The graphs shown in Appendix D.7 shows that B3 ancestor exhibits two peaks with a large proportion at the higher peak in the DAPI stain and this is reflected in the replicate. Upon analysis of the data as shown in Appendix D.7 Table D.7C, the fraction that makes up the lower peak, in DAPI, shows only line B5.5 changes statistically significantly and increases. For SYTOX lines B5.1, B5.2 and B5.3 all show a statistically significant increase.
In terms of peak location, the DAPI stained samples showed no statistically significant changes in the position of the lower peak, which is the case for the SYTOX stained samples too. For the higher peak location, only line B5.4 demonstrated statistically significant increase, while SYTOX showed statistically significant increase in lines B5.4 and B5.5. Overall these results do not seem to reflect anything consistent across the two stains other than possibly a change in the position of the higher peak as can be seen in Figure 5.8.
Figure 5.8: B5 Relative Change. Fraction of lower peak and position of both the lower and higher peaks in both DAPI and SYTOX as compared to the ancestor. The small peak position is missing due to there being no higher peak present in the SYTOX measurements.
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5.7.1.6Issatchenkia orientalis Kudravtsev C10
In the graph presented in the Appendix D.8 it would appear that C10 ancestor demonstrates two peaks in DAPI, this is supported by the replicate but to a lesser degree in SYTOX with a very small higher peak. Some of the evolved lines in DAPI appear to lose the lower peak which is not reflected in the SYTOX samples. When looking at the statistical results in Appendix D.8 Table D.8C, four evolved lines, C10.1, C10.2, C10.4 and C10.6, evolved towards a decrease in the fraction that was the lower peak in DAPI, save for line 4 which evolved towards an increase. In the SYTOX samples there were three lines where the lower peak fraction decreased statistically significantly, lines C10.1, C10.2 and C10.4. From Figure 5.9 it would appear that there is a common trend towards decreased lower peak fraction, each with one increasing line, however these do not occur in the same line and SYTOX does not show this increase to a statistically significant level.
From the analysis of the position of the peaks, the DAPI samples show two lines with statistically significant changes line C10.1 with a decrease and line C10.5 with an increase in position of the lower peak. SYTOX samples however show an all-round statistically significant increase in the lines for the lower peak. In the higher peak, lines C10.2, C10.3 and C10.6 all show statistically significant
Figure 5.9: C10 Relative Change. Fraction of lower peak and position of both the lower and higher peaks in both DAPI and SYTOX as compared to the ancestor.
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increases in the DAPI samples, while the SYTOX samples show no statistically significant changes. No consistent trend is apparent in these results from the evolved lines.
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5.7.1.7Kluyveromyces nonfermentans F8
The graph in Appendix D.9 shows the F8 ancestor as having what could be described as two peaks in the DAPI stained samples, however there is very little clear separation between the two peaks. In SYTOX samples while there is one clearly defined high peak in the ancestor, there also appears to be some similarity to the DAPI samples with a low level of signal between where a lower peak would be observed and the higher peak although not to a level that the statistical analysis registers it. When looking at the fraction that makes up the lower peak it is shown that for DAPI stained samples all evolved lines show a statistically significant increase. Looking at Figure 5.10, for SYTOX stained samples all lines show an increase in lower peak fraction, although this is only statistically significant at p=0.008 for line F8.5.
Appendix D.9 Table D.9C demonstrates that in terms of peak position, the lower peak in the DAPI samples all lines have statistically significant decrease in position compared to the ancestor. This
Figure 5.10: F8 Relative Change. Fraction of lower peak and position of both the lower and higher peaks in both DAPI and SYTOX as compared to the ancestor. The small peak position is missing due to there being no higher peak present in the SYTOX measurements.
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is not supported by the SYTOX samples due to the lack of lower peak in the ancestor to compare to. The position of the higher peak in the DAPI samples shows statistically significant increase in three evolved lines, F8.1, F8.4 and F8.6, while the SYTOX samples show statistically significant decrease in lines F8.2, F8.3, F8.5 and F8.6. From this data, there does not appear to be consistency in the changes in position of the peaks, but there does seem to be support for the changes in fraction that makes up the lower peak.
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5.7.1.8Saccharomyces cerevisiae SCE2
The graphs shown in Appendix D.10 show that the ancestor starts with two peaks for DAPI, but the consistency of the proportions of each peak between replicates is questionable. When it comes to SYTOX the higher peak appears to be very low in proportions and would not appear to count as a peak in the statistical analysis hence being assigned NA in Appendix D.10 Table D.10C. In terms of measuring the fraction of which makes up the lower peak, DAPI samples show an across the board statistically significant change towards increased fraction as the lower peak. However, SYTOX samples exhibit the exact opposite unanimous change in the evolved lines, statistically significantly decreasing in lower peak fraction.
If the peak position is considered, a number of notable trends can be observed and are demonstrated in Appendix D.10 Table D.10C and Figure 5.11. Firstly, the position of the lower peak in the evolved lines appear to increase in both DAPI and SYTOX stained samples, although this change Is only statistically significant in lines SCE2.5 and 6 for DAPI and SCE2.1, SCE2.3, SCE2.4 and SCE2.6 in SYTOX. For the higher peak, however, DAPI samples do not demonstrate the presence of a higher peak, while in SYTOX the ancestor does not have a double peak while the evolved lines do. This does mean that the statistical results cannot be used as there is no ancestor peak to compare to but the presence of these peaks in SYTOX evolved lines in itself suggests a change from the ancestor. From these results, we can conclude that there are no consistent changes across the two stains.
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5.7.1.9Summary
Here the DAPI and SYTOX DNA stain data was presented and analysed using linear models. From this analysis, observed number of observations may be made. Firstly, the populations presented different patterns, either demonstrating two observable single peaks or only one. The peak with the most counts also differed depending on yeast. Each yeast appeared to have a somewhat unique peak pattern. In most populations, the replicates would be similar and support the same pattern. However, there were often differences between the two staining methods, which, rather than adding robustness to the results, were frequently contradictory and even statistically significant ones, such as the change in the fraction that made up the lower peak in SCE2. Binomial tests were performed on the results from the two DNA stains to establish if there were any statistically significant difference from random chance in the results. As binomial tests require a minimum of six values, these results were not limited down to only statistically significant results like in previous chapters. From Table 5.12 it would appear that A1, A8 and C10 in DAPI staining do not have any statistically significant trends while in SYTOX this is the case for B3 and
Figure 5.11: SCE2 Relative Change. Fraction of lower peak and position of both the lower and higher peaks in both DAPI and SYTOX as compared to the ancestor. The large peak position is missing due to there being no higher peak present.
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B5. For the DAPI stain all evolved lines of B3, F8 and SCE2 show a movement of cell count fraction towards the lower peak, while A11 shows systematic movement of evolved lines towards the higher peak. When considering the position of the peaks, F8 evolved lines consistently show a decrease in the position of the lower peak, while SCE2 shows a consistent increase in the position of the lower peak in all evolved lines. In terms of the higher peak, both A11 and B5 show consistent increase in the higher peak location in all evolved lines.
SYTOX on the other hand shows some different patterns overall than the DAPI stain. The fraction of cells for A1, A8, A11 and SCE2 all appear to move towards an increase in the higher peak, while F8 shows a consistent move of the fraction of cells to the lower peak in all evolved lines. The position of the lower peak in A11, C10 and SCE2 increase consistently over the evolved lines, while no consistent changes are observed in the position of the higher peak.
YEAST Fraction P-value Pos. L
Peak P-value Pos. H Peak P-value A1 5/6 0.2188 2/6 0.6875 NA NA A8 3/6 1.3125 3/6 1.3125 NA NA A11 0/6 0.0313 5/6 0.2188 6/6 0.0313 DAPI B3 6/6 0.0313 3/6 1.3125 4/6 0.6875 B5 4/6 0.6875 3/6 1.3125 6/6 0.0313 C10 2/6 0.6875 1/6 0.2188 5/6 0.2188 F8 6/6 0.0313 0/6 0.0313 5/6 0.2188 SCE2 6/6 0.0313 6/6 0.0313 NA NA
YEAST Fraction P-value Pos. L
Peak P-value Pos. H Peak P-value A1 0/6 0.0313 4/6 0.6875 NA NA A8 0/6 0.0313 5/6 0.2188 NA NA A11 0/6 0.0313 6/6 0.0313 2/6 0.6875 SYTOX B3 2/6 0.6875 1/6 0.2188 5/6 0.2188 B5 3/6 1.3125 4/6 0.6875 4/6 0.6875 C10 1/6 0.2188 6/6 0.0313 4/6 0.6875 F8 6/6 0.0313 1/6 0.2188 1/6 0.2188 SCE2 0/6 0.0313 6/6 0.0313 2/6 0.6875
Table 5.12: Binomial Statistical Outcomes for all Populations. Testing the statistical significance of increases in fraction of data in the lower peak and increase in the position of each peak compared to the ancestor. Statistically significant results are highlighted in bold.
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While there is little consistency between DAPI and SYTOX there is consistency observed between replicates of each dye (see Appendix D for the graphs) and between evolved lines. Thus, there are clearly some consistent changes occurring, but the interpretation of these changes remains elusive.