PHYLOGENETIC ASSESSMENT OF LENGTH VARIATION AT A MICROSATELLITE LOCUS

Sixty-six haplotypes at a locus containing a simple dinucleotide (CA), microsatellite repeat were isolated by PCR-single-strand conformation al polymorphism from populations of the horseshoe crab Limulus polyphe mus. These haplotypes were sequenced to assess nucleotide variation di rectly. Thirty-four distinct sequences (alleles) were identified in a region 570 bp long that included the microsatellite motif, In the repe at region itself, CA-number varied in integer values from 5 to 11 acro ss alleles, except that a (CA)(8) class was not observed. Differences among alleles were due also to polymorphisms at 22 sites in regions im mediately flanking the microsatellite repeats, Nucleotide substitution s in these regions were used to estimate phylogenetic relationships am ong alleles, and the gene phylogeny was used to trace the evolution of length variation and CA repeat numbers. A low correlation between siz e variation and genealogical relationships among alleles suggests that absolute fragment size (as normally scored in microsatellite assays) is an unreliable indicator of historical affinities among alleles, Thi s finding on the molecular fine structure of microsatellite variation suggests the need for caution in the use of repeat counts at microsate llite loci as secure indicators of allelic relationships.