The Recombination Frequency of Sordaria with

the Introduction of 0.5 teaspoon of Sodium

Chloride
Introduction
Sordaria fimicola is an ascomycete fungus that is a saprotroph, meaning that it consumes
decaying organic material as its primary source of energy. Sordaria fimicola is frequently
utilized as a model organism because of its ease of study due to its rapid life cycle, easy
propagation, and its eight spore asci which are easily visible under a microscope.

The Sordaria Sexual Reproduction

Figure 1: Diagram of the Sordaria Life Cycle

Sordaria is haploid and can undergo both asexual and sexual reproduction. During sexual
reproduction, the antheridium, or haploid structure that produces male gametes, of one Sordaria
and the ascognium, or haploid structure that produces female gametes, of another Sordaria
undergo the fusion of cytoplasm in a process called plasmogamy (Antheridium-an overview;
Plasmogamy-an overview). After plasmogamy, the combined Sordaria cell undergoes
karyogamy, the fusion of cell nuclei, resulting in the formation of a diploid nucleus (Karyogamy-
an overview). This zygote then undergoes meiosis, resulting in four haploid nuclei that then
undergo mitosis to form eight ascospores.
The Effect of Environmental Stress on Genetic Diversity in Sordaria
Sordaria is haploid rather than diploid, so the synapsis and crossing over of genetic material
during prophase I of meiosis is responsible for the genetic variation rather than the independent
assortment of homologous chromosome pairs during metaphase I of meiosis. The crossing over
of Sordaria was studied in the “Evolution Canyons” of Israel. This location is accordingly titled
because it is composed of two mountain slopes with different climate conditions, enabling the
study of evolution in progress. The south facing slope (SFS or “African Slope”) is exposed to
higher solar radiation, resulting in higher temperatures and drought, while the north facing slope
(NFS or “European Slope”) has a more temperate climate that is cooler and more humid (Nevo,
2009). Researchers crossed the wild type and tan Sordaria from the same slope and different
slopes to study the impact of environmental stressors on the recombination frequency of
Sordaria (Lamb et al., 1998; Saleem et al., 2001). The study found that there was a significant
difference in the recombination frequencies between the two slopes, indicating that harsh
environmental stressors can potentially lead to changes in the recombination frequency and thus
genetic diversity of Sordaria (Lamb et al., 1998; Saleem et al., 2001). We decided to focus on
the environmental stressor of salt concentration, theorizing that a higher environmental salt
concentration would yield Sordaria with a lower recombination frequency.

Methods & Materials

Obtaining and Maintaining Sordaria
The Sordaria fungus used in this study was supplied by Carolina Biological Supply Company-
Burlington, NC. All treatments were performed at room temperature, approximately 21°C, on
October 19, 2021. Agar was poured into two petri dishes until the bottoms were covered. In the
treatment dish, ½ teaspoon of salt was thoroughly mixed into the liquid agar. These two plates
were then left to solidify. Once they did, the petri dishes were divided into four quadrants and the
covers were marked with whether the dish was the control or treatment. Before adding the
Sordaria to the plates, the work surface and scalpel were disinfected with a disinfectant wipe.
Afterwards, two small squares of agar blocks from the wild Sordaria culture (approximately
0.5cm square) containing fungal hyphae, were placed in diagonal quadrants with the hyphae side
down. This procedure was repeated for the tan Sordaria culture after the scalpel was disinfected.
After disinfecting the scalpel once again, this entire procedure was repeated for the second agar
plate. The scalpel was disinfected after the setup of both agar plates was completed. Surgical
tape was then carefully wrapped around the edge of each plate to not dislodge the Sordaria.
 Figure 2: Images of the Control Sordaria plate (left) and the Treatment Sordaria plate (right)
after setting up the mating plates.

Determining Recombination Frequency

After the control and treatment plates were left alone at room temperature, approximately 21°C,
for two weeks, the resulting mating cultures were observed under a microscope. The mated
perithecia were placed on a wet microscope slide using a sterilized scoop. The intact perithecia
were then carefully ruptured through pressing the eraser end of a pencil down on the microscope
cover of this slide. The resulting ruptured perithecia were then observed under 10X
magnification. The asci are clearly visible after the perithecia are ruptured, enabling us to note
the color of the asci of eight ascospores that result from the mating of the wild and tan Sordaria
cultures. The color patterns of the asci were recorded as well as the total number of intact asci
lines observed. The asci color patterns of 2:4:2 and 2:2:2:2 indicate that recombination events
have occurred, so the total of observations of these patterns divided by the total observations
noted equals the recombination frequency of the mating of the wild and tan Sordaria cultures.

Results
Due to experimental error and probable contamination, there were no Sordaria perithecia to
score the asci of. However, there was a control plate of Sordaria that was uncontaminated, and
we scored the asci of that plate. To score the asci, we used a microscope to look for an intact,
singular asci: a line of eight spores which were either black or tan in color. To be scored as non-
recombinant, the eight spores appeared in a pattern of four tan cells to four black cells (or vice
versa). Subsequently, we determined that events of recombination occurred in the asci when
these eight spores appeared in a color pattern of 2:4:2 or 2:2:2:2. On the other hand, we
determined that no events of recombination occurred when there were patterns of 4:4. An
example of what was observed under the microscope when scoring the asci is displayed by
Figure 3. 
 Figure 3: Example Image of a Perithecia of Asci. 

Figure 4 displays the observed asci scores of the 18 asci of the control group observed under the
microscope. 
Figure 4: Table of Asci Scores of the Control Group. 

Non- Recombinant Recombinant Total # Recombination Frequency (#

recombinant (2:4:2) (2:2:2:2) Asci recombinant/total # asci) X 100
(4:4)

9 6 3 18 50%

Figure 5 displays the data of Figure 4: Table of Asci Scores of the Control Group for a better
visualization of the results. 
 Figure 5: Graph of the Asci Scores of the Control Group

Despite having an uncontaminated control plate to score the asci of, no such plate of the
Sordaria treated with 0.5 teaspoon of Sodium Chloride was available. Accordingly, there was no
treated perithecia to score the asci, and therefore we have no data to share regarding the observed
recombination frequency of the treatment group. 

Discussion
Due to the contamination of the mating plates with an unidentified mold, the difference between
the recombination frequencies and crossing over of Sordaria in the 0.5 teaspoon salt
concentration treatment group and the control cannot be accurately analyzed. Although there was
an uncontaminated control plate available to observe the recombination frequency of, there was
no such treatment plate, and therefore no data is available for that treatment group.

Figure 6: Images of contaminated Control Sordaria plate (left) and Treatment Sordaria plate (right).
During the process of creating the Sordaria mating plates, there are several potential ways of
contamination. Since the original Sordaria cultures were shared amongst fellow researchers also
studying the relationship between various environmental stressors and Sordaria crossing over, it
is a possibility that the plates were contaminated prior to our extraction of the Sordaria for our
own mating plates. For instance, simply leaving the lid off the container provides ample
opportunity for environment to get into the cultures. Furthermore, contamination could’ve also
arisen from the improper sterilization of forceps in the extraction process. It is unlikely that the
agar pour was the primary source of contamination as we didn’t pour the control Sordaria plate,
meaning that if the agar pour was responsible, only the treatment Sordaria plate should be
contaminated.
Despite being unable to observe differences in the crossing over of the two treatments of
Sordaria, we did however observe a difference between the contamination levels of the two
treatments. The 0.5 teaspoon salt Sordaria plate displayed a significantly lower propagation of
the contaminate mold, leading to the theorization that the higher salt concentration inhibited the
growth and reproduction of the contaminate mold. However, further study would be necessary to
provide evidence that truly supported this hypothesized explanation for the observation.

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