Expanding the known haemosporidian parasite diversity in Eurasian bluethroat (Luscinia svecica) subspecies through amplicon sequencing

[Display omitted] •Cryptic diversity of haemosporidian parasites was found in European bluethroats.•High-throughput sequencing revealed more lineages than previous Sanger sequencing.•Geographic distance and habitat differences influenced distribution of parasites.•Parasite diversity in a near-extinct bluethroat population was similar to others. Monitoring haemosporidian parasites in birds is essential to comprehend the dynamics of avian malaria, a disease that significantly affects bird populations worldwide. This study concentrated on the prevalence and diversity of haemosporidian parasites in 198 specimens from two subspecies of the Eurasian bluethroat (Luscinia svecica), aiming to explore the genetic diversity and species richness of haemosporidian fauna across the host populations. By utilizing next-generation amplicon high-throughput sequencing (NGS), we observed a marked increase in the detection of haemosporidian diversity, revealing cryptic variants and species previously unidentified by Sanger sequencing. A high prevalence of Plasmodium was seen in all studied sites, accompanied by a less frequent Leucocytozoon infection in the red-spotted subspecies and minimal occurrence of Haemoproteus. Both previously known and new, low prevalence cryptic variants were detected, underscoring the complexity of haemosporidian infections in avian hosts. The use of species delimitation tools provided a detailed understanding of haemosporidian species diversity, their coexistence within hosts, and their phylogenetic relationships. Despite the varying ecological characteristics of the study sites, no significant difference in haemosporidian alpha diversity among populations was found. However, significant differences in beta diversity were identified, suggesting that habitat characteristics and geographic distance influence parasite distribution. These findings highlight the importance of advanced molecular techniques in revealing the hidden diversity of parasites, offering valuable insights into the ecology and evolution of haemosporidian infections. Given the threatened status of one of the host’s populations, knowledge on local diversity of haemosporidian parasites also has implications for possible conservation strategies.