Molecular phylogeny and diagnosis of species of the family Protostrongylidae from caprine hosts in Uzbekistan

Protostrongylids are important pulmonary parasites of ungulates, particularly caprines. Their complex life cycles involve terrestrian gastropods as intermediate hosts. Morphological discrimination of larvae in feces and snails is impossible to the species level, and molecular data are missing for many species. To improve diagnosis and epidemiology of protostrongylids, this study describes internal transcribed spacer 2 (ITS-2) sequences for five protostrongylid and one metastrongylid species from Uzbekistan. Morphological and molecular analyses identified Protostrongylus rufescens, Protostrongylus hobmaieri, Protostrongylus sp., Spiculocaulus leuckarti, and Cystocaulus ocreatus. Sequence differences between ITS-2 were sufficient to allow species identification, e.g., in the Protostrongylinae intraspecific and interspecific genetic distances ranged between 0-0.01 and 0.05 and 0.16, respectively. There was one exception in the Elaphostrongylidae with identical ITS-2 sequences in Elaphostrongylus cervi and Elaphostrongylus rangiferi questioning their status as valid species. Maximum likelihood analysis sequences largely supported the currently assumed phylogenetic relationships among Protostrongylidae as predicted using morphological characters. The monophyly of the subfamilies Varestrongylinae, Elaphostrongylinae, and Protostrongylinae was corroborated with support values in Shimodaira-Hasegawa or Bayesian modifications of the approximate likelihood ratio test (aLRT) >= 97 %, but support for Muellerinae was weak (8 and 52 %, respectively) since Muellerius capillaris differs significantly from the other Muellerinae. On the genus level, paraphyly of the genus Protostrongylus was confirmed since the members of the genera Spiculocaulus and Orthostrongylus were located within the Protostrongylus cluster (aLRT >= 99 %). Maximum likelihood unequivocally assigned every unique sequence to the correct species confirming suitability of ITS-2 regions for diagnosis protostrongylids except of E. cervi and E. rangiferi.