Distribution of the Northern Star Coral Distribution of the Northern Star Coral ((Astrangia poculataAstrangia poculata) and its Symbiotic ) and its Symbiotic Zooxanthellae on New Jersey Artificial Zooxanthellae on New Jersey Artificial

Reefs.Reefs.

Peter F. Straub, Professor of Biology, DSOTara Harmer, Assistant Professor of Biology

School of Natural Sciences & MathRichard Stockton College of NJ

Collaboration-Undergraduate Collaboration-Undergraduate Research and TeachingResearch and Teaching

Stockton Scientific Diving Stockton Scientific Diving Program- Straub -DSOProgram- Straub -DSO

BIOL 2175- Scientific BIOL 2175- Scientific Diving-Straub Fall 07Diving-Straub Fall 07

BIO 4211- Molecular BIO 4211- Molecular Evolution- Harmer Fall 07Evolution- Harmer Fall 07

BIOL 4215-Biotechnology- BIOL 4215-Biotechnology- Straub, Spring 08Straub, Spring 08

BIOL 4800- Biology BIOL 4800- Biology Research- Harmer Research- Harmer Fall/Spring 07/08Fall/Spring 07/08

Links

BIOL 2175 Fall 2007

Astrangia poculata Astrangia poculata (syn. (syn. A.A. danaedanae)) -Northern Star Coral -Northern Star Coral

Cnidaria › Anthozoa › Hexacorallia › Scleractinia › Faviina › Cnidaria › Anthozoa › Hexacorallia › Scleractinia › Faviina › Rhizangiidae › Astrangia Rhizangiidae › Astrangia

Found from the sub-tropics to the temperate zone <5C. Most corals 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 live in tropical to sub-tropical waters above 20 C and cannot tolerate high turbidityhigh 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, The individual coral skeletons or corallites are well defined, circular, compact, and up to 10 mm in diametercompact, and up to 10 mm in diameter

Living coral tissue may be translucent to dark brown depending on the Living coral tissue may be translucent to dark brown depending on the presence and number of zooxanthellae 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/

HypothesisHypothesis

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

MethodsMethods

Site selectionSite selection Collect corals by scubaCollect corals by scuba Extract chromosomal DNAExtract chromosomal DNA Amplify ssRNA (18S) Amplify ssRNA (18S)

sequence by PCRsequence by PCR DNA SequenceDNA Sequence Align sequences (Vector Align sequences (Vector

nTi) nTi) Look for relatedness in Look for relatedness in

coral pops (MacClade and coral pops (MacClade and PHYLIP)PHYLIP)

Coral Collection sites7 sites, 15 dives

NJ

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 MarvinClam DredgeSunk 1/16/199270 ft.

http://njscuba.net/sites

Wreck of the Almirante (the flour wreck) off Wreck of the Almirante (the flour wreck) off Atlantic City NJ (approx 8 mi) at 70 ft. Atlantic City NJ (approx 8 mi) at 70 ft.

United Fruit Co. vessel (378 ft x 50 ft) sunk in United Fruit Co. vessel (378 ft x 50 ft) sunk in collision Sept 6, 1918 w/ Navy tanker USS Hisko, collision Sept 6, 1918 w/ Navy tanker USS Hisko,

later wire dragged 2x, depth charges, fairly later wire dragged 2x, depth charges, fairly broken upbroken up

Underwater photos David Roche

http://njscuba.net

Coral collections 8/25/07, 8/29/07 and 10/6/07

Northern Star CoralAstrangia poculata

Sea Girt, Clam Dredge, Depth 70 ft sunk 8/1990. Coral Collection- 7/28/07

Photos Pete Straub

MV Atlantus Charters

Almirante (Flour)

Mike

Dave

Pete

Almirante (Flour)

Almirante

Coral plus mussels

Coral plus anemoneCoral heads Coral heads

Photos P. Straub & T. Harmer

Coral polyps extended

Individual coral polyps

Photos P. Straub & T. Harmer

Kept 10 gallon aquaria, filtered,Feed zooplankton, commercialzooplankton (preserved).

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

1. PCR Reaction Conditions100 ng chromosomal DNAQiagen TAQ polymeraseQuiagen coraload buffer0.2 uM dNTP’sForward Primer NS1*Reverse Primer Euk 516R**

1 cycle 94 C, 1:00 min35 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

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

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

Sequence Identification

Upload sequence to BLAST server at National Center for Biotechnology information server- “www.ncbi.nlm.nih.gov”

ID Astrangia danae

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

Inserted A in all NJ samples

A>G transition 1 car float sample

A>C transversion1 Jet Trader sample

A>G transition1 Lemuel Burrows sample

Closeup of selected Alignments

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Montastraea_annula GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTTGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CATTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATACorynactis_califor GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CATTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATAEnallopsammia_rost GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CATTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATACirrhipathes_lutke GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTTGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATAFungia_scutaria GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCCCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATAPhyllangia_mouchez GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATATubastraea_coccine GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CATAACTACT TGGATAACCG TAGTAATTCT AGAGCTAATAPavona_varians GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTTGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CATTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATAAstrangia_poculata GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA1-1-1, 1811 bases, GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA1-2-1, 1811 bases, GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA1-20-2-R-S44, 1811 GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA2-2-1, 1811 bases, GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA2-2-3, 1811 bases, GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA2-2-4, 1811 bases, GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA3-1-1, 1811 bases, GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA3-5-1, 1811 bases, -TAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTAT AGAGCTAATA3-5-3, 1811 bases, GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA4-8-2, 1811 bases, GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA4-8-3, 1811 bases, GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA4-20-1-R-S38_rc, 1 GTAGTCCTAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA5-20-2-R-S32, 1811 GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTGGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA5-21-1-F-S33_rc, 1 GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA5-21-1-R-S34, 1811 GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA5-21-2-F-S35_rc, 1 GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA5-21-2-R-S36, 1811 GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA6-20-2-F-S23_rc, 1 GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA6-22-1-R-S28, 1811 GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA7-21-2-R-S-8_rc, 1 GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA7-23-2-F-S15_rc, 1 GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA7-24-1-R-S18, 1811 GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA7-24-2-R-S20, 1811 GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA10_RMAD, 1811 base GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTC TAAGTATAAG CACTAGTACT GTGAAACTGC GAATGGCTCA TTAAATCAGT TATCGTTTAT TTGATTGTAC CTTTACTACT TGGATAACCG TAGTAATTCT AGAGCTAATA

160 170 180 190 200 210 220 230 240 250 260 270 280 290 300. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Montastraea_annula CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGAGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCACorynactis_califor CATGCGAAAA GTCCCGACTT CTTGGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTGTTTGGT GATTCATAGT AACTTCACGA ATCGCACGGC CTCGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCAEnallopsammia_rost CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCACirrhipathes_lutke CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTGCTTTGGT GATCCATAAT AACTGATCGA ATCGCATGGC GTTGAGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCAFungia_scutaria CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCAPhyllangia_mouchez CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCATubastraea_coccine CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCAPavona_varians CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTCATTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCAAstrangia_poculata CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA1-1-1, 1811 bases, CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA1-2-1, 1811 bases, CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA1-20-2-R-S44, 1811 CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA2-2-1, 1811 bases, CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA2-2-3, 1811 bases, CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA2-2-4, 1811 bases, CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA3-1-1, 1811 bases, CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AGCTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA3-5-1, 1811 bases, CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA3-5-3, 1811 bases, CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA4-8-2, 1811 bases, CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA4-8-3, 1811 bases, CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA4-20-1-R-S38_rc, 1 CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA5-20-2-R-S32, 1811 CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA5-21-1-F-S33_rc, 1 CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA5-21-1-R-S34, 1811 CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA5-21-2-F-S35_rc, 1 CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA5-21-2-R-S36, 1811 CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA6-20-2-F-S23_rc, 1 CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA6-22-1-R-S28, 1811 CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA7-21-2-R-S-8_rc, 1 CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA7-23-2-F-S15_rc, 1 CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA7-24-1-R-S18, 1811 CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA7-24-2-R-S20, 1811 CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA10_RMAD, 1811 base CATGCGAAAA GTCCCGACTT CT-GGAAGGG ATGTATTTAT TAGATTAAAA ACCAATGCGG GTTCTGCCCG GTTCTTTGGT GATTCATAGT AACTGATCGA ATCGCACGGC CTTGCGCTGG CGATGTTTCA TTCAAATTTC TGCCCTATCA

310 320 330 340 350 360 370 380 390 400 410 420 430 440 450. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Montastraea_annula ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGTCorynactis_califor ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTACAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGATT CAGG-GAGGTEnallopsammia_rost ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACC CAGG-GAGGTCirrhipathes_lutke ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTACAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGTFungia_scutaria ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATCACCC AATCCTGACT CAGG-GAGGTPhyllangia_mouchez ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGTTubastraea_coccine ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGTPavona_varians ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAATCGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGTAstrangia_poculata ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTG-CGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT1-1-1, 1811 bases, ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT1-2-1, 1811 bases, ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT1-20-2-R-S44, 1811 ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT2-2-1, 1811 bases, ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT2-2-3, 1811 bases, ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT2-2-4, 1811 bases, ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT3-1-1, 1811 bases, ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT3-5-1, 1811 bases, ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT3-5-3, 1811 bases, ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAGTGAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGGTGAGGT4-8-2, 1811 bases, ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT4-8-3, 1811 bases, ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAGTGAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT4-20-1-R-S38_rc, 1 ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT5-20-2-R-S32, 1811 ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT5-21-1-F-S33_rc, 1 ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT5-21-1-R-S34, 1811 ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT5-21-2-F-S35_rc, 1 ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT5-21-2-R-S36, 1811 ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT6-20-2-F-S23_rc, 1 ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT6-22-1-R-S28, 1811 ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGGGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT7-21-2-R-S-8_rc, 1 ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT7-23-2-F-S15_rc, 1 ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT7-24-1-R-S18, 1811 ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT7-24-2-R-S20, 1811 ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT10_RMAD, 1811 base ACTGTCGATG GTAAGGTAGT GGCTTACCAT GGTTGCAACG GGTGACGGAG AATTAGGGTT CGATTCCGGA GAGGGAGCCT GAGAAACGGC TACCACATCC AAG-GAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACT CAGG-GAGGT

460 470 480 490 500 510 520 530 540 550. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . .

Montastraea_annula AGTGACAAGA AATAACAATA CAGGG-CTTT TCCAA-GTCT TGTAATTGGA ATGAGTACAA C-TTAAATCC TTTAACGAGG ATCCATTGGA GGGCAAGTCT GGTCorynactis_califor AGTGACAAGA AATAACAATA CAGGG-CTTT TACAA-GTCT TGTAATTGGA ATGAGTACAA C-TTAAATCC TTTAACGAGG ATCCATTGGA GGGCAAGTCT GGTEnallopsammia_rost AGTGACAAGA AATAACAATA CAGGG-CTTT TCCAA-GTCT TGTAATTGGA ATGAGTACAA C-TTAAATCC TTTAACGAGG ATCCATTGGA GGGCAAGTCT GGTCirrhipathes_lutke AGTGACAAGA AATAACAATA CAGGG-CTTT TTTAA-GTCT TGTAATTGGA ATGAGTACAA C-TTAAATCC TTTAACGAGG ATCCATTGGA GGGCAAGTCT GGTFungia_scutaria AGTGACAAGA AATAACAATA CAGGG-CTTT TCCAA-GTCT TGTAATTGGA ATGAGTACAA C-TTAAATCC TTTAACGAGG ATCCATTGGA GGGCAAGTCT GGTPhyllangia_mouchez AGTGACAAGA AATAACAATA CAGGG-CTTT TCCAA-GTCT TGTAATTGGA ATGAGTACAA C-TTAAATCC TTTAACGAGG ATCCATTGGA GGGCAAGTCT GGTTubastraea_coccine AGTGACAAGA AATAACAATA CAGGG-CTTT TCCAA-GTCT TGTAATTGGA ATGAGTACAA CTTAAATTCC TTTAACGAGG ATCCATTGGA GGGCAAGTCT GGTPavona_varians AGTGACAAGA AATAACAATA CAGGG-CTTT TACAA-GTCT TGTAATTGGA ATGAGTACAA C-TTAAATCC TTTAACGAGG ATCCATTGGA GGACAAGTCT GGTAstrangia_poculata AGTGACAAGA AATAACAATA CAGGG-CTTT TCCAA-GTCT TGTAATTGGA ATGAGTACAA C-TTAAATCC TTTAACGAGG ATCCATTGGA GGGCAAGTCT GGT1-1-1, 1811 bases, AGTGACAAGA AATAACAATA CAGGG-CTTT TCCAA-GTCT TGTAATTGGA ATGAGTACAA C-TTAAATCC TTTAACGAGG ATCCATTGGA GGGCAAGTCT GGT1-2-1, 1811 bases, AGTGACAAGA AATAACAATA CAGGG-CTTT TCCAA-GTCT TGTAATTGGA ATGAGTACAA C-TTAAATCC TTTAACGAGG ATCCATTGGA GGGCAAGTCT GGT1-20-2-R-S44, 1811 AGTGACAAGA AATAACAATA CAGGG-CTTT TCCAA-GTCT TGTAATTGGA ATGAGTACAA C-TTAAATCC TTTAACGAGG ATCCATTGGA GGGCAAGTCT GGT2-2-1, 1811 bases, AGTGACAAGA AATAACAATA CAGGG-CTTT TCCAA-GTCT TGTAATTGGA ATGAGTACAA C-TTAAATCC TTTAACGAGG ATCCATTGGA GGGCAAGTCT GGT2-2-3, 1811 bases, AGTGACAAGA AATAACAATA CAGGGGCTTT TCCAAAGTCT TGTAATTGGA ATGAGTACAA CCTTAAATCC TTTAACGAGG ATCCATGGGA GGGCAAGTCT GGT

All aligned sequencesCoded and transferred to Phyllip for phylogenetic analysis

4-9-1 Jet

2-2-3 Sea

3-5-3 John

4-8-3 Jet

5-20-2-R-S

3-5-1 John

1-1-1 Glor

1-2-1 Glor

2-2-1 Sea

4-8-2 Jet

5-21-1-R-S

5-21-2-R-S

7-21-2-R-S

7-24-2-R-S

Astrangia

Phyllangia

1-20-2-R-S

2-2-4 Sea

5-21-1-F-S

5-21-2-F-S

6-20-2-F-S

7-24-1-R-S

7-23-2-F-S

6-22-1-R-S

4-20-1-R-S

3-1-1 John

Fungia scu

Tubastraea

Enallopsam

Corynactis

Pavona var

Montastrae

Cirrhipath

Acknowledgments-

Richard Stockton College- Faculty Summer Research SupportRichard Stockton College- NAMS Division Development FundNational Science Foundation- Instrumentation Support- DBI-0116165

Sunrise on station

Conclusion

Detected genetic differentiation

Low within and between areas

Nt 343 diagnostic NJ vs MACorals

Further analysis within NJSamples needed

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