Environmental DNA Analysis of Bivalve

Restoration in New York City

Source: http://www.huffingtonpost.com/2013/11/26/freshkills-park-solar-energy-new-york-ci ty-images -_n_4343185.html

By: Joshua Seidman

May 2016

Abstract:

Bivalves are important members of many ecosystems on the United States’ east coast.

Unfortunately, there has been a global decline of bivalve populations. Oyster reefs can work as

natural energy absorbers of wave and storm energy. Their ability to act as storm energy

absorbers in addition to filter play key roles in the overall health of their habitats and makes

them essential for healthy ecosystems. On Staten Island, NY, the Fresh Kills landfill opened in

1947 and closed in 2001 due to local pressure. Since then, restoration efforts have been in

effect, leading to what is now known as Freshkills Park, the largest landfill-to-park restoration in

the world. Currently, native ribbed mussels (Geukensia demissa) that play key roles in water

filtration and storm protection are recolonizing the park’s salt marshes. Eastern oysters

(Crassostrea virginica) also carry out these functions, and are being restored to selected

saltwater habitats, including at Soundview Park, Bronx River. This study implements

environmental DNA (eDNA) analysis of DNA extracted directly from water and sediment at

these sites, in order to better understand their overall eukaryotic biodiversity and restoration.

My research evaluates three salt marshes at Freshkills Park during different points in the

restoration, as well as the oyster reef at Soundview, Bronx River and two control sites. By

sequencing the DNA from our water and sediment samples, we have the power to run

statistical analysis (such as rarefaction, Bray-Curtis, and Jaccard) to better understand the true

effect of these bivalves on their particular systems. Upon completion of one cycle of sample

collections, it is clear that more samples will be necessary due to rarefactions curves.

Additionally, alpha diversity analysis revealed seasonal changes in biodiversity and a clear

difference between water and sediment.

Introduction:

The Atlantic coast of North America supports native bivalves, including Geukensia

demissa (ribbed mussels) and Crassostrea virginica (Eastern Oysters). For nearly 300 years

(1600-1900) bivalves played key roles in the economy of New York (Nigro, 2011). However,

because of human activities such as overharvest and pollution, bivalve populations have

plummeted. My research employs eDNA analysis to conduct a eukaryotic biodiversity

assessment of both Soundview Park (Figure 1) and Freshkills Park (Figure 2). Environmental

DNA analysis is potentially both effective and less expensive (Yu 2012) then previous methods,

and can serve as a useful complementary approach to classical morphological investigation.

This facilitates researchers to carry out this kind of survey on the biodiversity of different sites.

The concepts behind eDNA analysis, it’s applications, and the advantages of using it collectively

with next generation sequencing can be seen outlined in Figure 3.

The re-colonization of mussels (Figures 4 and 5) and the man-made oyster reefs provide

ecosystem services such as storm protection and water filtration at Freshkills Park and

Soundview Park, thus sheltering residents, as well as cleaning the water (Grabowski, 2012).

These bivalves, especially eastern oysters, are ecosystem engineers, which are organisms that

naturally build habitats for other species (Morin, 2011). These reefs are home to many

predators such as blue crab (Callinectes sapidus), white-fingered mud crab (Rhithropanopeus

harrisii), Atlantic oyster drill (Urosalpinx cinerea), and other organisms such as common shore

shrimp (Palaemonetes vulgaris). The importance of bivalves in these ecosystems is tremendous

because they are suspension feeders, feeding on algae, free-floating sediment, and potentially

even toxins. Their presence allows for healthier and clearer water (Ehrich et al. 2014, Fitzgerald

2013, Lotze et al. 2006, Newell 1988). However, eastern oyster populations have been reduced

drastically, dropping slightly below 1% of their historic abundance throughout the northwestern

Atlantic coast over the last 120 years (Beck et al. 2011, Frakenburg 1995, Lotze et al. 2006). As a

result there has been a decline in native species in these reef communities. Additionally, there

has been an increase in susceptibility due to loss of genetic diversity, invasion, and a loss of

food webs (Segan, Murray, and Watson 2016).

In addition to the services offered by bivalves, spartina grasses (Spartina alterniflora)

grow along the salt marshes trap debris and decaying matter when the tide rises. This debris

builds up and creates nutrient-rich mud called detritus, which helps create an ideal

environment for other organisms to thrive.

Environmental DNA analysis offers an effective and efficient way to carry out

biodiversity assessment. Traditionally, to monitor biodiversity researchers relied on observation

and morphological identification techniques. This would consist of time intensive planning,

raising funds, time, and work (Bohmann et al. 2014) and the outcome of these countless hours

of labor would often present limited results. Environmental DNA offers a time saving, cost

effective, and less invasive data collection protocol (Barnes & Turner 2015, Beja-pereira et al.

2009, Bohmann et al. 2014).

My objective is to conduct a comprehensive biodiversity analysis of 3 salt marshes at

Freshkills as well as Soundview Park and it’s two controls. I hypothesize that differences in

diversity will be apparent between areas with larger ribbed mussel and oyster populations, and

those without these populations. I hypothesize that there will not only be noticeable

differences in diversity between sites, but also that the greatest diversity will be found in areas

with restored oyster reefs and ribbed mussel populations.

Materials and Methods:

Field collections at Freshkills Park were carried out on October 23, 2015. My colleagues

carried out collections at Soundview on 08/03/15, 08/31/15, 09/28/15, and 10/25/15. Samples

were collected from both control areas (which are unrestored at Soundview), Oyster Reefs at

Soundview, and from three salt marshes at Freshkills.

Soundview Park:

Once water and sediment samples had been collected, DNA filtration and extraction

were carried out using MOBIO Powersoil and Powerwater kits. DNA was then sent to Molecular

Resources, LP, for sequencing and analyzed using the supercomputer pipeline via Quantitative

Insights into Microbial Ecology (QUIIME) (Caporaso, 2010). Once sequenced, samples were

identified down to order level because available online reference databases frequently don’t

have matches of all DNA fragments since many microorganisms have not been discovered yet

(Leray & Knowlton, 2014).

For these samples, the package Vegan in R-Studio was used to calculate rarefaction

curves to determine the presence of rare species and if there was adequate sampling. Next, R

was used to generate alpha diversities (using Shannon Diversity Index) and Jaccard plots at the

control and restored sites. Alpha diversities were then plotted in R to compare the data

(Whittaker, 1972). Calculating the alpha and beta diversities was a key step in analyzing the

biodiversity of this site. The alpha diversity is the species richness of a given location (the total

number of species present in an ecosystem) and the species evenness (the frequency of

individuals of each species in an ecosystem) (Morin 2012, Allen et al. 2009). The beta diversity is

the species composition across given locations. This was visualized using Bray-Curtis

dissimilarity and Jaccard Indexes. Bray-Curtis Dissimilarity is a test that was used in order to

better understand the dissimilarities between samples from different sites by using it to reflect

beta diversity (Clarke, 2006). It is a statistic that measures the compositional variation between

two sites, based on counts at each site. Bray-Curtis graphs that show greater distance between

two sites illustrate greater differences in their biodiversity, and vice versa. The Jaccard

Similarity Index can be defined as the ratio of the sum of species shared by two locations at

distance d to the number of species present in either one of them (Azaele, 2016).

Morphological observations were also used when assessing Soundview Park. We had

taken core samples back to our lab and worked on identifying a number of organisms that had

been found in the sediment of the cores.Upon analyzing the organisms, some were easily

identified using A Field Guide to the Atlantic Seashore: From the Bay of Fundy to Cape Hatteras.

Others were too difficult to identify or too disfigured to identify. In order to get a better look at

organisms for accurate morphological identification, a dissecting microscope was used.

Freshkills Park:

DNA extraction followed the methods described above for Soundview, and the extracts

were sent out for professional sequencing. These DNA sequences will later be evaluated to

remove any errors. Our data can then be used to run a number of different statistical tests as

described above (rarefaction, alpha diversity: Shannon Diversity Index, beta diversity: Jaccard

Index, Bray-Curtis Dissimilarity). These tests will allow us to compare sites to one another, as

well as the same sites at different points in time. In addition to our statistical analysis,

morphological observation was used to determine if there were any noticeable differences

between species abundance and landscape at different sites .

Results:

Soundview Park:

1. Environmental DNA Analysis

In total, there were 18 eDNA samples (6 water samples and 12 sediment samples) that

were collected in the summer and fall of 2015. They were all sequenced to the level of order for

biodiversity analysis of Soundview Park and the two control sites: Hunts Point Riverside Park

and non-restored oyster reef. In all, 270+ orders were recognized and nearly 760,000

sequences in total. The ten most abundant orders, from this list, accounted for 70% of all

sequences that were recognized.

Ostreoida, the order to which eastern oyster belongs, was not part of the top ten orders

and had very low detection (52 total counts). Another important detection was of the oyster

parasitic disease Dermo (order Perkinsida), which has also been recently documented to be

present in New York Harbor Aquaculture facilities (Levinton et al. 2013), and P. marinus

(counted more then 75 times).

2. Statistical Analysis

Rarefaction Curves (Figures 6 and 7) graphs portrayed species richness over sampling

trials. Each curve on the graph represents the number of species over time. Curves should

plateau, which indicates that sampling was sufficient and that no additional samples are

required. The fact that our curves did not plateau indicates that additional sampling is required.

Shannon Index was used to calculate alpha diversity (Figures 8 and 9). Alpha diversities

for sediment and water samples were similar, which is a good indicator that mixing is occurring

due to river-flow. The graphs also display obvious seasonal biodiversity patterns, which can

further be confirmed during sample collections that are to be carried out during summer and

fall. Additionally, sediment samples proved to contain much more DNA than water samples.

Bray-Curtis and Jaccard (Figures 10, 11, 12, and 13) computations focused on the more-

abundant species, meaning organisms that are not relatively abundant, were ignored. Jaccard

calculations only revealed the presence of organisms and did not focus on their relative

abundance in each location. Bray-Curtis and Jaccard plots show that there is a gap between

sites, which reflects an obvious seasonal biodiversity pattern. This was confirmed by the results

given by Shannon Index, which also display seasonal variation. Additionally, Bray-Curtis and

Jaccard results from water and sediment samples suggest that there is a distinct difference

between the two.

3. Morphological analysis

As can be seen in Table 1, a number of morphological identifications were made. In

addition to their identification, listed is an evaluation of whether or not they were present in

our samples that underwent eDNA analysis. No families of crab (Epialtidae, Varunidae,

Panopeidae) or orders (Deapoda) that were morphologically identified were confirmed using

eDNA analysis.

Freshkills Park:

Our research will give us the opportunity to understand the biodiversity that is present

at these sites, and also give us the opportunity to see if the restoration efforts are effective. Our

research will use the same methods and materials that were used at Soundview Park to carry

out a comprehensive biodiversity analysis of the park. Hopefully, upon completing our

experiments, we can verify that the restoration efforts are valuable, and that these newly

restored sites are healthy for organisms to thrive.

Discussion:

This study sought to utilize eDNA analysis to compare the biodiversity of Soundview

Park’s restored oyster reef with two control sites as well as carry out a comprehensive

biodiversity investigation of Freshkills Park. Initially, I had proposed that there would be an

obvious difference between the biodiversity of restored sites at Soundview and the unrestored

sites. I had hypothesized that the greatest diversity would be found in areas that have been

restored and the least diversity in those that are unrestored.

As supported by the absence of a plateau on rarefaction curves, there was insufficient

sampling. This suggests that additional samples should be collected, and so more samples will

be collected in the spring, summer, and fall to expand the study. Bray-Curtis and Jaccard Index

also failed to support this hypothesis. The large gaps between sites on our graphs illustrate the

seasonal variation between sites, which is consistent in both Bray-Curtis and Jaccard, which is

good indication that these are reliable statistical tests for such an investigation. However,

despite disproving my hypothesis, this evaluation has proven to be effective in other way. It

informed us of over 273 orders that are a part of these communities at Soundview Park and

other sample sites. The data also revealed that there were significant discrepancies in

biodiversity between seasons (summer and fall) and between sediment samples and water

samples.

The differences between summer and fall and between water and sediment that were

observed can be seen in Bray-Curtis and Jaccard Index evaluations. This data reflects the

differences caused by breeding, migration, and death (Morin 2011). Additionally, the results of

this study suggest that perhaps Soundview Park’s oyster reef may need more time to establish

itself. The fact that it is not fully established may be reflective of why there was no statistically

significant difference between sites. Beck et al. (2011) states that for a restoration of oyster

reef to be successful, the population must increase by 10% or more over its historical high.

Therefore, once the reef has established itself, repetition of this project will likely to result in

significant statistical differences. Sediment and water samples from Soundview Park were also

dissimilar; the differences result from the different kinds of organisms that inhabit these two

different elements. Organisms like Polychaetes are usually found in sediment, whereas

zooplankton and phytoplankton are commonly identified in water samples .

The values showed in Shannon Index demonstrate the doubts associated with eDNA,

especially the concerns about the origin from which DNA is being located. I believe that runoff

from neighborhoods and the change in tides, and river flow may have resulted in mixing. For

example, eastern oyster DNA was found at Hunts Point, despite being very unlikely due to the

fact that there is no hard substrate for the oyster’s larvae to latch on to.

This study also clearly demonstrates the differences between morphological evaluation

and eDNA analysis. A large list was produced from our eDNA analysis of microscopic and

macroscopic organisms that inhabit this ecosystem. However, despite sifting through sediment

samples and finding many organisms, like crabs, many of the morphological identifications

didn’t show up on our eDNA analysis.

References

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New York Public Library. 2 June 2011. Web. 13 Apr. 2016. 17. “Oyster Restoration Program." NYNJ Baykeeper. Web. 29 Apr. 2016.

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Figure 1: Map of Freshkills Park

(Source: http://freshkillspark.org/the-park/the-park-plan)

Figure 2: Map of Soundview Park

(Source: Google Maps)

Figure 3: (A) the idea of environmental DNA (eDNA), (B) eDNA applications, and (C) the

advantages of combining eDNA with next generation sequencing.

(Source: Bohmann et al.)

Figures 4 and 5: Ribbed mussels that were observed at

Freshkills Park on October 23, 2015.

(Source: Joshua Seidman)

Figure 6: Calculated rarefaction curves created in R-Studio for water samples. Each curve represents a water sample. Each sample began to level off towards the right but never

plateaus. The absence of a plateau for each curve suggests that more sample collections are necessary. Please refer to abbreviation key.

Abbreviations Key

W- water A- Aug 3, 2015

S- soil B- Aug 31, 2015

HP- Hunts Point C- Sept 28, 2015

BRC- Control site D- Oct 25, 2015

BRO- Restored Site

Figure 7: Calculated rarefaction curves created in R-Studio for sediment samples. Each curve represents a sediment sample. Each sample began to level off towards the right but never plateaus. The absence of a plateau for each curve suggests that more sample collections are necessary. Please refer to abbreviation key.

Abbreviations Key

W- water A- Aug 3, 2015

S- soil B- Aug 31, 2015

HP- Hunts Point C- Sept 28, 2015

BRC- Control site D- Oct 25, 2015

BRO- Restored Site

Figure 8: Calculated and plotted alpha diversities in R-Studio of sediment samples. As can be seen by comparison of Shannon Index for sediment samples and water samples, the sediment samples exhibited a lot more biodiversity during eDNA analysis. Please refer to abbreviation key.

Abbreviations Key

W- water A- Aug 3, 2015

S- soil B- Aug 31, 2015

HP- Hunts Point C- Sept 28, 2015

BRC- Control site D- Oct 25, 2015

BRO- Restored Site

Figure 9: Calculated and plotted alpha diversities in R-Studio of water samples. As can be seen by comparison of Shannon Index for sediment samples and water samples, the sediment samples exhibited a lot more biodiversity during eDNA analysis. Please refer to abbreviation key.

Abbreviations Key

W- water A- Aug 3, 2015

S- soil B- Aug 31, 2015

HP- Hunts Point C- Sept 28, 2015

BRC- Control site D- Oct 25, 2015

BRO- Restored Site

Figure 10: Calculated relative abundance plot of species created in R-Studio for water samples

using average abundance. Bray-Curtis computations ignore less abundant species. It focuses on orders that are most prevalent. Greater distances between sites can be interpreted as the

greater the dissimilarity between their biodiversity. The large gaps between water samples and sediment samples are what illustrate the difference between the two element’s biodiversity. Please refer to abbreviation key.

Abbreviations Key

W- water A- Aug 3, 2015

S- soil B- Aug 31, 2015

HP- Hunts Point C- Sept 28, 2015

BRC- Control site D- Oct 25, 2015

BRO- Restored Site

Figure 11: Calculated presence/absence plot of species created in R-Studio for water samples

using average relatedness. Jaccard calculations show the orders that are present/absent at each site. It does not pay attention to how many of each order is present; instead it only focuses on

whether or not the order was present at all. Like Bray-Curtis computations, the larger the gap between two sites, the less orders they have in common. When compared to Jaccard for

sediment, clear distinctions can be made between the biodiversity of the two elements. Please

refer to abbreviation key.

Abbreviations Key

W- water A- Aug 3, 2015

S- soil B- Aug 31, 2015

HP- Hunts Point C- Sept 28, 2015

BRC- Control site D- Oct 25, 2015

BRO- Restored Site

Figure 12: Calculated relative abundance plot of species created in R-Studio for sediment samples using average abundance. Bray-Curtis computations ignore less abundant species. It focuses on orders that are most prevalent. Greater distances between sites can be interpreted as the greater the dissimilarity between their biodiversity. The large gaps between water samples and sediment samples are what illustrate the difference between the two element’s biodiversity. Please refer to abbreviation key.

Abbreviations Key

W- water A- Aug 3, 2015

S- soil B- Aug 31, 2015

HP- Hunts Point C- Sept 28, 2015

BRC- Control site D- Oct 25, 2015

BRO- Restored Site

Figure 13: Calculated presence/absence plot of species created in R-Studio for sediment samples using average relatedness. Jaccard calculations show the orders that are present/absent at each site. It does not pay attention to how many of each order is present; instead it only focuses on whether or not the order was present at all. Like Bray-Curtis computations, the larger the gap between two sites, the less orders they have in common. When compared to Jaccard for sediment, clear distinctions can be made between the biodiversity of the two elements. Please refer to abbreviation key.

Morphologically Identified Organisms

Organisms Found in Sediment Cores (#)

Positive Morphological Identification

Order and Family identified in eDNA results

Clam Worm, Nereis sp. No Yes Order: phyllodocida

Family: NA Capatellid, Thread Worm, Capitella

genera

Yes (6) Yes NA

Common Slipper Shell

Crepidula fornicata

Yes (1) Yes NA

Dumeril’s clam worm Platynereis dumerilii

Yes (5) Yes NA

Eastern Oyster, Crassostrea virginica No Yes Order: ostreoida Family: ostreidae

Species: Crassostrea virginica.

Mud Dog Welk, Nassarius obsoletus Yes (1) Yes Family: Nassariidae

Spring Worm

Lycastopsis pontica

No Yes NA

White Fingered Crab,

Rhithropanopeus harrisii

No Yes Na

Table 1: This table represents the morphological identifications made at Soundview Park.

Column one lists the names of organisms that were identified during fieldwork at the park. Columns two and three list whether or not the organisms were found in sediment cores and if

positive identification was made morphologically. Lastly, column four lists the order or family of the organism as listed by the results of eDNA analysis.