Combining DNA sequences and morphology in systematics: testing the validity of the dragonfly species Cordulegaster bilineata

Morphological and molecular techniques are rarely combined when answering questions of taxonomic validity. In this study, we combine morphological techniques with DNA sequences to determine the validity of the dragonfly species Cordulegaster bilineata . The two dragonfly species C. bilineata and C. diastatops are very similar in size, body color, and morphological characters, and due to these similarities, the status of C. bilineata as a valid species is in question. In this study we compare morphological measurements of males and internal transcribed spacer 1 (ITS-1) sequences of rDNA between the two taxa. The hamule measurements (where copulation occurs) of males show little difference between the taxa in question, but the anal appendage measurements (where the male first contacts the female) show marked divergence between the two taxa. Cluster analysis with these anal appendage measurements correctly assigns almost all individuals measured into their respective taxon. PCR amplification products of ITS-1 display a ∼50 bp size difference between C. bilineata ( n = 4) and C. diastatops ( n = 5) regardless of collection site. Sequence data for these amplifications show 51 bp missing in one locus in the ITS-1 of C. bilineata relative to C. diastatops . A lone population of C. diastatops from Wisconsin has three individuals with ITS-1 products that match the size of both C. bilineata and C. diastatops . One individual from this population appears to yield two ITS-1 amplification products that match both C. bilineata and C. diastatops . Although this population may be evidence for hybridization between the two taxa, such hybridization is not necessarily sufficient to disqualify the validity of a separate species designation for C. bilineata . Morphology and ITS-1 sequences depict a high degree of divergence that is consistent with species-level differences.