Merten et al.: Genetic structure and dispersal capabilities of Coryphaena hippurus in the western Atlantic 
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Table 1 
Genetic diversity and summary statistics of dolphinfish (Coryphaena hippurus) based on mitochondrial nicotinamide ad- 
enine dinucleotide (NADH) dehydrogenase subunit 1 (ND1) sequences by region in the western central Atlantic. N=number 
of samples; Nh=number of haplotypes per location; /r=haplotype diversity; 7i=nucleotide diversity; 0 s =Watterson’s theta; 
F s =Fu’s F s \ // ri =Harpending’s raggedness index; SSD=sum of squared differences from mismatch analysis. Bold values in- 
dicate significance at P<0.05. In this study, the region of the southeastern United States included North Carolina, South 
Carolina, and Florida; the northeastern Caribbean Sea consisted of the northern and southern shores of Puerto Rico; the 
eastern Caribbean Sea comprised Dominica, Barbados, and Trinidad and Tobago; and the central North Atlantic consists of 
the Azores Islands. 
Regions 
N 
Nh 
h 
K 
0s 
P's 
H ri 
SSD 
Southeastern United States 
90 
59 
0.9763 
0.0040 
17.75 
- 25.46 
0.010 
0.001 
Northeastern Caribbean Sea 
183 
100 
0.9768 
0.0035 
19.02 
- 25.42 
0.017 
0.050 
Eastern Caribbean Sea 
43 
33 
0.9623 
0.0046 
16.41 
- 24.59 
0.009 
0.002 
Central North Atlantic 
8 
7 
0.9643 
0.0026 
4.24 
- 2.87 
0.065 
0.017 
Mean 
L=324 
1=199 
0.9699 
0.0038 
14.35 
- 19.58 
0.025 
0.175 
an Sea); and 4) the Azores Islands (central North At- 
lantic). At the smaller scale, comparisons were made 
within regions (e.g., within the northeastern Carib- 
bean Sea between the northern and southern coasts 
of Puerto Rico). The results from these investigations 
were used to examine how stock structure and stock 
connectivity of this species in relation to Puerto Rico 
are reflected in the variation of the ND1 gene around 
the western central Atlantic and the central North 
Atlantic. 
Materials and methods 
Field sampling 
Dolphinfish were primarily sampled at various loca- 
tions in the western central Atlantic (Table 1, Fig. 1). 
Tissue samples (~1 g; fin clip) were collected from land- 
ing sites, when fish at recreational fishing tournaments 
were weighed, or in situ with hook-and-line techniques 
along the northern and southern coasts of Puerto 
Rico from 2010 through 2013, along the southeastern 
United States in 2012, and from Barbados in 2014. 
Tissue samples were taken individually, immediately 
preserved in individual vials containing >95% ethanol 
solution, and stored at -20°C once in the laboratory. 
Additional samples from the Azores Islands (N=8) and 
Barbados (N=5) collected in 1998 were obtained from 
the South Carolina Department of Natural Resources; 
these samples included fin clips or heart tissue stored 
in a Sarkosyl-urea solution (1% Sarkosyl, 6 M urea, 
100 mM Tris at pH 6. 8-7.0). The samples in Sarkosyl- 
urea solution were stored at room temperature. Ad- 
ditional sequence data from the southeastern United 
States (N=l) and eastern Caribbean Sea (N= 12 ) were 
acquired from public databases (GenBank accession 
numbers: AF272054-AF272061, AF290386-AF290390, 
AF256056). 
DNA extraction, PCR amplification, and sequencing 
Total genomic DNA was extracted through the use of 
a DNeasy 1 kit (Qiagen Inc., Valencia, CA) according to 
the manufacturer’s instructions. Polymerase chain re- 
action (PCR) was used to amplify a fragment, with 1437 
base pairs (bp) of the mitochondrial genome consisting 
of the ND1 gene and portions of the flanking trans- 
fer RNAs (tRNAs) by using the L3324 (5'-GTCCTAC- 
GTGATCTGAGTTCAG-3”) and H4716 (5-TACAT- 
GTTTGGGGTATGGGC-3') primers (Chapman 2 ). After 
quality assessment and trimming to a common length, 
a 1288-bp fragment was used for all analyses contain- 
ing 126 bp of the 16S RNA upstream, the entire tRNA 
Leu (72 bp) and tRNA lie (69 bp) genes, a portion of 
the tRNA Gin (49 bp) gene downstream, and the en- 
tire ND1 gene (972 bp). PCR amplifications in 25-pL 
volumes were prepared with BioMix Red solution (Bio- 
line USA Inc., Taunton, MA) according to the manufac- 
turer’s instructions with the addition of 10 ng of DNA 
and 5 pM of each primer. Thermal cycling consisted of 
an initial denaturation step for 2 min at 94°C, followed 
by 35 cycles of 94°C for 30 s, 53°C for 25 s, and 72°C 
for 45 s, with a final 10 min extension at 72°C. Sanger 
sequencing in both forward and reverse directions was 
performed by the High Throughput Genomics Center in 
Seattle, Washington. All sequences and final alignment 
have been submitted to GenBank (accession numbers: 
KP057921-KP058244). 
Data analysis 
For visualization, quality assessment, contig (contigu- 
ous) assembly, and editing, DNA trace files were im- 
1 Mention of trade names or commercial companies is for iden- 
tification purposes only and does not imply endorsement by 
the National Marine Fisheries Service, NOAA 
2 Chapman, R. 2012. Personal commun. Hollings Marine 
Lab, Charleston, SC 29412. 
