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Background  The tsetse fly  Glossina fuscipes s.l.  is responsible for the transmission of approximately 90% of cases of human African trypanosomiasis (HAT) or sleeping sickness. Three  G. fuscipes  subspecies have been described, primarily based upon subtle differences in the morphology of their genitalia. Here we describe a study conducted across the range of this important vector to determine whether molecular evidence generated from nuclear DNA (microsatellites and gene sequence information), mitochondrial DNA and symbiont DNA support the existence of these taxa as discrete taxonomic units.    Principal Findings  The nuclear ribosomal Internal transcribed spacer 1 ( ITS1 ) provided support for the three subspecies. However nuclear and mitochondrial sequence data did not support the monophyly of the morphological subspecies  G. f. fuscipes  or  G. f. quanzensis . Instead, the most strongly supported monophyletic group was comprised of flies sampled from Ethiopia. Maternally inherited loci (mtDNA and symbiont) also suggested monophyly of a group from Lake Victoria basin and Tanzania, but this group was not supported by nuclear loci, suggesting different histories of these markers. Microsatellite data confirmed strong structuring across the range of  G. fuscipes s.l. , and was useful for deriving the interrelationship of closely related populations.    Conclusion/Significance  We propose that the morphological classification alone is not used to classify populations of  G. fuscipes  for control purposes. The Ethiopian population, which is scheduled to be the target of a sterile insect release (SIT) programme, was notably discrete. From a programmatic perspective this may be both positive, given that it may reflect limited migration into the area or negative if the high levels of differentiation are also reflected in reproductive isolation between this population and the flies to be used in the release programme.

Cryptic Diversity within the Major Trypanosomiasis Vector Glossina fuscipes Revealed by Molecular Markers