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Using only those individuals with complete multilocus genotypes (MLGs) from the microsatellite flanking regions, genotypic diversity Duvelisib supplier was quantified using Nei��s unbiased estimate of genotypic diversity ( Nei, 1987). Regional differences in MLG diversity were tested using bootstrap sampling with replacement as implemented in GenoDive ( Meirmans and vanTienderen, 2004) and regional genetic differentiation was tested using AMOVA in Arlequin v 3.01 ( Excoffier et al., 2005) with 1000 randomizations of the data. Recombination was tested pairwise between loci using linkage disequilibrium based on likelihood as implemented in GenePop on the Web ( Raymond and Rousset, 1995). All loci amplified successfully in each of the five P. marinus clonal cultures. All clonal isolates were homozygous except for those deriving from Maryland and New Jersey, both of which were heterozygous for the same four loci (Pm2903, Pm8517, SOD1-short, and SOD2-short). In spite of multiple attempts to generate outgroup sequence, no other Perkinsus species amplified for any locus. Sequence data were collected for a total of 474 amplicons from all samples, with the final analyzed sequence covering a total of 3928 bases per individual across the seven P. marinus loci ( Table 3). For the microsatellite flanking loci, 70.4% of sequencing attempts were successful for both strands. Similar to microsatellite flupentixol genotyping results ( Thompson et al., 2011), the distribution of missing sequences varied randomly among samples and locations for each locus, resulting in 54 samples (52%) with complete multilocus genotypes. Only 13 heterozygous genotypes (2.7%) were encountered among all sequences. For these heterozygous sequences, all PHASE inferences were highly significant (p?PFI-2 solubility dmso each locus, but marked divergence occurred between observed alleles (Fig. 1, Table 3). An average of 130 (stdev?=?38.5) chromosomes were sequenced at each locus, but no more than 4 alleles were found at any locus. One common (frequency?=?72 to 94%) and one alternate haplotype was observed at every locus, with singletons also observed at some loci (Fig. 1). The two major alleles differed in DNA sequence by an uncorrected average of 3.8% (stdev?=?2%). The largest allelic difference (7.4%, Table 3) was observed at the 257?bp SOD2-short locus. Locus Pm2232 had the lowest sequence difference (2.2%, Table 3). Insertions or deletions (indels) were observed at two loci, with a three nucleotide indel in Pm4488, and six different indels over a total of 14 nucleotide positions within SOD2 noncoding regions.