Download Longwood High School Campus DNA Barcodes: Correlation of

Longwood High School Campus DNA Barcodes: Correlation of Identified Samples to
Species Commonly Found in Carolina Bays Terrestrial Microhabitats
Authors: Kyra Philp, Yunjie Su
Mentor: Lucinda Hemmick
Longwood High School
Abstract
The campus at Longwood High School has several interesting geological features that our students have been studying for a
number of years. There is a region of the campus that forms a depression resembling the geological form of a Carolina bay.
The Carolina bays are a system of shallow depressions, mainly along the Eastern seaboard, that are now thought to represent
secondary impact craters of a major impact event that occurred near Quebec about 10,900 YBP (1-5). Because students in our
research program designed a museum exhibit at the Southold Indian Museum several years ago to illustrate this story, we have
a longstanding interest in the natural history of the Carolina bays. The purpose of this project was to identify species
inhabiting the terrestrial Carolina bay-like region on our school property, and to explore whether the species are common to
other terrestrial Carolina bays along the eastern seaboard. Plant, fungus, and invertebrate samples were collected from the
region and used to provide DNA samples that were sequenced in collaboration with the Cold Spring Harbor DNA Learning
Center. Identified species were then analyzed with regard to their appearance at other terrestrial Carolina bay habitats. Our
results indicate that a number of the species identified in our sample set exhibit a preference for the type of microhabitat
created in the terrestrial Carolina bays of North America.
Introduction
This project ties together our research class’s interests in citizen science, our local environment, a past project involving
investigating the origin of the Carolina bays, and special habitat features of the bays. Scientific evidence supports the theory
that Carolina bays were created as secondary impact craters from a major impact event near Quebec approximately 10,900
YBP, linked to the extinction of North American megafauna and the rapid decline of the Clovis culture (1-5). These shallow
depressions exist all along the Eastern seaboard and also somewhat to the west. The bays are both terrestrial and aquatic,
and those on Long Island have been mapped by the use of LIDAR imaging and examined regarding the presence of a unique
pebbly loess surface deposit (5,6). . A depression that begins on our school property may be part of a bay, although it can be
difficult to judge using the available maps. After researching what is known about terrestrial Carolina bays as microhabitats,
we became interested in using the opportunity given by the DNA Barcoding Project to investigate whether the species
inhabiting the depression might resemble others found in the Carolina bays on Long Island and other nearby areas.
Materials & Methods
All samples were collected from a large, shallow depression in the rear campus of Longwood High School. Twenty samples,
including plants, fungi, and insects, were collected. The samples were photographed, and the insects were euthanized using
ethanol fumes. Samples were kept frozen until analysis. For analysis, DNA was collected using protocols developed by the
Barcoding project leaders. Briefly, small portions of the samples were pulverized in extraction solution, and the DNA was
recovered from the supernatant using DNA-binding silica. The DNA was eluted from the silica and then PCR was performed
using the appropriate primers for the plants (rbcL), fungi (ITS) or insects (COI). PCR products were run on agarose gels to check
the size and quality of the amplified DNA as a quality control for identifying candidates for sequencing. If the size of the
amplified band was correct, and there was sufficient quantity of DNA, samples were submitted to GENEWIZ for sequencing.
The sequenced samples were analyzed using the DNA Subway routine, in which the genus and species of each sample was
identified. Our pictures of the original samples were used to confirm the likelihood of a correct match, and also the Bit scores
and number of mismatches were taken into consideration for identification.
Results
DNA sequences were identified using the DNA Subway database. To be more certain of a match to the correct genus and
species, we combined the tests of comparing photographs of our original samples with the database photographs, high Bit
scores, and selecting the top matches from the database. From our DNA samples (Figure 1), we have identified several plant
and animal species that are known to prefer habitat conditions common to terrestrial Carolina bays (shallow depressions
covered with a sandy, loose, pebbly mix called pebbly loess (5,6)). The Carolina bays as a unique microhabitat has been
examined in previous studies focusing on plant distribution (7). We identified some plant species that may prefer the Carolina
bays habitat. The Verbascum thapsus plant, known as Mullein, likes well-lit disturbed soil that is preferably dry and sandy (8).
Atrichum altecristatom is a common plant found often in roadside ditches (8). The fairy ring mushroom, Marasmios oreades,
likes grassy areas and even dunes (sandy soil) (9). Aporrectodea calignosa (known as the gray worm) prefers loosely packed or
sandy soil, similar to that seen in Carolina bays (10). The remainder of our identified plant and insect species inhabit a very
wide variety of habitats, so their presence in the area does not indicate a habitat preference.
5
4.5
3.5
3.5-4
2
3-3.5
2.5-3
2-2.5
1.5
1.5-2
9
1
1-1.5
11
0.5
0
0.5-1
0-0.5
Series9
Series5
Series1
15
Series3
13
Series7
7
Aporrectodea calignosa
4-4.5
2.5
5
LIDAR Image of Carolina Bay on
Long Island
4
3
1
3
4.5-5
3D Contour Map of the depression on the
Longwood Campus
Atrichum altecristatom
Verbascum thapsus
Google Map of All the Samples Collected
Marasmios oreades
Discussion
While many of the plant and animal species that were identified in this study occupy a wide variety of habitats around the world, there
were several present that are known to prefer the particular soil conditions commonly found in terrestrial Carolina bays. Based on this
preliminary data, we are interested in broadening our study to include examining a soil sample from this area of our campus. If this
region is actually a Carolina bay, we would expect a core sample of about three feet in depth to contain some traces of the impact event
that produced the “black mat” described by many researchers (1-5). Deposited at about 10,900 YBP, this thin blackened layer contains
nanodiamonds, glassy spherules, carbon, and other evidence of an impact event. An electron microscope will be needed to verify the
presence of these particles, so our future work will concentrate on obtaining a clean core sample from the area and gaining access to an
electron microscope to locate and identify these impact clues on soil from our school campus.
References
1. Firestone, R.B., et al. 2007 October 9. “Evidence for an extraterrestrial impact 12,900 years ago that contributed to the megafaunal extinctions and the younger Dryas cooling.” PNAS Vol. 104 (41), 16016-16021.
2. Wittke, J.H., et al. 2013. “Evidence for deposition of 10 million tonnes of impact spherules across four continents 12,800 y ago.” PNAS E2088–E2097, doi: 10.1073/pnas.1301760110.
3. Petaev, M. I., et al. 2013. “Large Pt anomaly in the Greenland ice core points to a cataclysm at the onset of Younger Dryas”. PNAS Vol. 110 no. 32, 12917–12920, doi: 10.1073/pnas.1303924110.
4. Israde-Alcantara, I., et al. 2012. “Evidence from central Mexico supporting the Younger Dryas extraterrestrial impact hypothesis.” PNAS vol. 109 no 13, E738–E747, doi: 10.1073/pnas.1110614109.
5. Tvelia, S. “Structure of Long Island’s Carolina Bays and Their Potential Relationship to the Proposed Younger Dryas Impact Event”. Available from: www.Geo.sunysb.edu/lig/Conferences/abstracts 15.
6. Gill, G. “Documenting the Occurrence of Carolina Bays on Long Island”. Available from: Pbisotopes.ess.sunysb.edu/lig/Conferences/abstract 13.
7. De Steven, D. 2007. “Local and Landscape Influences on the Plant Communities of Carolina Bays”. Southeastern Section, 56th Annual Meeting, Paper No. 2-9.
8. Floras of North America Internet Database. Available from: http://www.efloras.org/index.aspx.
9. “Mushroom Expert.com”. Available from: http://www.mushroomexpert.com/marasmius_oreades.html.
10. Aporrectodeo calignosa (Savigny, 1826). Available from: http://earthworms.elte.hu/Israel/species/ap_caliginosa.htm.