Plants employ a large array of strategies, both direct and indirect, to minimize the effect of seed predators. These include alternative strategies of regeneration, seed banks, abundant seed crops, altering phenology, spatial and temporal variation in seed productions and increasing plant defenses . It has also been demonstrated that pre-dispersal predation can facilitate the stratification of the seeds facilitating germination . A hybrid-derived lineage that has already successfully invaded a new habitat represents a useful model because, when combined with its progenitors, it allows for a replicated study of the invasion process. Here, I focus on the hybrid-derived California wild radish, which is an invasive lineage in western North America . Its progenitors, the cultivated radish Raphanus sativus and the wild radish R. raphanistrum, were introduced in western United States around mid 1800 . These two lineages naturally hybridize, and there is evidence for the hybridderived lineage to have originated from interspecific bidirectional hybridization between them . Genetically based differences between both progenitor lineages and a partial and temporary reproductive isolation during the first generations of hybrids results from a single reciprocal translocation . The polymorphism in fruit, flower color and shape, root morphology, chemical and structural defenses found in Raphanus lineages has been the focus of numerous studies in ecology, evolution, genetics and agricultural and food chemistry . Anecdotal observations have reported that bird pre-dispersal predation of seeds can be extensive in all three Raphanus lineages and birds are arguably the primary consumers of pre-dispersed seeds over invertebrate granivory . When granivory is excluded, the hybrid-derived California wild radish exhibits superior fitness compared to its parental lineages in common garden experiments across its Californian distribution . However, black plastic plant pots wholesale the hybrid’s relative fitness and that of one of the progenitors in the presence and absence of granivores, is unknown.

Our aim was to answer the following questions: what species of bird granivore is the main consumer of radish seeds? how much does the bird granivore affect relative fitness and relative potential fitness? are the variables likely to affect the birds’ selection of individual plants such as days to germination, plant final weight, total fruit production and potential reproduction, correlated with fruit damage? and are there viable seeds in the debris due to granivory resulting from the bird foraging behavior under damaged plants? In addition, the comparison among lineages allowed us to better understand novel biotic interactions in a successful invasive hybrid-derived lineage and to propose a mechanism that led to the replacement of both progenitors .Seed sources – The seeds used to breed the mother plants in the present study came from plants reared in a common garden during Spring 2005 and Winter 2006. The seed sources for the first generation of maternal plants are described in table 2.1. The second generation seeds are the result of natural open pollination in common gardens at the Agricultural Operations fields at the University of California-Riverside . More details on how the first generation plants were grown can be found in Ridley and Ellstrand . Common garden and experiment design – The common garden experiment took place during Spring and Summer seasons of 2010 at AgOps-UCR. Three replicate sites, each one consisting of two plots of 7 m by 7 m, were planted with 36 plants placed in a 6 x 6 grid with 1 m spacing in rows and columns. One of the plots at each of the three sites was covered with 3/4″ x 3/4″ orchard mesh to exclude above ground vertebrate damage while the other plot remained unprotected. These two conditions created two different treatments for the plants to grow in: protected from vertebrate seed predators and unprotected to vertebrate seed predators. In both cases the plants were exposed to open pollination, invertebrates and potentially underground vertebrates. All plots were oriented in the same North-South direction. We selected at random 8 seeds from 4 different mothers within each of the 9 above-mentioned populations for a total of 288 seeds. These seeds were divided in groups of 36, such that all mothers were represented in those 8 groups by 2 seeds. That is, each population had 4 seeds, for a total of 12 seeds per lineage.

These 8 groups of seeds were germinated in Petri dishes at the beginning of March and transplanted into seed starting trays filled with sterilized UC Soil Mix III at a climate controlled greenhouse. Once the seedlings had attained a three-leaf stage, 6 of the 36- grouped seeds were transplanted to the pre-water and plowed field plots. The two additional groups of seeds were used to replace any seed that did not germinate or any seedlings that did not survive the transplanting process. The plants were watered once daily for 10 min with a sprinkler system until most of the plants had started to flower. To maintain favorable abiotic conditions for the plants that flowered later, the watering persisted only every-other-day for 5 min. Granivores – We visited the sites at AgOps at least every two days to ensure that the experimental conditions were kept consistent during the entire length of the study. During those visits I also spent time observing the foraging behavior of the birds that began when fruits had attained a fully formed size. Once I became familiar with the birds foraging patterns, I spend an afternoon filming their behavior. Videos were captured with a digital video camera on a tripod. Videos are available as supplemental information.Fruit damage, fecundity, fitness related values and debris due to granivory – Variables related to morphology, damage and fitness were recorded before planting, during the experiment, and after the surviving plants were collected. All seeds were weighed to within 0.01 mg with an analytical balance . The germination and growth of seeds in the dish was recorded daily. At the end of the experiment when the plants were dry and had senesced, I recorded the final plant weight to within 0.001 g. To calculate fecundity and fruit damage, I counted total number of: damaged fruits that included all fruits with clear signs of missing or damaged sections, whole dropped fruits that were found detached from the dry plant, and whole attached fruits. With these variables I calculated fruit damage and fecundity. We counted total numbers of: flower buds, flowers, whole empty pedicels , and broken or pedicel scars on the stems. We also collected the fruit material or debris accumulated under heavily damaged plants, herein referred to as “debris due to granivory”, to discern what was discarded during the birds foraging behavior.

Potential seed viability was determined by visually inspecting the seed coat and by putting pressure on each seed between the thumb and the index fingers; when unviable, seeds had black and/or wrinkled seed coats and crumbled easily. All the previously described values and those in table 2 allow us to calculate relative fecundity and relative potential fecundity of plants in unprotected and protected plots. Because I did not count number of seed per fruits, I calculated the number of seeds based on the average number of seeds per fruit per populations. These average values,listed in table 2, were obtained from a previous study where I counted total number of seeds from 884 fruits that belonged to the same populations represented here . We consider these values appropriate to extrapolate the number of seed in our study because: the plants that produced them developed from pure lineage seeds from the same populations represented in our study listed in table 2.1, the plants were grown under similar conditions to the present study, and the plants were exposed to open pollination . Total number of seeds were extrapolated for a given plant by: multiplying total number of whole fruits per plant by the average value of seeds in table 2.2 according to the population of origin, black plastic plant pots bulk followed by multiplying total number of damaged fruits by the 2/3 of the average number of seeds according to the population of origin in table 2.2, and finally by adding the numbers obtained for whole and damaged fruits. Fecundity and female fitness values were calculated as follows. The average number of extrapolated seeds per population was calculated by dividing the total number of extrapolated seeds divided by the total number of fruits per population. Relative fecundity is the average number of extrapolated seeds divided by the highest average number of extrapolated seeds. Potential reproduction was calculated by adding flower buds, flowers, whole empty pedicels broken to whole, damaged and dropped fruits for a given plant. The average potential reproduction was calculated by adding the potential reproduction for a given population or lineage and dividing by the total number of plants and multiplied by 100. Finally, the percentage of the relative potential fecundity was calculated by dividing a given average potential reproduction to the highest average one among for populations and lineages separately and then multiplying by 100. Our fitness values did not explicitly include male fitness. Nevertheless I know based on prior studies in plants of the Raphanus lineage that male fertility is highly influenced by environmental factors and weakly correlated with female fertility values . Data analysis – Data were normalized as needed either with log-normal or Box Cox transformations using functions in R . Significant P values were adjusted a posteriori with sequential Bonferroni test to adjust for type I error . We used one-way analysis of variance to tests the effects of treatments and lineages on total fruit production. Variables related with fruit damage and with fitness were compared in pairs among lineages and between treatments with Wilcoxon tests. The effect of the treatments on relative fecundity and relative potential fecundity as well as average number of fruits and seeds were tested for significance with Fisher exact tests. These tests were performed to individually compare CAwr values to its progenitors. We also compared fecundity values to the highest ones with chi-square tests. Total number of fruit damaged was correlated using Spearman correlation coefficients and covariance to variables possibly related to final fruit production and general performance. Those variables included: days to germination, final plant weight as well as total number of fruits and total potential reproduction. In this case each lineage was tested independently.Flower buds, flowers and pedicels – No differences were found between lineages and treatments in average number of flower buds and open flowers . With respect to the average number of pedicels, values for CAwr from protected and unprotected plots are significantly different and higher than both progenitors under protected treatment as well as for the cultivar under unprotected treatment, respectively . Fruits with and with no damage – We only found damaged fruits in plants that were collected in unprotected plots . Consistent with our previous results, average numbers of fruits with damage are significantly different among lineages and treatments as revealed by Wilcoxon tests . Whole undamaged fruits were categorized as either attached to the dry plant or detached and on the ground. The cultivar Rs differs significantly from CAwr and Rr on lower average number of whole dropped fruits, whereas both wild lineages, CAwr and Rr, are comparable . No differences are found in the average number of whole attached fruits among lineages with the exception of CAwr and Rs from protected plots . The average proportions of damage, calculated as total number of damaged fruits over the total fruits produced for each lineage and population, are listed in Table 2.3. When the damage is estimated based on seeds removed, calculations of damage per population are reduced by at least 33 % and at most by 60% relative to the damage calculated based on fruit damaged. Damage, based on seeds removed, was calculated as the total number of seed removed divided by total number of seeds produced. As mentioned earlier, I did not count the total number of seeds per fruits during this experiment. Fruits from Cst-CAwr suffer higher damage than interior populations . Fruit production – Total fruit production does not differ under protected or unprotected treatments but does differ among lineages . The cultivated Rs lineage produced fewer fruits in protected treatments relative to both wild lineages, significantly differing from both CAwr and Rr . However, under unprotected conditions, Rs only substantially differs from unprotected and protected CAwr fruit production . Fecundity and fitness related values – CAwr has significantly higher fecundity in protected plots than in unprotected ones, as shown in table 2.4.