Single-strand conformation polymorphism (SSCP) analysis detects single point mutations in DNA molecules. We demonstrate that SSCP analysis of mitochondrial ribosomal DNA (rDNA) genes is a sensitive taxonomic tool because these genes often differ at numerous sites among closely related species. Using conserved primers, portions of the 12S or 16S rDNA genes were amplified using the polymerase chain reaction (PCR) in congeneric species of ticks, leafhoppers, mosquitoes, and closely related endoparasitic wasps. SSCP was performed and products were visualized with silver staining. Species-specific patterns were observed in all taxa. Intraspecific variation at the level of single nucleotide substitutions was detected. SSCP diagnostics are less expensive and time consuming to develop than PCR with species-specific primers, and, unlike PCR with arbitrary primers, there is minimal concern with DNA contamination from non-target organisms.

RAPD-PCR polymorphisms were used to examine breeding structure in three species of snow pool Aedes mosquitoes across three river drainages in northern Colorado. Larvae were collected from four snow pools for Aedes cataphylla Dyar, seven pools for A. hexodontus Dyar and six pools for A. pullatus (Coquillet). Polymorphisms were scored at 47-48 RAPD loci in each species. To test for isolation by distance, F ST /(1F ST ) values between pairs of pools were plotted against geographical distances and subjected to a Mantel test with 1000 random permutations. F ST values were independent of geographical distances in A. cataphylla and A. hexodontus but were somewhat correlated in A. pullatus suggesting isolation by distance in this species. A cluster analysis was performed on pair-wise F ST values among pools including seven pools that were sampled in both 1994 and 1995. Bootstrap analysis indicated that pools clustered across drainages and generally independently of geographical proximity. However, there was consistent support for clustering of larvae collected from the same pool across years in A. cataphylla and in high altitude collections of A. pullatus. Mountains do not appear to act as major barriers to gene flow in any of these species. Instead, seasonal differences in adult emergence may serve as barriers to migration among A. pullatus and A. cataphylla populations. Larvae of A. hexodontus are distributed continuously in grassy pools along the banks of rivers and genetic drift probably occurs through random larval mortality when these pools are washed out during spring run-off.

Snow pool Aedes mosquitoes are identified easily and accurately by microscopic examination of 4th instars or male genitalia. Early instars and females cannot be identified accurately. We demonstrate that restriction fragment-length polymorphisms (RFLP) in the internal transcribed spacer (ITS) of the nuclear ribosomal DNA cistron are specific to each of 13 snow pool Aedes species found in northern Colorado. The ITS was amplified by the polymerase chain reaction (PCR) and digested with AluI and MspI restriction endonucleases. Differences in the sizes of digested fragments among the 13 species were so slight that they could only be resolved with polyacrylamide gel electrophoresis and visualized with silver staining. A key to species found in the Rocky Mountains of northern Colorado was constructed using the PCR-RFLP patterns. Three of the species were collected in California and had identical PCR-RFLP patterns, indicating that restriction sites in the ITS may be conserved within some species.

RAPD-PCR polymorphisms were used to examine breeding structure in three species of snow pool Aedes mosquitoes across three river drainages in northern Colorado. Larvae were collected from four snow pools for Aedes cataphylla Dyar, seven pools for A. hexodontus Dyar and six pools for A. pullatus (Coquillet). Polymorphisms were scored at 47-48 RAPD loci in each species. To test for isolation by distance, F ST /(1 F ST ) values between pairs of pools were plotted against geographical distances and subjected to a Mantel test with 1000 random permutations. F ST values were independent of geographical distances in A. cataphylla and A. hexodontus but were somewhat correlated in A. pullatus suggesting isolation by distance in this species. A cluster analysis was performed on pair-wise F ST values among pools including seven pools that were sampled in both 1994 and 1995. Bootstrap analysis indicated that pools clustered across drainages and generally independently of geographical proximity. However, there was consistent support for clustering of larvae collected from the same pool across years in A. cataphylla and in high altitude collections of A. pullatus. Mountains do not appear to act as major barriers to gene flow in any of these species. Instead, seasonal differences in adult emergence may serve as barriers to migration among A. pullatus and A. cataphylla populations. Larvae of A. hexodontus are distributed continuously in grassy pools along the banks of rivers and genetic drift probably occurs through random larval mortality when these pools are washed out during spring run-off.