Unexpected ‘relationships’ among species were identified in two instances. The first is represented by the sisterspecies relationship between Prosimulium frohneiand the Colorado population of P. t r a v is i(=P. t r a v is i2), as opposed to between the latter population and P. t r a v is i1. Although closely related, P. fr o h n e iandP. t r a v is i(both within the Prosimulium hirtipesspecies group) are easily distinguishable morphologically. A similar situation was found between Prosimulium formosumand the more widespread P. t r a v is i sibling (=P. t r a v is i1). In this case, P. fo r mo s u mgrouped with P. t r a v is i1, clustering specifically within populations from southern Alberta (data not shown in Fig. 1). In this instance, very little genetic divergence was observed between this pair of species. This situation possibly indicates transfer of mtDNA elements through hybridization between these two species. Both species are included in
the P. h ir t ip e sspecies group but are easily distinguished morphologically. This presumed hybridization might
compromise the ability of COI to discriminate among species pairs. Further studies, including additional members of the P. h ir t ip e sspecies group, will be needed to uncover potential limitations of the COI gene for recovering species identity in this speciose lineage. Incomplete sampling is known to compromise the performance of COI in other organisms (Meyer & Paulay 2005) and more samples are needed to more fully understand the limitations, if any,of the barcoding gene for black fly identification