## 2 Results
### Nutrient-Limited Growth: Differential Growth Rates Impact Global Organization of Yeast Colonies
#### 2.2.4 Nutrient Limitation Further Promotes Subcolony Connectivity  
Next we investigated the impact of nutrient limitation together with budding division on subcolony organization and connectivity. To do this, we first analyzed the number of connected components between budding and non-budding colonies in nutrient-limited conditions. We find that nutrient-limited growth results in a significant decrease in the number of connected components for both budding and non-budding colonies compared to nutrient-rich growth. (Compare Figure 6A to Figure10A.) However, we also observe that the number of connected components for each of the first five subcolonies is significantly lower in budding colonies (Figure 10A). In addition, we see that the average time elapsed from creation of a given subcolony until it splits into more than 5 connected components is significantly lower in non-budding colonies (Figure 10B). These results not only reveal that nutrient limitation has a large impact on subcolony structure and connectivity, but also confirm that budding division maintains its role in promoting subcolony connectivity under nutrient limitation. To further investigate the impact of nutrient limitation and budding division together on subcolony structure and connectivity we considered the number of cells contained in small connected components (i.e., less than 10 cells). We find that nutrient limitation results in a large decrease in the total number of cells belonging to a small connected component (i.e., less than 10 cells) for both budding and non-budding colonies at the 24 h time point (compare Figure 6C with Figure 10C). This further highlights the strong impact of nutrient limitation on subcolony structure. In addition, we observe a significant difference in the number of cells in a small connected component between budding and non-budding colonies at the 18, 24, and 28 h time points. Moreover, we find that the survival curves for the time elapsed from creation of a subcolony to when it splits into more than 5 components are different between budding and non-budding colonies (Figure 6D), indicating that the probability that a given subcolony remains connected (i.e., less than 5 connected components) for longer than \(t\) hours is greater for budding colonies. Similarly as before, we considered this a separate event for each of the first 5 subcolonies and each of our 50 simulations. These results further support our observation that budding division acts as a mechanism increasing spatial adjacency within subcolonies in both nutrient-rich and nutrient-limited conditions. 2.2.5 Nutrient Limitation Changes Global Colony Organization by Driving Variation in Subcolony Sizes  
Results from our previous metrics provide compelling evidence that nutrient limitation has a large impact on spatial organization of growing yeast colonies. However, the strongest demonstration of the impact of nutrient-limited growth on morphological properties of growing yeast colonies is its effect on emergent patterns of subcolony structure and organization for which we do not yet have an explicit quantitative metric. Specifically, simulations of nutrient-limited colonies result in the appearance of  
Figure 10: Subcolony Structure and Organization in Nutrient-Limited Colonies. (**A**) Comparison of the number of connected components for the first five subcolonies between budding (red) and non-budding (blue) colonies at the 28 h time point. We observe a statistically significant difference in the number of connected components for subcolony 1 (\(p=3.39\times 10^{-10}\)), subcolony 2 (\(p=4.454\times 10^{-17}\)), subcolony 3 (\(p=8.537\times 10^{-11}\)), subcolony 4 (\(p=2.884\times 10^{-11}\)) and subcolony 5 (\(p=3.783\times 10^{-06}\)). (**B**) Comparison of time until each of the first five subcolonies splits into 5 connected components.