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 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.

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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, 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 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.

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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.

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.

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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.

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.

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

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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 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.

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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).

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.

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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).

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 AnalysisWe 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, 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).

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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/.

ResultsOur 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 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

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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).

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

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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 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.

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

Figure 4a. The total new growth (cm) of Ship Harbor eelgrass

shoots over a period of a month.

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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 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 Figure 5. Padilla Bay and Friday Harbor

temperatures from Novemer 2015 to October 2016.

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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 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

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increases. When thinking about restoration transplantation, we must keep the donor

location in mind. The eelgrass 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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