1. How Thickness of Chlorophyll in Aspen Bark is dependent on Height and Stand type. Lizabeth Rodriguez Winter Ecology Spring 2014 CU Mountain Research Station

2. Introduction It is important to understand where the chlorophyll is located within the bark and what affects it, because it is how Aspen can survive, and compete with conifers. -Bark can provide up to 15% of the photosynthetic surface of a tree (Schaedle and Foote 1971). Anatomy: Lenticels are breaks in the outside of the bark that are used for gas exchange, however they can not make up for the loss of stomata and carbon recycling must occur (Aschan et al. 2001) Outside of the bark is called the periderm Under the periderm is the cortex which contains chloroplasts. Layer of chloroplast containing cells is called Chlorenchyma (Aschan et al 2001) Chloroplast density dramatically decreases with depth (Schaedle et al. 1968) (Populus tremuloides michx.)

3. Previous studies Temperature (Schaedle and Foote 1971) Some bark photosynthesis occurs well below 0 degrees Rate of photosynthesis increases drastically between 10-25 degrees Elevation (Covington 1975) Trees appeared greener at higher elevations and more white at low elevations due to dead peridem build up Chlorophyll content of bark decreases as altitude increases Age (Aschan et al 2001) Chlorophyll content decreases with age None have considered height.  Since the top of trees get more sun, does the bark higher up on aspen trees have more or less chlorophyll when compared to lower levels? Hypothesis: There will be a thinner layer of chlorenchyma cells in bark that is at a shorter height; less light penetrates lower, especially underneath the snow therefore is not useful for the tree to spend energy up keeping a large amount of photosynthetic cells at lower heights. Null: The chlorenchyma layer will be a similar thickness throughout each tree

4. Does stand type (aspen only vs mixed with conifers) effect chlorophyll thickness? Hypothesis: Conifer trees shade the area, so aspens in mixed stands will need a thicker chlorenchyma to be able to produce as much glucose as aspens in stands that have no conifers.

5. Field Five 2x2m plots were sampled No sapling tissue was collected For each Aspen, three bark samples were taken according to height above the soil 225cm 155cm 30cm - underneath a significant amount of snow in every case. All samples are from the south side of the tree and stored in Zip-lock bags to minimize oxidation damage and drying out overnight.

6. 30 m 155225 Lab Cross section slices were observed under a compound microscope, and a photograph was taken Length measurements were made using AxioCam software multiple measurements were made for each image and averaged for each tree. Data Analysis Unpaired T-tests and a Two way anova were ran using R

7. P-value: 0.014 P-value: 0.598 There was a general trend of increasing chlorenchyma thickness with height Unpaired T-Tests were performed. There is a 59.89% chance there is no difference between 225 and 155 There is only a 1.41% chance there is no difference in chlorophyll thickness between 30 and 155 Results

8. Results separated by plot The correlation between height and chlorophyll thickness is strong in plots 1, 4 and 5 2&3 do not follow this pattern, these plots were noticeably sunnier and located in the aspen grove where no conifers were growing  2 nd Question: Does stand type (aspen only vs mixed with conifers) effect chlorophyll thickness?

9. F-value = mean of squares /mean residual variance (unexplainable error) Larger F-value means more significance Inversely related to P-value How thickness is affected by Height F=19.323 -what I looked at during t-test P=7.637e-05 Stand type F=27.793 P=4.658e-06 Mean thickness in plots 1,4&5 compared to mean thickness in plots 2&3 Interaction between height and stand type F=20.235 P=5.539e-05 Two way ANOVA-Thickness vs Height and Stand type In mixed stands there is a larger range of thickness between the heights, a different pattern of thickness depending on stand type.

10. Summary of results There was a general trend of increasing chlorenchyma thickness with height, this increase was especially evident between the below snow samples and the above snow samples. Aspen only stands have thinner chlorenchyma layers than Aspen trees that are mixed in with conifer trees In mixed stands there is a larger range of thickness between the heights, while aspen only stands do not have much variation, they exhibit a different pattern.

11. Strong reflection off of the snow explains why in plot number 3 the 155cm height was strongest. Plot 2 and 3 in Aspen grove (no conifers) - Discussion The 30cm height is not thinner, in this area because enough sunlight reaches through the snow to the bark allowing enough photosynthesis to occur offsets the energy cost of maintaining the specialized chlorophyll cells. Hypothesis: Conifer trees shade the area, so aspens in mixed stands will need a thicker chlorenchyma to be able to produce as much glucose as aspens in stands that have no conifers.

12. Discussion – summary The null hypothesis was rejected, the alternate hypothesis that Aspen trees do not have as much use for chlorophyll underneath the snow is supported. However, in Aspen only stands enough sunlight penetrates the snow and reaches the bark, at 30cm off the ground, allowing enough photosynthesis to occur to offset the energy cost of maintaining the specialized chlorophyll cells. Second null hypothesis was rejected, a difference between stand types was seen. Although rate of photosynthesis is increased in temperatures above zero, the insulation from the snow does not mean increased photosynthesis, because of the lack of available sunlight.

13. Further Research Do fungal infections effect how well Aspens photosynthesize? Hypothesis: Parasitic fungi steal nutrients and energy from plants, aspen trees that are victim s of a fungal infection will have a difference in chlorenchyma layer thickness when compared to healthy, uninfected trees Does the pattern of thicker chlorenchyma with increased height also apply to saplings?

14. Acknowledgements Thanks to Tim Kittel for data processing help Derek Sweeney for field equipment And Stephanie Meyer for letting me use her lab and microscope equipment

15. Literature cited: Aschan, G., Wittmann, c. &pfanz, H. (2001). Age-dependent bark photosynthesis of aspen twigs, Trees-Structure and Function, 15, 431-437 Covington, Wallace W. (1975) Altidudinal Variation of Chlorophyll Concentration and Reflectance of the Bark of Populous tremuloides. Ecology, 56, 715-720 Pearson, L.C. & Lawrence, D.B. (1958). Photosynthesis in Aspen Bark. American Journal of Botany, 45, 383-387. Schaedle, M. & Foote, K.C. (1971). Seasonal Changes in Photosynthetic Capacity of Populus Tremuloides Bark. Forest Science, 17, 308-&. Schaedle, M., Iannacco.P & Foote, K.C. (1968). Hill Reaction Capacity of Isolated Quaking Aspen Bark Chloroplasts. Forest Science, 14, 222-&.

16. Apendix – R console, script