Analysis

Phylogenetic Tree

Looking at our pollen morphology, the Rhododendron and and Kangaroo Paw shared more similarities leading us to believe that they were closer in relation. However, when looking at the protein sequences we found that the Kangaroo Paw was actually closer in relation to the Kris Kringle cactus. This makes sense because the Kangaroo Paw and Kris Kringle cactus are both plants that live in warm climates while the Rhododendron lives in temperate climates.

Original Scientific Question

"If our hypothesis is supported, we expect to find pollen which live in wet climates to have more protrusions or "spikes"while ones in dry climates have a smooth outside.
If our hypothesis is rejected, we will not see these physical features on the pollen grain which help it maneuver it's environment."

Upon beginning this experiment we believed that wet climate plants would have "clingy" pollen while dry climates had "airborne" pollen. Our hypothesis was semi correct. Kangaroo Paw which comes from a hot climate, while shriveled up appeared to be round/ airborne without any spikes. This matched our hypothesis for pollen and dry climates.The Kris Kringle cactus which resides naturally in a warm but humid climate displayed protrusions in the pollen grain. At first we did not understand why the Kris Kringle would have spikes, however after learning that they are from humid climates, their pollen morphology made sense.
The morphology of the rhododendron however did not show anything that looked "clingy" which is why we decided to group it with Kangaroo Paw in our initial tree. We thought that it would have spikes considering that it came from a wet climate. The rhododendron did not match our initial hypothesis.    

Pollen trees

Here is a link to our morphology pollen tree

Graph credit: Morgan

Above is our phylogenetic tree. Kalmiopsis_leachiana is the Rhododendron (We chose this genus out of the selection on uniprot due to it's nativity in Oregon) and the Anigozanthos_flavidus is the Kangaroo paw (We chose this genus due to the fact that it was yellow, like our sample).

What Our Pollen Morphology Shows

We have observed that the Schlumbergera has Echinate and Foveolate pollen texture, while The Rhododendron and Kangaroo Paw have different surface textures than the Schlumbergera, so we gave  Schlumbergera it’s own branch on the tree. Rhododendron and Kangaroo Paw both have a similar surface texture, therefore we have them branching off together. The Kangaroo Paw branches from the Rhododendron due to the fact that it has a Verrucate texture rather than the Scabrate texture found on the Rhododendron. The reasoning for their connection is the fact that they both have a Psilate surface texture.

What our data shows so far:
For the "Kris Kringle" schlumbergera truncata, we observe that the pollen is characterized as echinate and foveate, because of its spike and hole features. From that we can possibly conclude that the pollen has adapted to its environment to stick to animals in the desert. It could also be airborne, because its shape is   symmetrical in the way that it looks aerodynamic.
The kangaroo paw is more angular and multidimensional. It does not have the disk-like shape of the schlumbergera. Its surface appears smooth. From that we may be able to conclude that is is easier picked up by a pollinator, though it does not appear to be sticky.
Lastly, the rhododendron, is circular from the top view but we cannot conclude whether it is spherical, or more disk like. The resolution on the pictures of the resolution is low and the inconclusive.
Both the rhododendron and the kangaroo paw seem to share a psilate surface. They are neither flat nor disk-like and neither have apertures, like the schlumbergera truncata does.

These observations support our conclusion that the kangaroo paw and rhododendron are more closely related.

Procedure

Sample Procedure

1. Collect pollen samples from three different flowers.
2. Take fresh samples and place into dehydrator until dry.

Sem Procedure

1. When samples are dry prepare SEM stub.
2. Peel carbon tape and align with stub.
3. Place stub firmly on carbon tape and remove.
4. Take tweezers or a straight stick and divide it into thirds. 
5. Mark one third | the second third || and the third third |||.
6. Create a diagram showing where each sample will go. (Ex. |Sample 1||)
7. Place the first sample under under a dissecting microscope.
8. Locate the pollen grains under the microscope.
9. With a clean paint brush, collect the pollen grains on your brush. 
10.  Take the paint brush with the pollen grains and place it to the side.
11. Place the stub underneath the dissecting microscope. 
12. With the brush containing the pollen, carefully brush onto the stub in the correct section.
13. Repeat steps 9-14 with pollen sample 2 and 3.
14. Retrieve can of concentrated oxygen.
15. With the stub in one hand, point the concentrated oxygen 1 1/2 feet away. 
16. In a tornado motion, release oxygen directed at the stub until 2 inches away.
17. Repeat. 
18. Check under dissecting microscope to ensure pollen grains stayed in place.
19. Push stub into place of the SEM cup.
20. Turn it clockwise until it is no longer visible at eye level. 
21. Turn it an additional two notches.
22. Lift SEM door and slide cup into place until you hear a click.
23. Slide door down slowly.
24. Press maze icon to map image. 
25. Click setting> label and label selected sample With the first three letters of the sample, period and first initials of teammates. (Ex. Kanp4avmm) 
26. Locate the selected sample and zoom in at 2000x
27. If image is blurry, press contrast and auto focus until clear. 
28. Take pictures and 2000x or greater if wanted.
29. Go to archives.
30. Select 2000x picture and click ruler icon.
31. Tap one end of pollen and then the next. Record measurement. Save.
32. Repeat with 2 other samples.

Leica Procedure

1. Take first sample and place on the stage.
2. Locate pollen grain and focus. 
3. Capture picture at 35x. Repeat with other 2 samples.

Pollen Tube Growth Procedure

1. Gather pollen from one sample and brush onto concave slide.
2. Drop 1-2 drops of pollen growth media (0.1% Boric acid and 30% Sucrose) onto slide. Cover with cover slip.
3. Locate pollen under  compound microscope at 400x and look for tube growth.
4. Every 5 minutes record length of growth.  (Not all pollen will display tube growth)
5. Draw pollen or take a picture. Repeat.  

Pollen data table

Table comparison of the three pollen grains.

Recordings from SEM Microscope 12/12

12/12/13
Took photos of Schlumbergera (Cactus) and Kangaroo Paw but didn't get to take photos of Rhododendron.

cacp4ammv: Some pollen has one mark and some pollen has two marks. At 2000x the measured width is 47.8 mm. We took pictures at 5000x, 13000x and 20000x.

rhop4ammv: Took several pictures of this pollen under the label of cacp4ammv by accident, need to adjust later. At 2000x the measured width was 29.6 mm.

Background

Research on the morphology and development of pollen and tapetum has contributed to understanding the evolution of these characters and elucidating phylogenetic relationships among seed plants.
Over evolutionary time, the morphology of angiosperm pollen has evolved toward an increasing number of apertures, among other things. From a neo-Darwinian point of view, this means that (i) some polymorphism for aperture number must exist and (ii) there must be some fitness increase associated with increasing the aperture number. Pollen types with different aperture numbers often occur in the same species.
Although the majority of flowering plants achieve pollination by exploiting the food‐seeking behavior of animals, some use alternative ploys that exploit their mate‐seeking behavior. Sexual deception is currently known only from the Orchidaceae and almost always involves pollination by male hymenoptera.
Pollen grains are the carriers of the male gametes or their progenitor cell, in higher plants. They also are important tools for paleoclimatic reconstruction. They reflects the ecology of their parent plants and their habitats and provide a continuous record of their evolutionary history.