Molecular analysis of polymorphic species of the genus Marshallagia (Nematoda: Ostertagiinae)

Background: The genus Marshallagia (Family Haemonchidae, subfamily Ostertagiinae) contains multiple species of nematodes parasitising the abomasum (or duodenum) of ruminants, in particular of Caprinae. Male specimens have been described to be polymorphic with the frequent/major morphotype initially described in the genus Marshallagia while the minor/rare morphotype was initially often placed in the genus Grossospicularia. Due to common morphological features, certain pairs of morphotypes were suggested to belong to the same species such as Marshallagia marshalli/M. occidentalis. However, molecular evidence to confirm these pairs of morphotypes belonging to the same species is missing. Methods: In the present study, Marshallagia sp. were collected from domestic sheep in Uzbekistan. Male specimens were morphologically described with particular emphasis on the structure of the bursa copulatrix. After DNA isolation from morphologically identified specimens, PCRs targeting the ribosomal internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) regions were conducted. After Sanger sequencing, maximum likelihood phylogenetic analyses and pairwise identities between sequences were calculated. Results: The major morphotypes of M. marshalli, M. schumakovitschi and M. uzbekistanica and the minor morphotypes M. occidentalis, M. trifida and M. sogdiana were identified and their morphology was documented in detail. ITS2 sequences showed little variation and did not allow diagnosing species. In contrast, phylogenetic analysis of cox1 sequences identified highly supported clusters and verified that M. marshalli, M. occidentalis and M. uzbekistanica are different morphotypes of the species M. marshalli while M. schumakovitschi and M. trifida represent distinct morphotypes of M. trifida. For M. sogdiana no corresponding major morphotype could be identified in the present study. Due to a large barcoding gap, comparison of cox1 sequences in terms of percent identity was sufficient to reliably assign the sequences to a particular species without phylogenetic analysis. Conclusions: The data presented here create a framework that will allow the classification of other members of the genus in the future and underline that parallel morphological and molecular analysis of specimens is crucial to improve the taxonomy of polymorphic species.