Scartella cristata - Amazon S3

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18 Mitochondrial DNA D-Loop Analysis Genetic analysis was performed on a subsample of Scartella cristata to identify relationships between sample sites and determine the origin(s) of individuals on Texas jetties. The null hypothesis that Texas jetties represented a single undifferentiated population originating from a single source population was tested. To this end, individuals ranging from 40 to 50 mm TL were selected from Galveston (n=20), Port Aransas (n=19), and South Padre Island (n=19) samples to minimize variation that may occur between age classes (Fig. 4). An additional nine individuals were collected from Gulf Islands National Seashore on Santa Rosa Island, Florida in December 2001 for use in this analysis (Fig. 5). DNA was extracted from these individuals following protocols described by Greig (2000) with minor modification. Galveston Island, Texas n=20 Santa Rosa Island, Florida n=9 Port Aransas, Texas n=19 South Padre Island, Texas n=19 Fig. 5. Capture locations of Scartella cristata used in mtDNA an alysis. Briefly, approximately 0.05 g of epaxial muscle tissue was added to a labeled 1.5 μL microfuge tube with 200 μL of TENS solution (50 mM Tris-HCl [pH 8.0], 100 mM EDTA, 100 mM NaCl, 1.0% SDS) and 20
19 μL of Proteinase K (10mg/ml) and incubated 10-12 hours at 55 C on a heat block. Nucleic acids were precipitated by adding 20 μL of 5 M NaCl and then spun at 14,000 g for 10 minutes. The sample supernatant was pipetted into a second microfuge tube and approximately 400 μL of 100% cold ethanol was added to precipitate DNA. The sample was spun again at 14,000 g for 10 minutes and the resulting supernatant discarded. DNA pellets were washed by adding 300 μL of 70% cold ethanol, spun again at 14,000 g, and the resulting supernatant was discarded. Pellets were allowed to air-dry overnight. The DNA was resuspended in 100 μL of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) and placed on a heat block at 65 C until the pellet fully dissolved. The presence and quality of extracted DNA was verified by electrophoresis of 3 μL of resuspended DNA through a 1.0% agarose gel at 100 V for approximately 30 minutes. DNA was visualized using ethidium bromide. Polymerase chain reaction (PCR) was performed to amplify a 380 base pair segment of the mtDNA D-loop region using the heavy strand primer CSBD-H (5’-CCGGTCTGAACTCAGATCACGT- 3’) and light strand primer L15998-PRO (5’-TACCCCAAACTCCCAAAGCTA-3’). PCR was conducted in Eppendorf Mastercycle Gradient (Brinkmann Instruments Inc., Westbury, NY) with the following reaction profile: initial denatuaration for 2 minutes at 94.0 C followed by 35 cycles (denaturation, 30 seconds at 94.0 C; annealing, 45 seconds at 50.0 C; extension, 1 minute at 72.0 C) and a final extension for 3 minutes at 72.0 C. Electrophoresis through a 1.2 % agarose gel at 100 V for approximately 30 minutes was used to examine the quality of PCR product and check for contamination. Again, ethidium bromide was used for visualization. Upon successful PCR, the product was cleaned of excess dNTP, primers, and AmpliTaq ® using ExoSAPIT ® (USB Corporation, Cleveland, OH) following manufacturer’s recommendations. Clean PCR products were prepared for cycle sequencing using a BigDye ® terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA). Cycle sequencing was performed in an Eppendorf Mastercycler Gradient with the following reaction profile: 25 cycles (denaturation, 10 seconds at 96.0 C; annealing, 5 seconds at 50.0 C; extension, 4 minutes at 60.0 C). Upon completion of cycle sequencing, the product was cleaned using RapXtract ® II dye terminator removal kit (Prolinx, Bothell, WA) and diluted in 30 μL of dd H 2 O. Single strand nucleotide sequences were obtained
Page 1 and 2: TEMPORAL AND SPATIAL VARIABILITY OF
Page 3 and 4: 3 ABSTRACT Temporal and Spatial Var
Page 5 and 6: 5 ACKNOWLEDGMENTS No journey of dis
Page 7 and 8: 7 and Moore (1998) indicate that Hy
Page 9 and 10: 9 and larvae of organisms that must
Page 11 and 12: 11 METHODS Study Areas Blenny assem
Page 13 and 14: 13 29° 18.8’ 29° 16.2’ Galves
Page 15 and 16: 15 each worked a different 15-m tra
Page 17: 17 for the same time period by the
Page 21 and 22: 21 where n is the total number of b
Page 23 and 24: 23 RESULTS Habitat Characterization
Page 25 and 26: 25 the Galveston beachfront were la
Page 27 and 28: 27 Species Composition A total of 4
Page 29 and 30: 29 and declined quickly by mid wint
Page 31 and 32: 31 60 n=195 50 TL (mm) 40 30 n=1867
Page 33 and 34: 33 Hypsoblennius hentz and Hypsoble
Page 35 and 36: 35 0.8 0.7 H’ J’ H’ and J’
Page 37 and 38: 37 Mean age of individuals from the
Page 39 and 40: 39 2.5 2.0 1999 n=52 1.5 1.0 0.5 %
Page 41 and 42: 41 100 80 SLOPE INT ERCE PT R 2 Gal
Page 43 and 44: 43 elated haplotypes (B, D, E) acco
Page 45 and 46: 45 A H I H F D D C F Florida n=9 B
Page 47 and 48: 47 45 Florida n=9 45 Port Aransas n
Page 49 and 50: 49 during early life stages of thes
Page 51 and 52: 51 on these jetties exhibited a fai
Page 53 and 54: 53 Hypsoblennius ionthas=2.82 mm (C
Page 55 and 56: 55 Scartella cristata is a wide-ran
Page 57 and 58: 57 source for Port Aransas and Sout
Page 59 and 60: 59 The subpopulations Scartella cri
Page 61 and 62: 61 more robust population. The catc
Page 63 and 64: 63 The last comprehensive survey in
Page 65 and 66: 65 on why this species is so abunda
Page 67 and 68: 67 Crabtree RE, Middaugh DP (1982)
Page 69 and 70:
69 Hubbs CL (1939) The characters a
Page 71 and 72:
71 Santos RS, Nash RDM (1996) Seaso
Page 73 and 74:
APPENDIX A 73
Page 75 and 76:
75 100 100 75 NOVEMBER 2000 n=7 75
Page 77 and 78:
77 100 100 75 NOVEMBER 2000 n=3 75
Page 79 and 80:
79 100 100 75 MAY 2000 n=23 75 AUGU
Page 81 and 82:
81 100 JUNE 2001 n=525 100 AUGUST 2
Page 83 and 84:
83 100 100 75 FEBRUARY 2001 n=7 75
Page 85 and 86:
85 100 75 JULY 2000 n=81 100 75 OCT
Page 87 and 88:
87 100 100 75 MARCH 2001 n=1 75 JUN
Page 89 and 90:
89 100 100 75 JUNE 2001 n=7 75 AUGU
Page 91 and 92:
91 APPENDIX B. Sequences for each h
Page 93 and 94:
93 HAPLOTYPE K CTGAC_TTGAAAAGTTGGGA
Page 95 and 96:
95 HAPLOTYPE V CTGCC_TTTAAAAGTT_GGA
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