Anderson and Karel: Limited genetic structure of Brevoortia patronus revealed by microsatellite markers 
73 
40°N 
35°N - 
30°N - 
25°N - 
20°N - 
7 
Sample 
no. 
Location 
n 
Species 
1 
Laguna Madre, TX 
29 
Gulf Menhaden 
2 
Matagorda Bay, TX 
30 
Gulf Menhaden 
3 
Sabine Lake, TX 
22 
Finescale Menhaden 
4 
Lake Charles, LA 
29 
Gulf Menhaden 
5 
Apalachicola, FL 
30 
Gulf Menhaden 
6 
Charlotte Harbor, FL 
19 
Yellowfin Menhaden 
7 
Bogue Sound, NC 
8 
Atlantic Menhaden 
8 
Sandy Hook Bay, NJ 
30 
Atlantic Menhaden 
— i — “ 
95°W 
! 
90°W 
0 
210 420 
J I I I L 
J L 
840 
_ I— L 
1260 
J I I L 
1680 km 
j i I 
— i — 
85°W 
1 
80°W 
1 
75°W 
1 
70°W 
1 
65°W 
Figure 1 
Map of sampling locations in the Gulf of Mexico and Atlantic Ocean where the samples of Gulf Menhaden ( Brevoortia 
patronus), Finescale Menhaden ( B . gunteri), Yellowfin Menhaden (B. smithi ), and Atlantic Menhaden ( B . tyrannus) were 
collected for this study. Samples used for microsatellite identification were collected with a seine net along the shoreline 
near Freeport, Texas, in 2012. All other samples used for power analysis and analysis of population genetics were taken as 
described in a previous study (Anderson, 2007) in 2002-04. 
Positive colonies were amplified through polymerase 
chain reaction (PCR) with M13 primers (Life Technolo- 
gies Corp.) before sequencing. Each reaction included 1 
pL of template DNA (50 ng/pL), 1 Ready-To-Go® PCR 
bead (GE Healthcare Life Sciences, Piscataway, NJ), 
and 24 pL of forward and reverse primer cocktail (0.4- 
pM standard primer concentration of each primer), for 
a total of 25 pL. The following cycling regime was used 
for PCR: 1) 1 cycle of 2 min at 95°C; 2) 10 cycles of 
95°C for 30 s, 55°C for 30 s decreasing 1°C each cycle, 
and 72°C for 1 min; 3) 20 cycles of 95°C for 30 s, 50°C 
for 30 s, and 72°C for 1 min, adding 3 s of extension 
per cycle; and 4) a final extension period of 7 min at 
72°C. Aliquots of each PCR product were purified with 
Exo-Sapit® PCR purification reagent (Affymetrix, Inc., 
Santa Clara, CA). Purified amplicons were sequenced 
with M13 primers with the following reaction condi- 
tions: 30 cycles of 96°C for 20 s, 50°C for 20 s, and 60°C 
for 4 min. Sequencing reactions were carried out in 10- 
pL volumes with DTCS Quick Start Master Mix (Beck- 
man Coulter Inc., Brea, CA) according to the manufac- 
turer’s instructions. Sequences were separated with a 
CEQ 8000 capillary sequencer (Beckman Coulter, Inc.). 
Raw sequences were trimmed and edited manually 
and overlapping forward and reverse traces for each 
sequence were aligned with the software package Se- 
quencher (vers. 4.2; Gene Codes Corp., Ann Arbor, MI). 
Primer design and locus characterization 
Among 573 positive clones examined, 91 contained 
repeated fragments, meaning the microsatellite motif 
was repeated >5 times. From the 91 prospective loci, 
primers were designed for the loci that had the follow- 
ing characteristics: 1) the repeated motif was 3 bases 
or more in length, 2) no interruptions occurred within 
