Salinity Tolerances and Nitrogen Requirements of Native and … · 2018. 2. 8. · Fox 1 Salinity Tolerances and Nitrogen Requirements of Native and Invasive Gracilaria species in

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Salinity Tolerances and Nitrogen Requirements of Native and Invasive

Gracilaria species in New England

Lydia Fox, Elena L. Peredo, Lauren Hamm

Abstract

In this work, I explore how physical and chemical factors affect the growth under in vitro

conditions of the native species of red algae Gracilaria tikvahiae and the invasive species

Gracilaria vermiculophylla. I determined the specific and relative growth rate over 24 days of

exposure to different environmental conditions. In vitro growing conditions mirrored realistic

ecological parameters of nitrate and salinity, such as those found at Waquoit Bay National

Estuarine Research Reserve. Under low nitrate and low salinity, the invasive G. vermiculophylla

outcompetes the native G. tikvahiae. Under extremely low salinity (0 ppt), G. tikvahiae dies.

However, in intermediate levels of nitrate and salinity, neither will outcompete the other. I also

evaluated the tolerance to short periods of desiccation in both species. G. vermiculophylla is able

to survive exposure to dry air for periods of at least one hour while G. tikvahiae dies after 30

minutes. My results predict that the distribution in a model estuarine ecosystem, such as Waquoit

Bay (Cape Cod, MA), the native and invasive will be found near each other except for areas

where the native cannot persist, the tidal zones, and in low salinity waters near the north end of

the Bay. Those areas will be dominated by the invasive G. vermiculophylla.

Introduction

Gracilaria is a cosmopolite genus of red macro-alga (Rhodophyta). In the United States,

Gracilaria tikvahiae is native from the East Coast. An invasive species, G. vermiculophylla, is

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commonly found on both the East and West coasts of North America. It was first introduced to

the United States, in Virginia, in the 1990s and arrived in New England in 2000s (Nettleton et

al., 2013) where it rapidly expanded. G. vermiculophylla is considered one of the most invasive

marine species in Europe (Nyberg, 2007). Originating from Southeast Asia, it has rapidly

expanded to Europe, North Africa, and North America.

Gracilaria vermiculophylla on the East Coast of US has become a nuisance. During

normal growing conditions, it attaches through its holdfast to different substrates and remains

anchored. However, during storms or disturbances, it is ripped loose and rafts in the water,

eventually washing up onshore (Gorman et al., 2017). For houses and residents on the shores of

water bodies, this is disruptive because the decomposing algae on the shore have an unpleasant

smell and attract many insects. Ecologically, the rafting algae can occur in high abundances,

shading other aquatic plants and consuming nitrogen that could be available to other species

(Gorman et al., 2017). While it can have positive effects on systems with bare and muddy

substrate by increasing the diversity of the system (Ramus et al., 2017), it generally leads to a

decrease in seagrass and other submerged aquatic vegetation (Martínez-Lüscher & Holmer,

2010; Nyberg, 2007). The distribution of native and invasive species of Gracilaria is difficult to

determine as G. vermiculophylla is phenotypically similar to the native species G. tikvahiae, so

introductions of the invasive species can go unnoticed for long periods of time.

The dispersion of G. vermiculophylla is thought to have occurred after multiple human-

mediated independent introductions, probably through inadvertent introduction with commercial

cultures of Japanese Pacific oysters (Krueger-Hadfield et al, 2017). G. vermiculophylla presents

all the characteristics of a successful colonizer in new and diverse environments, including a

broad tolerance to temperature, salinity, nutrients, grazing and sediment burial. It is able to

withstand temperatures between 2oC and 35

oC, with optimal growth between 11

oC and 30

oC

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(Yokoya et al. 1999). It can also survive and grow in salinities as low as 2 ppt and as high as 60

ppt, which is a broader range than most marine algae (Nyberg, 2007). G. vermiculophylla

withstands an emerged humid, dark environment for more than five months, simulating

transportation in fishing nets or in a dredger. All of these characteristics explain the rapid

expansion of G. vermiculophylla across northern latitudes, particularly Sweden, Denmark, and

the Northeast coast of the United States.

In my research, I will be exposing invasive and native Gracilaria species to a broad range

of salinities, from fresh water conditions to marine, and to different levels of nitrate, from

optimal for mass multiplication to minimal levels in ocean seawater. The results of my research

can be applied to predict patterns of Gracilaria distribution and growth in multiple estuarine

environments. If we can predict how an invasive species spreads, we can optimize management

strategies. Currently, there is little data on how the native Gracilaria reacts towards the invasive

species. Because they look very similar, there are no observations on whether the invasive

Gracilaria outcompetes the native or if each can live together in the same ecosystem.

Under my experimental set up, if one species grows more than the others, this will

provide an indication of what the dynamics of G. tikvahiae and G. vermiculophylla are when the

latter spreads in an ecosystem. As test system, I will be predicting the distribution of both species

in Waquoit Bay National Estuarine Research Reserve for which decades of environmental data

are readily available (Valiela et al., 2016). It is known that at one point the native species, G.

tikvahiae, and the invasive G. vermiculophylla coexisted in the area (Charles Yarish, UCONN,

pers. comm. to E.L. Peredo). There are no published studies determining the distribution or

abundance of these two species in Waquoit Bay because these two are phenotypically identical

and therefore impossible to identify in the field. My goal will be to use physiological

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characteristics to predict where G. vermiculophylla will be dominant in the Bay, which will

inform management on this invasive species.

Methods

Algal material: I cultured three lineages of Gracilaria isolated in nature and maintained

in culture (kindly provided to E.L. Peredo by Charles Yarish, UCONN). These cultures include

G. tikvahiae, a native species collected in Rhode Island; G. vermiculophylla; an invasive species

from Massachusetts which represent the diversity found in Long Island Sound; and, for

comparative purposes, I also included a non-invasive lineage of G. vermiculophylla collected in

Korea, where it is native. All of the samples were clipped from material in ongoing cultures to

minimize variation and prevent past environmental factors from affecting the conclusion about

growth or nitrogen uptake.

Nitrate uptake: We tested the rate of nitrate uptake within the three different lineages of

Gracilaria. To be able to measure the independent uptake of each individual species, thalli from

the same species were transferred into a separate jar (3 jars, 6 thalli per jar). The thalli were

maintained in seawater without no additional nitrogen supplementation for 72-hours, to ensure a

visible uptake rate. After this period, thalli were placed in 500 mL of fresh sea water

supplemented with 60 µM of nitrate. Corresponding nutrients described in von Stosch’s

enrichment medium were also added, to allow for maximum uptake of nitrate. Nitrate levels

were determined from seawater samples collected at the bottom of the jar. Sampling points were

collected after adding the nitrate (time 0) and then in a time sequence of 30 minutes, 1 hour, 4

hours, 8 hours, 12 hours, 24 hours, and 48 hours. Nitrate was analyzed with a Lachat Flow

Injection Analyzer (FIA) according to a modification of Wood et al. (1967). The uptake rate was

calculated as µg N hr-1

gg dry biomass, using the formula:

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𝑈𝑝𝑡𝑎𝑘𝑒 𝑟𝑎𝑡𝑒 =∆((𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑎𝑡 𝑇𝑛 − 𝑇0) ∗

14 𝑔 𝑁1 𝑚𝑜𝑙 𝑁

∗ 𝑉𝑜𝑙𝑢𝑚𝑒)

𝐷𝑟𝑦 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 (𝑔)

𝑈𝑝𝑡𝑎𝑘𝑒 𝑟𝑎𝑡𝑒 = (𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑎𝑡 𝑇0 ∗ 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑁 ∗ 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛)

− (𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑎𝑡 𝑇𝑛 ∗ 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑁 ∗ 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛)

with T0 the initial sample and T0 subsequent sampling times.

Culturing experiment: All algae were grown in 32-ounce jars (~900 mL) with 700 ml of

the appropriate culturing medium. In each jar, all three lineages were grown (two thalli per

species) to evaluate responses under competitive conditions. Each thallus was wrapped in color-

coded thread for individual identification and collection of growth data. The temperature was

kept constant at room temperature (around 25oC) and external lights simulated growing

conditions. These lights were on for a long day, 16 hours, and a short night, or 8 hours and

provided approximately 40 µE/ m*s of light. The algae were grown in von Stosch’s enriched

seawater medium (von Stosch, 1963) which includes NaNO3 (variable in nitrogen and

nitrogen/salinity gradient), Na2HPO4, FeSO4, MnCl2, Na2EDTA, Thiamine-HCl, Biotin, and

B12. Media was replaced weekly over the growing period (4 weeks). The basal levels of nitrate

in the seawater used to prepare the medium were determined for each water change, because the

seawater is collected from a natural environment already containing nitrogen. During the week,

distilled water was added to account for evaporation. Distilled water is required to ensure no

changes for initial conditions. There was a constant rotation of the beakers to avoid position

shading and I disturbed and moved the algal thalli to measure them on a regular schedule.

Nitrate gradient (Figure 1): The effect of nitrate was tested in a five-point gradient, (0, 5,

60, 250, 500 µM), with 500 µM being the optimal concentration for mass growth and 0 µM of

added nitrogen, or the basal levels in oceanic water, being the most stressful condition. Each

treatment was carried out by duplicated. To ensure that changes in nitrogen are the sole

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manipulator of growth, all nutrient concentrations except for NaNO3 was held constant. To

simulate algal conditions in natural systems, the 0 µM, 5 µM, and 60 µM gradients tested in this

experiment of concentrations of NaNO3 are correlated with natural levels of total nitrogen

detected in Waquoit Bay, which is a highly nitrogen-rich system (Valiela et al., 2016). However,

in our experimental setting it is expected the algae will be able to consume during the week the

initial nitrate present in these medium-low concentrations. 500 µM levels should not be limiting

during the course of the experiment.

Salinity gradient (Figure 1): Salinity was tested in a five-point gradient test (0, 8.75, 17.5,

26.25, 35 ppt), with 35 ppt being the optimal concentration for mass growth and 0 ppt being the

most stressful condition. To modify salinity, media was prepared by mixing different amounts of

filtered seawater and mineral spring water to ensure that trace elements will not be diluted as

well. To confirm that salinity levels are the only cause for changes in growth rate, all nutrients

were added at full concentrations and held constant. While sodium is an element associated with

the nutrient additions in Von Stosch’s medium, the concentration is negligible compared to the

total amount of sodium in seawater, a difference of milligrams to grams.

Nitrate-Salinity gradients (Figure 1): To simulate the conditions that the algae face in

natural systems, the simultaneous effect of the two variables, nitrogen and salinity, was tested.

Nitrogen was added at the concentrations described above. Based on previous data which

suggested extreme sensitivity to low salinity in G. tikvahiae, the salinity gradient defined as 10,

16.25, 22.5, 28.75, 35 ppt to assure the survival of all algal lineages. While nitrate and salinity

were variable (see Figure 1) all other conditions were maintained constant.

Growth measurements and statistical analysis: Growth was expressed as changes of

biomass for each Gracilaria thallus. Each thallus was weighed once a week. To prevent

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desiccation, samples were measured in seawater. Specific growth rate was estimated using the

formula

𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝐺𝑟𝑜𝑤𝑡ℎ 𝑅𝑎𝑡𝑒 (𝑑−1) =ln 𝑆2−ln 𝑆1

𝑇2−𝑇1

where S1 and S2 are the fresh weight at days T1 and T2, respectively (Gorman et al., 2017).

Additionally, the relative growth rate (RGR) was measured for each species across the gradient

experiments. To determine the RGR, the natural log of the biomass of each thallus over each

time for each treatment was calculated. Those values were then plotted on a graph, and adjusted

to a regression. The slope of that line was calculated. The slope of the line was then averaged

across all replicates of the experiment, and plotted as a scatter plot.

Gracilaria growth is highly plastic; different conditions provoke different growth patterns,

colors, and shapes. Photos were taken of the Gracilaria over the four-week period to document

color and branching pattern changes. While this is not a quantifiable measure of Gracilaria

growth or an exhaustive study, it was interesting to observe differences in growth patterns and

color.

Desiccation: An experiment was conducted to evaluate the desiccation tolerances of G.

tikvahiae and G. vermiculophylla. The tolerances to desiccation were tested by removing sets of

thalli from seawater and maintained them exposed to dry or damp air for intervals of 1 minute,

10 minutes, 30 minutes, and 60 minutes. The effect of damp conditions was tested with one set

of thalli sandwiched between two dampened towels. The effect of dry conditions was tested at

the same time intervals with another set of thalli resting on dry towels, exposed to the dry air. For

each time interval and condition, three thalli were tested. After exposure, the thalli were placed

in beakers with seawater and observed over the next 48 hours. The experiment was stopped after

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48 hours to ensure that desiccation was the sole cause of death among the thalli, as opposed to

nutrient deficiencies or anoxia due to decaying material.

Results

Nitrate uptake: Nitrate uptake was determined for each lineage of Gracilaria. The uptake

rate of the invasive G. vermiculophylla was double the uptake rate of the native G. tikvahiae.

Nitrate uptakes in the Massachusetts was 431 µg N hr-1

g-1

dry biomass, Korean was 450 µg N

hr-1

g-1

dry biomass, and Native was 213 µg N hr-1

g-1

dry biomass (Table 1).

Culturing experiment (Nitrate): After the first week in the nitrate gradient, all specific

growth rates were relatively high. The rates were highest in the Native species at the 500 µM,

250 µM, and 60 µM gradient points, ranging from 6-7% growth per day. The two invasive

lineages had roughly the same pattern and rate of growth, around 4-5% per day. The 0 µM

gradient point was the lowest for both the Native species and Korean lineage, at 1.5% and 2.8%,

respectively, but the Massachusetts lineage did not experience the same drop in rate at that point.

Instead, the Massachusetts’ lowest rate was 2.6% at 60 µM (Figure 2).

Between the first and second week, the specific growth rates drop across all jars, ranging

from 1-5% instead of 2-8%. The highest rate of growth was the Massachusetts at the gradient

point 500 µM, with 5.2% growth. Across all lineages, as the nitrate gradient decreased, the

specific growth rate generally decreased. However, there was some variance; the Native lineage

increased at 60 µM, with a higher growth rate of 4.5%. The Korean had the lowest overall

growth, ranging from 1% in the 0 µM to 3% in 500 µM (Figure 3).

In Week 3, the Native continued the trend in Week 2 of decreasing specific growth rates

as the nitrate gradient decreases. The highest growth rate was in the Korean at 5 µM, with a rate

of 4.3%. In the Korean and the Massachusetts, a new trend started to emerge, of increasing

specific growth rates from 500-5 µM, with a decrease at 0 µM (Figure 4).

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The relative growth rate for nitrate didn’t vary through the five gradient points. There

was a slight upwards trend from 0 µM to 60 µM, then a slight decrease at 250 µM, with a larger

increase at 500 µM. The three lineages were all relatively equal to each other; there wasn’t an

outlier in the gradient points. The RGR remained within 0.02-0.04 for all five gradient points

(Figure 6).

Culturing Experiment (Salinity): After the first week in the salinity gradient, all specific

growth rates were relatively high. The rates were highest in the Native lineage at the 28 ppt and 8

ppt gradient points, with 6.6% and 7.8% growth per day, respectively. The two invasive lineages

had roughly the same pattern and rate of growth, around 4-6% per day. The 0 ppt gradient point

caused nearly instant death in the native G. tikvahiae, with all four thalli dying over the first

week of the experiment (Figure 7).


from 0-3.4% instead of 3-7%. The highest rate of growth was calculated for the invasive

Massachusetts at the gradient point 8 ppt, with 3.4% growth. In the Native lineage, as the salinity

gradient decreased, the specific growth rate generally decreased. This trend was followed in the

first three points of the Korean as well, decreasing, until 8 ppt, where the highest growth for that

week was observed. There was no growth at 8 ppt in the Massachusetts. No growth at was

observed at 0 ppt in the Korean or the Massachusetts G. vermiculophylla (Figure 8).

In Week 3, the Korean had high growth from 35-18 ppt and then dropped to 0.5% at both

8 and 0 ppt. The Native maintained a stable growth rate across the four gradient points. The

Massachusetts decreased from 26 ppt to 8 ppt, but had a low growth rate of 1.7% at 35 ppt. The

Massachusetts had no growth at 0 ppt. (Figure 9).

The relative growth rate for salinity displayed a clear upwards trend, from 0 ppt to 26 ppt.

The Native lineage was dead in Week 1 at 0 ppt, and the Massachusetts did not display a growth

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rate until 26 ppt. The Korean had a clear upward trend, from 0 ppt to 26 ppt, where it then

decreased slightly at 35 ppt. This decrease at 35 ppt was seen across all lineages. The values for

the RGR ranged from 0 to 0.03 (Figure 11).

Culturing Experiment (Combination): After the first week in the combination gradient,

inverse levels of salinity and Nitrate (Fig. 1), all specific growth rates were relatively high. The

rates were highest in the Native species at the 16 ppt, 5 µM gradient point and the 28 ppt, 60 µM

gradient point, ranging from 7-10% growth per day. The Korean lineage overall had a higher

growth rate than the Massachusetts, ranging from 5-10% as opposed to 4-6%. (Figure 12).


from 0-2% instead of 4-10%. Also, across all three lineages, the gradient points 16 ppt, 250 µM

and 10 ppt, 500 µM did not see any growth. The highest rate of growth was the Korean at the

gradient point 22 ppt, 60 µM, with 1.8% growth (Figure 13).

In Week 3, the growth rate ranged between 0-3.5%. All three lineages show a small trend

of increasing specific growth rate in medium salinity and nitrate increases (22 ppt, 60 µM and 28

ppt, 5 µM), which decreased in higher salinity and lower nitrate concentrations (35 ppt, 0 µM).

In low salinity (

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unexpected, as it survived (and grew) at 0 and 8 ppt in the salinity gradient. We believe this

unexpected result could be attributed to experimental causes and not to the intrinsic tolerance to

salinity of the material. There was a large amount of variance within the combined salinity and

nitrate levels experiment, as can be observed in the Relative Growth rate (Figure 16), with values

ranging from 0 to 0.06.

Desiccation tolerance of native and invasive species of Gracilaria: Desiccation tolerance

was evaluated under two circumstances, removal from sea water while maintaining humidity

‘damp condition’ and removal from seawater and exposure to dry air ‘dry condition’. Evaluation

of desiccation tolerance was conducted in a time sequence of exposure, of 1 minute, 10 minutes,

30 minutes, and 60 minutes, with a control of 0 minutes. After exposed material was returned to

seawater. The experiment was stopped after 48-hours, to ensure that the initial desiccation was

the sole cause for death. All G. tikvahiae and G. vermiculophylla thalli survived in the control, 1

minute, and 10 minutes exposure, regardless of the treatment of ‘damp’ versus ‘dry’. This was

also the result for all material exposed for 30-minute or 60-minute under ‘damp” conditions. On

the contrary, in the 30-minute and 60-minute exposure under dry conditions, all the G. tikvahiae

thalli died, turning a bright pink color after 24-hours and white after 48-hours, while the invasive

G. vermiculophylla showed no signs of stress.

Discussion

The nitrate uptake results showed a clear difference between the native Gracilaria

tikvahiae and invasive Gracilaria vermiculophylla (Table 1). The Native had an uptake rate of

213 µg N hr-1

g-1

dry material, while the Korean and Massachusetts G. vermiculophylla had

double the Nitrate uptake rate, with an average of 441 µg N hr-1

g-1

dry material, (Table 1). Our

results allow us to predict where the invasive species will be in a physical setting because they

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indicate that G. vermiculophylla will be more efficient in low nitrate areas, outcompeting the

Native species. This prediction is substantiated by the growth data generated at the nitrate

gradient and comparing specific growth rates between the native and invasive species in the low

nitrate points.

We can look at the nitrate gradient to see how Gracilaria reacts in differing nitrate

conditions. While the nitrate uptake experiment showed that the invasive lineage had double the

uptake rate of the Native, this faster rate was not immediately obvious in the gradient results.

Both the invasive and native lineages preferred higher nitrogen conditions, but between 500 µM,

250 µM, and 60 µM, there wasn’t a gradient point with a much larger growth rate. However, at 5

µM and 0 µM, there was a general decline in growth rate. Only the Massachusetts sustained

growth at 0 µM added Nitrate (Fig. 5). We had predicted a bigger influence of the competition

with invasive species in the growth of the native Gracilaria tikvahiae. The competitive

conditions may have influenced the uptake rate of the invasive lineage, causing it to decrease its

rate, or maybe the effects of this competition would require a longer experiment to be

immediately obvious. Additionally, there may have been other missing nutrients, which could

have prevented total uptake over the seven-day period (Figure 5). There was no death in this

experiment, which was expected because, while the lack of nitrate prevents growth, the algae can

still survive in seawater, based on preliminary data and observations.

The gradient with the strongest trend was the salinity gradient. Both G. vermiculophylla

and G. tikvahiae are euhaline species (Nyberg, 2007; Bird et al. 1979; Bird and McLachlan,

1986; Kim et al., 2016), so it was surprising when the salinity gradient below 15 ppt did not

cause a large death event. At 8 ppt, despite the Native species showing no growth, it did not

show external signs of death. The invasive lineages showed growth at this extremely low salinity

(8 ppt), with the Korean rate higher than the Massachusetts. At these levels of salinity, if enough

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nitrate is present, the invasive can grow while the Native cannot. At 0 ppt (fresh water), the

Native died within 24 hours, while the invasive can live and grow, provided with sufficient

levels of nitrate. This broad tolerance allows the potential range of distribution of the invasive to

be larger than the native in estuaries and brackish water. Originally, we believed that G.

vermiculophylla (Mass) would have a larger range of tolerances because it is an invasive lineage,

while G. vermiculophylla (Korea), being a collected in the native range of the species may not

have adapted to survive in as large of a range of environments. However, based on the salinity

gradient, this hypothesis was not supported. Despite having a similar growth rate at 0 ppt, the

Korean lineage had approximately twice the growth rate at 8 ppt, compared to the Massachusetts.

This suggests that the Massachusetts lineage tested may have lost some of its range to salinity

tolerances, potentially because in New England there may be more ecosystems with higher

salinity levels compared to ecosystems in Korea, which influenced the ability of the algae to

overcome osmotic potentials. Additionally, the relative growth rate showed a decrease in growth

at 35 ppt in all three lineages, suggesting that G. tikvahiae and G. vermiculophylla prefer

estuarine environments, with a lower salinity level than a fully marine environment (Figure 10).

Finally, the inverse gradient of nitrate and salinity was tested to try to understand the

interaction between environmental parameters and better predict where G. tikvahiae and G.

vermiculophylla will be found in an ecosystem with this naturally occurring inverse gradient,

such as Waquoit Bay. Under these conditions, the largest growth was found at the 16 ppt, 250

µM point. However, this growth rate, compared across the Native lineage, to the salinity at a

similar point, and the nitrate at the same point, is much lower throughout the experiment, from

around 2% in the salinity gradient and 3% in the nitrate gradient, compared to around 1.3% in

the combination gradient. In this gradient, a point of osmotic stress was reached at 10 ppt in the

Massachusetts and the Native, but growth remained high in the Korean. At 16 ppt, both the

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Korean and the Native had high rates of growth. The Massachusetts had no growth at either of

those two points, despite having high levels of nitrate. Without sufficient nitrate to fulfill their

metabolic requirements, the algae did not grow as well, in the 35 ppt, 0 µM and 28 ppt, 5 µM.

Overall, this treatment did not react as expected. The Massachusetts had unexpected low growth

rates at low salinity, high nitrate conditions, where both the Native and the Korean were able to

grow. This may indicate a problem with the algal material, which may have been influenced by

past environmental stressors. Additionally, much like in natural ecosystems, plant behavior

depends on multiple variables and can often happen on levels which are on the micro-scale

instead of the macro-scale (Figure 15).

In all three gradient experiments, across all three lineages, there was a large difference in

specific growth rates between the first and second week, while from the second week onward the

specific growth rate was relatively constant, without the large fluctuations. In the nitrogen

gradient, the Korean gradient point 35 ppt changed from 6.1% to 4.3% from Week 1 to Week 2.

Because this occurred at the beginning of the experiment, and was not repeated in subsequent

weeks, we believe that this high growth was due to previous, optimal conditions in the medium

and environment of the container in which the algae were initially grown. The algae may have

stored nutrients in their biomass and used both the surrounding nutrients and provided nutrients

the first week, to grow at such a high rate. After the first week, those nutrient storages were

exhausted, so the algae had to rely on the added nutrients in sub-optimal conditions, which

stunted growth. When calculating the relative growth rate, we decided to calculate without the

first week of data to remove outliers and abnormal growth patterns (Figures 5, 10, 15).

However, before I was able to collect data in the gradients experiment, preliminary data

was collected and used to modify the experiment for accurate measurements and analysis. In the

first attempt at culturing these thalli, G. tikvahiae began to die within 24-hours of exposure in all

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jars, including the control treatments where optimal growth was expected. After observation, we

hypothesized that this die-off was probably due to accidental desiccation during collecting,

weighing and imaging of the thalli. Also, we hypothesized that the native G. tikvahiae is more

intolerant to desiccation and that the two lineages of G. vermiculophylla used in the experiment,

from Korea and Massachusetts. This hypothesis was supported with the results of our experiment

of desiccation tolerance between the Native G. tikvahiae and G. vermiculophylla thalli, where we

found that the Native died after a period of exposure of 30 minutes to dry conditions while the

Korean recovered once returned to seawater (produced oxygen, pers. observation). Ecologically,

this suggests that G. vermiculophylla is able to survive closer to shore, where it could be

periodically exposed to desiccation due to tidal action. G. tikvahiae is only able to survive in

constant submersion, outside of the tidal zone. The second time the gradient experiment was

attempted, the Native again began to die after being transferred to the gradients. After some

investigation, it was discovered that the medium was accidentally created with freshwater,

instead of seawater. The Korean and Massachusetts thalli were not affected as strongly as the

Native. This confirms our results from the salinity gradient. Invasive Massachusetts and Korea

are better able to survive in a broader range of salinities, further proving that G.

vermiculophylla’s physiology is that of a strong invasive species.

With all of this information about G. tikvahiae and G. vermiculophylla, I evaluated areas

of potential presence of native and invasive Gracilaria, specifically tailored to the physical

system of Waquoit Bay (Cape Cod, USA). I found no evidence of one species outcompeting the

other in areas where salinity and nitrate levels are sufficient, so G. tikvahiae and G.

vermiculophylla will potentially coexist in most of the bay (Figure 23). I expect G. tikvahiae to

be found in most of the bay, in areas with high-medium salinity. G. tikvahiae will also be found

in deep water, in areas with little chance to be exposed to desiccation. My results indicate that,

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because G. vermiculophylla is more tolerant to lower salinities, grows well in all nutrient

concentrations, and can be desiccated without death, G. vermiculophylla will be found in areas

where G. tikvahiae cannot survive, including river areas in the north of Waquoit Bay. It will also

be able to colonize areas within the main bay where native G. tikvahiae is not present,

particularly near the coastline.

Applying in vitro results to a physical system is important, but not perfect. My

experimental design was a highly controlled system, with no influence of external elements such

as varying temperatures, pH, light, and other organisms. When predicting the distribution of

Gracilaria, it is merely meant as a tool towards understanding one aspect of their patterns of

growth and not as a perfect tool as it does not reflect the full complexity of a natural system.

Field work and genetic identification is a complementary way to gain information and insight

into these two species. The results of one can inform the other; for example, if the experimental

design shows that G. tikvahiae will not grow in freshwater, if Gracilaria sps. is found in an area

of low salinity or exposed to desiccation, the fieldwork researchers are now able to confidently

assume that the sample will correspond to the invasive G. vermiculophylla. Pairing in vitro

experiments with field work and corroborating filed data with molecular identification would

further strengthen our understanding of the patterns of growth and distribution of G. tikvahiae

and G. vermiculophylla.

Collaboration

While I will determine the tolerances, preferences, and functional characteristics of

Gracilaria, my colleague, Lauren Hamm, will be determining the distribution and genetic

composition of Gracilaria in Waquoit Bay. Our work will be complementary; in Waquoit, I will

predict where each species will be, while Lauren will be able to support or deny that. She is

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working directly with a site and developing a comprehensive view, while my work can be

applied to both that site and regionally.

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Table Appendix

Table 1: The nitrate uptake of G. tikvahiae and G. vermiculophylla (Mass) and G.

vermiculophylla (Korean).

Nitrate Uptake (µg N hr-1

g-1

dry biomass)

Native 213

Invasive (Mass) 431

Invasive (Korean 450

Figure Appendix

Figure 1: The experimental design of the nitrate, salinity and combined gradients performed in

this work.

NitrateGradient(uM)

SalinityGradient(ppt)

NitrateSalinityGradient

500 250 60 20 0

0 8.75 17.5 26.25 35

G.verm iculophylla-Korea1G.verm iculophylla-Korea2

G.verm iculophylla-MA1G.verm iculophylla-MA2

G.tikvahiae-RI1

G.tikvahiae-RI1

500,10 250,16.25 60,22.5 5,28.75 0,35

5

(uM,ppt)

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Figure 2: Specific growth rate under different Nitrate concentrations from Week 0 to Week 1,

with each bar representing the average and standard deviation calculated from four thalli at one

gradient point. Data are ordered from highest to lowest in the gradient for each lineage.




-2%

0%

2%

4%

6%

8%

10%

12%

1

Nitrogen Week 1-Week 0

Korean

Native

Mass

500 250 60 5 0 500 250 60 5 0 500 250 60 5 0 µM

µM

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Figure 5: Specific growth rate under different Nitrate levels over the entire experiment, with each

bar representing the average and standard deviation calculated from four thalli at one gradient

point. Nitrate concentration are represented from highest to lowest in the gradient.

µM

µM

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Figure 6: The relative growth rate of the nitrate gradient during the 24 days of the experiment. In

the Figure is represented the average RGR of the two replicas and the standard deviation. For

clarity, X axis is represented in log scale.

Figure 7: Specific growth rate under different salinity levels from Week 0 to Week 1, with each


point. Data are ordered from highest to lowest in the gradient for each lineage. In black is

0

0.01

0.02

0.03

0.04

0.05

0.06

1 10 100 1000

Re

lati

ve G

row

th R

ate

Nitrate (µ M)

Nitrate Relative Growth Rate

RGRJar1NativeAVE

RGRJar1KoreanAVE

RGRJar1MAInvAVE

ppt

0 5 60 250 500

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represented death events.




represented death events. No bar indicates no growth.




represented death events.

ppt

ppt

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Figure 10: Specific growth rate under different salinity levels over the entire experiment (24

days), with each bar representing the average and standard deviation calculated from four thalli

at one gradient point. Data are ordered from highest to lowest in the gradient for each lineage. In

black is represented death events.

Figure 11: The relative growth rate of the salinity gradient during the 24 days of the experiment.

In the Figure is represented the average RGR of the two replicas and the standard deviation.

0

0.01

0.02

0.03

0.04

0.05

0.06

0 5 10 15 20 25 30 35

Re

lati

ve G

row

th R

ate

Salinity (ppt)

Salinity Relative Growth Rate

RGRJar1NativeAVE

RGRJar1KoreanAVE

RGRJar1MAInvAVE

ppt

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Figure 12. Specific growth rate under combination of different Nitrate and salinity levels from

Week 0 to Week 1, with each bar representing the average and standard deviation calculated

from four thalli at one gradient point. Data are ordered from highest to lowest in the gradient for

each lineage.

Figure 13: Specific growth rate under combination of different Nitrate and salinity levels from



each lineage.

ppt,

µM

ppt,

µM

Fox 25

Figure 14: Specific growth rate under combination of different Nitrate and salinity levels from



each lineage. In black is represented death events.

Figure 15: Specific growth rate under combination of different Nitrate and salinity levels over

the experiment (24 days), with each bar representing the average and standard deviation

calculated from four thalli at one gradient point. Data are ordered from highest to lowest in the

gradient for each lineage. Black indicates death events.

ppt,

µM

ppt,

µM

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Figure 16: The relative growth rate of the combined nitrate and salinity gradient during the 24

days of the experiment. In the Figure is represented the average RGR of the two replicas and the

standard deviation.

0

0.01

0.02

0.03

0.04

0.05

0.06

0 5 10 15 20 25 30 35

Re

lati

ve G

row

th R

ate

Combo (high salinity, low nitrogen) shown salinity in ppt

Combination Relative Growth Rate

RGRJar1NativeAVE

RGRJar1KoreanAVE

RGRJar1MAInvAVE

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Figure 17: Predictions for the distribution of G, tikvahiae and G. vermiculophylla in Waquoit

Bay based in the tolerance to low salinity and nitrate requirements of each species. The

underlying map shows the salinity in ppt, from marine to freshwater (red to blue) (Waquoit Bay

Reserve).

References

Bird C.J., Chen L.C.M. & McLachlan J. 1979. Effects of temperature, light and salinity on

growth in culture of Chondrus crispus, Furcellaria lumbricalis, Gracilaria tikvahiae

(Gigartinales, Rhodophyta), and Fucus serratus (Fucales, Phaeophyta). Botan- ica Marina

MASSACHUSETTS ESTUARIES PROJECT

127

Figure VI-7. Contour plots of modeled salinity (ppt) from the model of the Waquoit Bay system.

G.tikvahiaeG.vermiculophylla

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22: 521-527.

Bird C.J. & McLachlan J. 1986. The effect of salinity on distribution of species Gracilaria Grev.

(Rhodophyta, Gigartinales): an experimental assessment. Botanica Marina 29: 231 —

238

Fox, S. E., Stieve, E., Valiela, I., Hauxwell, J., & Mcclelland, J. (2008). Macrophyte Abundance

in Waquoit Bay: Effects of Land-Derived Nitrogen Loads on Seasonal and Multi-Year

Biomass Patterns. Estuaries and Coasts, 31(3), 532-541.

Gorman, L., Kraemer, G. P., Yarish, C., Boo, S. M., & Kim, J. K. (2017). The effects of

temperature on the growth rate and nitrogen content of invasive Gracilaria

vermiculophylla and native Gracilaria tikvahiae from Long Island Sound,

USA. Algae, 32(1), 57-66.

Hammann M, Rempt M, Pohnert G, Wang G, Boo SM, Weinberger F. 2016a. Increased

potential for wound activated production of Prostaglandin E2 and related toxic

compounds in non-native populations of Gracilaria vermiculophylla. Harmful Algae 51:

81–88.

Hammann M, Wang G, Boo SM, Aguilar-Rosas LE, Weinberger F. 2016b. Selection of

heat-shock resistance traits during the invasion of the seaweed Gracilaria

vermiculophylla. Marine Biology 163: 1–11.

Kim, J. K., Yarish, C., & Pereira, R. (2016). Tolerances to hypo-osmotic and temperature

stresses in native and invasive species of Gracilaria (Rhodophyta). Phycologia, 55(3),

257-264.

Kim, S. Y., Weinberger, F., & Boo, S. M. (2010). Genetic Data Hint at A Common Donor

Region for Invasive Atlantic And Pacific Populations of Gracilaria Vermiculophylla

(Gracilariales, Rhodophyta)1. Journal of Phycology, 46(6), 1346-1349.

Fox 29

Koutsovoulos G, Kumar S, Laetsch D, Stevens L, Daub J, Conlon C. 2016. No evidence for

extensive horizontal gene transfer in the genome of the tardigrade Hypsibius dujardini.

Pnas 113: 1–6.

Krueger-Hadfield SA, Kollars NM, Strand AE, Byers JE, Shainker SJ, Terada R, Greig

TW, Hammann M, Murray DC, Weinberger F, et al. 2017. Genetic identification of

source and likely vector of a widespread marine invader. Ecology and Evolution 7: 4432–

4447.

Martínez-Lüscher J, Holmer M. 2010. Potential effects of the invasive species Gracilaria

vermiculophylla on Zostera marina metabolism and survival. Marine Environmental

Research 69: 345–349.

Nettleton, J. C., Mathieson, A. C., Thornber, C., Neefus, C. D., & Yarish, C. (2013). Introduction

of Gracilaria vermiculophylla (Rhodophyta, Gracilariales) to New England, USA:

Estimated Arrival Times and Current Distribution. Rhodora, 115(961), 28-41.

Nyberg CD. 2007. Introduced marine macroalgae and habitat modifiers: their ecological role

and significant attributes.

Ramus AP, Silliman BR, Thomsen MS, Long ZT. (2017). An invasive foundation species

enhances multifunctionality in a coastal ecosystem. Proceedings of the National Academy

of Sciences: 201700353.

S. Yokoya, Nair & Kakita, Hirotaka & Obika, Hideki & Kitamura, Takao. (1999). Effects of

environmental factors and plant growth regulators on growth of the red alga Gracilaria

vermiculophylla from Shikoku Island, Japan. Hydrobiologia. 398-399.

Valiela, I., Owens, C., Elmstrom, E., & Lloret, J. (2016). Eutrophication of Cape Cod estuaries:

Effect of decadal changes in global-driven atmospheric and local-scale wastewater

nutrient loads. Marine Pollution Bulletin, 110(1), 309-315.

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von Stosch, H. (1963). Wirkungen von Jod un Arsenit auf Meeresalgen in Kultur. Proc. Int.

Seaweed Symp. 4:142-150.

Waquoit Bay National Estuarine Research Reserve, www.waquoitbayreserve.org/.

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Salinity Tolerances and Nitrogen Requirements of Native and … · 2018. 2. 8. · Fox 1 Salinity Tolerances and Nitrogen Requirements of Native and Invasive Gracilaria species in