The effects of environmental stress on meiosis and genetic diversity in the model organism, Sordaria fimicolaIntroductionThe experiment is performed to know if the environment plays a role in meiosis during sexual reproduction to create genetic diversity. Genetic Diversity is defined as the amount of genetic information within and among individuals of same species. It allows species to strive through the harsh environment and reduce the likelihood of inheritance of unfavorable traits. It is known that Meiosis produces gametes with various genotypic features. The process of recombination, where allele pairs trade genetic information prior to Metaphase I, is the biological process researchers are specifically looking at (Heredity and Life Cycle). It provides a source for genetic diversification.To study how the environmental conditions may affect gene expression, a research group from the Imperial College of Science, Technology, and Medicine in England were studying a species of fungi, Sordaria fimicola, in the natural habitat called Evolution Canyons. the Evolution Canyons are in Israel; they have a South Facing Slope (SFS) and North Facing Slope (NFS). The SFS has higher temperatures and more arid while the NFS is more humid, and temperate (Singaravelan et al. 2010) (See figure 1). S. fimicola lives in both regions and releases either dark-colored asci (wild-type) or tan-colored asci.S. fimicola was selected as a model organism because of its short life cycle, abundance, and production of patterned asci that depends on recombination (Hass and Ward 2010). Depending on what slope they live on, the environmental conditions may pressure them into expressing color pattern differences, providing evidence of changing crossover frequencies. A part of its life is spent as a haploid (n) while the other part of its life is spent as a diploid (2n) (Fungi I). (See Figure 2.)  Through Meiosis and followed by Mitosis, the fungi forms 8 asci that’s either dark or tan in the case of S. fimicola. Meiosis is the sexual production part of the life cycle in which haploid cells are formed from the original diploid cells. On the other hand, Mitosis guides cell replication and growth.Crossing over occurs during Meiosis. During crossing over, two homologous chromosomes in a tetrad exchange genetic information on the parts of one of the non-sister chromatids (Hereditary and Life Cycle). The crossover frequency is the amount of times crossing over occurs. Various types of asci patterns found in S. fimicola are the result of crossing over.During the process of Meiosis and following Mitosis, S. fimicola produces three types of asci patterns—2:4:2, 2:2:2:2, and 4:4. The 2:4:2 and 2:2:2:2 patterns are the result of recombination, whereas, no recombination takes place in 4:4 pattern. For the non-recombinant strain, a chromosome for dark asci pattern pairs with its tan homologous counterpart during Meiosis. They then separate three times; two times during Meiosis and then one time more during Mitosis. As no recombination occurred, the dark-colored coding chromosome will form four dark asci while the tan-colored chromosome will also form four tan asci, ending with a 4:4 pattern. For the 2:2:2:2 asci pattern, recombination occurs between the two inner, non-sister chromatids. And for the 2:4:2 asci, recombination occurs in one of the inner chromatids with the homologous outer, non-sister chromatid.The purpose of the research is to see if environmental stress influences recombination frequencies. It will be found out if the recombination frequency is more in the sample exposed to low ultraviolet radiations and the one that is exposed to higher ultraviolet radiations. Then, both the results will be compared to the control sample that was not exposed to ultraviolet radiations. To accomplish these tasks, three strains of S. fimicola is prepared in agar cultures for mating. Then, the perithecia that includes asci would be examined under a microscope. Finally, data will be collected by counting the asci, giving results that will be used to calculate the recombination frequency. Methods and MaterialsTwo treatments were set up to observe the environmental stress on Sordaria fimicola. The fungus was tested under low radiation of 100 Gy and a high radiation of 500 Gy. The control group of the fungus was not exposed to any kind of radiations. Our group performed low radiation treatment on the S. fimicola.The first step in the lab was to allow S. fimicola to mate. To do this, samples of control, as well as the treated S. fimicola (grown on their own agar dishes), were provided in the lab on November 7, 2018.  Newly-made, plain agar plates were provided in the lab to grow the samples and perform the experiment. On the bottom of the plates, four equal quadrants were drawn with the marker to clearly define the space for each type of the sample (See figure 3). A sterilized scalpel was then used to cut out small square parts of the agar from both the control as well as the treated sample provided. The cut-out portions were placed carefully upside down onto the fresh agar plates. Wild-type samples were placed diagonally to get better results of the cross between the wild and the tan type samples. After a week of treatment, the agar plates were reexamined to find newly formed perithecia bordering each quadrant. Recently formed perithecia were used to examine the recombination under the microscope. The microscope slide was prepared by placing a drop of distilled water on the slide. Then, a sterilized inoculation loop was to scrap-off a small sample of perithecia from one of the bordering samples on the agar plate. The sample was then placed on the drop of distilled water on the microscope plate. The slide was completely prepared by placing a coverslip on the water droplet containing the sample. Once the slides were prepared, a small amount of pressure was applied on the coverslip in order to break the perithecia and release the asci. The slides were observed under the microscope with a magnification of 400x to score asci (See Figure 4).Data Analysis Three patterns of asci combinations (4:4, 2:2:2:2, 2:4:2) were looked and counted for. The frequency of recombinant asci was calculated by adding the number of asci of Type B (2:4:2) and Type C (2:2:2:2) and diving the sum by the total number of asci found in the sample. Overall recombination frequency of the organism was found by the formula: [(frequency of recombinant asci) / 2 ] * 100. ResultsTreatmentNon -recombinantRecombinant Total # of AsciTotal # of recombinant Asci (B+C)# of TYPE A Asci (4:4)# of TYPE B Asci (4:2:4)# of TYPE C Asci (2:2:2:2)Control1254219Table 1: Individual data for control.TreatmentNon -recombinantRecombinant Total # of AsciTotal # of recombinant Asci (B+C)# of TYPE A Asci (4:4)# of TYPE B Asci (4:2:4)# of TYPE C Asci (2:2:2:2)Control26874115Table 2: Lab group data for Control.TreatmentNon -recombinantRecombinant Total # of AsciTotal # of recombinant Asci (B+C)# of TYPE A Asci (4:4)# of TYPE B Asci (4:2:4)# of TYPE C Asci (2:2:2:2)Low Radiation (100 Gy)7582013High Radiation(500 Gy)6592014Table 3. Lab group data for Treatment.TreatmentNon -recombinantRecombinant Total # of AsciTotal # of recombinant Asci (B+C)# of TYPE A Asci (4:4)# of TYPE B Asci (4:2:4)# of TYPE C Asci (2:2:2:2)Control1014562208107Table 4. Combined Section data for Control.TreatmentNon -recombinantRecombinant Total # of AsciTotal # of recombinant Asci (B+C)# of TYPE A Asci (4:4)# of TYPE B Asci (4:2:4)# of TYPE C Asci (2:2:2:2)Low Radiation (100 Gy)3021298050High Radiation(500 Gy)51314913180Table 5. Combined Section data for Treatment.TreatmentNon-recombinantRecombinantTotal # of AsciTotal # of recombinant Asci (B+C)Frequency of Type B Asci (B/TOTAL)Frequency of Type C Asci (C/TOTAL)Frequency of Recombinant Asci (B+C/TOTAL)# of TYPE A Asci (4:4)# of TYPE B Asci (4:2:4)# of TYPE C Asci (2:2:2:2)Control10145622081070.51440.2160.724Table 6. Combined Section Data Analysis for ControlTreatmentNon-recombinantRecombinantTotal # of AsciTotal # of recombinant Asci (B+C)Frequency of Type B Asci (B/TOTAL)Frequency of Type C Asci (C/TOTAL)Frequency of Recombinant Asci (B+C/TOTAL)RatioB/C# of TYPE A Asci (4:4)# of TYPE B Asci (4:2:4)# of TYPE C Asci (2:2:2:2)Low Radiation (100 Gy)302129 80500.6250.2630.5800.4534High Radiation(500 Gy)513149131800.6110.2370.3740.6337Table 7. Combined Section Data Analysis for Treatment. Figure 1: Overhead and side images of Evolution Canyon depicting the SFS and NFS (Singaravelan  et al. 2010)Figure 2: Alternation of Generation for the life cycle of Fungi (Cyr 2002)  Figure 3: Placement of Wild-Type (dark-colored) and Tan-Type S. fimicola. Notice how Wild-Types are placed diagonally opposite from each other. Tan Types follow the same assortment. Figure4: Sample of S. fimicola asci patterns under microscope. The asci are usually formed in patterns of 8 and researchers score only the 2:4:2, 2:2:2:2, and 4:4 patterns. (Singaravelan  et al. 2010)DiscussionOverall, recombination frequency was higher in both radiations as compared to the control data. Recombination was observed between two strains (2:2:2:2 and 2:4:2) of S. fimicola in all the treatments and the control. In table 1-7, the recombination frequency was calculated. The frequency of recombinant asci in control group was 72.4%. Whereas, the frequency of recombinant asci increased in the treatment sample, 58.0% in low radiation treatment and 37.4% in high radiation treatment.The trend observed from each table reveals that recombination occurs more frequently in low radiations compared to high radiations. The hypothesis suggested was wrong as the recombination frequency was little higher in low radiation as compared to the high radiation environment. This suggests that S. fimicola may also have cross over more in the North Facing Slope of Evolution Canyon. As more results came in, it was noted how the numbers became more uniform, providing more solid evidence.However, despite the evidence illustrated in Tables 1-7, it should be highlighted that there may be sources of error that might have changed the actual results. First, errors might have occurred while preparing agar plates. On the agar plates, there might be growth of some different fungus on agar plate due to contamination.  Contamination could have resulted from the improper use of laboratory material. Through agar preparation step for mating S. fimicola, scalpels were instructed to be disinfected. Second, the negligence of disinfecting the scalpels may have caused the contamination. Third, improper sealing of the agar plate in the end could also make the culture of S. fimicola vulnerable to other contaminations.Problems like contamination leads to other errors. Forth, the improper breakage of perithecia to release the asci can also lead to improper results. By tapping too hard or too gentle, the asci may get destroyed or not enough asci would be released.From this experiment, a sample baseline for S. fimicola was prepared in optimal lab setting. This means that if no environmental effects like radiations were at work, then S. fimicola would cross over at the frequency of around 72.4%. For the other group of researchers who are experimenting at the Evolution Canyons, they can use this lab as a baseline to compare how S. fimicola mate in the wild.ReferencesBurpee, D., Cyr, R., Hass, C., Ward, A. and D. Woodward.2018. A Laboratory Manual for Biology 110 Biology: Basic Concepts and Biodiversity. Department of Biology, The Pennsylvania State University, University Park, PA.Cyr, R. 2018. Fungi I – Evolution and Diversity, Phyla Chytridiomycota and Zygomycota. In, Biology 110: Basic Concepts and Biodiversity Course Website. Department of Biology, The Pennsylvania State University. https://courses.ed.science.psu.edu/biol110/node/4257.Cyr, R. 2002. Meiosis, Heredity and Life Cycles. In, Biology 110: Basic Concepts and Biodiversity. Course Website. Department of Biology, The Pennsylvania State University.               https://courses.ed.science.psu.edu/biol110/node/4256.Lidzbarsky GA, Shkolnik T, Nevo E (2009) Adaptive Response to DNA-Damaging Agents in               Natural Saccharomyces Cerevisiae Populations from ‘‘Evolution Canyon’’, Mt. Carmel, Israel. PLoS ONE 4(6): e5914. doi: 10.1371/journal.pone.0005914Genetic Diversity Definition| Biodiversity A-z. http://biodiversitya-z.org/content/genetic-diversitySingaravelan, N., Pavlicek, T., Beharav, A., Wakamatsu, K., Ito, S., & Nevo, E. (2010). Spiny mice modulate eumelanin to pheomelanin ratio to achieve cryptic coloration in “evolution canyon,” israel. PLoS One, 5(1) doi: http://dx.doi.org/10.1371/journal.pone.0008708.