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