Background

Avian haemosporidians are single-celled eukaryotic parasites of vertebrates that require dipteran vectors for transmission. The genera Plasmodium, Haemoproteus and Leucocytozoon currently comprise over 5000 parasite lineages based on a 478-bp section of the mitochondrial cytochrome b gene, which is the standard DNA barcode for avian haemosporidians. The mitochondrial genomes of apicomplexan parasites are highly condensed, with a length of approximately 6000 bp, containing three coding genes (cytochrome c oxidase subunit I, cytochrome c oxidase subunit III and cytochrome b) and dispersed fragments of the small and large ribosomal RNA (rRNA) genes. Since the mitochondrial genomes are relatively conserved, they are valuable markers for studying the phylogenetic relationships between haemosporidian parasites. However, until recently, mitochondrial genomes were unavailable for parasites of the Haemoproteus nisi and Leucocytozoon toddi species groups, which are exclusive to accipitriform raptors and strongly diverged from other Haemoproteus and Leucocytozoon parasites.

Methods

We screened 171 accipitriform raptors from Austria and Germany using new primers targeting the cytochrome b gene of a previously neglected L. toddi clade. We also developed a new primer assay that enables the amplification and sequencing of the complete mitochondrial genomes of haemosporidian parasites. This process involved long-range PCRs with lineage-specific primers placed within the cytochrome b gene, followed by five nested PCRs targeting conserved sequence regions.

Results

Screening the accipitriform raptors revealed 10 new L. toddi group lineages. We sequenced 18 mitochondrial genomes belonging to five H. nisi group, nine L. toddi group, and two other Leucocytozoon lineages. Phylogenetic analyses based on mt genome sequences placed the L. toddi lineages within the genus Leucocytozoon, but the results did not support a monophyly of the genus Haemoproteus.

Conclusions

The new nested PCR approach with lineage-specific primers used for the long-range PCRs described herein successfully enabled the sequencing of the complete mitochondrial genomes, even in samples with mixed infections. The mitochondrial genomes of the H. nisi and L. toddi group lineages are highly valuable for resolving the phylogenetic relationships of the order Haemosporida since these parasites belong to clades distinct from other Haemoproteus and Leucocytozoon parasites.