1. Distribution of the Northern Star Coral (Astrangia poculata) and its Symbiotic Zooxanthellae on New Jersey Artificial Reefs. Peter F. Straub, Professor of Biology, DSO Tara Harmer, Assistant Professor of Biology School of Natural Sciences & Math Richard Stockton College of NJ

2. Collaboration-Undergraduate Research and Teaching Stockton Scientific Diving Program- Straub -DSO Stockton Scientific Diving Program- Straub -DSO BIOL 2175- Scientific Diving-Straub Fall 07 BIOL 2175- Scientific Diving-Straub Fall 07 BIO 4211- Molecular Evolution- Harmer Fall 07 BIO 4211- Molecular Evolution- Harmer Fall 07 BIOL 4215-Biotechnology- Straub, Spring 08 BIOL 4215-Biotechnology- Straub, Spring 08 BIOL 4800- Biology Research- Harmer Fall/Spring 07/08 BIOL 4800- Biology Research- Harmer Fall/Spring 07/08 Links BIOL 2175 Fall 2007

3. Astrangia poculata (syn. A. danae) -Northern Star Coral Cnidaria › Anthozoa › Hexacorallia › Scleractinia › Faviina › Rhizangiidae › Astrangia Cnidaria › Anthozoa › Hexacorallia › Scleractinia › Faviina › Rhizangiidae › Astrangia Found from the sub-tropics to the temperate zone <5C. Most corals live in tropical to sub-tropical waters above 20 C and cannot tolerate high turbidity Found from the sub-tropics to the temperate zone <5C. Most corals live in tropical to sub-tropical waters above 20 C and cannot tolerate high turbidity Colonies can be encrusting, massive (mounding) or branching Colonies can be encrusting, massive (mounding) or branching The individual coral skeletons or corallites are well defined, circular, compact, and up to 10 mm in diameter The individual coral skeletons or corallites are well defined, circular, compact, and up to 10 mm in diameter Living coral tissue may be translucent to dark brown depending on the presence and number of zooxanthellae Living coral tissue may be translucent to dark brown depending on the presence and number of zooxanthellae May contain zooxanthellate, or not, given local light conditions. May contain zooxanthellate, or not, given local light conditions. http://www2.aims.gov.au/coralsearch/html/

4. Hypothesis Since the distribution of northern star coral in NJ is patchy, only found on hard surfaces such as wrecks, is there any detectable genetic differentiation among local collection sites and between disjunct populations. Since the distribution of northern star coral in NJ is patchy, only found on hard surfaces such as wrecks, is there any detectable genetic differentiation among local collection sites and between disjunct populations.

5. Methods Site selection Site selection Collect corals by scuba Collect corals by scuba Extract chromosomal DNA Extract chromosomal DNA Amplify ssRNA (18S) sequence by PCR Amplify ssRNA (18S) sequence by PCR DNA Sequence DNA Sequence Align sequences (Vector nTi) Align sequences (Vector nTi) Look for relatedness in coral pops (MacClade and PHYLIP) Look for relatedness in coral pops (MacClade and PHYLIP)

6. Coral Collection sites 7 sites, 15 dives NJ

7. Lemuel Burrows, torpedoed, 3/14/1942, 80 ft water Jet Trader, yard oiler, 95 ft., artificial reef, sunk 9/2/05 Car float, 60 ft John Marvin Clam Dredge Sunk 1/16/1992 70 ft. http://njscuba.net/sites

8. Wreck of the Almirante (the flour wreck) off Atlantic City NJ (approx 8 mi) at 70 ft. United Fruit Co. vessel (378 ft x 50 ft) sunk in collision Sept 6, 1918 w/ Navy tanker USS Hisko, later wire dragged 2x, depth charges, fairly broken up Underwater photos David Roche http://njscuba.net Coral collections 8/25/07, 8/29/07 and 10/6/07

9. Northern Star Coral Astrangia poculata Sea Girt, Clam Dredge, Depth 70 ft sunk 8/1990. Coral Collection- 7/28/07 Photos Pete Straub MV Atlantus Charters

10. Almirante (Flour)

11. Mike Dave Pete Almirante (Flour)

12. Almirante

14. Coral plus mussels Coral plus anemone Coral heads Photos P. Straub & T. Harmer

15. Coral polyps extended Individual coral polyps Photos P. Straub & T. Harmer Kept 10 gallon aquaria, filtered, Feed zooplankton, commercial zooplankton (preserved).

16. DNA Extractions Remove tissue from single corallite using scalpel blade tip (like cutting grapefruit) Qiagen DNAeasy (column method) Kits Eluted DNA from columns with Sterile water Measure DNA content at A260 with spectrophotometer Analyze by agarose gel electrophoresis- High MW Adjust concentration for PCR reaction

17. 1. PCR Reaction Conditions 100 ng chromosomal DNA Qiagen TAQ polymerase Quiagen coraload buffer 0.2 uM dNTP’s Forward Primer NS1* Reverse Primer Euk 516R** 1 cycle 94 C, 1:00 min 35 cycles 94 C, 1:00 min 52 C, 1:30 min 72 C, 2:00 min 1 cycle 72 C, 10:00 min Polymerase chain reaction (PCR) w/universal eukaryotic small subunit (ss)RNA- (18S) ~550 bp *White et al., 1991 **Di’ez et al., 2001 3. Purify PCR products using Promega SV gel and PCR product cleanup colums kit 2. Analyze by agarose gel electrophoresis

18. Cloning amplified products from Coral PCR Purified coral PCR products were cloned using a pGemT cloning kit (Promega kit) simplifies cloning as PCR products may have an “A” base overhang and is simple to ligate to pGemT plasmid vector. Ligation products were transformed into E. coli. E. coli colonies were selected on ampicillin. DNA plasmid minipreps were performed on individual E. coli cultures Plasmids concentration measured by spectrophotometer A260

19. Automated Fluorescent DNA Sequencing Use ~200 ng coral plasmid DNA, Beckman Coulter DCTS master mix With forward (T7) and reverse (sp6) sequencing primers Separation Beckman Coulter CEQ 8000 Genetic Analyzer Export sequence trim out plasmid vector sequences

20. Sequence Identification Upload sequence to BLAST server at National Center for Biotechnology information server- “www.ncbi.nlm.nih.gov” ID Astrangia danae

21. Align 24 NJ artificial Reef samples with 1 full length Astrangia poculata (MA) and 9 other Scleractinian species (only part of alignment shown).

22. Inserted A in all NJ samples A>G transition 1 car float sample A>C transversion 1 Jet Trader sample A>G transition 1 Lemuel Burrows sample Closeup of selected Alignments

23. All aligned sequences Coded and transferred to Phyllip for phylogenetic analysis

25. Acknowledgments- Richard Stockton College- Faculty Summer Research Support Richard Stockton College- NAMS Division Development Fund National Science Foundation- Instrumentation Support- DBI-0116165 Sunrise on station Conclusion Detected genetic differentiation Low within and between areas Nt 343 diagnostic NJ vs MA Corals Further analysis within NJ Samples needed Effective collaborative project Linking Scientific Diving, Research and Teaching