Analysis of Phylogenetic Trees

We hypothesized that Asian Lily and Day lily would be more closely related because of their pollen morphology similarities (monocolpates and reticulate-like surfaces.) In contrast, the Calla Lily was inaperturate and had a psilate surface. Our hypothetical tree disagreed with our protein-sequence-based phylogeny tree. The protein sequence showed that the Calla Lily and the Asian Lily were more closely related. This discrepancy between our research and the unipro tree makes us question the validity of the information we obtained from unipro. In addition, the unipro did not have a protein sequence for the Asian Lily; consequently, a protein sequence of its subspecies was used. To check our results, we researched the origins of each lily and discovered that the Asian Lily and Day Lily both originate in Asia, whereas the Calla Lily originates in Africa. Since the results we got from the unipro was again inconclusive with our data and research, we hypothesize that the protein-sequence-based phylogeny tree is wrong.

Jalview Data Table

Unit Prot Data Table

Hypothetic Phylogeny Tree Based on Pollen Morphology

We hypothesize that the Asian Lily and Day Lily are more closely related due to their similar reticulated surfaces. The Asian Lily has a reticulate surface, while the Day Lily has more of a foveolate surface. In addition, they are both monocolpates. In contrast, the Calla Lily is inaperturate and has a psilate surface.

Experimental Question Analysis

We were testing three different stages of an asian lily to see if pollen morphology changes when a flower develops. We thought samples one and two would be bigger than the third sample, thinking the last sample would be older and get smaller. Our hypothesis was proved wrong. We thought that the second sample would be the longest, but the third sample turned out to be the longest (125 micro meters). The second sample was the shortest (109 micro meters), and the first sample was 120 micro meters. It is hard to analyze this data compared to our hypothesis because we didn't test a lot of data, only three different grains of pollen.

Procedure

1. Preparation for the SEM:

To prepare a pollen sample of the Lilium asiatica (Asian Lily), we first cut the anthers of three different lilies within the bouquet. Once dehydrated, we prepared the samples for the SEM. First, we placed a piece of carbon tape on the stub. Then, we used a different brush for each pollen sample to place a small amount of pollen onto the stub. After that, we set the sample in the tray. Before placing the sample into the SEM, we used liquid air to blow off any loose pollen--moving from low to high pressure. Once this was completed, we placed the sample in the cup. The next step was crucial: we clicked the top notch of the SEM four times to the right. Then we placed the cup into the slot and once the green light turned on, we pulled down the door. We devoted the remainder of our time to mapping specific areas of the sample and taking measurements. 

2. LEICA Procedure: 

Stage 1 Asian Lily (35X)

Stage 2 Asian Lily (35X)

Stage 3 Asian Lily (35X)

We used the Leica EZ4D Microscope to obtain images of our pollen at a magnification of 35X. With its 7 LEDS, the Leica is a microscope that provided us with high quality illumination and contrast in the images of our pollen samples.








After putting small samples of pollen at each stage in separate dishes, we placed it under the microscope. Three people were needed to operate the Leica and obtain the images of our sample. Two were needed to adjust and focus the lens onto the specimen and one was needed to capture the images at the right moment. We experimented by adjusting the light at various angles to optimize our view of the sample.







Unfortunately, we were unable to recognize any noticeable differences between the pollen grains at their various stages. We expected to see a greater distinguishing characteristics at the SEM.

Julia preparing the sample for the Leica. 

3. Compound Microscope:

At Station 8, we created wet mounts of the pollen samples we had left over of our Asian Lilies. We used the growth media solution in place of water. The image below is the pollen of our Asian Lily at Stage 2.

Asian Lily Stage 2 (400X)

 From the image, we can see one aperture. There are also distinguishable features on the surface of the pollen grains.

Christina observing the pollen sample!

4. SEM Work:

While working with the SEM Phenom Pro by FEI, we were looking between fiduciary marks to focus on different stages of our pollen samples. After many adjustments, we worked to focus on individual pollen grains and captured their image at a magnification of 2000X. (See SEM photos on sidebar.) 

Kent focusing on SEM images!

Addendum 12/19

Julia: Add addendum and data table onto blog
Kent: Edit SEM photo onto seashore
Christina: Add procedure onto blog