Artificial Selection 1 Running Head: Artificial Selection

Effects of Artificial Selection on Brassica rapa NAME

Artificial Selection 2 Effects of Artificial Selection on Brassica rapa

Evolution is a theory made popular by Charles Darwin that suggests all species are descendents of a common ancestral organism and have undergone changes in allele frequencies over time; hence the repeatedly used phase descent with modification (Freeman, 2005). Natural selection, genetic drift, gene flow, and mutation are examples of processes by which allele frequencies can change over time (Freeman, 2005). Darwins theory of evolution via natural selection, explained by Ernst Mayr What Evolution is (2001), follows a logical sequence. Natural selection requires a) variation, b) heritability, c) pressure to change, and d) change must improve fitness of parent and offspring (Mayr, 2001). First, for natural selection to occur, genetic variation must be present within a population. Second, the genetic variation must be heritable between individuals of that population. Third, the heritable trait must be beneficial to the individuals survival when some type of competition or environmental pressure is applied. Lastly, the favored trait must improve the species fitness. Thus, in the processes of natural selection, particular heritable traits possessed by individuals within a population have the ability to pass environmental favored genes to their offspring. Therefore, evolution via natural selection results when differential reproductive success is based on heritable variation (Freeman, 2005). Humans have been manipulating the process of natural selection before Charles Darwin observed the patterns and processes of evolution. Approximately 11,000 years before Darwin wrote On the Origin of Species (1842), native peoples of Southeast Asia were cultivating crops of rice and beans by selecting seeds from the heartiest food in order to continuously produce the increased yield (Warren, 1909). A more drastic application of humans manipulating the process

Artificial Selection 3 of natural selection is written in the history of ancient Greece, in particular, Spartans used to select infants by a series of physical tests and were known to discard those individuals that could not endure or pass the tests (Behreandt, 2006). The Spartans aimed to artificially breed the most outstanding warriors. Artificial selection is a process by which humans deliberately manipulate hereditable phenotypes of a population by allowing only certain individuals with favorable phenotypes to reproduce (Freeman, 2005). Evolution via natural selection is testable; therefore, it is logical to assume that evolution via artificial selection is also testable (Freeman, 2005). Since the natural selection process takes centuries to complete or observe (Mayr, 2001), most studies and experiments are conducted through artificial selection and the results are extended to natural selection. One way to accomplish this is to use organisms that have been artificially bred to grow fast. An example of this is from the Brassica genus, which is a plant from the mustard family (Walker & Hoese, 2007). The Brassica genus has been artificially selected to create many vegetables that we eat (Walker & Hoese, 2007). Brassica oleracea and Brassica juncea have been artificially selected to create common foods such as kale, cauliflower, broccoli, and mustard (Walker & Hoese, 2007). Brassica rapa is also known as a turnip. B. rapa is a widespread outcrossing angiosperm that grows annually in weedy populations (Siemens et al, 1998). B. rapa, or the Wisconsin Fast Plant, has been artificially selected to grow faster than its naturally grown ancestors (Walker & Hoese, 2007). Due to their ability to grow fast, B. rapa offer biologist opportunities to study genetic changes in a short period of time; thus, making it economically beneficial for agricultural and genetic studies (Newell, 2006). This model organism was used to

Artificial Selection 4 observe the workings of Mendels Laws of Inheritance to determine the genotype of an unknown B. rapa. The purpose of this experiment was to artificially select for longer first internode length of the Brassica rapa to determine if the length of the first internode is a heritable genetic trait. The hypothesis was made that if the length of the first internode of Brassica rapa is the expression of a heritable genetic trait, then a population of B. rapa can be artificially selected to inherit that trait. In previous studies, Brassica rapas overall length was measured under the pressure of competition. This experiment showed that competition increased overall height, size, and flower production (Miller, 1994). This suggests that in competitive environments B.rapa can evolve through changes in plant development (Miller, 1994). If the length of first internode is indeed a heritable trait, the second generation of B. rapa will have longer first internode lengths than their parent population. The results, regardless of their outcome, will be of importance to the scientific community. Individuals with the longest first internode length were artificially selected and breed to obtain a population of Brassica rapa with longer first internode lengths.

Materials and Methods Planting the Parent Generation The experiment followed the materials and methods outlined in BIOL 171L: Evolution and Biodiversity Course Pack (2007) provided by doctors Walker and Hoese. For the experiment, the soil composition was 50% Organic fraction (6 parts peat moss and 9 parts forest humus) and 50% Inorganic fraction (6 parts washed plaster sand and 9 parts pumice). The fertilizer components of the soil had a 20:20:20 mixture of nitrogen, phosphorus, and potassium and contained finely ground dolomite. Fill 1 X 1 X 3 plants cells with the soil. The planting

Artificial Selection 5 cell flats should be filled with loosely packed soil. Place the soil filled tray and place inside the watering tray. One Brassica rapa was seed 1.0 cm deep into each soil filled cell using marked forceps. This process was repeated 98 times until all (n=98) seeds were planted. The planted trays were placed under the artificial sunlight on the holding rack. 3.0cm of water was added to the watering tray to moisten the soil. The plants were watered continuously with .5cm 1.5cm of water throughout the experiment with watering frequency as follows: Mondays and Wednesdays water 1.0cm, 1.5cm on Fridays, and more as needed for increased room temperatures. All data on watering, room temperature (in degrees Celsius), and other general observations about growth were recorded on the data sheet at each watering period. Selection Event 14 days after the planting, using a ruler, the length of the parent populations first internode length of (n=96) plants was measured and recorded the data. Using Microsoft Excel 2003, the average length, standard deviation, and frequency were calculated to create a population frequency histogram. Based on the results of the graph, (n=10) individuals with the longest first internode length were selected and staked. The first generation acted as our control population; therefore, the unselected individuals were thrown away. The plants were returned to the same position on the holding rack under the artificial light. Pollination 3 days after selection, the selected individuals were artificially pollinated. A cotton swab and gently rubbed it on the anthers and the stigma of the selected flowering individuals for about ten seconds each flower. The pollen saturated cotton swab was rubbed it in the same manner to the flowers of the next selected individual. This pattern continued until all selected individuals were artificially pollinated. After the plants have been pollinated twice, the used cotton swab was

Artificial Selection 6 disposed and the plants were returned to the holding rack. Watering continued in same manner for four more weeks. Planting Second Generation Watering of the plants was stopped four weeks after pollination. The plants must go one week without water to dry the legumes. With same marked forceps, the siliques were removed from the plants and gently broke them open and placed the seeds into a small glass bowl. The larger brown seeds were selected to plant while the other seeds, along with the soil and selected individuals, were disposed of. The planting cell flats were refilled with soil of the same composition. The selected seeds were planted 1.0cm deep into soil same as before until all (n=98) were planted. The trays were returned to same location under the artificial sunlight on the holding rack and filled the watering tray with 3.0cm of water. Watering and recording continued for 14 days as before. Statistics 14 days after planting, the length of the first internode of all plants (n=58) were measured and recorded. The information was recorded into a new data table on Microsoft Excel 2003. Again the average, standard deviation, and frequency, using bins of .03cm, were calculated and graphed; a new frequency histogram based on the new data of the Second Generation (F1 generation), or Offspring specimens. After the plants and soil were disposed of, a comparison was made of the internodel length of the First Generation (P1 population) and the Second Generation (F1 generation). Two-tailed t-tests assuming equal variances were conducted using Microsoft Excel 2003 to test for differences between trays within generations and between P1 and F1 data.

Artificial Selection 7 The selection differential (S) was calculated by using the formula: S = | Xp Xs |; where Xp is the average phenotype of the parents and Xs is the average phenotype of the selected population (Walker & Hoese, 2007). The response to selection (R) was calculated by using the formula: R = | Xp Xs offspring |; where Xs offspring is the average phenotype of the offspring of the selected population. Once that information was obtained, heritability (h2) was calculated for length of first internode by using the formula: h2 = R/S.

Results Evaluation of Individuals in P1 & F1 Populations A frequency histogram was used to evaluate the significance of the data when comparing P1 population (n=96) and F1 generation (n=58) (Figure 1). P1 population had a higher percent of individuals with first internode lengths that ranged from 0.0 cm to 1.6 cm. After 1.6 cm to 1.9 cm ranges; the F1 generation had a higher percent of individuals in ranges 2.0 cm to 6.5 cm (Figure 1). Therefore, the percent of individuals with longer first internode lengths came from F1 generation. The t-test was used to compare the P1 and F1 generation and showed a significant increase in first internode length of F1 generation over P1 generation (p<0.01, T(1,153) = -2.59) (Figure 2). Evaluation of P1 & F1 Populations The average lengths of the first internode in the P1 generation (n=96) was 1.31 cm and 1.72 cm in the F1 generation (n=58) (Figure 2). The P1 generation had an average length of 1.21 cm in tray 1 and 1.41 cm in tray 2; the P1 population had a standard deviation of .70 cm in tray 1 and .81 cm in tray 2 (Figure 3). The F1 generation had an average length of 1.10 cm in tray 1 and

Artificial Selection 8 2.34 cm in tray 2; the F1 generation had a standard deviation of .66 cm in tray 1 and 1.20 cm in tray 2 (Figure 4).

P1:(Parent) & F1: (Offspring) Generations

30.00% 25.00%

1st Generation (Parent) 2nd Generation (Offspring)

Percent of Individuals

20.00% 15.00% 10.00% 5.00% 0.00% 0.0 - 0.3 0.4 - 0.7 0.8 - 1.1 1.2 - 1.5 1.6 - 1.9 2.0 - 2.3 2.4 - 2.7 2.8 - 3.1 3.2 - 3.5 6.2 - 6.5

Length of 1st Internode (cm)

Figure 1. Frequency histogram between P1 & F1 generations

Offspring population had increased 1st Internode Length

3.00

1st Internode Length (cm )

2.50 2.00 1.50 1.00 0.50 0.00

Parent

Offspring

Figure 2. Mean comparison of first internode length between P1 & F1 generations

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P1 Generation: 1st Internodel Lengths

2.50

1st Internode Lenght (cm)

2.00

1.50

1.00

0.50

0.00 Tray 1 Tray 2

Figure 3. Mean & standard deviation of first internode length between trays 1 & 2; 1.21 cm & 1.41 cm respectively.

F1 Generation: 1st Internodel Lengths

4.00 3.50

1st Internode Lenght (cm)

3.00 2.50 2.00 1.50 1.00 0.50 0.00 Tray 1 Tray 2

Figure 4. Mean & standard deviation of first internode length between trays 1 & 2; 1.10 cm & 2.34 cm respectively.

Artificial Selection 10 Evaluation of Tray Effect A two-tailed t-test assuming equal variances was used in both P1 and F1 populations to test for a possible tray effect. The t-test in the P1 generation showed no significant difference (p<.19, T(1,95) = -1.31) between trays 1 and 2 (Figure 3). The t-test of the F1 generation showed significant differences (p<.00, T (1,57) = -4.84) between trays 1 and 2 (Figure 4). Evaluation of Heritability The heritability of the first internodel length was measured by determining the response to selection and dividing the results by the selection differential result. Selection differential (S), and response to selection were, 1.36 cm and .41 cm respectively (Table 1). Heritability (h2) was calculated to be .30 (Table1).

Table 1. Calculation of Selection Differential, Response to Selection, and Heritability of P1 and F1 generations. Parent (Xp) S= 1.31 cm Parent (Xp) R= 1.31 cm Response ( R ) H= 0.41 1.36 0.3 1.72 cm Selection ( S ) .41cm Result 2.61 cm Offspring (Xo) 1.36 cm Result Selected (Xs) Result

Artificial Selection 11 Discussion The present study observed the effects of artificial selection on offspring populations of Brassica rapa. As predicted, this study has demonstrated that the length of first internode is indeed a heritable trait in this species. The offspring generation had significantly longer first internodel lengths than their parent population (Figure 2). Expectedly, the dramatic shift occurred as internode lengths surpassed the P1 generation average of 1.6 cm and reached 6.5 cm (Figure 1); which were lengths the P1 individuals never reached. These results are consistent with previous research and provided additional support for the idea of length as a heritable phenotype of the Brassica rapa. The results of this study support the working hypothesis that artificial selection, as an extension of natural selection, is a factor of evolution. As previously mentioned, measurable results through the process of natural selection take centuries to observe (Mayr, 2001). In this study, the artificial selection process achieved significant results in just a few months. The heritability (h2 = .3) of this trait, implies that, if left to natural selection, internodel length is heritable, but slow to observe. However, other factors, such as the tray effect phenomenon, as well as other non-incorporated environmental factors, could have contributed to change first internode length between generations and therefore altering the results. Tray effect may have played a significant role in the results of the F1 generation (p<.00, T (1,57) = -4.84) (Table 2). It was found that first internode lengths were much longer in tray 2 than in try 1 in the F1 generation (Figure 4). These results suggest that there were more factors contributing to the internode length than genetic variation. This information should be taken into account for future studies.

Artificial Selection 12 One limitation of the present study was the absence of environmental controls, such as room temperature and water. There were days when the room temperature reached lows of 22.5C and highs of >30.0C (Appendix A). This variation in temperature could have affected the growth of the plants. The plants also received inconstant and infrequent amounts of water throughout the experiment (Appendix A). I hypothesize that if room temperature and watering factors where controlled, a higher heritability result could have been achieved (Table 1). If this study were to be replicated, I would suggest a tighter control of environmental factors. The present study opens new questions about the nature of evolution via natural selection. In particular, why, if artificial selection is a derivative of natural selection, did the offspring generation yield 40% less individuals Brassica rapa than parent generation? Was it because the first internode length phenotype conflicts or inhibits fertilization and only the strongest survived? If that be the case, then increased internodel length would not be a factor of natural selection because the trait did not increase fitness. Or, was the decreased germination a result of artificial pollination? If it were, then artificial selection may not be a conclusive solution to observe evolution via natural selection. Further studies should determine other environmental factors that influence evolution via natural and artificial selection.

Artificial Selection 13 Work Cited Behreandt, Dennis, (2006). Freedom in Sparta and Athens. The New American. Working paper 29 May 2006. Accessed 13 Nov 2007. http://www.thenewamerican.com/node/1972. Mayr, Ernst, (2001). What Evolution is . New York, NY: Basic Books. Miller, T.E. (1994). Evolution of Brassica rapa Population in Intra-and Interspecific Competition. Department of Biological Science, Florida State University. Tallahassee, FL. Newell, Sandra J. (2006). Does Herbicide Resistance Have a Cost in Brassica rapa? The American Biology Teacher. 68, 530-535. Siemens, D.H. & Mitchell-Olds, T. (1998). Evolution of pest-induced defenses in Brassica plants: Tests of theory. Ecology. 79, 632-646. Walker Ph.D, S. & Hoese Ph.D, W. (2007). BIOL 171L: Evolution and Biodiversity Course Pack. University Readers, Inc. Warren Ph.D., G.F., (1909). Elements of Agriculture. London, England: The MacMillan Company. Working paper 21 June 1909. Accessed 13 Nov. 2007. http://www.thenewamerican.com/node/1972.

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Appendix A