival since the last census and sex (once the plant had flowered) for each plant. Total plant leaf area was estimated twice during the growing season for the same randomly selected subset of plants (25 of each treatment in each of the four blocks). Leaf area was estimated by measuring leaf length and width of each leaf, then using a previously derived regression to calculate area. This regression explains 99% of the variation in leaf area (N = 63, P < 0.0001). Total leaf area was calculated as the sum of the areas of all leaves on a plant. Growth rate was estimated as the proportional difference in total leaf area between the two total leaf area estimates. Because males and females have different growth rates (Decker, 1997
) we calculated the residual growth rate of each plant after the effect of sex had been removed in an ANOVA, and then averaged the residual growth rate over all plants in each treatment. Mean residual growth rates in each treatment were compared by Tukey's studentized range test (GLM procedure; SAS, 1990
) to determine whether treatments had resulted in different environments for C. texensis seedlings. Because an intense hail storm during the flowering period severely damaged many plants (Decker, 1997
), fitness components were not measured for plants in this experiment. The effect of the treatments on sex ratio was analyzed by maximum likelihood ANOVA for categorical data (CATMOD procedure; SAS, 1990
).
Environmental sex determination, field experiment
We performed a field experiment at Arapaho Prairie in 1996 in order to determine (1) whether the sex ratio varied with natural variation in any of several environmental variables, and (2) whether any environmental variable showed a sex-by-environment interaction for fitness. Forty 4-m2 plots were located at apparent extremes of environmental variation experienced by C. texensis: 20 plots were located near survey points where C. texensis was abundant and/or large plants had been present in 1995, and 20 plots were located near survey points where C. texensis was present but rare in the previous year. Although plot quality, measured as mean fitness of planted C. texensis, varyed dramatically, these differences were not correlated with population size at the nearest survey marker in 1996 (data not presented). One hundred ungerminated seeds were planted at ~20-cm intervals into each of the 40 plots. On 24–25 April 1996, 50 seeds were planted into each of 36 plots, and on 10 May 1996, 50 additional seeds were planted into the first 36 plots, and 100 seeds were planted into four additional plots. Seeds used in this experiment were from the bulk lot described above. Each seed was planted 5–7 cm deep in a small (~7.5 cm diameter), uniform disturbance made using a bulb planter, and marked with a plastic pot tag. Although emergence rates differed between cohorts (cohort 1: 45%, cohort 2: 12.5%), there were no differences in sex ratio or fitness gain patterns between cohorts; therefore the two cohorts are combined in the analyses presented below.
Plots were censused approximately every 10 d for date of emergence, survival since the last census, and sex (once the plant had flowered). All plants were harvested on 13–14 September 1996, and flower number (on males), seed number (on females), and aboveground biomass were determined for each plant. The sex ratio of each plot was calculated as the proportion of individuals surviving to flower that were male. Twenty-seven percent of the 4000 seeds planted emerged as seedlings, and of these, 78% survived to flower. The number of flowering plants per plot ranged from three to 33, with a mean of 20.4.
We used total flower number as an estimate of male fitness and seed number as an estimate of female fitness. Male flower number was estimated by counting remaining flowers and scars left by senesced flowers, using a dissecting microscope. This estimate of male fitness assumes that all flowers are equally attractive to pollinators and that the amount of pollen does not vary among flowers. We have no data with which to evaluate the first assumption. However, while male plants do differ in the amount of pollen produced per flower, among-plant variation in flower number is much greater; thus, including pollen counts would have a negligible effect on estimates of male fitness (Decker, 1997
). Using the number of mature seeds as an estimate of female fitness assumes that all seeds are equally viable and have the same chance of dispersal to suitable sites. Mean absolute fitness for each sex (mean flower number for males and mean seed number for females) was calculated over all males or all females in the experiment, and the relative fitness of each plant in the experiment was calculated by dividing its absolute fitness by the mean absolute fitness for its sex. Thus, across all plants in the experiment mean male fitness and mean female fitness are both equal to one. For each of the 40 plots male fitness was the average of the relative fitnesses of males present in the plot. Similarly, plot female fitness was the average of the relative fitnesses of females present in the plot.
Environmental variation among the 40 plots was measured in several ways. Soil water availability was estimated three times during the growing season (on 25 June, 7 July, and 3 August 1996). Soil samples were collected by combining six 2.5 cm diameter x 20 cm deep cores taken around the perimeter of each plot. Samples were weighed, dried at 50°C for 72 h, and weighed a second time. These data were used to estimate the percentage water present in each sample. Chemical characteristics of the soil in each plot were also measured. Soil samples were collected as above on 27 June 1996 and frozen until analysis by the University of Nebraska Department of Agronomy Soil and Plant Analytical Laboratory. Soil samples were analyzed for pH, potassium, phosphorus, sodium, and organic matter (using methods presented in North Dakota Agricultural Experiment Station, 1988
), and ammonium and nitrate (using methods of Keeney and Nelson, 1982
).
Environmental variation among plots was measured in two additional ways. First, overall plot quality was estimated by using the mean aboveground biomass for each plot as a bioassay. Presumably, plots with consistently larger plants were of higher quality. Because females are on average larger than males, we calculated the residual biomass of each plant after the effect of sex had been removed in an ANOVA, and then averaged the residual biomass over all plants in each plot. To meet the assumptions of ANOVA, biomass data were log transformed before analysis. Second, plot quality was estimated by using the number of planted seeds (out of 100) that emerged from the soil. In using this bioassay as a measure of plot quality we are assuming that germination and/or early survival rates are greater in high-quality plots. Because the proportion of planted seeds that emerged differed between the first and second cohorts, only the 36 plots with seeds from both cohorts were included in this analysis.
If sex is determined by environmental factors, one would expect a relationship between the sex ratio and one or more of these environmental variables (soil measures and/or bioassays). Pearson product-moment correlation coefficients were calculated between environmental variables and the sex ratio in each plot (CORR procedure; SAS, 1990
). Sex ratio and environmental variables were normally distributed without transformation.
Finally, the differential effect of plot environment on the fitness of each sex was analyzed by analysis of covariance (ANCOVA). Mean plot fitness for males and females was analyzed in ANCOVAs, which included sex, one of the environmental variables (the covariate), and the sex-by-environmental variable interaction as predictors. Separate ANCOVAs were performed for each of the soil variables, residual aboveground biomass, and number of seedlings emerged. To meet the assumptions of ANCOVA, fitness data were log transformed before analysis. In these analyses a significant sex-by-environmental variable interaction would indicate that the relative fitness of one sex increases more rapidly across an environmental gradient. In the presence of this interaction, ESD, if present, is favored by natural selection.
RESULTS
Population sex ratios
Population sex ratios at Arapaho Prairie varied among years (Table 1). Over all three habitat types the sex ratio was significantly female biased in 1994 (G1 = 6.78, P < 0.009). In 1995 and 1996 the direction of bias was reversed, and over all habitat types the sex ratio was significantly male biased (1995: G1 = 30.42, P << 0.001; 1996: G1 = 79.85, P << 0.001). In 1997 and 1998 the sex ratio did not differ from 1 : 1 (1997: G1 = 0.29, P > 0.50; 1998: G1 = 0.00, P > 0.90). However, in 1998 the sex ratio was significantly male biased in the slope habitat (Table 1). Although sex ratios in the three habitats (ridge, slope, valley) varied in the degree of departure from 1 : 1 (Table 1), there was no evidence that sex ratios differed among habitat types (1994: 
22 = 0.83, P < 0.6565; 1995: 22 = 4.19, P < 0.1231; 1996: 22 = 1.16, P < 0.5606; 1997: 22 = 0.88, P < 0.6444; 1998: 22 = 3.35, P < 0.1871). Total population size was extremely variable among years (Table 1).
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Effect of soil characteristics and overall plot quality on sex ratio at Arapaho Prairie
Sex ratios in the 40 plots at Arapaho Prairie varied from 0.363 to 0.769. There were no significant correlations between plot sex ratio and any of the soil variables measured (Table 4). Furthermore, there was no evidence of an effect of mean residual biomass on sex ratio in the Arapaho Prairie experimental plots (Table 4; Fig. 2). However, plots with more seedlings emerging tended to be more female biased (Table 4). P values for these correlations have not been corrected for multiple comparisons, however, and thus, this correlation may be spurious.
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The results of the population survey at Arapaho Prairie indicate that this population of C. texensis does exhibit biased adult sex ratios and that the direction of bias changes between years. However, there is little evidence that the observed sex ratio bias in C. texensis is due to environmental sex determination. Population sex ratios at Arapaho Prairie did not differ between habitat types, and in the field experiment at Arapaho Prairie sex ratio bias was weakly correlated with only one of the environmental variables measured in this study (number of seedlings emerged). In the common garden experiment, the sex ratio was only marginally biased in the watered treatment. Moreover, while there is evidence for a strong effect of the environment on fitness, the effect is the same for males and females, indicating that there is no reason for environmental sex determination to be favored by natural selection if it should arise in this species. The absence of selection favoring ESD is probably the strongest evidence we have for the absence of ESD in C. texensis. Taken together, these data suggest that sex is probably genetically determined in this species.
Thus, explanations other than ESD for fluctuating sex ratios must be sought. The mechanism of sex determination in C. texensis has, to our knowledge, not previously been investigated. However, there are some data addressing sex determination in other euphorbs and other Croton species. Mercurialis annua (Euphorbiaceae) is an annual with both dioecious and androdioecious populations. In dioecious populations sex is determined by three unlinked loci (Louis, 1989
; Durand and Durand, 1991
). In androdioecious populations the adult sex ratio in M. annua is influenced by both genetics and the amount of intraspecific competition experienced by plants (Panell, 1997
). Specifically, the frequency of males increases with density, suggesting density-dependent gender choice. In Croton suberosus and C. bonplandianum, both monoecious perennials, allocation to male vs. female function changes with changing environmental conditions (Shaanker and Ganeshaiah, 1984
; Dominguez and Bullock, 1989
). Thus, at least in some species genetic and environmental factors appear to act together to determine sex, and ultimately, the population sex ratio. It is possible that the environment and genetics together determine sex in C. texensis.
However, we were unable to identify an environmental variable clearly correlated with adult sex ratio. In addition to the analyses reported above for individual environmental variables, we also used principal components analysis to summarize the environment in our Arapaho Prairie plots. Correlations between the principal component scores and plot sex ratio were not significant (not presented). Furthermore, there were no sex by principal component interactions for fitness (not presented), indicating that male and female fitness changed at the same rate across these composite environmental variables. These analyses are consistent with our conclusion that sex is probably genetically determined in C. texensis.
Nonetheless, there are several intriguing observations that taken together suggest some effect of the environment on the adult sex ratio at Arapaho Prairie. First, in the common garden experiment sex ratio was marginally male biased in the watered treatment. Second, more seedlings emerged in plots in which the sex ratio was female biased. And finally, early-season soil temperature and potential evapotranspiration appear to be correlated with sex ratio. None of these results alone is especially compelling, but taken together they suggest some unidentified effect of the environment on sex ratio. Moreover, there appear to be no strictly genetic explanations that can account for the year-to-year fluctuations in sex ratio that we observe (Bull, 1983
).
Conn and Blum (1981)
, Willson (1983)
, and Allen and Antos (1993)
discuss possible causes of biased sex ratios, some of which cause correlations with environmental factors. Of these, differential vegetative growth and apomixis can be ruled out in the case of C. texensis, the first because it is an annual lacking clonal growth, and the second because the sex ratio is male biased in two of the five years of this study. Maternal control of the sex ratio, usually thought of as a response to environmental or physiological factors, also seems unlikely, given the lack of a sex-by-environment interaction for fitness.
Biased sex ratios can also be produced if male- and female-determining gametes differ in their ability to fertilize ovules, although laboratory results have been difficult to replicate in the greenhouse or field (Correns, 1928, cited in Conn and Blum, 1981
; Lloyd, 1974
; Carroll and Mulcahy 1993
). If biased sex ratios in C. texensis were due to pollen competition, one would expect to see ratios of ~1 : 1 when pollen was limiting. While the lack of apparent attractive features of female C. texensis flowers could indicate that pollen receipt is normally low, this mechanism cannot explain the shift from female bias to male bias observed over the course of this study.
We believe the most likely explanation for the biased adult sex ratios observed in the natural population at Arapaho Prairie is differences between males and females in germination requirements and/or early survival. Differences between males and females in later survival seem unlikely in this system because most plants that attain the four-leaf stage survive to flower. However, many seeds do not germinate but apparently remain viable (Pilson and Decker, unpublished data), and up to 40% of germinated seeds die before emerging from the soil. If we assume that the underlying genetic basis of sex determination results in a primary sex ratio of 1 : 1, differences in both germination requirements and early survival probably affect the adult sex ratio. This is because the sex ratio fluctuates from female biased to male biased. For example one environmental condition might favor germination of females, leading to a female biased sex ratio, while another might favor survival of males, leading to a male-biased sex ratio. Although the adaptive value of such sex differences is not obvious, a similar situation was clearly documented by Purrington and Schmitt (1995)
in Silene latifolia. These authors found that mortality of male seeds was greater between 0 and 5 mo after burial, but that female seeds had a higher probability of remaining dormant at 5 mo.
While we do not have data that allows a definitive test of the hypothesis that differential germination and/or survival leads to biased adult sex ratios in C. texensis, some of our data are suggestive. For example, the sex ratio was marginally male biased in watered cups, and 5 yr of census data from Arapaho Prairie suggest that the correlation between the proportion of males and May evapotranspiration is negative. These observations suggest that males have a higher probability of survival in low-water-stress environments. However, some of our data are more difficult to interpret. For example, the number of seedlings emerging was negatively correlated with the proportion of males in field plots, but Arapaho census data suggest that population size is positively correlated with the proportion of males in the population. Further experimental work is required to determine whether the environment has sex-differential effects on germination and/or mortality in C. texensis.
An environmental influence on sex expression (either permanent or changing during an individual's life span) has been reported for 50 dioecious or subdioecious plant species from 25 families (Freeman, Harper, and Charnov, 1980
), and in some species this produces spatial segregation of the sexes across an environmental gradient. Bierzychudek and Eckhart (1988) reviewed the literature and found that biased sex ratios correlated with an environmental gradient had been reported in 21 of 32 investigated species. Similarly, in a literature survey Shea, Dixon, and Sharitz (1993)
found evidence of spatial segregation of the sexes in 15 of 26 species. For example, locally biased adult sex ratios in dioecious species are frequently observed in relation to environmental gradients such as moisture (Freeman, Klikoff, and Harper, 1976
; Fox and Harrison, 1981
; Waser, 1984
; Freeman and Vitale, 1985
), elevation (Grant and Mitton, 1979
), soil pH (Cox, 1981
), light availability (Lovett Doust and Cavers, 1982
; Zimmerman 1991
), salinity (Freeman, Klikoff, and Harper, 1976
; Vitale and Freeman, 1986
), water depth (Williams, 1995
), density of conspecifics (Lovett Doust, O'Brian, and Lovett Doust, 1987
; Pannell 1997
) as well as with other, less clearly defined environmental variables (Lloyd and Bawa, 1984
; Korpelainen, 1991
; Shea, Dixon, and Sharitz, 1993
). However, the cause of these local sex ratio biases is usually unknown, and sex allocation theory predicting the evolution of ESD is generally untested. It is not clear, for example, if the cause of these biased sex ratios is ESD or differential mortality of the sexes.
If a variable environment does cause differences between the sexes in germination or early mortality, then the sex ratio should vary among cohorts. However, this hypothesis is difficult to test with data from the literature because most studies only report data from one year. Of the 32 species listed by Bierzychudek and Eckhart (1988)
in which the possibility of locally biased sex ratios was investigated, only four include data from more than one year of population surveys (Hancock and Bringhurst, 1980
; Meagher, 1980
; Lovett Doust and Cavers, 1982
; Waser, 1984
). None of these four species showed a complete reversal of direction of sex ratio bias during the period of the survey such as we found in C. texensis, although Waser (1984)
found that sex ratios of Simmondsia chinensis were male biased in two of four years, and not significantly different from 1 : 1 in the other two years. More importantly, the majority of dioecious species for which sex ratio biases have been reported are long-lived perennials, often with lengthy prereproductive periods (Wheelwright and Bruneau, 1992
; Garcia and Antor, 1995
), clonal growth patterns (Lovett Doust and Cavers, 1982
; Korpelainen, 1991
), or intermittent flowering habits (Meagher, 1980
). Thus, these data provide little information on whether sex ratios vary between cohorts, as would be expected if the environment affected germination or early survival differently in the two sexes.
Sex differences in germination requirements or early mortality, such as that reported by Purrington and Schmitt (1995)
and suggested by the data reported here, are contrary to the conventionally predicted scenario for sex-differential mortality. Specifically, it is usually assumed that females exhibit higher mortality because they experience greater stress and resource demand during seed maturation than males do during pollen production (e.g., Allen and Antos, 1993
). Despite this prediction, clear examples of sex differences in late mortality are rare. For example, in most of the examples of spatial segregation of the sexes reported in Bierzychudek and Eckhart (1988)
and Shea, Dixon, and Sharitz (1993)
the underlying cause of the sex bias is unknown. Thus, it is not clear how frequently early mortality differs among the sexes and leads to biased sex ratios. In addition, the adaptive explanation, if one exists, for sex differences in germination or early mortality is elusive.
In conclusion, results presented here show that our study population of the dioecious annual C. texensis exhibits biased sex ratios, but that these biases are not caused by environmental sex determination. We also have no evidence that the rate of fitness gain across an environmental gradient differs between the sexes. Thus, environmental sex determination is not favored by natural selection. Some of our data suggest that the cause of fluctuating sex ratios in C. texensis is environmentally determined sex differences in germination or early mortality, although we have not demonstrated this definitively.
FOOTNOTES
1 The authors thank Alexandra Basolo, Kathy Keeler, Colin Purrington, and an anonymous reviewer for comments on the manuscript, and Christopher Nordstrom Griffiths, Tony Joern, Robert Kaul, Nanci Ross, Eric Sundvall, and Melanie Traxler for insightful conversation, help in the field or laboratory, and/or the loan of equipment. Kathy Keeler first noticed biased sex ratios in Croton texensis. This work was funded in part by grants from the Jesse Lee Fund of the School of Biological Sciences and the University of Nebraska Research Council. 
2 Current address: 2088 Mead Drive, Boulder, Colorado 80301 USA.
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Allen, G. A., and J. A. Antos. 1993 Sex ratio variation in the dioecious shrub Oemleria cerasiformis. American Naturalist 141: 537–553.
Alward, R. D., and A. Joern. 1993 Plasticity and overcompensation in grass response to herbivory. Oecologia 95: 358–364. [CrossRef][ISI]
Barnes, P. W., A. T. Harrison, and S. P. Heinisch. 1984 Vegetation patterns in relation to topography and edaphic variation in Nebraska sandhills prairie. Prairie Naturalist 16: 145–158.
Bawa, K. S., and P. A. Opler. 1977 Spatial relationships between staminate and pistillate plants of dioecious tropical forest trees. Evolution 31: 64–68. [CrossRef][ISI]
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