Uncovering haemosporidian diversity in urban Feral Pigeons (Columba livia domestica) in Lisbon, Portugal: integrated molecular and morphological insights

Abstract Avian haemosporidian parasites ( Plasmodium , Haemoproteus , and Leucocytozoon ) are widespread haemoparasites that can impact avian health and fitness. Feral Pigeons ( Columba livia domestica ) are abundant in urban environments, yet the diversity and prevalence of haemosporidians in this host remain poorly studied, particularly in Europe. This study assessed the prevalence, genetic diversity, and morphological characteristics of haemosporidian parasites in 40 Feral Pigeons sampled from two sites in Lisbon, Portugal. It represents the first application of an integrated molecular and microscopy approach to haemosporidian research in Portugal. All individuals were infected with Haemoproteus columbae , and four distinct lineages were detected (COLIV03, COQUI05, CXNEA02, HAECOL1), all corresponding to this morphospecies, representing new geographic records for Portugal. Moreover, all lineages (except HAECOL1) were reported for the first time in Europe. Morphological examination, together with phylogenetic analysis and low genetic divergence (0.21–0.64%) from reference lineages, supported the assignment of lineage CXNEA02 to H. columbae , establishing its vertebrate host and morphospecies for the first time. Parasitaemia ranged from 0.05% to 8.11%, spanning infection intensities from low (≤ 1%) to high (> 5%) levels, and showed only a weak association with host body condition. In contrast, the presence of double gametocyte infections (DGIs) emerged as a significant predictor of reduced body condition, independent of parasitaemia, indicating increased physiological costs to the host. Male–female DGIs were detected exclusively within the dominant lineage HAECOL1, suggesting a potential role of this infection pattern in transmission dynamics and contributing to the exceptionally high prevalence observed in this system. The combined use of PCR and microscopy enabled the identification of molecular and morphological false negatives, the linkage of genetic lineages with morphospecies, and the identification of biologically meaningful infection traits not detectable by molecular screening alone. These findings underscore the underexplored diversity of avian haemosporidians, even in highly abundant host species, and highlight the value of integrated methodological approaches for avian parasite ecology.