In search of mitochondrial markers for resolving the phylogeny of cyclophyllidean tapeworms (Platyhelminthes, Cestoda) - a test study with Davaineidae

The most species rich order of tapeworms is the Cyclophyllidea and prior to wide-scale sampling of these worms for phylogenetics, we wished to develop reliable PCR primers that would capture fragments of mitochondrial (mt) DNA with phylogenetic utility across the order. Nuclear ribosomal RNA gene sequences are well-established and valuable markers for resolving flatworm interrelationships spanning a wide range of taxonomic divergences, but fail to provide resolution amongst recently diverged lineages. Entire mt genomes of selected cyclophyllidean tapeworms are available on GenBank, and we used these to design PCR primers to amplify mtDNA from cox1, rrnL and nad1 for a range of cyclophyllideans (7 davaineids, 1 hymenolepidid and 1 dilepidid) and selected outgroups (Tetrabothrius sp. and Mesocestoides sp.). A combined nuclear and mt gene data set was used to estimate a reference phylogeny and the performance of the individual genes was compared to this. Although nuclear and mt genes each contributed to the structure and stability of the phylogenetic estimate, strongest nodal support was provided by nuclear data amongst the basal lineages and by mt data amongst the most recently diverged lineages. The apparent complementarity afforded by combining nuclear and mt data was compromised by these data partitions providing conflicting signal at poorly supported nodes. Nevertheless, we argue for a combined evidence approach. PCR primers that amplify rrnL were designed and tested successfully against a diversity of cyclophyllideans; rrnL and nad1 appeared to be more informative than the fragment of cox1. The genus Raillietina was not supported by molecular evidence. The new primers will likely provide considerable resolution to estimates of cyclophyllidean interrelationships in future studies.