Partial ribosomal non-transcribed spacer sequences distinguish the snowberry fly, Rhagoletis zephyria Snow from the apple maggot, R. pomonella (Walsh)

The apple maggot, Rhagoletis pomonella (Walsh), has been introduced into the apple growing regions of the Pacific Northwest over the past 60-100 years.  Apple maggot (larvae and adults) is difficult to distinguish from its morphologically similar sister species, the snowberry fly, R. zephyria Snow, which is native and abundant in the Pacific Northwest. While morphological identifications are common practice, a simple, inexpensive assay directly based on genetic differences would be very useful.  Here we report nucleotide substitution and insertion-deletion mutations in the non-transcribed spacer (NTS) of the ribosomal RNA gene cistron of R. pomonella and R. zephyria that appear to be diagnostic for these two fly species.  Insertion-deletion variation is substantial and results in a 49 base-pair difference in PCR amplicons from R. zephyria and R. pomonella that can be scored using agarose gel electrophoresis. PCR amplification and DNA sequencing of 766 bp of the NTS region from 38 R. pomonella individuals and 35 R. zephyria individuals from across their geographic ranges led to the expected PCR fragments of approx. 840 bp and 790 bp, respectively, as did amplification and sequencing of a smaller set of 26 R. pomonella and 16 R. zephyria flies from sympatric sites in Washington State.  Conversely, 633 bp mitochondrial COI barcode sequences drawn from this same set of flies were polyphyletic with respect to R. pomonella and R. zephyria. The ribosomal NTS region has been underutilized in insect taxonomy and systematics, and may prove useful in a number of systems where identifications are problematic.