418 
Fishery Bulletin 109(4) 
Map of mutton snapper ( Lutjanus analis) collection sites and known sites of spawning aggregations in 
the Caribbean Sea and Florida Keys. Collection sites are represented by stars; known aggregation sites 
are represented by triangles. 
was extracted by using a standard phenol-chloroform 
protocol (Sambrook et al., 1989). 
Seventeen microsatellites were surveyed in three 
multiplex panels: panel 1 ( Lan6 , Lan 11, Lan 12, Lan 13, 
Och4 , and Ra6), panel 2 (Lan9, Lca22, Lca64, Lsy8, 
Lsy 13, and Ra 2), and panel 3 {Lan 3, Lea 20, Lsy 4, 
Prs248, and Ral). Touchdown polymerase-chain-reac- 
tion (PCR) protocols were as follows: 95°C for 3 min; 7 
cycles at 95°C for 30 sec, annealing at T A1 for 1 min, 
and 72°C for 4 min; 7 cycles at 95°C for 30 sec, anneal- 
ing at T A2 for 1 min, and 72°C for 4 min; and 28 cycles 
at 95°C for 30 sec, annealing at T A3 for 1 min, and 72°C 
for 4 min, with a final extension of 10 min at 72°C. An- 
nealing temperatures T A1 , T A2 , and T A3 , respectively, 
were 55°C, 53°C, 51°C (Panel 1), 54°C, 52°C, and 50°C 
(Panel 2), and 52°C, 49°C, and 46°C (Panel 3). PCR 
primer sequences for individual microsatellites may be 
found as follows: Ral, Ra2, and Ra6 (Bagley and Geller, 
1998); Lca20, Lca22, Lca6 4, and Prs248 (Gold et al., 
2001); and Lan3, Lan6, Lan9, Lanll , Lan\2, Lanl3, 
Lsy4, Lsy8, Lsyl3, and OchA (Renshaw et al., 2007). 
Details regarding fluorescent labeling of primers and 
amplification are described in Renshaw et al. (2006, 
2007). An ABI Prism 377 DNA sequencer (Applied Bio- 
systems Inc., Foster City, CA) was used to separate 
and visualize amplification products. Gel analysis was 
performed with Genescan Analysis, vers. 3.1.2® (Ap- 
plied Biosystems), with allele-calling performed with 
Genotyper® software, vers. 2.5 (Applied Biosystems). 
Genotypes at the 16 polymorphic microsatellites as- 
sayed may be found at http://wfsc.tamu.edu/doc/, ac- 
cessed July 2011, under the file name “Microsatellite 
genotypes of mutton snappers.” 
A 590 base-pair (bp) fragment of the mitochondri- 
al NADH-dehydrogenase subunit 4 (ND-4) gene was 
amplified and sequenced for 134 individuals (25-29 
from each locality). The primers NAP-2 (Arevalo et al., 
1994) and ND4LB (Bielawski and Gold, 2002) were 
used for fragment amplification, and ND4BL was used 
for sequencing. Polymerase chain reaction amplifica- 
tions were run in 30 pL reaction volumes, with -100 ng 
whole genomic DNA, lx GoTaq Flexi Buffer (Promega, 
Madison, Wisconsin), 1.5 mM MgCl 2 , 0.5 pM of each 
primer, 250 pM of each dNTP, and 1.7U GoTaq Taq 
polymerase (Promega). The PCR protocol consisted of a 
95°C initial denaturation, followed by 35 cycles of 95°C 
for 30 sec, 50°C for 45 sec, and 72°C for 1 min, with 
a 10 minute final extension at 72°C. Sequencing reac- 
tions were carried out with a Big Dye terminator kit®, 
vers. 3.1 (Applied Biosystems), according to the manu- 
facturer’s recommendations; products were separated 
and visualized on an ABI 3100 capillary sequencer 
