We anticipate that the amount of potentially viable seeds found in CAwr debris due to granivory with various degrees of fruit wall coverage affect: seed dormancy and thus widen the range of germination in the seed bank from the cohort of seed that will fall all at once with the senesced plant and, availability to post-dispersal seed predation with opportunities for dispersal. Our results, combine with all previous evidence of the hybrids high genetic polymorphism, suitable life-history traits and higher fecundity both inside and outside its distribution range, provide a compelling argument for the hybrid to have displaced its progenitor lineages and for its capacity to be a successful invader .During the invasion process, the ability of a species to successfully persist and evolve depends on spreading its propagules . These offspring carry the resulting effects of biotic and abiotic selective forces including both natural and sexual selection. High reproductive rate characterizes successful invaders because it translates into thriving demographics allowing dispersal and range expansion . However, reproductive investment alone does not always explain invasiveness such as when natural selection has the potential to modify this over time . In multi-seeded fruits, understanding the effects of within-fruit seed characteristics is an opportunity to explore the effects of sexual selection. Sexual selection effects, procona buckets coupled with natural selection, may influence the success of an invasive lineage .
Seeds are the propagules of sexually reproduced plants and the majority of plant invaders rely on their dispersal . Most of the studies on seed or fruit traits in invasive plants either emphasize the effects of high fecundity or the rate of dispersal and life history traits . However other seed characteristics such as seed size , seed paternity and maternity and within-fruit seed position are variables that have been shown to influence seedling establishment, plant growth, adult plant size, ecology and reproductive output in noninvasive species . Intraspecific seed size variation is considered to be stable and tends to vary under density-dependent competitive situations . Variation in seed size also occurs within fruits and this is particularly apparent when the ovules are arranged in a linear arrangement within the fruits. In cucumber , the higher the fruit position on the mother plant and the within-fruit peduncular seeds were the slower to reach maturity and lowest dry weight . However, differences in fruit and ovule positions were compensated over time, when seeds had more time to mature inside the fruit . Seed size, from a broad phylogenetic to specific ecological interactions is a significant trait in most plant’s lifetime performance.Therefore, within-fruit seed size and other characteristics such as seed position and paternity, can have a relevant effect on invasive hybrid derived lineages. Therefore, I investigated how those within-fruit seed characteristics compare between a hybrid lineage relative to its progenitors. In particular, the present study compares the within-fruit seed characteristics and fitness among the invasive hybrid-derived California wild radish and its cultivated Raphanus sativus and wild R. raphanistrum progenitors in a noncompetitive setting. At the seeded portion, Raphanus fruits hold up to 13 seeds.
A relatively fine seed coat covers Raphanus seeds and its integral presence induces dormancy . Some of the features of the hybrid-derived CAwr reproductive biology may strongly influence its ability to adapt to new environments. The hybrid derived lineage fruits have multiple paternity and multiple lines of evidence suggest that the seed siring occurs in a non-random mating manner . Thus, the identity of the pollen donor is an important determinant in the sequence of the fertilization and seed placement within the pod-like fruit . Inside the fruit, seeds vary in size , with a general tendency to be heavier at the attached or peduncular end . The order of ovule fertilization in the Raphanus raphanistrum follows two patterns: ovules in stylar section first, followed by middle ones and lastly peduncular ones, or middle ovules first followed by stylar ones and peduncular ones. These patterns are explained by gamete selection at prezygotic mechanisms level as well as by the gynoecium internal structure . A central transmitting tissue structure, called septum, allows compatible pollen tubes to grow and by-pass ovules at stylar positions . However, I do not know if all three lineages have the same within-fruit seed characteristics. Our objectives are to compare seed weight among all three Raphanus lineages and their populations, to determine if within-fruit seed positioning influences seed weight, fecundity, and other morphological as well as fitness related characteristics, to compare if single and to mixed hand pollination crosses influence fitness values, to document the occurrence of multiple paternity in all three lineages, and to assess maternal and paternal effect on seed weight and other fitness related variables.Seed sources – The plants that produced the seeds used to breed the mother plants and the control fruits in these experiments were reared in a common garden during Spring 2005 and Winter 2006. The seed sources for those maternal and control plants are described in table 3.1.
The seeds are the result of natural open pollination in common gardens at the Agricultural Experiment Station at the University of California-Riverside . More details on how the plants were grown can be found in Ridley and Ellstrand . Control vs. mixed and single crosses – We measured seed characteristics from seeds extracted from: fruits obtained from open pollinated plants hereincalled control plants: at random I selected five mothers within each of three populations for each lineage and randomly chose three fruits for each mother plant for a total of 135 fruits, and from the offspring resulting hand pollinations from mix and single pollen hand pollination crosses performed in 2010. In total I performed 595 crosses, 336 mixed pollen crosses and 259 single pollen crosses, in a total of 83 plants: 23 CAwr, 30 Rr and 30 Rs, for more details on these crosses see “Paternity”. Viable seeds common garden – Fruit from control, mixed and single crosses were carefully opened with a cutting knife . Inside each opened fruit, seeds in all positions were examined. We counted the total number of seeds or seed set, including viable and unviable seeds as well as empty seed compartments. Seed viability was initially 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 disintegrated easily. Before planting in soil, all “viable” seeds were soaked in 600-ppm giberellic acid solution and germinated in large trays lined with distilled water damped Whatman filters. The trays were placed in the dark of the laboratory and visited twice daily to record germination. After 5 days all seeds were transplanted in seedling trays with UC Soil mix III and moved into a temperature-controlled greenhouse. The greenhouse was visited daily to continue monitoring for germination or any other changes. After 53 days, the plants that survived were transplanted to common garden plots at the University of California-Riverside Agricultural Operations , where I monitored daily for floral buds emergence and first opening flower as well as any changes in the plant condition including signs of herbivory. The common garden design consisted of four plots of 11 m x 11 m at AgOps-UCR, where I planted the seedlings spaced by 1 m in all directions. The plants were watered once daily for 5 min with a sprinkler system until they all started to flower. Plants that survived to the end of the experiment were measured for total number of fruits produced and final plant height , procona florida container and collected to obtain final plant weight .Starting from the peduncular end , each viable seed was weighed to the 0.01 mg with an analytical balance .
The seeds were always extracted in the same order, and sequentially placed by columns in a labeled 96 well plate recording the row and column in a spreadsheet. Once all “viable” seeds of a fruit were placed in a plate, the section assigned to the fruit was securely taped and labeled with the lineage, population, mother plant and fruit ID’s to be stored for further analysis. This arrangement kept the seeds in order and prevented any translocations among positions and fruits. Lastly, the seeds were planted in a common garden to test fitness, always maintaining the same order of extraction from peduncular, middle, to stylar ends.Three within-fruit seed characteristics were tested: seed weight, within-fruit seed weight percentages, and relative withinfruit seed fecundity. Within-fruit seed weight percentages were calculated by dividing a given seed weight by the sum of all seed weights in a given fruit and multiplied by 100. Within-fruit relative fecundity was calculated by dividing the seed fecundity by the overall fruit fecundity where fecundity is defined by the total number of fruits produced. Within-fruit seed position was considered in two different ways: the seed positions per se recorded during the seed extraction process, and as seed position bins. To make the different fruit positions comparable, I divided fruit positions into three seed position bins corresponding to the peduncular, middle and stylar positions as described in figure 3.1. These seed position bins were based on our own observations and on the way I divided the fruits as I opened and removed the seeds. Within the seeded portion of the fruit, I divided the fruit in two. This central point was defined as the middle portion . Next, 2/3 away from that central point toward the basal part of the fruit I marked the peduncular portion . Then, 2/3 away from the same central point, this time towards the distal part of the fruit, I determined the stylar portion of the fruit. These variables included days to germination after planting, which constituted the time baseline for all other life cycle variables, such as, the days to floral bud emergence, and the days to first opened flower. Paternity – To assess the occurrence of multiple paternity on the three lineages I performed mix and single pollen crosses on mature unopened flower buds. Any mature flower bud with a corolla that was not tightly closed or that presented any opening as a result of damage was discarded. Immediately after performing hand pollinations , pollinated stigmata, fully covered with pollen, were covered with labeled tulle bags until the end of the experiment. The purposes of the tulle bags were to avoid unwanted pollen transfer, to protect the fruit from any predation and to allow proper maturation without losing the fruit once dried.At the beginning of May 2010, I collected the pollen sources plants for our hand pollinations from a natural well-established population of CAwr. The five already flowering plants were found in Hemet in the San Jacinto Valley in Riverside County, California. The chosen plants had different flower colors looked healthy, and had no pests or signs of herbivory. These plants were individually transplanted to gallon pots and then placed in a temperature-controlled greenhouse at UCR. In the greenhouse the five plants were watered daily, trimmed to prolong their flowering and maintained pest free until the end of the experiment. Mix pollen load crosses included different combinations of four pollen donors, and in one case three, out of the set of five described above. At a given time, the mixture of pollen donor plants depended on: 1) the outcome of initial single crosses that verified compatibility with the pollen receivers and also 2) pollen donor flower availability at a particular time. Single crosses were also used as a reference through the microsatellite analyses. Once a mother plant was pollinated with a particular pollen mix, the same mix was used to pollinate the rest of the flowers on that same mother plant. Despite evidence for lack of effect in the amount of pollen on reproductive output , I equalized amounts of pollen applied per donor across crosses as follows . For single crosses, I always removed the pollen from a single new flower by gently tapping all six anthers against the bottom of a clean Petri dish. For mixed crosses, I selected two anthers per father, totaling eight anthers for all four fathers. All eight anthers were dissected from new flowers with tweezers and tapped against the bottom of a clean Petri dish. The pollen collected from all anthers was blended by gently swirling the pollen with a clean piece of folded lab tissue held with tweezers.