SAS Analytics Experience 2016

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Presenter: Reuben Hilliard
Faculty Advisors: Dr. Paula Jackson & Dr. Brad Barney
Kennesaw State University
INTRODUCTION
Surviving Climate Change
Comparing drought & fungicide response in two riparian tree species for use in ecological restoration
Riparian zones fulfill many ecosystem functions and occur at
all elevations near rivers, streams, and in floodplains. They
function as a natural buffer against erosion in river and
stream banks, filter downstream pollution, and provide
increased habitat complexity (Wildlife, 2006). Due to
development, logging, and expanding agriculture, many
riparian zones have been destroyed or depleted. This has a
profound ecological effect that leads to increased
sedimentation and pollution in natural water systems
(Hernandez-Santana, 2011). These zones often undergo
rehabilitation to restore health back into the surrounding
environment. Salix nigra (Black Willow) and Platanus
occidentalis (American Sycamore) are two common riparian
species of trees (Conger, 1996). Of these, Salix nigra is
frequently used to restore these areas, however not much
information exists on the ability to use Platanus occidentalis
for this purpose. This research is part of a larger study
looking at the ecology and physiology of both of these
species, with the overarching aim of comparing the behavior
of Platanus occidentalis to the more widely studied Salix
nigra, and determining the feasibility of using Platanus
occidentalis in restoration processes. Of additional
importance is the fungal biota which inhabit the soil
beneath these trees. Mycorrhizal fungi have been reported
to improve plant growth in many crops through enhanced
root growth and function (Westphat et al, 2008). It also
improves early plant establishment and increased the most
valuable early fruit yield under some environmental stress
conditions. This is of ecological importance and will be
incorporated in this study.
Salix nigra
leaves (left)
and
Salix nigra
tree (right)
Platanus
occidentalis
leaves (left)
and
Platanus
occidentalis
tree (right)
(Image:
bioimages.vanderbilt.edu/baskauf
/15370.htm)
(Image:
bioimages.vanderbilt.edu/baska
uf/23004.htm)
(Image:
bioimages.vanderbilt.edu/baska
uf/29666.htm)
(Image:
http://bioimages.vanderbilt.ed
u/baskauf/13574.htm)
STATISTICAL MODELS
Randomized Complete Block Design
yij = µ + αi + bj + εij
Mixed Effects Model
y = Xβ + Zu + ε
Unstructured Covariance Matrix
Autoregressive Heterogeneous Covariance Matrix
One-way ANOVA
yij = µ + τi + εij
Sum of Mean
Squares df Square F
Treatment SSR / dfF = MSR MSR/MSE
Error SSE / dfE = MSE
Total SST dfT
Both species can be found in overlapping geographical
regions of the Southeast.

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One cause for data errors in greenhouse experiments can be due to microclimate differences, such as light, airflow and
heat among saplings, within different planters. As stated by Brien et al. (2003), sound statistical design and analysis is
better than rearranging the position of plants during the experiment itself. In our experiment, Platanus occidentalis and
Salix nigra saplings were planted using a Randomized Complete Block Design, as seen in Table I. It involved a complete
experimental treatment within each planter series, allowing for homogenous growing conditions within a single tray,
regardless of differences among planters themselves. To account for variability in sapling size, each tray had a similar
distribution of size classes. All the saplings were tagged with unique identifiers, such as PO-01 or SN-02. 15-17
individuals of each species were subjected to a control, inundation, or drought condition; and drought with and
without the addition of mycorrhizal spores, a fungal biota, which was controlled with the addition of a fungicide,
Benomyl. In total, 31 Platanus and 34 Salix cuttings received sufficient nutrients in the form of a slow-release fertilizer
and after taking baseline measurements, were allowed to grow in planters through the spring of 2015. From May 18th
until August 23rd, a team of myself and 3 undergraduate research assistants, took anatomical and physiological
measurements.
Block 1 Block 2
A B B A
D C C D
Treatments
A: Control; No Fungi (Benomyl added) & Wet
B: Fungi & Drought
C: Fungi & Wet
D: No Fungi (Benomyl added) & Drought
Saplings planted; fertilized
Saplings measured; tagged
METHODS
For the anatomical measures, an indicator of growth rate, both the circumference and the height were taken for each
plant on a weekly basis. For the physiological data, a LICOR LI-6400 Infrared Gas Analyzer (IRGA) was used to measure
photosynthetic rate from leaves repeatedly over the period of weeks, systematically moving through the plants,
selecting a predefined leaf from a randomly selected plant from each treatment and block. This was a tedious process,
with each leaf taking up to 16min for a full measurement run. The results were used to build light response curves. Net
Photosynthetic rate, or CO2 assimilation (µmol CO2 m-2 leaf area s-1) from several trials were plotted against light
intensity, or Absorbed Photosynthetically Active Radiation (αPAR, µmol photons m-2 leaf area s-1). The slope of the
linear phase of the response curve is a measure of "photosynthetic efficiency" of the plant, or how efficiently solar
energy is converted into chemical energy. Different plants show differences in the shape of their light response curves,
which reveals characteristics of the underlying photosynthesis processes, including the efficiency at which light is
utilized by photosynthesis and the rate of O2 uptake.
In this longitudinal study, both the anatomical and the physiological data were analyzed using SAS 9.4 with the MIXED
Procedure, which models mixed effects over time.
Because a drought stress condition increases root to shoot ratio (Xu et al, 2015), to test this, 16 trees were randomly
selected from each of the treatment and species groups. After drying in the oven overnight, the dry weight of each
tree was used to calculate the root to shoot ratio. The cumulative total leaf area was taken using the LI-COR 3100C. To
detect the presence of arbuscular mycorrhizal root colonization, 160 root samples were collected from eight trees of
each species. Roots were stained using a 0.05% Trypan Blue solution and the presence of mycorrhizal structures was
quantified using the root piece method.
The LI-COR LI-6400 (left) is a portable
Photosynthesis System and a major tool for
ecological researchers in the field (LI-COR, 2013).
The function of the LI-6400 is based on detecting
differences in CO2 concentration of air before
(reference) and after (sample) it comes in contact
with the plant leaf. Differences in CO2
concentrations are detected through the use of
infrared gas analyzers. The LI-COR allows for in situ
measurements of photosynthetic rates and for
independent control of the leaf chamber CO2, H2O,
temperature, and light . Results from the LI-COR
may be downloaded into an Excel spreadsheet for
further analysis.
Physiological measurements (top left); Planters in grid – trial start date (top mid);
Anatomical measurements (top right); LI-COR 3100C Leaf Area Meter (bottom left);
Staining of greenhouse samples in 0.05% Trypan Blue solution (bottom mid); Colonized
root after staining (bottom right)

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RESULTS
Graphs, Boxplots and Bar Chart output
designed in SAS 9.4 and Tableau 9.2
Anatomical Results from the Mixed Effects Model.
Drought treatment highly significant (** p < 0.0001)

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CONCLUSIONS
The anatomical results indicate that Salix nigra and Platanus occidentalis do respond differently to drought
conditions. In fact, the interaction of the days count and the drought condition was highly significant (p < .0001).
This meant that as the experiment proceeded the drought condition became more pronounced. As seen in the
anatomical results, for the drought condition, Platanus was able to outperform Salix in the linear rate of growth
(corrected for errors), 0.86cm/Day and 0.74cm/Day, respectively. The fungicide treatment did not have a significant
effect in either species.
The physiological results indicated that the PAR level, or light intensity given to the leaf, was significant (p < 0.05). This
analysis had to be stratified by species, as none of the other factors were significant initially. When this was done,
Platanus was right on the cusp of being significant in the drought condition during period 2 (last 5 weeks of the
experiment). Even though Salix didn’t respond to the drought or fungicide treatments, this information is still of
biological interest to researchers, as it indicates that Salix can be stressed and still perform unhindered, with minimal
interruptions.
Of particular note is how well Platanus performed in the ‘No Fungicide/Drought’ (Treatment B), which can be observed
in both the light curve and the mean maxima photosynthetic output figures. But when an ANOVA was performed to
confirm the results, there was no significant difference at PAR 800 by Species and Treatment.
The ANOVAs from the Root to Shoot Ratio and Leaf Area showed no significant differences in means between Species
and Drought treatment, but of interest was the much larger variability among Salix plants. Larger Root to Shoot Ratios
among younger trees and greater variability in measurements, indicate that compared to Platanus, when Salix
scavenged for water, it struggled far more in the drought treatment.
Because Platanus performed as well or slightly better than Salix in this study, the overall results were quite positive
and allow future research to focus specifically on Platanus as a species to use in restoration of Southeastern US
riparian ecosystems.
RELEVANT SAS CODE
REFERENCES
*i) Coding the explanatory variables;
DATA research.anatomical_data;
SET together;
daysc = date - mdy(5,18,2015); dayscat = daysc;
tag = species; species = substr(tag,1,2);
fungicide = scan(treatment,1,"/");
drought = substr(scan(treatment,2,"/"),1,1);
tmt=1; if species="PO" then tmt=tmt+4;
if drought="D" then tmt=tmt+2;
if fungicide="Fungicide" then tmt=tmt+1;
RUN;
*ii) Optimal Anatomical model. Unstructured Covariance
Matrix;
PROC MIXED data = research.anatomical_data;
CLASS species drought fungicide tag blocknum dayscat;
MODEL height = species drought species|daysc
drought|daysc
/solution ddfm = kr;
REPEATED dayscat/ subject = tag(blocknum) type = un R
RCORR ; RUN;
*iii) Optimal Physiological model, stratified by species.
Autoregressive Heterogeneous Covariance Matrix;
PROC SORT data = research.master_photo; BY Species Tag
PAR; run;
PROC MIXED data = research.master_photo;
BY Species;
CLASS tag treatment blocknum PAR;
MODEL photo = treatment PAR ;
RANDOM blocknum;
REPEATED PAR /subject = tag type = arh(1) ;
LSMEANS treatment /pdiff tdiff ; RUN;
*iv) Data output for Mean Maxima Photosynthetic Rate
and Standard Errors;
ODS GRAPHICS ON;
PROC MIXED data = research.master_photo;
WHERE PAR = 800;
CLASS Tag blocknum fungicide drought species PAR
period;
MODEL photo = species*fungicide*drought
/ noint solution ddfm=kr;
RANDOM blocknum;
RANDOM int / subject=tag;
RUN;
ODS GRAPHICS OFF;
*iv) One-way ANOVA for Root to Shoot Ratio and Total
Leaf Area;
TITLE "Root to Shoot Ratio";
PROC ANOVA data=mass_area;
CLASS Fungi Drought Species ID Block drought_sps ;
MODEL Root_Shoot_Ratio = drought_sps Block;
MEANS drought_sps/Tukey;
RUN;
TITLE "Total Leaf Area";
PROC ANOVA data=mass_area;
CLASS Fungi Drought Species ID Block drought_sps ;
MODEL Leaf_Area = drought_sps Block;
MEANS drought_sps /Tukey;
RUN;
Oregon Department of Fish and Wildlife. 2006. Oregon Conservation Strategy. Oregon Department of Fish and Wildlife,
Salem, Oregon.
Hernandez-Santana, V., Asbjornsen, H., Sauer, T., Isenhart, T., Schilling, K., & Schultz, R. 2011. Enhanced transpiration
by riparian buffer trees in response to advection in a humid temperate agricultural landscape. Forest Ecology and
Management, 261(8), 1415-1427.
Conger, RM. 1996. Black willow (Salix nigra ) use in phytoremediation techniques to remove the herbicide bentazon
from shallow groundwater. Master’s thesis, Louisiana State University
Brien, C. J., Berger, B., Rabie, H., & Tester, M. 2013. Accounting for variation in designing greenhouse experiments with
special reference to greenhouses containing plants on conveyor systems. Plant Methods, 9(5), 1746-4811
Westphal, A., Snyder, N., Xing, L. 2008. Effects of Inoculations with Mycorrhizal Fungi of Soilless Potting Mixes During
Transplant Production on Watermelon Growth and Early Fruit Yield. HortScience, 43(2), 354-360
LI-COR Biosciences. (2013). The LI-6400 Portable Photosynthesis System. Retrieved from
http://envsupport.licor.com/index.jsp?m=Current&spec=LI6400,Brochures&menu= Photosynthesis%20Systems
Xu, W., Cui, K. , Xu, A., Nie, L., Huang, J., & Peng, S. 2015. Drought stress condition increases root to shoot ratio via
alteration of carbohydrate partitioning and enzymatic activity in rice seedlings. Acta Physiologiae Plantarum, 37:9

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