Herbivory defense and growth tradeoffs along a moisture gradient in Lupinus latifolius var. columbianus

Lilly Boiton1, James Powers2, and Jordan Waits3

1University of California, Los Angeles; 2University of California, Santa Barbara; 3University of California, San Diego

ABSTRACT

Hypotheses such as the plant stress hypothesis, apparency theory, and the resource availability hypothesis provide contrasting predictions to how plants respond to abiotic stress and their interactions with herbivores. In this paper we examined the effects of a water availability gradient on the morphological characteristics and herbivory of Lupinus latifolius var. columbianus. We measured the abundance of the leaf beetle, Galeruca rudis, and Aphididae as well as the amount of leaf herbivory damage on lupine individuals as a response to soil moisture, proximity to an adjacent stream, and thickness of their leaves. Positive trends were found for the leaf thickness of L. latifolius with distance from water and resulting drier soil. Thinner leaves, increased growth, and increased herbivory were positively associated with plants that were closer to the stream, where soil was moister. G. rudis was associated with lupines in moist soils while aphid activity was higher among plants farther away from our creek where soils were drier. Our findings corroborate the resource availability hypothesis, which predicts that plant species growing along a gradient of resource availability (ie. soil moisture) divert valuable resources from vegetative growth, towards constitutive plant defense when resources are scarce. Additionally, we have shown that the resource availability hypothesis can be applied intraspecifically, as well as interspecifically.

Keywords: resource availability hypothesis, phenotypic plasticity, herbivory, constitutive plant defense, Lupinus latifolius

INTRODUCTION in response to varying light availability. Furthermore, plants are able to Despite being sessile organisms, plants can ontogenetically develop shifts in phenotype respond to environmental gradients by across clines of resources or interacting altering a variety of morphological and organisms. This phenotypic plasticity for physiological characteristics in order to instance allows plants grown in sun to have increase fitness. For example, (Clausen et al. thicker leaf cuticles and contain more 1948) found that the height and leaf size of stomata than leaves from plants grown in Achillea millefolium growing along Sierra shade (Cooper and Qualls 1967, Boardman Nevada mountain ranges varies dramatically 1977). Thicker leaves are advantageous in

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water-limited or high temperature defense investment was dictated by environments because they reduce amounts of herbivory, the RAH points to evapotranspiration (Cooper and Qualls resource availability as the ultimate cause of 1967, Boardman 1977, Yeats and Rose 2013, defense investment, regardless of herbivore Yang et. al. 2016). In addition, herbivory on a pressure. This efficiency in resource plant can induce it to increase production of allocation allows plants on either end of a chemical defenses to protect against future resource gradient may display comparable attack (Chen 2008, Karban 1997). Thus, realized growth rates when accounting for there are several models describing how relative decreases in intrinsic growth rates phenotypic plasticity in plants allows them from investments in defense or losses to to make tradeoffs to compensate for abiotic herbivores (Endara and Coley 2011). In the stressors and herbivory. case of water stress, plants will decrease The plant stress and optimal defense growth while maintaining photosynthetic hypotheses predict that plants under abiotic activity per unit leaf area, leading to a build- stress (i.e. low water or nutrients) are up of carbohydrates that can be diverted harmed more by herbivory and are less able into developing thicker leaf cuticles or higher to produce defenses to combat herbivory tannin concentrations (Bradford and Hsiao (White 1969, Rhoades 1979). Alternatively, 1982, Herms and Mattson 1992). Effectively, the plant vigor hypothesis and apparency plants adopt a “two birds, one stone” theory argue that faster growing, healthier strategy when their plastic responses to shoots are more appealing to herbivores, are resource scarcity (ie. thicker leaves in dry “bound to be found,” and are consumed areas) also serve to make them less more (Price 1991, Feeny 1976). Price, appealing to herbivores. however, adds that there is a need for a These theories are somewhat limiting in diversity of hypotheses describing the their scope of plant-herbivore interactions, gradients of interactions between plants and as they primarily examined interspecific their herbivores in order to accurately variation. As a result, examining how well explain them and their complexity. The the RAH describes intraspecific variation in resource availability hypothesis (RAH) plant growth and defense has recently therefore provides a framework that become an important topic. Specifically, encompasses these interactions as a there seems to be disagreement on how well continuum, rather than placing importance the RAH predicts constitutive defense of on one side of the spectrum (White 1969, plants growing in resource-deficient Feeny 1976, Rhoades 1979). It argues that environments (Hahn and Maron 2016). plants grown in high resource environments Much work has been done discussing how grow quickly and allocate little energy to lupine, along with other nitrogen fixers, can constitutive defenses, adopting a strategy of cheaply produce alkaloids and other herbivory tolerance. On the other hand, nitrogen containing chemical defenses that plants grown in low resource environments can be induced by herbivory (Krischik et al. grow slowly and allocate more energy into 1991, Bernays and Chapman 2007). constitutive defenses, in order to resist However, lupine seems to have been herbivory (Coley et al. 1985). In contrast with overlooked as a model organism with which apparency theory, which argued that to test intraspecific variation in constitutive

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defenses along a gradient of resource stress. 2.2 Experiment 1: Field Survey Lupine contains lower concentrations of alkaloids in leaves than in reproductive parts To test the effects of various abiotic factors (Bernays and Chapman 1994). As a result, it’s on L. latifolius we measured the soil possible that it may protect its leaves with moisture and distance from stream of lupine more quantitative (constitutive) rather than plants as well as the leaf herbivory of each qualitative (induced) defenses as it becomes plant. Five transects were taken at Valentine more water limited. As discussed above, Camp; transects were run perpendicular to plants may accomplish this indirectly by Mammoth creek into the surrounding producing thicker leaves meant to limit sagebrush steppe vegetation in order to evapotranspiration. Such leaves may contain capture a moisture gradient. Each transect higher concentrations of tannins or thicker was 100 m in length and two m in width with layers of cellulose that may be less palatable their origin at Mammoth Creek and to phytophagous insects. In the eastern extending uphill in elevation. Lupines along Sierra Nevada, Lupinus latifolius var. each transect were surveyed with a columbiana is often found along a gradient minimum of three meters between each from moist, stream fed meadows into drier, plant recorded. Leaf thickness was sagebrush chaparral, making it a promising measured at the center of the midrib of lupine species with which to test this idea. three leaflets from three different leaves for Along this gradient, we expect to find that L. each plant, the average leaf thickness was latifolius is smaller, has thicker leaves, and recorded. The height of each plant was experiences less herbivory in drier soils. measured from the base of the plant to the highest stem. Plants were checked for the METHODS presence of aphids and the leaf beetle Galeruca rudis using a visual survey. Soil 2.1 Site samples were taken from the base of each plant surveyed and dried at 60 °C for 24 All field data and specimens were collected hours in order to calculate the percent mass at Valentine Eastern Sierra Reserve on the of moisture present in each sample. On each eastern slope of the Sierra Nevada in plant three leaves were photographed and California at roughly 2,500 m in elevation. analyzed for percent leaf herbivory damage Valentine Camp is a UC Natural Reserve and (quantified as missing leaf tissue) using the has been protected from grazing since the LeafByte application (Adam Campbell, vers. early 1900s. Surveys were carried out 1.2.0). adjacent to Mammoth Creek in mixed sagebrush and meadow habitats. Surveys 2.3 Experiment 2: Beetle Choice Trials were conducted between 10:00 AM and 4:00 PM on July 31 and August 1 and 2. Specimens of G. rudis, were collected from Weather conditions were clear throughout a mixed range of plants in Valentine Camp with temperatures ranging from 20–27 °C. Reserve. Cuttings of lupine plants were taken from within 20 m of Mammoth creek as well as 100 m uphill from Mammoth Creek in the sagebrush steppe. One lupine taken

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from both near and far from the creek were creek. We found that plants in drier soils had each placed in a plastic container of roughly thicker leaves than those in moist soils (N= 0.5 m by 0.5 m on opposing sides. Cuttings 49, R2=0.160, p=0.004, Figure 1). It was also were trimmed to approximate each other in found that plant height was higher for size and leaf count; flowers were removed lupines found in moist soil (N = 49, R2 = from all plants. Stems were wrapped in 0.137, p=0.009, Figure 2). A multiple linear dampened paper towels to prevent wilting regression showed that greater herbivory during the experiment. Fifty G. rudis were rates were associated with thinner leaves, placed into the enclosure and were left for higher soil moisture, and proximity of plants ninety minutes in lab conditions. At the end to Mammoth Creek (Whole Model: N = 49, of ninety minutes plants were removed and R2=0.362, F=8.52, p<0.001, Thickness: t= the number of G. rudis found on each plant -3.39, p=0.002, Moisture: t=-2.20, p=0.033, was recorded. This trial was repeated three Figure 3, Distance: t=-2.65, p=0.011). times with new clippings and G. rudis for a A multiple logistic regression for G. rudis total of one hundred and fifty beetles. showed their presence was associated with higher soil moisture but no pattern was 2.4 Statistical Analysis observed for beetles with either distance from stream or leaf thickness (Whole Model: Soil moisture and leaf herbivory (as a N=49, X2=10.8, p=0.013, Moisture: X2=3.97, percentage of leaf tissue missing) were log p=0.046, Figure 4, Distance: X2=0.12, p=0.73, transformed using excel in order to run Figure 5, Thickness: X2=1.20, p=0.27). In the parametric statistical tests in JMP Statistical case of aphids there was a strong association Software (SAS Institutive, vers. 14.0). Linear between greater distance from Mammoth regressions were run to determine the effect Creek and aphid presence but no of soil moisture on plant growth and relationship with leaf thickness or soil investment strategies such as height and leaf moisture (Whole Model: N=38, X2=7.72, thickness. A multiple linear regression was p=0.052, Moisture: X2=0.63, p=0.428, Figure also run on the effects of leaf thickness, soil 4, Distance: X2=5.39, p=0.02, Figure 5, moisture, and distance from the river on leaf Thickness: X2=0.17, p=0.678). herbivory. Multiple logistic regressions were run to determine the effects of distance, soil 3.2 G. rudis Choice Trials moisture, and leaf thickness on the presence of aphids and G. rudis. A chi-square analysis Of 150 beetles placed in enclosures, 82 was performed on choice trial data to were found on plants at the end of 90 determine if beetle choice deviated from the minutes. Forty-nine beetles were found on expected random results of 50% of beetles plants taken from near the creek while 33 choosing leaves from near vs. far plants. were found on plants taken uphill from Mammoth Creek. G. rudis were marginally RESULTS more likely to be found on plants taken from near Mammoth Creek than on plants taken 3.1 Field Survey uphill from Mammoth Creek (X2=3.08, p=0.079). In total, 53 plants were surveyed across five transects branching out from Mammoth

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Figure 1: Leaf thickness of Lupinus latifolius versus soil moisture. Leaf thickness is shown as the average Figure 3: Rate of log leaf herbivory of Lupinus measurement leaf thickness for Lupinus latifolius latifolius versus leaf thickness. Leaf herbivory is plants as measured at the midrib. Soil moisture represented as the proportion of leaf area eaten on represents the percentage of water in the soil when each lupine. Thickness represents the leaf thickness sampled as measured by wet mass lost. Leaf thickness of the lupin as measured at the midrib. We found a increases with decreasing soil moisture (N=49, negative trend between leaf thickness and rate of 2 R =0.16, p=0.004). herbivory (N=49, t=-3.39, p=0.0015).

Figure 2: Plant height versus soil moisture. Soil Figure 4: Aphid and G. rudis presence and absence moisture represents the mass percentage of water in by soil moisture. Soil moisture is reported as the the soil sample as measured by wet mass lost when percentage of mass lost in soil when dried. Bars dried. As soil moisture increases, we see an increase represent the average soil moisture of plants where in plant height as measured from the ground to the aphids and beetles were either present or absent. 2 highest stem (N=49, R =0.137, p=0.009). Beetles were more likely to be found on plants in more moist soil (N=49, X2=3.97, p=0.0463) no such effect was present for the distribution of aphids (N=38, X2=0.63, p=0.428).

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suber showed its high foliage plasticity, and this can also be seen in L. latifolius per our results on leaf thickness. Our findings that lupine in drier soils had decreased growth and thicker leaves corroborates the RAH’s key concept that plants in resource limiting environments invest more in defenses than in vegetative growth (Endara and Coley 2011). In addition, decreased herbivory on lupine in drier soils showed that leaf thickness was an effective deterrent of herbivory, thus maintaining fitness of lupine Figure 5: Aphid and G. rudis presence and absence despite stress from water scarcity. by distance. Aphids were found on plants that The defense that L. latifolius built to averaged a distance of approximately sixty meters enhance resistance to water stress also from Mammoth creek and were less likely to be found on plants near the river (X2=5.39, p=0.02). No effect served as a defense against herbivores. Our of distance to creek was found for beetle presence results show that leaf herbivory decreased (X2=0.12, p=0.73). as the thickness of the leaves increased, which supported our hypothesis that there DISCUSSION would be less herbivory away from Mammoth Creek. Plants under more The results of the observational study drought stress increase their defenses support our hypotheses on plant stress against herbivory because it is less costly to response, which was that there would be invest in defenses than lose foliage to thinner leaves and increased growth on L. herbivores (Endara and Coley 2011). The latifolius closer to Mammoth Creek. There plants farther from the creek had more was a negative correlation between soil resistance to herbivory, and our choice trials moisture and leaf thickness, as the further tested the effect of these defenses on the the plant was from the creek, the thicker its leaves were. Similarly, plant height positively preferences of G. rudis. The results of the associated with soil moisture. Lupine further choice trials provide some support for our from the stream were under more water original hypothesis that G. rudis would stress because water was not as easily choose stalks taken from near Mammoth accessed. The water gradient we studied at Creek, rather than further away from the Valentine Camp was comparable to the creek. Leaf beetle mandibles experience rainfall gradient focused on in another study more wear from tougher leaf cuticles on Quercus suber, an evergreen oak tree (Sanson et al. 2001), therefore it makes (Gouveia 2008). Leaf thickness of Q. suber sense that G. rudis would prefer the thinner increased with declining rainfall, which was leaves. attributed to an enhanced resistance to We concluded that aphids preferred lupine water stress because of high cell further from the creek, while G. rudis concentration in the leaves. These preferred lupine closer to the creek. The difference in the distribution pattern of each intraspecific changes in the foliage of Q. could be because the aphids were impacted

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by different factors than G. rudis, since they concerning how well this hypothesis applies are not leaf herbivores. It’s possible that in to the intraspecific level (Hahn and Maron drier soils, the phloem of lupine plants 2016). It is unlikely that a binary conclusion becomes more concentrated, making it can be made when considering the myriad of more appealing to aphids. Another interactions that exist between individual explanation may be found in lupine’s ability plant species and their abiotic and biotic to produce chemical defenses and where environments. However with this study, we they are most concentrated. L. latifolius is a support the RAH’s applicability to the nitrogen-fixer, which facilitates its intraspecific level and hope to contribute to production of alkaloids, toxic compounds a more complete conversation concerning that can act as a secondary deterrent to its scope. insect herbivory (Krischik et al. 1991). It has also been shown that these chemical ACKNOWLEDGMENTS defenses can be induced following herbivory, increasing their concentration in We would like to thank our advisors phloem and leaves (Bernays and Chapman Prahlada Papper, Tim Miller, and Krikor 1994). It would be interesting as a topic of Andonian for their guidance and dedication. future study to determine if these induced Our work was supported by the University of defenses decrease in drier areas, where California Natural Reserve System. This work lupine would have less water to mobilize its was performed at Valentine Camp, chemical defenses. Previous studies done on doi:10.21973/N3JQ0H. apple plants and drought stress concluded REFERENCES that drought stress had differing effects on constitutive and induced insect herbivore Bernays, E. A., and R. F. Chapman. 1994. Host-plant resistance responses (Gutbrodt 2011). A selection by phytophagous insects. Chapman & tradeoff between induced and constitutive Hall, New York. defenses along moisture gradients could Boardman, N. T. 1977. Comparative photosynthesis help explain the pattern of aphid distribution of sun and shade plants. Annual Review of Plant seen in our results. Physiology 28:355–377.

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