1. UNIVERSITY OF WASHINGTON, SEATTLE
How Eelgrass Origin
Affects Performance in
Warm Water
______________________________________________________________________
Gigi Gaultier1
, Joanna Luse2
, Malise Yun3
1. Gigi Gaultier. University of Washington, Seattle. gigipeaches@gmail.com.
2. Joanna Luse. Eastern Washington University. joannaluse@gmail.com
3. Malise Yun. University of Washington, Seattle. my.malisey@gmail.com
December 9, 2016

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Abstract
The local eelgrass species in the Salish Sea, Zostera marina, serves as a
nursery home to countless organisms in its environment. Understanding the
response of eelgrass in different temperature environments can teach us about
restoration transplantation throughout the Salish Sea and assure its health for
the eelgrass and the organisms in and around it. Zostera marina generally thrives
in an environment with a temperature range of 6-17 C but begins to decline in its
performance once reaching a temperature of 25 C. Within the Salish Sea, the
temperature of the water varies from location and throughout the seasons,
ranging from 3 C in the winter to 23 C in the summer in just Padilla Bay, WA. We
looked at the response in eelgrass shoots within two sites in the Salish Sea:
Padilla Bay, a warm water location, and Ship Harbor, a cold water location. We
tested these shoots in an environment with a gradual temperature increase from
14 C to 19 C for one month and a short term temperature increase to 30 C for 6
hours within that month. We found that growth in Zostera marina of Padilla Bay
and Ship Harbor can be negatively affected in an environment with gradually
increasing temperature for a long period of time. The short term temperature
increase did not have as much of an affect due to the natural temperature
variations during the summer in these two locations. Due to Padilla Bay eelgrass
shoots coming from a warm location, we noticed that the shoots took a longer
period of time to respond negatively than Ship Harbor eelgrass shoots. We think
that this is because Padilla Bay is a warmer location so the shoots were more
acclimated to that warmer temperature, but too long of a period of time can
stress out the shoots too much that their performance begins to decrease.
Keeping restoration transplantation in mind, the donor location matters. The
temperature of the donor location must be similar to the environment in which it
is being moved to or it will not survive.

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Question
How does eelgrass from different locations in the Salish Sea respond to
being warmed as predicted in 100 years and spiked with a higher temperature to
model extreme weather?
Introduction
Eelgrass, Zostera marina, plays an important plant role in ecosystems around the
Salish Sea. They are extremely vital when it comes to providing a habitat and a food

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source for many organisms that live around them (Lee, 2007).Doing research on
eelgrass is important so that we can anticipate responses we might see in the natural
world. This underwater plant is very useful for ecological and economic values. Eelgrass
provides as a nursery for fish which can help support entire fisheries (Raun, 2013).
Eelgrass is found in most places across the globe of all the seagrass species (Thom,
2014) and without its presence, entire environments would change greatly in response.
Although so important, scientists have been seeing effects on this plant due to human
stressors increasing through CO2, pollution and temperature changes of global
warming. The average rate of decline in seagrass has increased by 9% from 1940s to
1990 due to these human stressors (Waycott, 2009).
Eelgrass has experienced a wide die off in high temperatures during the summer
(Nejrup 2008). Although many seagrasses are found throughout the world, most all
seagrass also increase growth through spring and summer and decrease in fall and
winter (Lee, 2007). Generally, once the summer temperatures hits their high
temperatures, eelgrass begins to decline and the process starts over again. But with
global warming speeding up this process, recent studies have shown that with a 1°C
increase, 5 to 6 days in the eelgrass growing season is impacted on them (Brodeur,

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2015). Many experiments have been conducted on the stressors from temperature
change for

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eelgrass. The effects of temperatures higher than 30 C are where eelgrass shoots in
particular begin to decrease. This can include an increase of decay, bleaching and
diseases found on the leaves of the eelgrass (Neckles, 1999). Decaying disease can be
found through a darkening brown/black streaking and bleaching is a more white tone
that spreads throughout the leaf. The density and biomass of eelgrass shoots also
decreases as more shoots start dying off with higher temperatures extremes (Thom,
2014).The optimal temperature for eelgrass survival is between 15-20 C (Nejrup 2008),
(Lee, 2007) but with global warming upon us, that is not always what we get. Eelgrass
over the world has decreased by 29% since 1998 (Waycott, 2009).
Finding the perfect temperature for Zostera marina can be difficult. In the Salish
Sea we have a range of temperatures that fluctuate throughout the year. In the winter,
the temperatures of Friday Harbor water can go down to 8 degrees C and up to 15
degrees C in the summer (NOAA, 2016). This is the water that we will be using to flow in
for our water tanks, giving the eelgrass a slightly lower temperature than what is most
suitable for them. This will also make drastic changes in the water temperature more
noticeable. With global warming, water temperatures are supposed to increase at a

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slightly faster rate, giving summer temperatures “higher highs” than normal. With
spiked increases from “the blob”(an unusual drastic increase of temperature hitting
the pacific northwest), areas in the Puget Sound have increased as high as 2.2 degrees C
higher than normal (Seattle Times, 2015). This spike in temperature can shock the
eelgrass and possibly give a different response to eelgrass than a gradual increase. This
leads us to our main question of looking at the effect on Zostera marina from gradual
temperature increases vs extreme spike temperature increases.
Looking at the effects of temperature rises, whether that be gradual or a heat
spike, is important for understanding the adaptability of these plants. With global
warming currently

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increasing the temperature of the oceans, we want to know if there is enough time for
eelgrass to be able to adapt to their changes and survive. Collecting shoots from two
different locations; Padilla Bay and Ship Harbor, allows us to see different environments
within the Salish Sea and how they each respond individually. When slightly increasing
the temperature of the water by a few degrees (C) in which the shoots are in, it is a
possibility that eelgrass will respond negatively to just an overall increase. But testing an
extreme spike increase, much like a heat wave, is a short term effect that could make the
eelgrass respond negatively from being exposed to just an extreme even if for a short
period of time. Comparing two different temperature increases will show us a more
specific response that we are looking for so we can better prepare for how these
eelgrass shoots might respond in their natural environment of the Salish Seas with real
weather patterns.
Methods
1. Prepare 10 tanks for placing our eelgrass inside. Label these tanks 1-10 and
randomize which side of the room they will start on.

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2. Collect 20 9” tall tubs to put in the tanks. Label each tub on the outside so
that 10 tubs are A and 10 tubs are B. Randomize which letter tub will be one which side
of the room. Place one A tub and one B tub in each tank.
3. Place one bubbler above each tank and split the tube so that it can reach into
each tub within the tank. Duct tape the tube to the side of the inside of the tub.
4. Make tags for each shoot containing the tank number (1-10), tub letter (A or B),
location by first letter (P or S), shoot letter (Y or Z) and whether it will be spiked or
unspiked (S or U) (example: 5APYS). Print on water proof paper and hole punch. Tie tags
onto string.
5. Collect 80 rocks to attach to the rhizomes of the eelgrass shoots.
6. Collect the eelgrass - 60 plants from the two locations; Padilla Bay, Ship Harbor.
Although only 40 from each location will be needed, we have extras just in case
something goes wrong. To do this process, we will visit these locations and carefully
uproot the shoots to place into two different buckets (one per location). Bring these
shoots back to the lab to prepare.

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7. Place our eelgrass shoots in a tank with bubble snails for about three hours to
eat off most of the epiphytes. Wipe off any excess epiphytes before collecting them in
buckets.
8. Prepare the 2 treatments:
- Ambient Control Tanks (5)
Tanks 1, 5, 6, 8 and 9 (randomized dependent on plumbing)
Water flowing in from Friday Harbor
Each tank contains 2 tubs
Both locations (2 shoots from each location) in each tub
- Warmed Control Tanks (5)
Tanks 2, 3, 4, 7 and 10 (randomized dependent on plumbing)
Air temperature warmed, water not flowing
Each tank contains 2 tubs
Both locations (2 shoots from each location) in each tub
9. Cut the rhizomes of each shoot (80 total) to 4 cm.
10. Tie the tags onto the rhizomes of the shoots and tie around rocks. Each shoot
should have the appropriate tag to its location and a rock to help weigh it down.

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11. Measure the initial shoot length (cm), sheath length (cm), sheath width (mm),
and # of leaves for each shoot. For each leaf on the shoot, measure the total length (cm)
and decay percentage (0%, 1%, 10%, 20%, 50% or 100%) based on the wasting index
chart. Record all of this data in the lab notebook measuring chart. Prick all of the Y
shoots at the end of the sheath length.
12. Place the shoots in the tubs according to their tag. Four shoots total should
be in each tub: 2 from each location, and eight per tank.
13. Check temperature and light of each tub every day between 8:30-11:30 am
and 5:30-7:30 pm. For temperature: hold thermometer in the tub for 10 seconds and let
stabilize. For light: hold the knob above the water in the middle of the tub and let
stabilize. Record measurements on “Temp Check Sheet”in lab notebook. Take notes
of process and any abnormalities throughout the experiment in the lab notebook.
14. After six days, Fill 9 5-gallon buckets with water to let sit over night in the lab.
This will let the water reach the same air temperature as the stationary tanked tubs.
15. The next day, examine (collect and measure) the pricked “Y” eelgrass shoot
from both locations per tub and record shoot length (cm), sheath length (cm), sheath

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width (mm), # of new leaves. On each leaf measure the new growth and the decay
percentage.
16. Place all shoot back in their correct tub based on their tag.
17. Replace the water in each tub to clean them out. For the circulating tanks,
refill the tubs with water pumped in from Friday Harbor. For the stationary tanks, refill
them with water from the buckets that have been sitting over night to reach the same
air temperature as they are in the tubs.
19. After week 2, Measure and record for all “Y” and “Z” shoots (shoot length
(cm), sheath length (cm), sheath width (mm), # of new leaves, growth (cm) on each leaf
and decay percentage).
20. Prick all “Z” shoots after measuring them.
21. Place all shoots back in their correct tubs based on their tag.
22. Starting at 10:30 am, place aquarium heaters into tubs 1B, 2A, 5A, 8B, and 9B.
Spike the eelgrass tub water to 30 C for 13 hours in these five tubs. (We noticed the
temperature was not increasing as we liked so we took the tubs out of their tanks to
place on boards above their tank. We did this at hour 6 which is when we really saw
temperature increases).

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23. Record temperatures of each tub (even unspiked ones) every hour of the
spike starting at 10:30 am.
24. At hour 13, turn off and take out the aquarium heaters. Put the tubs back into
their correct tanks.
25. The next day, starting at 1:15 pm, place aquarium heaters into tubs 3A, 4A, 6B,
7B and 10A. Take the tubs out of their tanks to sit on boards above their tank. Spike the
eelgrass tub water to 30 C for 6 hours in these five tubs.
26. Record temperatures of each tub (even unspiked ones) every hour of the
spike starting at 1:15 pm.
27. At hour 6, turn off and take out the aquarium heaters. Put the tubs back into
their correct tanks.
28. After 5 days, Fill 9 5 gallon buckets with water to let sit over night in the lab.
This will let the water reach the same air temperature as the stationary tanked tubs.
29. The next day, measure and record all of the “Y” eelgrass shoots (shoot
length (cm), sheath length (cm), sheath width (mm), # of new leaves, growth (cm) on
each leaf and decay percentage).

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30. Replace the water in each tub to clean them out. For the circulating tanks,
refill the tubs with water pumped in from Friday Harbor. For the stationary tanks, refill
them with water from the buckets that have been sitting over night to reach the same
air temperature as they are in the tubs. While cleaning out the tubs, scrub off the
diatoms that were growing on the side, especially in the circulating tanks.
31. The next day, measure and record all of the “Z” eelgrass shoots (shoot
length (cm), sheath length (cm), sheath width (mm), # of new leaves, growth (cm) on
each leaf and decay percentage).
32. After 5 days, Measure and record all of the “Y” and “Z” eelgrass shoots
(shoot length (cm), sheath length (cm), sheath width (mm), # of new leaves, growth (cm)
on each leaf and decay percentage).
33. Take down experiment and clean tanks. Dispose of eelgrass.
Statistical Analysis
We are running a 3-way ANOVA and a split plot.
The response variables we are running statistical analysis on are shoot length,
sheath length, sheath width, new growth for the leaves, and decay percentage. Our
explanatory variables are temperature, acclimated and unacclimated treatment, location,

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and spike (short term temperature increase) or no spike. These last variables are the
fixed variables.
We are using the 3-way ANOVA to compare Location (Padilla Bay - warm
location, and Ship Harbor - cold location), Spiked (yes/no), and acclimated (yes/no).
We used a split plot because we have two tubs within one tank.
The random effects are the tanks, tubs, repeatedly measuring shoots.
Functions:
aov()
summary()
Citing R: (R Core Team, 2016)
Core Team (2016). R: A language and environment for statistical computing. R
Foundation for Statistical Computing, Vienna, Austria. URL.https://www.R-project.org/.
Results
Our analysis of the response of eelgrass in a gradual temperature increase
showed us that Padilla Bay took a longer period of time to decline in shoot length (cm)
than Ship Harbor. The general trend of Ship Harbor shoot length (cm) shows us that
after week two, the growth began to decline. Both of the Ship Harbor warmed tanks

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responded worse than the cool shoots. The warmed treatment eelgrass shoots lost
about 7-12 cm of growth since our initial
measurements while the cool treatment eelgrass
shoots only lost about 1 or 2 cm since the initial
measurements (Figure 2a). In contrast, Padilla Bay
eelgrass shoots did not decline nearly as much as
the Ship Harbor eelgrass shoots. The warmed
treatment eelgrass shoots declined the most as well, but only by about 5 cm since the
initial measurements. The cool treatment eelgrass
shoots from Padilla Bay varied a bit between
whether or not
the short term
temperature increase was put in effect on them.
The cool treatment with the short term
temperature increase grew about 3 cm while the
cool treatment without the short term temperature increase lost about 3 cm since the
initial measurements (Figure 2b).
Figure 2a. The change in shoot
length (cm) of Ship Harbor eelgrass
shoots over a period of a month.
Figure 2b. The change in shoot
length (cm) of Padilla Bay eelgrass
shoots over a period of a month.

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The decay of Ship Harbor eelgrass shoots also trended toward more percentage
than Padilla Bay eelgrass shoots. In the warmed
treatment of Ship Harbor eelgrass shoots, the
decay percentage increased by 30% (Figure 3a).
The warmed treatments of the Padilla Bay
eelgrass shoots only increased decay by about
12% (Figure 3b). The cool treatment eelgrass
shoots for both location did not increase decay
by nearly as much. For Ship Harbor, the cool
treatment eelgrass with a short term
temperature increase decayed by 15% at the
most while the Ship Harbor cool treatment
without a short term temperature increase
decayed by 5% at the most. Padilla Bay eelgrass shoots also varied a bit here. The cool
treatment eelgrass shoots with the short term temperature increase seemed to decline
Figure 3a. The change in decay
coverage (%) of Ship Harbor eelgrass
shoots over a period of a month.

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in decay, meaning it started at 25% decayed and ended the experiment with about 20%
decay. The cool treatment without the short
term temperature increase increased only by 5%
from the beginning of the experiment to the
end.
The final analysis of eelgrass response on
increasing water temperature we looked at was the
new growth (cm). The Ship Harbor eelgrass shoots
grew much more than the Padilla Bay eelgrass
shoots. The warm treatment shoots from Ship
Harbor without a short term temperature increase
seemed to grow the most, increasing by almost 45 cm.
Figure 3b. The change in decay
coverage (%) of Padilla Bay eelgrass
shoots over a period of a month.
Figure 4a. The total new growth
(cm) of Ship Harbor eelgrass
shoots over a period of a month.

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All of the other treatment eelgrass shoots from
Ship Harbor only grew about 19 to 21 cm (Figure
4a). Padilla Bay eelgrass grew about the same with
an exception of the warm treatment without the
short term temperature increase. All of the Padilla
Bay eelgrass shoot treatments grew about 15 to
25 cm with an exception of the cool treatment without the short term temperature
increase which grew 30 cm (Figure 4b).
Discussion
Padilla Bay and Ship Harbor took different amounts of time to respond negatively
to gradual temperature increases in the water. We noticed that overall, Padilla Bay
eelgrass shoots were more susceptible to changes in the water temperature than Ship
Harbor eelgrass shoots were because Padilla Bay shoots took a week longer to begin
declining (Figure 2a). These results go in hand with real life temperatures at these two
locations. Padilla Bay receives much higher temperature fluctuations throughout the
year with temperature differences from 3 C in the winter to 23 C in the summer. In
Figure 4b. The total new growth
(cm) of Padilla Bay eelgrass
shoots over a period of a month.

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addition, temperature
increases of more
than 10 C happen for
about half a month in
the summer time,
putting the eelgrass
through a bigger stress of
temperature difference for a
longer period of time. Ship Harbor on the other hand, only changes from about 8 C in
the winter to 14 C in the summer throughout the year. In the summer, the short term
temperature increases happen for only a week with about a 3 C temperature increase
instead (Figure 5.). While Padilla Bay is having enormous temperature increase for a
longer period of time, Ship Harbor is not as used to large temperature increases and
especially not for a long period of time.
Eelgrass from Padilla Bay reacts better, with less decay, growth stunt and stop of
leaf growth, than Ship Harbor Eelgrass shoots because it is more accustomed to stressful
temperature conditions. Ship Harbor consisted of a faster decaying response throughout
Figure 5. Padilla Bay and Friday Harbor
temperatures from Novemer 2015 to October 2016.

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the shoots in the tubs that were going through gradual temperature increase of 14 C to
19 C because it wasn’t used to reaching higher temperatures for such a long period of
time. The spike of 30 C for 6 hours did not affect the Ship Harbor shoots as much as the
gradual increase because they are somewhat used to short period temperature
increases. The new growth increase on the warm treatment Ship Harbor eelgrass shoots
is a possible response to stress as well. The increase water temperature could potentially
have stressed the eelgrass to a point of wanting to grow because it was decaying so
much.
We thought that Padilla Bay would do better because of the temperature they are
accustomed to in their natural environment. We also thought that acclimated tubbed
eelgrass would do better because they get a gradual increase from 14 C to 19 C before
being spiked to 30 C which made for less of a shock in temperature. This was not the
case. We noticed most of the shoots that were acclimated had a higher mean decaying
percent and a decrease in shoot length. This would most likely be because it added
stress because they were “spiked” twice instead of once. Our responses show us that
eelgrass cannot withstand long period of temperature increases. When thinking about
restoration transplantation, we must keep the donor location in mind. The eelgrass

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shoots from a colder location responded worse in a warm location, suggesting to us that
eelgrass prefer a similar temperature environment to live in when moving.
Acknowledgements
I would like to thank the University of Washington, Friday Harbor Labs for hosting
the Eelgrass research class and allowing us to conduct our experiments here. Sylvia
Yang, for teaching the class and sharing all of her knowledge, helping us through all the
ups and down and of course, giving us an incredible amount of support. Will King, for
putting so much dedication into our projects and helping us abundantly with coding
and creating our statistical analyses. The entire FHL 470 eelgrass research class for
supporting each other and sharing knowledge throughout the course. For all of the love
and support from my parents, advisors and close friends for keeping me focused. Lastly,
of course for the amazing communication, cooperation and dedication from my team
mates Joanna Luse and Malise Yun. They were the best team I could have asked for in
this project, constantly keeping us on task while making this project one of the best
experiences.

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