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Barcoding Benthic Invertebrates in Green's Creek to Assess Biodiversity and Water Quality
Sohum Sheth and Alex Urmaza assisted by Mrs. Maria Brown, Sayville High School
Figure 3: Station 6 has three times
the number of species observed at
Station 4. Figure 4: Effective Number of
Species (e Shannon beta Index )
calculation provides a
biodiversity metric and suggest
that the biodiversity at Station 6
is greater (6X) than at Station 4.
Figure 5: Station 4 (n=54) community
structure represents species present that
are pollution and/or disturbance tolerant
and Station 6 (n=64) has a community
structure representing good – excellent
water quality conditions .
1.093
6.652
0 2 4 6 8
Station 4
Station 6
ENS
ENS
4
13
Total species number
Station 4
Station 65
4
3
0
1
2
3
4
5
6
Poor Fair Good
Nu
mb
er
of
Ind
ivid
uals
per
Bio
ind
icato
r C
ate
go
ry
Water Quality Category
Variation in Bioindicator Species per Station
Station 4
Station 6
Station 6
Station 4
Station 6
Station 4
Results
Materials & Methods Permits & Field Methods
NYSDEC
SCDP&R
Great South Bay Audubon Society
Laboratory Methods
DNA Extraction (Qiagen™ Dneasy Kit)
PCR/Gel Electrophoresis
PCR Purification (Qiagen™ PCR Purification Kit)
Sequencing (Genewiz, Inc.)
Analytical Methods
DNA Subway – Blue Line
Mega 6.0
Shannon Biodiversity Calculator
Water Quality Rating Scale
Study Location
Figure 2: Station 6 is located at Brookside County Park Preserve and Station 4 is
located just south of Brook Street, West Sayville, NY.
Figure 1: Materials and methods schematic
Expected (E)
Category
(P,F,G) by
Station (4,6)
Expected
Frequency
EP4 0.166
EF4 0.166
EG4 0
EP6 8.166
EF6 4.166
EG6 4.00
WQ
Category Station
4 Station
6 TOTAL
Poor 1 5 6
Good 2 4 6
Fair 1 3 4
TOTAL 4 12 16
Observed X2 = 16.664
Rules for Testing: X2 Critical
alpha at 0.05 = 5.99,
at 0.01 = 9.21, at 0.001 = 13.82.
There is a statistically
significant difference between
bio-indicator species between
Stations 4 and 6. Station 4
(Poor) and Station 6 (Fair).
Abstract
Introduction
Table 1: Water quality rating scale
for biotic community
Structure and Station results. Source:
http://www.ydae.purdue.edu/natural_resources/Resources/BioindicatorWQ/tabl
e.jpg
Green’s Creek, located in West Sayville, NY feeds directly into Long Island’s Great South
Bay. Previous studies begun in 2000 show that the water quality in the freshwater reaches
was of fair to poor quality. Benthic invertebrates are good bio-indicators for water quality
while DNA barcoding provides confirmation to benthic community structure and can
increase species inventory six-times that of methods using only standard taxonomic keys.
Two Stations were sampled at Brookside County Park; Station 4 had a course-grained
sandy substrate located downstream of the weir, and Station 6 had fine-grained,
organically rich substrate and was upstream of the weir. Benthic invertebrates were sorted
using a dissecting microscope and photographed using a Vernier™ Proscope and
classified using taxonomic keys. DNA were extracted using a Qiagen™ DNeasy kit,
underwent PCR using the 12S primer adopted from Saux, Simon & Spicer (2000), and
were purified using a Qiagen™ PCR Purification kit and sent to Genewiz, Inc. for
sequencing. DNA Subway Blue Line was used as a bioinformatics tool for assessing novel
barcodes. The Shannon beta biodiversity index and the effective number of species (ENS)
were calculated to allow for a comparison of biodiversity between the two Stations. A chi
square test suggests that the two locations are significantly different in their biodiversity
and that Station 6 supports greater biodiversity than Station 4. This is likely due to the weir
that separates the two sampling locations and prevents nutrient - rich sediment from
moving downstream to Station 4. Station 6 also showed better water quality (Fair) than
Station 4 (Poor-Fair) when using species as bio-indicators. The 12S primer proved
effective for aquatic invertebrates as 29% of samples had a success rate for sequencing,
whereas the COI/CO2 cocktail produced no results using multiple extraction methods. All
five organisms analyzed produced novel barcodes.
DNA barcoding can be used to provide improved descriptions of community structure
within aquatic ecosystems (Sweeney, Battle, Jackson, & Dapkey 2011 and can be used to
assess water quality conditions and biodiversity within lotic and lentic systems.
GOALS:
Determine species richness of macrobenthic invertebrates (independent variable) habitat
types (independent variable) above a weir which supports moving water, fine grained
sediments and is rich in organics, and below the weir which supports moving water, course
grained sediments, and little organic materials in Green’s Creek (Figure 1).
Use barcoding techniques to confirm water quality bio-indicator species (dragonfly,
damselfly, stonefly, mayfly, etc.) and/or to identify species that cannot be identified using
standard taxonomic keys and to identify novel barcodes in Genbank.
Figure 6: Barcode and ML phylogenetic tree
for chironimids. All 4 organisms (ANM7,
SA1, SA4, and SA5) appear to be novel.
Table 2: Chi Square Contingency Table
Figure 7: Barcode and ML phylogenetic tree for left spiral snail.
Sample SA2 appears to have a novel barcode.
Cold Spring Harbor Laboratory (2014). Using DNA Barcodes to Identify and Classify Living Things. pp1-41.
New York State Department of Environmental Conservation (1996). Water Quality Regulations; Surface Water and Ground Water Classification and Standards.
Title 6, Chapter X Parts 700 – 705.
Saux,C., Simon C., & Spicer,G.,S. (2003). Phylogeny of the dragonfly and damselfy Order Odonata as inferred by mitochondrial 12S ribosomal RNA sequences.
Ann. Entomol. Soc. Am, 96(6), 693-699.
Smith, D., G. (2001). Pennak’s freshwater invertebrates of the United States (2001). John Wiley & Sons., New York.
Sweeney, B.W., Battle, J.M., Jackson, J.K., & Dapkey, T. (2011). Can DNA barcodes of stream macroinvertebrates improve descriptions of community structure
and water quality? Journal of North American Benthological Society 30 (1): 195-215.
References
Acknowledgements: Mrs. Maria Brown, our Families, DNALC Staff
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