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Water and nitrogen manipulations of the desert shrub Larrea divaricata subsp. tridentata (Zygophyllaceae)
Journal of Arid Environments (1994)28:139-146
Water and nitrogen manipulations of the desert shrub Larrea diraricata subsp, tridentata (Zygophyllaceae)
A. Gonzalez-Coloma*, C. S. Wisdom, M. R. Sharifi& P. W. Rundel Laboratory of Biomedical and Environmental Sciencest, Universityof California, 900 VeteranAvenue, Los Angeles, California 90024, U.S.A. (Received 26 November 1992, accepted 19 March 1993) Water supplementation of Larrea divaricata subsp, tridentata bushes growing under natural conditions affected the amount of resin, nordihydroguaiaretic acid (NDGA) and soluble protein present in the leaves. Nitrogen fertilization had no effect on these parameters. The total resin and NDGA content also varied with the leaf production dates, but the NDGA concentration in the resin was not affected by either the treatment or the time of production. Herbivore activity, measured as a percentage of the leaves eaten, was not affected by these manipulations, varied with season (highest during the summer) and had a negative correlation with the resin content of the plant.
Keywords: Larrea divaricata subsp, tridentata; nitrogen fertilization; irrigation; resin; nordihydroguaiaretic acid; herbivory
Introduction T h e availability of resources to plants has been frequently proposed to influence the relationship between plants and their associated arthropods. Plant nitrogen and water content can have a direct influence on host plant choice due to their concentrations in plant tissue (Mattson, 1980; White, 1984; Mattson & Haack, 1987; Lighffoot & Whitford, 1987) and indirectly by changing plant secondary compounds (Bryant et al., 1987; Abrahamson et al., 1988). To examine the effect of changes in the water and nitrogen levels on the chemistry that mediates the plant-insect relationships of arid land plants, we manipulated water and nitrogen concentrations available to the creosote bush (Larrea divaricata subsp, tridentata) (DC.) Felger & Lowe. T h e creosote bush is an evergreen xerophytic shrub abundant in warm desert regions of the south-western United States and Mexico. Its drought resistance allows it to remain metabolically active and produce new leaves and shoots under extremely dry conditions (Sharifi et al., 1988). The abundance of this shrub and its perennial growth habits make Larrea one of the most predictable plant resources in many desert environments (Barbour et al., 1977). T h e leaves are covered with a resinous coating which functions as a barrier to prevent water loss from leaf surfaces, a solar-ultra-violet filter and an insect feeding deterrent (Chapman et al., 1988; Meinzer et al., 1990; Rhoades, 1977). * Address for correspondence: Centro de Ciencias Medio-ambientales, CSIC, Sevrano 115-dpdo., 28006, Madrid, Spain. t Operated for the U.S. Department of Energy by the University of California under contract No. DE-ACO3-76-SF00012.
0140-1963/94/020139 + 08 $00'00/0
© 1994AcademicPress Limited
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This resin is composed of a complex mixture of phenolics, saponins, terpenoids and wax esters that account for 10-20% of the leaf dry weight. Over 80% of the resin is composed of phenolic aglycones, with the major component being norihydroguaiaretic acid (NDGA), a catechol lignan. The remaining resin is a complex mixture of partially o-methylated flavones and flavonols along with small amounts of acid and basic components (Mabry et al., 1977). NDGA is a potent antioxidant (Oliveto, 1972) and also exhibits important bactericidal, fungicidal and antiherbivore properties (Rhoades, 1977; Greenfield & Shelly, 1987; Chapman et al., 1988; Gonzalez-Coloma et al., 1988). Nitrogen content is an important regulator of insect populations on plants (Mattson, 1980). Likewise, variation of nutrient availability within the creosote bush is correlated with the variation in the number of foliage arthropods (Lighffoot & Whitford, 1987), which exhibit various degrees of specificity for Larrea (Schultz et al., 1977; Chapman et al., 1988). Little is known, however, about how nutrient variation affects the plant's quantity of secondary defensive compounds. We report on how creosote bush resin, NDGA, protein content and quantity of herbivory changed with season, how these parameters were affected by the variation of nitrogen and water availability to plants growing under natural conditions, and how these parameters correlated with herbivore quantity on Larrea foliage. Materials and methods
The study site was a sandy wash woodland located in Living Desert Reserve near Palm desert, California (33°44'N, l16°2YW, elevation 60 m), where Larrea divaricata subsp. tridentata is the dominant perennial plant. The average annual precipitation (149 mm) is mostly of frontal origin and falls between December and March. Late summer precipitation (July to September) occurs as localized thunder storms and is highly variable. The average July maximum temperature exceeds 40°C. Five groups of 12 Larrea bushes were selected at the beginning of 1984, and divided into three replicated blocks. The following five treatments were randomly assigned to these blocks: (1) control (C), (2) water alone (W), (3) nitrogen applied to the foliage (FN), (4) nitrogen applied to the soil under the canopy (SN), and (5) combination of water and fertilizer (WN). Plants were irrigated and fertilized as described by Sharifi et al. (1987), and the quantity of herbivory was estimated as the percentage of leaves with herbivore damage on over 25% of the surface of a leaf from marked branches. Fully expanded mature leaves were collected seasonally between March of 1984 and December of 1985 from around each bush and were classified by their production date according to Sharifi et al. (1987). All plant samples were oven dried (60°C) within 48 h from the time of collection and the dried material stored at 4°C for further analysis. The resin was extracted with acetone, gravimetrically measured and its NDGA content quantified by HPLC as described by Gonzalez-Coloma et al. (1988). The protein content of the dried leaves was measured in duplicate following a modification of the Bradford method (Howard, 1987) and the samples calibrated against ribulose 1,5-diphosphate carboxylaseoxygenase standard (Sigma Chemical Co.). Data for resin, NDGA, protein content and quantity of herbivory on the creosote bush foliage produced at the begining of the experiment (spring of 1984) were analysed by ANOVA analysis to test for initial site-dependent variations. Treatment effects were evaluated by non-parametric Kruskall-Wallis analysis by the percentage ranking of the 1984 spring data, since significant initial site-dependent variations were detected. Differences between levels of class variables were tested by using 95% confidence intervals for means, or Mann-Whitney paired tests when appropriate. Measurements of insect damage were arcsine-transformed (Zar, 1984) before analysis because they were collected as percentages.
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T h e relationships b e t w e e n creosote b u s h foliar resin, N D G A , p r o t e i n content, a n d q u a n t i t y o f h e r b i v o r y for each collection date were e x a m i n e d by linear regression analysis. Regressions were p e f o r m e d using the m e a n q u a n t i t y of h e r b i v o r e activity p e r plot against the m e a n leaf resin, N D G A , p r o t e i n content a n d total biomass (according to Sharifi et al., 1987) values p e r plot.
Results T a b l e 1 shows the results o f the n o n - p a r a m e t r i c A N O V A analysis p e r f o r m e d on the data classified b y leaf p r o d u c t i o n date and t r e a t m e n t . T h e data were t r a n s f o r m e d as p e r c e n t o f the spring 1984 values to test for t r e a t m e n t effects. F o l i a r resin and N D G A content o f the p l a n t ( m g g -1 d r y weight) were affected by b o t h t r e a t m e n t and season, the N D G A c o n c e n t r a t i o n in the resin (mg N D G A g-1 resin) d i d not vary with these factors and the soluble p r o t e i n c o n t e n t showed a significant variation with t r e a t m e n t . T a b l e 2 shows the m e a n values o f the p a r a m e t e r s classified by t r e a t m e n t . T h e resin and N D G A content of Larrea foliage was lower in both irrigated g r o u p s ( W and W N ) when c o m p a r e d to the d r y ones (C, F N and SN). T h e p r o t e i n content was h i g h e r for the nitrogen
Table I. A N O V A results for leaf resin content and its concentration in NDGA, leaf soluble protein content and quantity of herbivory of Larrea shrubs by treatment and season Treatment Variable Resint (rag g-1 dry weight) NDGAt (rag g - 1 dry weight) NDGA (rag g - 1resin) Proteint (mg g-a dry weight)
Test statistic
df.:~
83"917
4 (129)
69"551
Season Level of significance
Test statistic
df.~
Level of significance
H.
5-349
4 (160)
*
4 (129)
***
5-832
4 (160)
H
1"115
4 (160)
NS
1"503
4 (160)
NS
9-571
3 (57)
*
0'253
3 (89)
NS
*, p < 0.05; *% p < 0.0005; ***, p < 0-0001;NS, not significant. t Treatmenteffectsevaluatedon the percentageofspring84 transformedvaluesto correctfor site-dependentdifferencesat the b~'nnlng of the experiment.Kruskan-Wallisanalysisby ranks. :[:Degreesof freedombetweengroupsand withingroups(). Table 2. Average values (standard error) of foliar resin, N D G A and protein content of manipulated Larrea tridentata bushes. Data is correctedfor initial
site-dependent differences Treatment Control Foliar nitrogen Soilnitrogen Water Water + nitrogen
Resin (mg g - t dry weight)* 238"9 (23" 1)°* 205"6 (29"3) a 204"0 (17"9) ~ 141"3 (1"50) b 131" 1 (12"7) b
NDGA (mg g- 1 dry weight)* 52-60 47"22 60"57 21"83 22"46
(7"50) a (9-19) ° (10"44) a (0"02) ~ (3"36) b
Protein (mg g- 1 dry weight)* 74"87 (3"97) a -110-9 (16"17)b 108"34 (3"32) ~ 72"83 (6"22) °
* Valuesin columnfollowedby the sameletter are not statisticallydifferent(Mann-Whitneypairedtests).
142
A. GONZALEZ-COLOMA E T AL. 400]
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I
Spring
Summer
I
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Fall Date
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Figure 1. Amountoffoliar resin ofmanipulated creosote bush in 1984-5. Treatmentsareasfollows: unwatered group (--0-) and water group ( - V - ) . Data are represented as mean + 1 S.E.M.
(N) and water (W) groups when compared to either the control (C) or the water plus nitrogen ( W N ) ones. Figure 1 shows the seasonal variation of the resin content. W e grouped the data in two sets, irrigated and non-irrigated plots, according to the results of the M a n n - W h i t n e y test. For the non-irrigated group (C, F N and SN) the resin levels increased in both springs, while it peaked in the fall of 1984 and the summer of 1985 in the irrigated plots (W and
WN). 120,
100
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/
80
/
/
://T
60 < Z 40
E 20
0
/ V
I
Spring
i
Summer
I
Fall Date
i
Spring
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Figure 2. NDGA content of manipulated creosote bush mature leaves in 1984-5. Treatments are as in Figure 1. Data are represented as mean + 1 S.E.M.
RESIN AND CHEMISTRY OF LARREA DIVARICATA
143
10
/
-t- 6
::4 co 2 co
I July
I Aug
I Nov
~0 ~''I Dec Date
I Mar
Aug
I Sep
Figure 3. Quantity of herbivory on manipulated creosote bush plants in 1984-5. Dates represent time of collection. Data are represented as mean percent eaten of total leaves + 1 S.E.M.
Figure 2 shows the seasonal variation of the foliar N D G A levels. The data were grouped as above. T h e amount of N D G A peaked in the fall of 1984 and the spring of 1985 for the non-irrigated group and in the summer of 1985 for the irrigated one. The quantity of herbivory on Larrea bushes did not show a significant variation with the treatments applied to these plants (p > 0"05), but a significant seasonal variation was detected (p < 0"0001) (Fig. 3). The results of the linear regressions between quantity of herbivory, Larrea chemistry and total biomass showed that none of the parameters measured had any relationship with the quantity of herbivory except for the amount of resin that had a negative, but not significant, correlation coefficient ((9 = - 0 " 8 3 , %r: = 0"69, p = 0"63).
Discussion
We will first discuss the seasonal variations of the parameters studied and then the effects that the different treatments had on these parameters in the light of the carbon/nutrient balance hypothesis.
Seasonal variations of Larrea chemistry and herbivory The protein content of mature foliage in creosote did not vary seasonally. This supports previous studies in which Larrea divaricata exhibited relatively constant proportions of proteins through time (Oechel et al., 1972). T h e average amounts of leaf resin varied seasonally. They were significantly higher in the spring than in summer or fall in the non-irrigated bushes. D o w n u m et al. (1988) observed a similar pattern of foliar resin variation in creosote bush leaves sampled in the same area during 1984.
1~
A. G O N Z A L E Z - C O L O M A E T AL.
The seasonal variation in NDGA content of Larrea leaves followed a different pattern than that observed for the resin content, suggesting differences in the control mechanisms responsible for their accumulation and/or variation in the plant. The variation that we observed in NDGA levels could also be affected by genetic and other environmental factors. Abiotic factors (such as ozone) affect NDGA levels in creosote (Gonzalez-Coloma et al., 1988) as does solar irradiance (Downum et al., 1988). There is also a high variability in NDGA content within and between Larrea populations (Greenfield & Shelly, 1987; Downum et al., 1988; Gonzalez-Coloma et al., 1988). The seasonal progression of herbivory was measured by counting the relative number of leaves which show 25% or more area consumed by herbivores. This is a conservative measure since we could not sample leaves totally consumed. Despite the conservative nature of these measurements they provide an interesting perspective on seasonal patterns of herbivory which varied with season according to the natural abundance of arthropods in the field (Schultz et al., 1977). The quantity of herbivory peaked in summer in both 1984 and 1985, being generally higher in 1985. This can be attributed to lower primary production during this year (Sharifi et al., 1987) and thus a smaller pool of resources available for Larrea herbivores. Resource manipulations effects on Larrea chemistry and herbivory Bryant et al. (1983) have suggested that the carbon-nutrient balance of individual plants strongly affects their allocation of resources to primary and secondary metabolism. They predict that fertilization with growth limiting nutrients will increase the nutrient content of leaves while decreasing their concentration in carbon-based secondary products. Nutrient levels of Larrea (proteins) We found that Larrea bushes treated with water plus nitrogen (WN) had the same soluble protein content as the control (C), while supplementation with either one separately increased this protein content. Previous studies have shown that Larrea tridentata supplementation with nitrogen (N) or water plus nitrogen (WN) increases its total nitrogen while the foliage production is only increased when the WN treatment is applied (Lighffoot & Whitford, 1987; Sharifi et al., 1987). This discrepancy between our results and these reports might be related to the different techniques used (i.e. they used Kjeldahl for total nitrogen; we used Bradford for proteins and/or to differences in the nitrogen metabolism between the treatment groups. Carbon-based defences (resin and N D G A ) The amount of resin was only affected by water supplementation. The watered group (W, WN) had more water (Meinzer et al., 1988) and less resin in the leaves than the unwatered one (C, FN, SN). Since the leaf resin coat acts as an ideal antitranspirant in Larrea (Meinzer et al., 1990) we propose a possible relationship between the lower resin content of the watered group and a decreased need to protect the leaves against water loss, although more research is needed to prove this hypothesis. The NDGA variation with treatment was similar to the variation observed for the resin (less NDGA in the watered group). Quantity of herbivory The nutrient supplementation of Larrea did not affect the quantity of herbivory. On the contrary, the results of Lighffoot & Whifford (1987) provide evidence that the increased water status of creosote plants in combination with higher levels of foliar nitrogen benefited populations of phytophagous insects, the sap-sucking insects being more responsive than leaf-chewing ones. It is important to emphasize that we only measured the impact of leaf-chewing insects.
RESIN AND CHEMISTRY OF LARREA DIVARICATA
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Additionally, carbon-based secondary products of Larrea (only resin) showed a negative relationship with the quantity of leaf-chewing herbivory. These results can be misleading because the pool sizes of leaves available for these herbivores varied between treatments and years (Sharifi et al., 1987). However, it has been shown that grasshopper herbivory on individual Larrea shrubs is highly variable; it is related to the variable quality of leaves for herbivores (resin and NDGA content) and has a strong genetic component within populations (Greenfield & Shelly, 1987; Greenfield et al., 1990) although there is evidence that other compounds in addition to NDGA, are important in this system (Greenfield & Gonzalez-Coloma unpublished results). Further exploratory chemical work will be necessary to fully understand this interaction.
Summary We measured the impact that water and nitrogen manipulation had on protein content, resin chemistry and quantity of herbivory and the seasonal variation of these parameters of naturally growing Larrea divaricata subsp, tridentata bushes. The soluble protein content of Larrea increased with either water of nitrogen supplementation, supporting the carbon-nutrient hypothesis. The carbon-based defences of creosote bush (resin and NDGA foliar content) were affected (lowered) by water supplementation, while the nitrogen fertilization did not have any effect on these parameters. The creosote bush NDGA concentration in resin was independent of the leaf production date and treatment. The amount of resin had a negative relationship with the quantity of leaf-chewing herbivory on creosote bush, although not significant under our experimental conditions. This research was supported by N.S.F. grant No. BSR82-16814, the Center for Intermedia Transport Research (EPA Contract CR-8117201) and the Ecological Research Division of the Office of Health and Enviornmental Research, U.S. Department of Energy. We gratefully acknowledge Dr Joseph Hoffmann for his help in improving this manuscript.
References Abrahamson, W. G., Anderson, S. S. & McCrea, K. D. (1988). Effects of manipulation of plant carbon nutrient balance on tall golddenrod resistance to a gall making herbivore. Oecologia (Berlin) 77: 302-306. Barbour, M. G., MacMahon, J. A., Bamberg, S. A. & Ludwig, J. A. (1977). Structure and distribution of Larrea communities. In: Mabry, T. J., Hunziker, J. H. & Difeo, D. R. (Eds), Creosote bush. Biology and Chemistry of Larrea in New World Deserts, Stroudsberg, Pennsylvania: pp. 177-251. Dowden, Hutchinson & Ross. Bryant, J. P., Clausen, T. P., Reichardt, P. B., McCarthy, M. C. & Werner, R. A. (1987). Effect of nitrogen fertilization upon the secondary chemistry and nutritional value of quaking aspen (Populus tremuloides Michx.) leaves for the large aspen (Chon'stoneura conflictana Walker). Oecologia (Berlin) 73: 513--517. Chapman, R. F., Bernays, E. A. & Wyatt, T. (1988). Chemical aspects of host-plant specificity in three Larrea-feeding grasshoppers. Journal of Chemical Ecology 14: 561-579. Downum, K. R., Dole, J. & Rodriguez, E. (1988). Nordihydroguaiaretic acid: inter- and intrapopulational variation in the Sonoran Desert creosote bush (Larrea tridentata, Zygophyllaceae). Biochemical Systematics and Ecology, 16:551-555. Gonzalez-Coloma, A., Wisdom, C. S. & Rundel, P. W. (1988). Ozone impact on the antioxidant nordihydroguaiaretic acid content in the external leaf resin of Larrea tridentata.Biochemical Systematics and Ecology, 16: 59-64. Greenfield, M. D. & Shelly, T. (1987). Variation in host-plant quality: implications for territoriality in a desert grasshopper. Ecology, 68: 828-838.
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Greenfield, M. D., Shelly, T. E. & Gonzalez-Coloma, A. (1989). Territory selection in a desert grasshopper: the maximization of conversion efficiency on a chemically defended shrub. Journal of Animal Ecology, 58: 761-771. Howard, J. J. (1987). Leafcutting ant diet selection: The role of nutrients, water and secondary chemistry. Ecology, 68: 503-515. Lightfoot, D. C. & Whitford, W. G. (1987). Variation in insect densities on desert creosote bush: is nitrogen a factor? Ecology, 68: 547-557. Mabry, T. J., Difeo, Jr., D. R., Sakakibara, M., Bohnstedt, C. F. & Siegler, D. (1977). Natural products chemistry of Larrea. In: Mabry, T. J., Hunziker, J. H. & Difeo, D. R. (Eds), Creosote Bush. Biology and Chemistry of Larrea in New World Deserts, pp. 115-134. Stroudsberg, Pennsylvania: Dowden, Hutchinson & Ross. Mattson, Jr., W. J. (1980). Herbivory in relation to plant nitrogen content. Annual Review of Ecology and Systematics, 11: 119-161. Mattson, W. J. & Haack, R. A. (1987). The role of drought in outbreaks of plant-eating insects. Drought's physiological effects on plants can predict its influence on insect populations. BioScience, 37:110-118. Meinzer, F. C., Sharifi, M. R., Nilsen, E. T. & Rundel, P. W. (1988). Effects of manipulation of water and nitrogen regime on the water relations of the desert shrub Larrea tridentata. Oecologia (Berlin), 77: 480-486. Meinzer, F. C., Wisdom, C. S., Gonzalez-Coloma, A., Rundel, P. W. & Shutltz, L. M. (1990). Effects of leaf resin on stomatal behaviour and gas exchange of Larrea tridentata (DC.) Cov. Functional Ecology, 4: 579-584. Oechel, W. C., Strain, B. R. & Odening, W. R. (1972). Tissue water potential, photosynthesis, 14C-labeled photosynthate utilization, and growth in the desert shrub Larrea divaricata. Ecological Monographs, 42: 127-141. Oliveto, E. P. (1972). Nordihydroguaiaretic acid. A naturally occurring antioxidant. Chemical Industry, 17: 677-679. Rhoades, D. F. (1977). Integrated antiherbivore, antidessicant and ultraviolet screening properties of creosote bush resin. Biochemical Systematics and Ecology, 5:281-290. Schultz, J. D., Otte, D. & Enders, F. (1977). Larrea as a habitat component for desert arthropods. In: Mabry, T. J., Hunziker, J. H. & Difeo, D. R. (Eds), Creosote Bush. Biology and Chemistry of Larrea in New World Deserts, pp. 126-208. Stroudsberg, Pennsylvania: Dowden, Hutchinson & Ross. Sharifi, M. R., Meinzer, F. C., Nilsen, E. T., Rundel, P. W., Virginia, R. A., Jarrel, W. M. & Herman, D. J. (1987). Effect of resource manipulation on the quantitative phenology of Larrea tridentata (creosote bush) in the Sonoran Desert of California. American Journal of Botany, 75: 1163-1174.
White, T. C. R. (1984). The abundance of invertebrate herbivores in relation to the availability of nitrogen in stressed food plants. Oecologia (Berlin), 63: 90-105. Zar, J. H. (1984). Biostatistical Analysis. Engelwood Cliffs, N J: Prentice Hall.
Water and nitrogen manipulations of the desert shrub Larrea diraricata subsp, tridentata (Zygophyllaceae)
A. Gonzalez-Coloma*, C. S. Wisdom, M. R. Sharifi& P. W. Rundel Laboratory of Biomedical and Environmental Sciencest, Universityof California, 900 VeteranAvenue, Los Angeles, California 90024, U.S.A. (Received 26 November 1992, accepted 19 March 1993) Water supplementation of Larrea divaricata subsp, tridentata bushes growing under natural conditions affected the amount of resin, nordihydroguaiaretic acid (NDGA) and soluble protein present in the leaves. Nitrogen fertilization had no effect on these parameters. The total resin and NDGA content also varied with the leaf production dates, but the NDGA concentration in the resin was not affected by either the treatment or the time of production. Herbivore activity, measured as a percentage of the leaves eaten, was not affected by these manipulations, varied with season (highest during the summer) and had a negative correlation with the resin content of the plant.
Keywords: Larrea divaricata subsp, tridentata; nitrogen fertilization; irrigation; resin; nordihydroguaiaretic acid; herbivory
Introduction T h e availability of resources to plants has been frequently proposed to influence the relationship between plants and their associated arthropods. Plant nitrogen and water content can have a direct influence on host plant choice due to their concentrations in plant tissue (Mattson, 1980; White, 1984; Mattson & Haack, 1987; Lighffoot & Whitford, 1987) and indirectly by changing plant secondary compounds (Bryant et al., 1987; Abrahamson et al., 1988). To examine the effect of changes in the water and nitrogen levels on the chemistry that mediates the plant-insect relationships of arid land plants, we manipulated water and nitrogen concentrations available to the creosote bush (Larrea divaricata subsp, tridentata) (DC.) Felger & Lowe. T h e creosote bush is an evergreen xerophytic shrub abundant in warm desert regions of the south-western United States and Mexico. Its drought resistance allows it to remain metabolically active and produce new leaves and shoots under extremely dry conditions (Sharifi et al., 1988). The abundance of this shrub and its perennial growth habits make Larrea one of the most predictable plant resources in many desert environments (Barbour et al., 1977). T h e leaves are covered with a resinous coating which functions as a barrier to prevent water loss from leaf surfaces, a solar-ultra-violet filter and an insect feeding deterrent (Chapman et al., 1988; Meinzer et al., 1990; Rhoades, 1977). * Address for correspondence: Centro de Ciencias Medio-ambientales, CSIC, Sevrano 115-dpdo., 28006, Madrid, Spain. t Operated for the U.S. Department of Energy by the University of California under contract No. DE-ACO3-76-SF00012.
0140-1963/94/020139 + 08 $00'00/0
© 1994AcademicPress Limited
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This resin is composed of a complex mixture of phenolics, saponins, terpenoids and wax esters that account for 10-20% of the leaf dry weight. Over 80% of the resin is composed of phenolic aglycones, with the major component being norihydroguaiaretic acid (NDGA), a catechol lignan. The remaining resin is a complex mixture of partially o-methylated flavones and flavonols along with small amounts of acid and basic components (Mabry et al., 1977). NDGA is a potent antioxidant (Oliveto, 1972) and also exhibits important bactericidal, fungicidal and antiherbivore properties (Rhoades, 1977; Greenfield & Shelly, 1987; Chapman et al., 1988; Gonzalez-Coloma et al., 1988). Nitrogen content is an important regulator of insect populations on plants (Mattson, 1980). Likewise, variation of nutrient availability within the creosote bush is correlated with the variation in the number of foliage arthropods (Lighffoot & Whitford, 1987), which exhibit various degrees of specificity for Larrea (Schultz et al., 1977; Chapman et al., 1988). Little is known, however, about how nutrient variation affects the plant's quantity of secondary defensive compounds. We report on how creosote bush resin, NDGA, protein content and quantity of herbivory changed with season, how these parameters were affected by the variation of nitrogen and water availability to plants growing under natural conditions, and how these parameters correlated with herbivore quantity on Larrea foliage. Materials and methods
The study site was a sandy wash woodland located in Living Desert Reserve near Palm desert, California (33°44'N, l16°2YW, elevation 60 m), where Larrea divaricata subsp. tridentata is the dominant perennial plant. The average annual precipitation (149 mm) is mostly of frontal origin and falls between December and March. Late summer precipitation (July to September) occurs as localized thunder storms and is highly variable. The average July maximum temperature exceeds 40°C. Five groups of 12 Larrea bushes were selected at the beginning of 1984, and divided into three replicated blocks. The following five treatments were randomly assigned to these blocks: (1) control (C), (2) water alone (W), (3) nitrogen applied to the foliage (FN), (4) nitrogen applied to the soil under the canopy (SN), and (5) combination of water and fertilizer (WN). Plants were irrigated and fertilized as described by Sharifi et al. (1987), and the quantity of herbivory was estimated as the percentage of leaves with herbivore damage on over 25% of the surface of a leaf from marked branches. Fully expanded mature leaves were collected seasonally between March of 1984 and December of 1985 from around each bush and were classified by their production date according to Sharifi et al. (1987). All plant samples were oven dried (60°C) within 48 h from the time of collection and the dried material stored at 4°C for further analysis. The resin was extracted with acetone, gravimetrically measured and its NDGA content quantified by HPLC as described by Gonzalez-Coloma et al. (1988). The protein content of the dried leaves was measured in duplicate following a modification of the Bradford method (Howard, 1987) and the samples calibrated against ribulose 1,5-diphosphate carboxylaseoxygenase standard (Sigma Chemical Co.). Data for resin, NDGA, protein content and quantity of herbivory on the creosote bush foliage produced at the begining of the experiment (spring of 1984) were analysed by ANOVA analysis to test for initial site-dependent variations. Treatment effects were evaluated by non-parametric Kruskall-Wallis analysis by the percentage ranking of the 1984 spring data, since significant initial site-dependent variations were detected. Differences between levels of class variables were tested by using 95% confidence intervals for means, or Mann-Whitney paired tests when appropriate. Measurements of insect damage were arcsine-transformed (Zar, 1984) before analysis because they were collected as percentages.
RESIN AND CHEMISTRY OF LARREA DIVARICATA
141
T h e relationships b e t w e e n creosote b u s h foliar resin, N D G A , p r o t e i n content, a n d q u a n t i t y o f h e r b i v o r y for each collection date were e x a m i n e d by linear regression analysis. Regressions were p e f o r m e d using the m e a n q u a n t i t y of h e r b i v o r e activity p e r plot against the m e a n leaf resin, N D G A , p r o t e i n content a n d total biomass (according to Sharifi et al., 1987) values p e r plot.
Results T a b l e 1 shows the results o f the n o n - p a r a m e t r i c A N O V A analysis p e r f o r m e d on the data classified b y leaf p r o d u c t i o n date and t r e a t m e n t . T h e data were t r a n s f o r m e d as p e r c e n t o f the spring 1984 values to test for t r e a t m e n t effects. F o l i a r resin and N D G A content o f the p l a n t ( m g g -1 d r y weight) were affected by b o t h t r e a t m e n t and season, the N D G A c o n c e n t r a t i o n in the resin (mg N D G A g-1 resin) d i d not vary with these factors and the soluble p r o t e i n c o n t e n t showed a significant variation with t r e a t m e n t . T a b l e 2 shows the m e a n values o f the p a r a m e t e r s classified by t r e a t m e n t . T h e resin and N D G A content of Larrea foliage was lower in both irrigated g r o u p s ( W and W N ) when c o m p a r e d to the d r y ones (C, F N and SN). T h e p r o t e i n content was h i g h e r for the nitrogen
Table I. A N O V A results for leaf resin content and its concentration in NDGA, leaf soluble protein content and quantity of herbivory of Larrea shrubs by treatment and season Treatment Variable Resint (rag g-1 dry weight) NDGAt (rag g - 1 dry weight) NDGA (rag g - 1resin) Proteint (mg g-a dry weight)
Test statistic
df.:~
83"917
4 (129)
69"551
Season Level of significance
Test statistic
df.~
Level of significance
H.
5-349
4 (160)
*
4 (129)
***
5-832
4 (160)
H
1"115
4 (160)
NS
1"503
4 (160)
NS
9-571
3 (57)
*
0'253
3 (89)
NS
*, p < 0.05; *% p < 0.0005; ***, p < 0-0001;NS, not significant. t Treatmenteffectsevaluatedon the percentageofspring84 transformedvaluesto correctfor site-dependentdifferencesat the b~'nnlng of the experiment.Kruskan-Wallisanalysisby ranks. :[:Degreesof freedombetweengroupsand withingroups(). Table 2. Average values (standard error) of foliar resin, N D G A and protein content of manipulated Larrea tridentata bushes. Data is correctedfor initial
site-dependent differences Treatment Control Foliar nitrogen Soilnitrogen Water Water + nitrogen
Resin (mg g - t dry weight)* 238"9 (23" 1)°* 205"6 (29"3) a 204"0 (17"9) ~ 141"3 (1"50) b 131" 1 (12"7) b
NDGA (mg g- 1 dry weight)* 52-60 47"22 60"57 21"83 22"46
(7"50) a (9-19) ° (10"44) a (0"02) ~ (3"36) b
Protein (mg g- 1 dry weight)* 74"87 (3"97) a -110-9 (16"17)b 108"34 (3"32) ~ 72"83 (6"22) °
* Valuesin columnfollowedby the sameletter are not statisticallydifferent(Mann-Whitneypairedtests).
142
A. GONZALEZ-COLOMA E T AL. 400]
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Figure 1. Amountoffoliar resin ofmanipulated creosote bush in 1984-5. Treatmentsareasfollows: unwatered group (--0-) and water group ( - V - ) . Data are represented as mean + 1 S.E.M.
(N) and water (W) groups when compared to either the control (C) or the water plus nitrogen ( W N ) ones. Figure 1 shows the seasonal variation of the resin content. W e grouped the data in two sets, irrigated and non-irrigated plots, according to the results of the M a n n - W h i t n e y test. For the non-irrigated group (C, F N and SN) the resin levels increased in both springs, while it peaked in the fall of 1984 and the summer of 1985 in the irrigated plots (W and
WN). 120,
100
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80
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I
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i
Summer
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i
Spring
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Figure 2. NDGA content of manipulated creosote bush mature leaves in 1984-5. Treatments are as in Figure 1. Data are represented as mean + 1 S.E.M.
RESIN AND CHEMISTRY OF LARREA DIVARICATA
143
10
/
-t- 6
::4 co 2 co
I July
I Aug
I Nov
~0 ~''I Dec Date
I Mar
Aug
I Sep
Figure 3. Quantity of herbivory on manipulated creosote bush plants in 1984-5. Dates represent time of collection. Data are represented as mean percent eaten of total leaves + 1 S.E.M.
Figure 2 shows the seasonal variation of the foliar N D G A levels. The data were grouped as above. T h e amount of N D G A peaked in the fall of 1984 and the spring of 1985 for the non-irrigated group and in the summer of 1985 for the irrigated one. The quantity of herbivory on Larrea bushes did not show a significant variation with the treatments applied to these plants (p > 0"05), but a significant seasonal variation was detected (p < 0"0001) (Fig. 3). The results of the linear regressions between quantity of herbivory, Larrea chemistry and total biomass showed that none of the parameters measured had any relationship with the quantity of herbivory except for the amount of resin that had a negative, but not significant, correlation coefficient ((9 = - 0 " 8 3 , %r: = 0"69, p = 0"63).
Discussion
We will first discuss the seasonal variations of the parameters studied and then the effects that the different treatments had on these parameters in the light of the carbon/nutrient balance hypothesis.
Seasonal variations of Larrea chemistry and herbivory The protein content of mature foliage in creosote did not vary seasonally. This supports previous studies in which Larrea divaricata exhibited relatively constant proportions of proteins through time (Oechel et al., 1972). T h e average amounts of leaf resin varied seasonally. They were significantly higher in the spring than in summer or fall in the non-irrigated bushes. D o w n u m et al. (1988) observed a similar pattern of foliar resin variation in creosote bush leaves sampled in the same area during 1984.
1~
A. G O N Z A L E Z - C O L O M A E T AL.
The seasonal variation in NDGA content of Larrea leaves followed a different pattern than that observed for the resin content, suggesting differences in the control mechanisms responsible for their accumulation and/or variation in the plant. The variation that we observed in NDGA levels could also be affected by genetic and other environmental factors. Abiotic factors (such as ozone) affect NDGA levels in creosote (Gonzalez-Coloma et al., 1988) as does solar irradiance (Downum et al., 1988). There is also a high variability in NDGA content within and between Larrea populations (Greenfield & Shelly, 1987; Downum et al., 1988; Gonzalez-Coloma et al., 1988). The seasonal progression of herbivory was measured by counting the relative number of leaves which show 25% or more area consumed by herbivores. This is a conservative measure since we could not sample leaves totally consumed. Despite the conservative nature of these measurements they provide an interesting perspective on seasonal patterns of herbivory which varied with season according to the natural abundance of arthropods in the field (Schultz et al., 1977). The quantity of herbivory peaked in summer in both 1984 and 1985, being generally higher in 1985. This can be attributed to lower primary production during this year (Sharifi et al., 1987) and thus a smaller pool of resources available for Larrea herbivores. Resource manipulations effects on Larrea chemistry and herbivory Bryant et al. (1983) have suggested that the carbon-nutrient balance of individual plants strongly affects their allocation of resources to primary and secondary metabolism. They predict that fertilization with growth limiting nutrients will increase the nutrient content of leaves while decreasing their concentration in carbon-based secondary products. Nutrient levels of Larrea (proteins) We found that Larrea bushes treated with water plus nitrogen (WN) had the same soluble protein content as the control (C), while supplementation with either one separately increased this protein content. Previous studies have shown that Larrea tridentata supplementation with nitrogen (N) or water plus nitrogen (WN) increases its total nitrogen while the foliage production is only increased when the WN treatment is applied (Lighffoot & Whitford, 1987; Sharifi et al., 1987). This discrepancy between our results and these reports might be related to the different techniques used (i.e. they used Kjeldahl for total nitrogen; we used Bradford for proteins and/or to differences in the nitrogen metabolism between the treatment groups. Carbon-based defences (resin and N D G A ) The amount of resin was only affected by water supplementation. The watered group (W, WN) had more water (Meinzer et al., 1988) and less resin in the leaves than the unwatered one (C, FN, SN). Since the leaf resin coat acts as an ideal antitranspirant in Larrea (Meinzer et al., 1990) we propose a possible relationship between the lower resin content of the watered group and a decreased need to protect the leaves against water loss, although more research is needed to prove this hypothesis. The NDGA variation with treatment was similar to the variation observed for the resin (less NDGA in the watered group). Quantity of herbivory The nutrient supplementation of Larrea did not affect the quantity of herbivory. On the contrary, the results of Lighffoot & Whifford (1987) provide evidence that the increased water status of creosote plants in combination with higher levels of foliar nitrogen benefited populations of phytophagous insects, the sap-sucking insects being more responsive than leaf-chewing ones. It is important to emphasize that we only measured the impact of leaf-chewing insects.
RESIN AND CHEMISTRY OF LARREA DIVARICATA
145
Additionally, carbon-based secondary products of Larrea (only resin) showed a negative relationship with the quantity of leaf-chewing herbivory. These results can be misleading because the pool sizes of leaves available for these herbivores varied between treatments and years (Sharifi et al., 1987). However, it has been shown that grasshopper herbivory on individual Larrea shrubs is highly variable; it is related to the variable quality of leaves for herbivores (resin and NDGA content) and has a strong genetic component within populations (Greenfield & Shelly, 1987; Greenfield et al., 1990) although there is evidence that other compounds in addition to NDGA, are important in this system (Greenfield & Gonzalez-Coloma unpublished results). Further exploratory chemical work will be necessary to fully understand this interaction.
Summary We measured the impact that water and nitrogen manipulation had on protein content, resin chemistry and quantity of herbivory and the seasonal variation of these parameters of naturally growing Larrea divaricata subsp, tridentata bushes. The soluble protein content of Larrea increased with either water of nitrogen supplementation, supporting the carbon-nutrient hypothesis. The carbon-based defences of creosote bush (resin and NDGA foliar content) were affected (lowered) by water supplementation, while the nitrogen fertilization did not have any effect on these parameters. The creosote bush NDGA concentration in resin was independent of the leaf production date and treatment. The amount of resin had a negative relationship with the quantity of leaf-chewing herbivory on creosote bush, although not significant under our experimental conditions. This research was supported by N.S.F. grant No. BSR82-16814, the Center for Intermedia Transport Research (EPA Contract CR-8117201) and the Ecological Research Division of the Office of Health and Enviornmental Research, U.S. Department of Energy. We gratefully acknowledge Dr Joseph Hoffmann for his help in improving this manuscript.
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