PCR-RFLP based diagnostic tests for moxostoma species in ontario

Six Moxostoma species are found in Ontario, including two federally listed species. Knowledge of their distribution and status has been limited by incorrect field identifications, collections that report Moxosotoma or sucker species, and new site records without voucher specimens. To develop species diagnostic genetic markers, we amplified and sequenced two mitochondrial genes (ATPase subunits 6 and 8). Species-specific sequence differences were easily identified (of 830 analyzed bases, 132 substitutions were phylogenetically informative). Based on sequence differences, four restriction enzymes were identified and tested for their reliability as species diagnostic tools. Restriction digests of previously sequenced samples matched predicted species-specific patterns. Overall, 90% of samples digested matched. Disagreement between 20% of samples provided by various government agencies and species-specific digest patterns indicate a need for greater care during field identifications.