Disentangling parasite diversity in the neotropics: Descriptions of two new species and shortcomings of molecular diagnostics for leucocytozoids.

Avian communities from South America harbor an extraordinary diversity of species (Haemosporida, Leucocytozoidae). Here, of 890 birds sampled, 10 (1.2%) were infected with parasites. Among them, two new species were discovered and described. sp. nov. and sp. nov. were found in non-migratory highland passeriforms belonging to the Grallaridae and Cotingidae, respectively. They both possess gametocytes in fusiform host cells. However, due to combining microscopic examination and molecular detection, it was revealed that these parasites were present in co-infections with other species, which gametocytes develop in roundish host cells, therefore exhibiting two highly distant parasite lineages isolated from the same samples. Remarkably, the lineages obtained by cloning the mtDNA genomes were not captured by the classic nested PCR, which amplifies a short fragment of cytochrome gene. Phylogenetic analyses revealed that the lineages obtained by the classic nested PCR clustered with parasites possessing gametocytes in roundish host cells, while the lineages obtained by the mtDNA genome PCR protocol were closely related to parasites possessing gametocytes in fusiform host cells. These findings suggest problems with the sensitivity of the molecular protocols commonly used to detect species. A detailed analysis of the primers used in the classic nested PCR revealed a match with DNA sequences from those parasites that possess gametocytes in roundish host cells (i.e., ), while they differ with the orthologous regions in the mtDNA genomes isolated from the samples containing the two new species. Since these are mixed infections, none of the lineages detected in this study can be assigned accurately to the new morphospecies that develops in fusiform host cells. However, phylogenetic analyses allowed us to hypothesize their most probable associations. This study highlights the need for developing detection methods to assess the diversity of parasites accurately.