show Abstracthide AbstractRecent studies on microRNA evolution focused mainly on the comparison of microRNA complements between animal clades. However, evolution of microRNAs within such groups is poorly explored despite the availability of comparable data that in some cases lack only a few key taxa. For flatworms (Platyhelminthes) microRNA complements are available for free-living flatworms and all major parasitic lineages, except for the Monogenea. We present the microRNA complement of the monogenean flatworm Gyrodactylus salaris that facilitates a comprehensive analysis of microRNA evolution in Platyhelminthes. Using the newly designed bioinformatics pipeline miRCandRef the microRNA complement was disentangled from Next-Generation-Sequencing of smallRNAs and genomic DNA without a priori genome-assembly. It consists of 39 microRNA hairpin loci of conserved microRNA families, and 22 novel microRNAs. A comparison with the microRNA complements of Schmidtea mediterranea (Turbellaria), Schistosoma japonicum (Trematoda), and Echinococcus granulosus (Cestoda) reveals a substantial loss of conserved bilaterian, protostomian, and lophotrochozoan microRNAs. Eight of the 46 expected conserved microRNAs were lost in all flatworms, 16 in Neodermata and 24 conserved microRNAs could not be detected in the cestode and the trematode. Such a gradual loss of microRNAs has not been reported before for other animal phyla. Currently, little is known about microRNAs in Platyhelminthes, and for the majority of the lost microRNAs there is no prediction of function. As suggested earlier they might be related to morphological simplifications. The presence and absence of 153 conserved microRNAs was compared for platyhelminths and 32 other metazoan taxa. Phylogenetic analyses support the monophyly of Platyhelminthes [Turbellaria+Neodermata((Monogenea (Trematoda+Cestoda)))].