Phylogenomic analyses reveal introgression and cryptic speciation in the globally distributed, vector-transmitted pathogen Plasmodium relictum

Establishing species limits is challenging, particularly for pathogens of wildlife. These pathogens can be difficult to sample and culture, and their genome sequencing must often be conducted in the presence of high levels of host DNA. Plasmodium relictum is a mosquito-vectored avian malaria pathogen that is a globally distributed host generalist, comprised of several genetic lineages. We used sequence capture data from 52 P. relictum infections originating from multiple continents to generate a genomic dataset of the pathogen. With this data, we established a robust phylogeny and determined species limits among P. relictum lineages. We generated phylogenomic trees by maximum likelihood and Bayesian methods with multi-species coalescent models and confirmed robustness of the topology by varying the amount of missing data in the analyses. Our results suggest the existence of two cryptic species among the infections we analyzed and provide evidence of genetic introgression between these species. One of the cryptic species, GRW4, devastated the endemic and immunologically naïve avifauna of Hawaii after its introduction to the islands ca. 100 years ago, and so was tested for positive selection in the GRW4 Hawaiian clade. Although we hypothesized it would be released from host selective pressures, we did not find evidence of positive selection in the Hawaiian GRW4 clade, and we discuss possible explanations. Overall, our results underscore the importance of genomic analyses for resolving pathogen species limits and understanding pathogen evolution.