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Background  To date, mitochondrial genomes of more than one hundred flatworms (Platyhelminthes) have been sequenced. They show a high degree of similarity and a strong taxonomic bias towards parasitic lineages. The mitochondrial gene  atp8  has not been confidently annotated in any flatworm sequenced to date. However, sampling of free-living flatworm lineages is incomplete. We addressed this by sequencing the mitochondrial genomes of the two small-bodied (about 1 mm in length) free-living flatworms  Stenostomum sthenum  and  Macrostomum lignano  as the first representatives of the earliest branching flatworm taxa Catenulida and Macrostomorpha respectively.    Results  We have used high-throughput DNA and RNA sequence data and PCR to establish the mitochondrial genome sequences and gene orders of  S. sthenum  and  M. lignano . The mitochondrial genome of  S. sthenum  is 16,944 bp long and includes a 1,884 bp long inverted repeat region containing the complete sequences of  nad3 ,  rrnS,  and nine tRNA genes. The model flatworm  M. lignano  has the smallest known mitochondrial genome among free-living flatworms, with a length of 14,193 bp. The mitochondrial genome of  M. lignano  lacks duplicated genes, however, tandem repeats were detected in a non-coding region.  Mitochondrial gene order is poorly conserved in flatworms, only a single pair of adjacent ribosomal or protein-coding genes –  nad4l-nad4  – was found in  S. sthenum  and  M. lignano  that also occurs in other published flatworm mitochondrial genomes. Unexpectedly, we unambiguously identified the full metazoan mitochondrial protein-coding gene complement including  atp8  in  S. sthenum  and  M. lignano . A subsequent search detected  atp8  in all mitochondrial genomes of polyclad flatworms published to date, although the gene wasn’t previously annotated in these species.    Conclusions  Manual, but not automated genome annotation revealed the presence of  atp8  in basally branching free-living flatworms, signifying both the importance of manual data curation and of diverse taxon sampling. We conclude that the loss of  atp8  within flatworms is restricted to the parasitic taxon Neodermata.    Electronic supplementary material  The online version of this article (doi:10.1186/s12864-017-3807-2) contains supplementary material, which is available to authorized users.

Atp8 is in the ground pattern of flatworm mitochondrial genomes