1. Environmental DNA Analysis of Bivalve
Restoration in New York City
Source: http://www.huffingtonpost.com/2013/11/26/freshkills-park-solar-energy-new-york-city-images-_n_4343185.html
By: Joshua Seidman
May 2016

2. 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.

3. 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

4. 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).

5. 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

6. 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.

7. 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

8. 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

9. 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

10. 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.

11. 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.

12. References
1. Azaele, Sandro et al. “Predicting Spatial Similarity of Freshwater Fish Biodiversity.”
Proceedings of the National Academy of Sciences of the United States of America 106.17
(2009): 7058–7062. PMC. Web. 19 May 2016.
2. Barnes, Matthew A., and Cameron R. Turner. "The Ecology of Environmental DNA and
Implications for Conservation Genetics." Conservation Genetics Conserv Genet 17.1
(2015): 1-17.
3. Beck, Michael W., Robert D. Brumbaugh, Laura Airoldi, Alvar Carranza, Loren D. Coen,
Christine Crawford, Omar Defeo, Graham J. Edgar, Boze Hancock, Matthew C. Kay,
Hunter S. Lenihan, Mark W. Luckenbach, Caitlyn L. Toropova, Guofan Zhang, and Ximing
Guo. "Oyster Reefs at Risk and Recommendations for Conservation, Restoration, and
Management." Bioscience 61.2 (2011): 107-16.
4. Beja-Pereira, Albano, Rita Oliveira, Paulo C. Alves, Michael K. Schwartz, and Gordon
Luikart. "Advancing Ecological Understandings through Technological Transformations in
Noninvasive Genetics." Molecular Ecology Resources 9.5 (2009): 1279-301.
5. Bohmann, Kristine, and et al. "Environmental DNA for Wildlife Biology and Biodiversity
Monitoring." Cell Press 2014
6. Clarke, Somerfield, & Chapman. (2006). On resemblance measures for ecological
studies, including taxonomic dissimilarities and a zero-adjusted Bray–Curtis coefficient
for denuded assemblages. Journal of Experimental Marine Biology and Ecology, 330(1),
55-80.
7. Ehrich, Melinda K., and Lora A. Harris. "A Review of Existing Eastern Oyster Filtration
Rate Models." Ecological Modelling 297 (2015): 201-12. ScienceDirect.
8. Fitzgerald, Allison Mass. "The Effects of Chronic Habitat Degradation on the physiology
and metal accumulation of Eastern Oysters (Crassostrea virginica) in the Hudson Raritan
estuary." ProQuest LLC (2013): 1-197.
9. Grabowski, Jonathan H., Brumbaugh, Robert D., Keeler, Andrew G., Opaluch, James J.,
Peterson, Charles H., Piehler, Michael F., Smyth, Ashley R. (2012). Economic valuation of
ecosystem services provided by oyster reefs.(Articles)(Report). BioScience, 62(10), 900.
10. J Gregory Caporaso, Justin Kuczynski, Jesse Stombaugh, Kyle Bittinger, Frederic D
Bushman, Elizabeth K Costello, Rob Knight. (2010). QIIME allows analysis of high-
throughput community sequencing data. Nature Methods, 7(5), 335.
11. Leray, Matthieu, and Nancy Knowlton. "DNA Barcoding and Metabarcoding of
Standardized Samples Reveal Patterns of Marine Benthic Diversity." Proceedings of the
National Academy of Sciences Proc Natl Acad Sci USA 112.7 (2015): 2076-081. PNAS.
12. Levinton, Jeffrey, Michael Doall, and BassemAllam. "Growth and Mortality Patterns of
the Eastern Oyster Crassostrea Virginica in Impacted Waters in Coastal Waters in New
York, USA." Journal of Shellfish Research 32.2 (2013): 417-27.
13. Lotze, H. K. et al "Depletion, Degradation, and Recovery Potential of Estuaries and
Coastal Seas." Science 312 (2006): 1806-809.

13. 14. Morin, Peter J. Community Ecology. 2nd ed. Malden, MA: Blackwell Science, 2012.
15. Newell, Roger I.E. "Ecological Changes in Chesapeake Bay: Are They Result of over
Harvesting The American Oyster?" Consortium Publication (1988): 29-31.
16. Nigro, Carmen. "History on the Half-Shell: The Story of New York City and Its Oysters."
New York Public Library. 2 June 2011. Web. 13 Apr. 2016.
17. “Oyster Restoration Program." NYNJ Baykeeper. Web. 29 Apr. 2016.
18. Segan, Daniel B., Kris A. Murray, and James E.m. Watson. "A Global Assessment of
Current and Future Biodiversity Vulnerability to Habitat Loss–climate Change
Interactions." Global Ecology and Conservation 5 (2016): 12-21.
19. "The Park Plan - Freshkills Park Alliance." Freshkills Park Alliance. N.p., n.d. Web. 13 May
2016.
20. Yu, D., Ji, Y., Emerson, B., Wang, X., Ye, C., Yang, C., & Ding, Z. (2012). Biodiversity soup:
Metabarcoding of arthropods for rapid biodiversity assessment and
biomonitoring. Methods in Ecology and Evolution, 3(4), 613-623.

14. Figure 1: Map of Freshkills Park
(Source: http://freshkillspark.org/the-park/the-park-plan)
Figure 2: Map of Soundview Park
(Source: Google Maps)

15. 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.)

16. Figures 4 and 5: Ribbed mussels that were observed at
Freshkills Park on October 23, 2015.
(Source: Joshua Seidman)

17. 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- RestoredSite

18. 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- RestoredSite

19. 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- RestoredSite

20. 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- RestoredSite

21. 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- RestoredSite

22. 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- RestoredSite

23. 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- RestoredSite

24. 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.

25. Morphologically Identified
Organisms
Organisms
Found in
Sediment
Cores (#)
Positive
Morphological
Identification
Order and Family identified in eDNA
results
ClamWorm, 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 clamworm
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.