Shulzitski et al.: Population connectivity among Dry Tortugas, Florida, and Caribbean populations of Lut/anus analis 
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of AmpliTaq Gold (Applied Biosystems, Foster 
City, CA). Cycling parameters began with a 
hot start of 10 minutes at 95°C, followed by 35 
cycles of 30 seconds at 94°C, 30 seconds at the 
optimized annealing temperature (see Table 1), 
and 60 seconds at 72°C, and a final extension of 
30 minutes at 72°C. Polymerase chain reaction 
products were visualized on an ABI 3100 genetic 
analyzer (Applied Biosystems, Foster City, CA) 
and examined further with GENESCAN 3.7 soft- 
ware (Applied Biosystems, Foster City, CA). In 
GENOTYPER 3.7 (Applied Biosystems, Foster 
City, CA) peaks were labeled and binned into 
allele-size categories. 
Data analysis 
Each population was tested for departures from 
Hardy-Weinberg equilibrium (HWE) at each locus 
with GENEPOP vers. 3.4 software (Raymond and 
Rousset, 1995a) by using a probability test and 
a Markov chain method to obtain the unbiased 
exact P-value (Guo and Thompson, 1992). In a 
similar manner, all loci and population pairs 
were tested for linkage equilibrium. Because 
sample sizes varied from 40 to 55 individuals, 
allelic richness (the number of alleles present 
in populations independent of sample size) was 
calculated for each population-locus combination 
and overall with FSTAT vers. 2.9.3 software 
(Goudet, 1995). This parameter is an estimate of 
the expected number of alleles for a subsample 
of genes equal in size to that of the smallest 
sample. 
In order to assess allelic and genotypic distri- 
butions across populations, an exact probability 
test (Raymond and Rousset, 1995b) and a log- 
likelihood based exact test (Goudet et al., 1996) 
were performed in GENEPOP to evaluate genic 
and genotypic differentiation, respectively. Both 
tests employed a Markov chain method to calcu- 
late an unbiased estimate of the P-value. 
An unbiased estimator, 9 (Weir and Cocker- 
ham, 1984), of Wright’s (1921) fixation index 
(F gT ), a measure of among-population subdivi- 
sion, was calculated with the GENETIX vers. 
4.02 program (Belkhir et al., 2001) for each 
locus, as well as for each pairwise population 
comparison; permutation tests (1000 random- 
izations) were used to estimate P-values. An 
estimator of P ST , p (Slatkin, 1995), was also 
calculated by using FSTAT (Goudet, 1995). This 
analog of P ST takes into account allelic size by 
assuming that alleles of a similar size are more 
closely related, given that loci adhere to a step- 
wise mutation model (Slatkin, 1995). An addi- 
tional measure of pairwise genetic differention, 
0 ST , has been reported to be more appropriate 
in the case of highly polymorphic loci such as 
microsatellites, and was calculated with Geno- 
