Background A defoliation event took place on May 26, 2010 on Bald Mountain in West Campton, NH. Martha Carlson observed a number of sugar maples that were partially or totally defoliated on May 27, Extensive analysis of spectral reflectance data, atmospheric chemistry, leaf anatomy, and meteorological conditions by Carlson supports the hypothesis of a PAN event as the atmospheric pollutant that caused the defoliation. Carlson developed a supervised classification from June 2, 2010 Landsat to map the regional extent of the damage (Carlson et al. 2013). This study focused on identifying evidence of damage to the sugar maples three years after the PAN event and begin to ground-truth the stress signature identified by Carlson from the Landsat data. Results 1.Anomalous Twig Morphology Photo A – No Damage Photo B – Partial Damage Photo C – Total Damage Discussion Sugar maples on Bald Mountain that were partially or totally defoliated from the 2010 PAN exhibited a higher frequency of anomalous twig morphology than the maples that were not defoliated. Most of the anomalous growth occurred in the 2010 growth year which provides historical evidence of the defoliation event. The chlorophyll concentrations of leaves collected in 2013 is still under investigation. Leaves collected from partially and totally damaged trees were thinner, which may be an indication that the trees are still recovering from the stress of the PAN event. Finally, anomalous growth was evident in the 2010 growth rings of the totally damaged trees. Multiple false growth rings were present indicating a response to significant stress in Conclusion Based on the results to date, the evidence indicates that there is post-event damage from the defoliation event that occurred on May 26, 2010 to the sugar maples located on Bald Mountain in West Campton, NH. The field and laboratory observations showed anomalies in the twig morphology, leaf anatomy, and growth rings on trees that were impacted by the 2010 event. Additional analysis is needed to explore the anomalous leaf anatomy and possible variations in chlorophyll concentrations to further document the post-event damage. This summer research experience has provided valuable insight into the process of science. I have had many opportunities to experience the highs and lows of working through the research process. I have realized how much time is needed to complete each step properly to ensure the evidence is accurate. The experience has also highlighted the collaborative nature of scientific research and the need for frequent discussion and questioning. I look forward to working with my colleagues to incorporate this experience into our high school science curriculum. Literature Cited Carlson, M., Rock, B. N., and Talbot, R. W. (2013). Documenting A Defoliation of Maples, May 26, (Manuscript in prep). Carlson, M. Chlorophyll Extraction Protocol (in prep) Acknowledgements This research was funded through the NSF NH EPSCoR Program # I would like to thank Dr. Barrett N. Rock and Martha Carlson for their support and guidance on this research experience. We would like to thank Hank Parker for allowing access to the Bald Mountain sugar maples. Photo A is a control sample (Field) showing normal twig morphology for growth. Photo B is from a partially damaged sugar maple (856) from the 2010 PAN event. Photo C shows a twig from a totally damaged maple (856A). Photos B and C show anomalous growth for Variations in Chlorophyll Concentrations 3. Anomalous Leaf Anatomy Figure 5: Wet Leaf Average Thickness Figure 6: Dry Leaf Average Thickness 4. SEM Analysis of Growth Rings Figure 7: No Damage (40x)Figure 8: Total Damage (36.2x) Figure 7 is a cross section of a twig from a tree that was not damaged (A856) in the 2010 event. This twig displays normal annual growth rings. The smaller 2013 growth year is a result of collecting the sample on July 12, Figure 8 is a cross section of a twig from a tree that was totally defoliated (856A) in May There is evidence of multiple false growth rings in the 2010 growth ring indicating that the tree experienced significant stress in the 2010 growth year. The two control samples had normal twig morphology except for occasional missing buds, missing first internodes, and presence of adventitious buds. The partially and totally damaged trees showed a higher frequency of missing buds, missing first internodes, and a higher number of buds present but not open. These trees also had examples of anomalous annual growth and branching that occurred at the 2010 growth year. These anomalies consisted of the following: 12 possible buds evident on 4 internodes (856-3) 1 cm of growth for 2010; 5-6 leaf scars as well as rings around the twig; possibly 5-6 short internodes; slight bend in twig (856A-1) 2010 and 2011 bud collars side by side with 1-2 mm of growth for 2010 (856A-5) Multiple branching at apical bud; three twigs and evidence of a fourth (856A-2) 1 internode and 2 leaf scars; bud collar with few rings; minimal growth (856B-4, 856B-2 & 856B-5) Figure 1: Absorbance Curves for 5 Sugar Maples Figure 2: Peak Absorbance for Leaf Pigments Figure 3: 2013 Leaf Cross Section from Undamaged Sugar Maple (530x) Figure 4: 2013 Leaf Cross Section from Totally Damaged Tree (530X) Figure 3 is a SEM image of a leaf cross section collected in 2013 from a maple that was undamaged (A856) in Figure 4 is a SEM image of a leaf cross section collected in 2013 from a totally damaged maple (856A). UE = Upper Epidermis, PL = Palisade Layer, SL = Spongy Layers, LE = Lower Epidermis Methods Branches were collected with a pole pruner on July 12, 2013 from five different sugar maples on Bald Mountain in West Campton, NH. Samples were collected from two control trees (Field & A856) that were not defoliated from the May 2010 event. Branches were also collected from one tree (856) that was partially defoliated and two trees that were totally defoliated (856A & 856B) in May Samples of leaves and twigs were placed in ziplock bags, transported in a cooler, and then stored in a refrigerator. Five twigs were analyzed for anomalous morphology from each of the five study trees using a dissecting scope and 10x hand lens. Characteristic features were also photographed. Chlorophyll extraction protocols suggested by Carlson produced enough material to run two replicates for each of the five trees. Light absorbance was measured using a HP UV Vis Spectrophotometer provided by the UNH Leitzel Center. A Scanning Electron Microscope (SEM) was used to analyze annual growth rings in twigs and leaf cross sections of leaves collected on July 12, The twigs and leaves were analyzed from A856, 856, and 856A to compare trees that experienced no defoliation, partial defoliation, and total defoliation from the PAN event. Fresh leaves were analyzed (wet) and then dried in an oven and sampled again (dry). Abstract A hypothesized peroxyacetyl nitrate (PAN) event occurred on May 26, As a result, a number of sugar maples (Acer saccharum) were partially or completely defoliated on Bald Mountain in West Campton, NH. A MultiSpec image was generated from June 2, 2010 Landsat data that identified the damaged vegetation as well as other regions in New Hampshire that exhibited the same stress signature. The purpose of this study was to design methods that could identify post-event damage, and assess the current health of the Bald Mountain sugar maples. The study revealed anomalous twig morphology on trees that were partially and totally damaged in These trees had shorter annual growth units in 2010, anomalous branching patterns, missing buds, and buds that were present but not open. Light absorbance curves were completed with the spectrophotometer and chlorophyll concentrations are still being calculated for analysis. Leaves collected in 2013 from the partially and totally damaged trees were thinner than leaves collected from trees that were not damaged in 2010 due to a thinner spongy layer and a disorganized palisade layer. Also, the twigs from the partially and totally defoliated trees showed evidence of anomalous 2010 growth rings. Future Work Ground-truth additional locations in NH that have been identified (11,000 acres) as areas impacted by the PAN event to confirm the accuracy of the stress signature indicated in Carlson’s paper. Develop additional analytical field measurements to document the effects of the PAN event and the annual recovery of different tree species. Design curriculum to be incorporated into high school biology courses in NH and pilot the curriculum at Winnacunnet High School in Hampton, NH. Figures 5 and 6 display the average leaf thickness with +1 and -1 standard deviations represented by the black bars. Both wet and dry samples were taken from the same leaf. SEM measurements were taken from the upper epidermis to the lower epidermis to determine leaf thickness. Six locations on each leaf were randomly selected for the measurements. The average leaf thickness from a tree that did not experience damage was 95.1 µm and 92.8 µm for wet and dry leaves respectively. A leaf from a partially damaged tree had an average thickness of 60.2 µm for wet and 75 µm for the dry sample. A leaf from a totally damaged tree had an average thickness of 42.3 µm for wet and 47.1 µm for the dry sample. SEM cross sections of leaves from 2010 damaged trees showed that the decreased thickness was a result of a disorganized palisade layer and a thinner spongy layer. This anomalous leaf anatomy may be an indication that the partially and totally damaged trees from 2010 may still be recovering from the stress of the PAN event. Figure 1 displays the spectrophotometer absorbance curves for the five maples in the study. The four vertical lines indicate the peak absorbance for the four pigments analyzed. Carotenoids are located at 470 nm, anthocyanins at 520 nm, chlorophyll b at 649 nm, and chlorophyll a at 664 nm. 856 B – Total Damage 856A – Total Damage Field – No Damage 856 – Partial Damage & A856 – No Damage Figure 2 shows the leaves from the totally damaged trees (856A & 856B) have the highest levels of light absorbance. Leaves from one undamaged tree (Field) had the next highest levels. Leaves from the other undamaged tree (A856) and the partially damaged tree (856) had the lowest absorbance levels. Concentrations of each pigment are still being calculated and analyzed to fully understand the significance of the data. UE PL SL LE Using Field and Laboratory Observations to Examine the Impact of a PAN Event on Sugar Maples in New Hampshire Michael Handwork, Martha Carlson, and Dr. Barrett N. Rock