Jaclyn Wu Multicellularity article analysis 3/1/12 Multicellularity has long been known to be a key element to evolution but the

mechanism and nature of its development have remained a mystery. Using Saccharomyces cerevisiae, a type of unicellular yeast, Ratcliff, Denison, Borello, and Travisano experimentally explored how multicellularity might evolve in an environment where selection favored fast-settling yeast (2011). Ratcliff et al. focused their experiments on the origin of multicellularity and the shift from being unicellular to forming clusters, as well as how multicellularity was then maintained. The scientists hypothesized that multicellularity would develop if they selected for larger size. To achieve this, each of the replicate populations was transferred into a culture tube and the bottommost layer was selected. Clusters settle faster than individual cells and those cells selected were then transferred, cultured again, and selected by the same manner. After 60 transfers, the scientists noted a snowflake-like appearance of attached cells in all populations; multicellular clusters had become predominant. This was used to show that if cells were exposed to an environment where multicellularity increased fitness, they would quickly evolve multicellular forms. Ratcliff et al. also examined whether or not the clusters arose from postdivision adhesion or aggregation. The snowflake clusters were digested to obtain individual cells, which then developed into more multicellular snowflakes. These clusters were stained to observe that divisions of existing cells formed new clusters, not aggregation of separate cells. This suggests that early multicellular organisms may have also evolved in this manner. Another hypothesis they tested was how the clusters were maintained and reproduced. Ratcliff et al. grew the yeast in a medium and saw that daughter clusters were released one-by-one as multicellular propagules, which then underwent a similar process. These cells were again exposed to selection for fast-settlers with varying time frames. Rapid-settling populations saw an increase in number of cells and size of the cluster; this showed the continued maintenance and evolution of the snowflake clusters. Because the phenotype of the whole was affected, they concluded that selection was now acting on the multicellular cluster rather than the individual cells. A final hypothesis tested by the scientists was that a larger cluster size favors the evolution of apoptosis. After observing higher frequencies of apoptosis with larger snowflake clusters, the scientists used video microscopy and noted that daughter propagules tended to break off at points with higher numbers of apoptotic cells. They drew the conclusion that a small percentage of cells become apoptotic in order to allow a larger number of propagules to break off more easily, which increases reproductive fitness. Ratcliff et al. explored a number of hypotheses in their experiments. Selecting for multicellularity by using the effect of size on settling rate ensured that multicellular clusters tended to be chosen and allowed to reproduce. This does, however, make the assumption that greater size alone is the reason for development of multicellularity, which may not be the case. Perhaps it was advantageous for cells in a harsh environment to cooperate to meet basic

Jaclyn Wu Multicellularity article analysis 3/1/12 needs. In their experiment, all populations were grown on a nutrient-rich medium, which meant that genotypes that may have been eliminated by the earths early environment survived in the lab. If cells that are more likely to be cooperative also tend to clump slower, they wouldve been eliminated by the strong selection methods used by Ratcliff et al. Their manner of selection skews the experiment towards selecting for the fastest clumping cells, which may not necessarily be the most fit in another environment. The scientists concluded that this shows the development of multicellularity may be simpler and quicker than originally thought but this is based solely on size and clumping of the cells. It is likely that with other conditions and without the strong selection imposed by the experiment, multicellularity would involve a variety of factors and not occur simply based on cluster size. The evidence for post-division adhesion as the mechanism for development of clusters was soundly tested and supported. Evidence for this was established in two different ways. One, the actual formation of clusters was observed as daughter propagules breaking off a parent and growing by division. Secondly, the individual cell that developed into a cluster was stained by a technique used to detect yeast budding, which is appropriate in detecting division since budding is also a process that originates from a single cell. Additionally, Hamiltons Rule can be invoked to support post-division adhesion, as the cells of the cluster would be highly related to one another. This explains why individual cells might sacrifice their reproduction in order to benefit the entire cluster because inclusive fitness is increased. Along those lines, this also provides support for the scientists findings that apoptosis co-evolves with size to increase inclusive fitness (via an advantage in propagule production) at the cost of a few individuals. An important implication of this is emerging division of labor. Ratcliff et al. were reasonable in concluding that programmed cell death places the apoptotic cells at the same level as somatic cells, in that they serve a function apart from passing on genetic information. Their experiments provide evidence that the apoptotic cells may have been the first somatic cells not used for reproduction. Overall, Ratcliff et al were able to provide solid experimental evidence for the rapid development, reproduction, and maintenance of multicellularity from unicellular yeast cells. There was a simplification in the manner in which they selected for the yeast but it does show that under certain conditions where size is adaptive, multicellularity can evolve much quicker, with less complexity, than previously imagined. Such a rapid change followed by consistent cluster form also may imply punctuated equilibrium is fully plausible for macroevolution.

Jaclyn Wu Multicellularity article analysis 3/1/12

Works cited: Ratcliff W, Denison R, Borello M, Travisano M. 2012. Experimental evolution of multicellularity. Proceedings of the National Academy of Sciences of the United States of America 109(5):1595-1600.

Jaclyn Wu Multicellularity article analysis 3/1/12 beginning: -two problems: origin as well as maintenance of multicellularity -first step likely evolution of genotypes that form simple cellular clusters; after clusters, selection among multicelled clusters has to be greater than selection among single cells within cluster -division of labor must occur within a cluster to increase cluster-level fitness -use experimental evolution to directly explore the evolution of early multicellularity, focusing on the mode of cluster formation (postdivision adhesion vs. aggregation), the shift from single-cell to cluster-level selection, and the evolution of among-cell division of labor -use of gravity to select for primitive multicellularity in unicellular yeast (clusters settle more quickly than single cells, allows for selection of clustering genotype) -10 replicate populations of initially isogenic yeast grown in nutrient-rich medium, then shaken and transferred to a fresh medium where they stayed for 45 min. then transferred to a fresh medium and allowed to settle to the bottom of the culture tube -cells in the lowest 100uL transferred to a fresh medium -later on, used centrifugations to settle yeast and pick which ones to transfer to fresh medium (this process allowed for selection of clusters of cells) -rapid increases in settling rate over course of selection -after 60 transfers, all populations had predominantly spherical snowflake-lke phenotypes that were composed of attached cells -snowflake phenotype has 34% fitness advantage over individual cells when selection is present but is 10% fitness cost if there is no settling selection -to test if clusters were due to aggregation or postdivision adhesion (results in more genetic similarity, possibly less conflict between cluster and single celled selection): -snowflake clusters were digested by an enzyme to get individual cells, which were then allowed to grow for 16h, each of which ended up forming new snowflake like clusters -aggregation not witnessed -cells were stained using a method that detects yeast bud scars, which allowed the scientists to see that the groups of cells that made up the snowflake were due to divisions of the existing component cells. -snowflake cells also phenotypically stable, transferred 35x without selection but maintained snowflake nature and no unicellular strains detected -transition from single celled to multicellular studied by looking at cluster reproduction and settling survival -clusters come from unicellular or multicellular propagules

Jaclyn Wu Multicellularity article analysis 3/1/12 -clusters were grown in a medium and shown to release daughter clusters al as multicellular propagules (the parent cluster did not completely dissolve into several daughter clusters.) -the unicellular ancestor divides into 2 daughter cells that are similar in size to the parent but the multicellular propagules released by the clusters were often less than half the size of the parent clusters and always at least a certain size. The scientists believe that this sugguest there is a youth and adult life stage for the multicellular yeast. -these again tested with selection with another 35 transfers with similar conditions except for varying the time allowed for gravitational settling (5, 15, 25min) (They used replicate populations where the snowflake phenotype had already evolved.) -35 transfers a day were performed and the populations that were only given 5 minutes to settle developed a setting rate that was 20% greater than those populations that were given more time. -other notable features for the rapidly settling clusters were that they were larger, produced larger daughter clusters, and had more cells -the average size of a cluster also more than doubled with the fastest settling time treatment compared to the original snowflake cluster -selection caused the phenotype to change so the scientists concluded that it acts on reproduction of the clusters as a whole, rather than the individual component cells themselves -to see if it would be beneficial for once cell to sacrifice its reproduction in order to benefit the whole cluster, the scientists incorporated Hamiltons rule, in that dividing labor provides opportunities that outweigh the loss of reproduction by the individual cells. (Also, because the clusters formed by post-division adhesion, they were all much more highly related to one another.) -programmed apoptosis was seen -propagule size had to be balanced between settling rate and how quickly the yeast could grow (Bigger and faster-settling snowflake yeast takes a longer time to grow than their smaller counterparts.) Alternatively, while smaller snowflake yeast propagules dont settle as quickly, they also grow faster and are able to produce more of the smaller propagules. (all clusters take time before they begin producing propagules) -they also had the hypothesis that large cluster size favors evolution of apoptosis -found that faster settlers also was correlated with frequency of apoptotic cells -apoptotic or dead cells are less strongly attached to daughter cells and through video microscopy, it was observed that the propagules often broke off from the oldest cells, where there was the highest frequency of death. This led them to believe that the dead cells were likely what cause the cells to separate where they did. This was tested by inducing increased apoptosis over transfers, which then resulted in cluster producing smaller and more daughter propagules. After 14 transfers, the clusters were about 40% of the parent

Jaclyn Wu Multicellularity article analysis 3/1/12 cluster but by 60 transfers, they were only about 20% of the size of the parent. The clusters in general increased in size as expected with selection during transfers, but proportionally, the daughter clusters were smaller. -This increase in apoptosis only is seen with larger snowflake phenotypes because their larger size allows them to create larger numbers of sufficiently sized propagules. -in large clusters, a small percentage of cells become apoptotic to be serve as points where propagules can break off, which allows for a greater number of propagules to arise from the cluster -because apoptotic cells do not leave descendents after their death, they are like somatic cells in that they do not pass on genetic information. The scientists believed this to be consistent with the hypothesis that early multicellular tissues would be mostly for reproductive use because otherwise the overall fitness would go down compared to if the cells were all reproducing individually. The divided labor must be worth the loss in reproduction. -shows that multicellularity can happen without much complexity and over a quicker period of time than originally thought. In the experiment, the snowflake yeast evolved in all the experimental populations within 60 days.