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Abstract Haemosporidians constitute a monophyletic group of vector-borne parasites that infect a wide range of vertebrate hosts, including Neotropical lizards. The remarkable diversity of these host-parasite associations and inadequate research on certain parasite groups have resulted in controversial haemosporidian taxonomy. Herein, we rediscover erythrocytic and non-erythrocytic haemosporidians infecting golden tegus (Tupinambis teguixin) from Brazil and Colombia. The erythrocyte-inhabiting parasite belongs toPlasmodiumsp., and the non-erythrocytic form was identified asSaurocytozoon tupinambi, previously attributed to the Family Leucocytozoidae. These non-pigmented haemosporidian parasites do not multiply in the blood. The relationships between theSaurocytozoonand Leucocytozoidae species were discussed for many years, especially during the 1970s. However, cytochrome b (cytb) sequences and the mitochondrial genomes recovered for this species strongly support classifying this parasite as aPlasmodiumtaxon. Therefore, we proposed a new combination for this parasite,Plasmodium(Saurocytozoon)tupinambicomb. nov., whereSaurocytozoonis retained as a subgenus due to its distinct morphology. These results reinforce that a broader definition of Plasmodiidae must include saurian parasites that develop non-pigmented leucocytozoid-like gametocytes. 

Abstract BackgroundStudies on haemosporidian diversity, including origin of human malaria parasites, malaria's zoonotic dynamic, and regional biodiversity patterns, have used target gene approaches. However, current methods have a trade-off between scalability and data quality. Here, a long-read Next-Generation Sequencing protocol using PacBio HiFi is presented. The data processing is supported by a pipeline that uses machine-learning for analysing the reads. MethodsA set of primers was designed to target approximately 6 kb, almost the entire length of the haemosporidian mitochondrial genome. Amplicons from different samples were multiplexed in an SMRTbell® library preparation. A pipeline (HmtG-PacBio Pipeline) to process the reads is also provided; it integrates multiple sequence alignments, a machine-learning algorithm that uses modified variational autoencoders, and a clustering method to identify the mitochondrial haplotypes/species in a sample. Although 192 specimens could be studied simultaneously, a pilot experiment with 15 specimens is presented, including in silico experiments where multiple data combinations were tested. ResultsThe primers amplified various haemosporidian parasite genomes and yielded high-quality mt genome sequences. This new protocol allowed the detection and characterization of mixed infections and co-infections in the samples. The machine-learning approach converged into reproducible haplotypes with a low error rate, averaging 0.2% per read (minimum of 0.03% and maximum of 0.46%). The minimum recommended coverage per haplotype is 30X based on the detected error rates. The pipeline facilitates inspecting the data, including a local blast against a file of provided mitochondrial sequences that the researcher can customize. ConclusionsThis is not a diagnostic approach but a high-throughput method to study haemosporidian sequence assemblages and perform genotyping by targeting the mitochondrial genome. Accordingly, the methodology allowed for examining specimens with multiple infections and co-infections of different haemosporidian parasites. The pipeline enables data quality assessment and comparison of the haplotypes obtained to those from previous studies. Although a single locus approach, whole mitochondrial data provide high-quality information to characterize species pools of haemosporidian parasites. 

Symbionts, including parasites, are ubiquitous in all world ecosystems. Understanding the diversity of symbiont species addresses diverse questions, from the origin of infectious diseases to inferring processes shaping regional biotas. Here, we review the current approaches to studying Haemosporida's species diversity and evolutionary history. Despite the solid knowledge of species linked to diseases, such as the agents of human malaria, studies on haemosporidian phylogeny, diversity, ecology, and evolution are still limited. The available data, however, indicate that Haemosporida is an extraordinarily diverse and cosmopolitan clade of symbionts. Furthermore, this clade seems to have originated with their vertebrate hosts, particularly birds, as part of complex community-level processes that we are still characterizing. 

Abstract Plasmodium species causing malaria in humans are not monophyletic, sharing common ancestors with nonhuman primate parasites. Plasmodium gonderi is one of the few known Plasmodium species infecting African old-world monkeys that are not found in apes. This study reports a de novo assembled P. gonderi genome with complete chromosomes. The P. gonderi genome shares codon usage, syntenic blocks, and other characteristics with the human parasites Plasmodium ovale s.l. and Plasmodium malariae, also of African origin, and the human parasite Plasmodium vivax and species found in nonhuman primates from Southeast Asia. Using phylogenetically aware methods, newly identified syntenic blocks were found enriched with conserved metabolic genes. Regions outside those blocks harbored genes encoding proteins involved in the vertebrate host-Plasmodium relationship undergoing faster evolution. Such genome architecture may have facilitated colonizing vertebrate hosts. Phylogenomic analyses estimated the common ancestor between P. vivax and an African ape parasite P. vivax-like, within the Asian nonhuman primates parasites clade. Time estimates incorporating P. gonderi placed the P. vivax and P. vivax-like common ancestor in the late Pleistocene, a time of active migration of hominids between Africa and Asia. Thus, phylogenomic and time-tree analyses are consistent with an Asian origin for P. vivax and an introduction of P. vivax-like into Africa. Unlike other studies, time estimates for the clade with Plasmodium falciparum, the most lethal human malaria parasite, coincide with their host species radiation, African hominids. Overall, the newly assembled genome presented here has the quality to support comparative genomic investigations in Plasmodium. 

Abstract The distribution of avian haemosporidians of the genusLeucocytozoonin the Neotropics remains poorly understood. Recent studies confirmed their presence in the region using molecular techniques alone, but evidence for gametocytes and data on putative competent hosts forLeucocytozoonare still lacking outside highland areas. We combined morphological and molecular data to characterize a newLeucocytozoonspecies infecting a non-migratory red-legged seriema (Cariama cristata), the first report of a competent host forLeucocytozoonin Brazil.Leucocytozoon cariamaen. sp. is distinguished from theLeucocytozoon fringillinarumgroup by its microgametocytes that are not strongly appressed to the host cell nucleus. The bird studied was coinfected withHaemoproteus pulcher, and we present a Bayesian phylogenetic analysis based on nearly complete mitochondrial genomes of these 2 parasites.Leucocytozoon cariamaen. sp. morphology is consistent with our phylogenetic analysis indicating that it does not share a recent common ancestor with theL.fringillinarumgroup.Haemoproteus pulcherandHaemoproteus cathartiform a monophyletic group withHaemocystidiumparasites of Reptilia, supporting the polyphyly of the genusHaemoproteus. We also discussed the hypothesis thatH. pulcherandH. cathartimay be avianHaemocystidium, highlighting the need to study non-passerine parasites to untangle the systematics of Haemosporida. 

Abstract Morphological traits from blood stages have been the gold standard for determining haemosporidian parasite species. However, the status of some taxa and the value of such traits in parasites from reptiles remain contentious. The scarce sampling of these species worsens the situation, and several taxa lack molecular data. A survey was performed in the Magdalena Department in Colombia, where 16 species of reptiles were captured. A peculiar haemosporidian parasite was found in the Turnip-tailed gecko Thecadactylus rapicauda . This haemosporidian does not show malarial pigment in blood stages under light microscopy; thus, it fits the Garnia genus's characters belonging to the Garniidae . However, the phylogenetic analyses using a partial sequence of cytochrome b and the mitochondrial DNA placed it within the Plasmodium clade. Our findings suggest that many putative Garnia species belong to the genus Plasmodium , like the one reported here. This study either shows that visible malarial pigment in blood stages is not a diagnostic trait of the genus Plasmodium or malarial pigment might be present in an undetectable form under a light microscope. In any case, the current taxonomy of haemosporidian parasites in reptiles requires revision. This study highlights the importance of using molecular and morphological traits to address taxonomic questions at the species and genus levels in haemosporidian parasites from reptiles. 

Abstract Delimiting and describing Plasmodium species in reptiles remains a pressing problem in Haemosporida taxonomy. The few morphological characters used can overlap, and the significance of some life-history traits is not fully understood. Morphologically identical lizard Plasmodium forms have been reported infecting different cell types (red and white blood cells) in the same host and have been considered the same species. An example is Plasmodium tropiduri tropiduri , a species known to infect erythrocytes, thrombocytes and lymphocyte-like cells. Here, both forms of P. t. tropiduri were analysed using light microscope-based morphological characteristics and phylogenetic inferences based on almost complete mitochondrial genomes of parasites naturally infecting lizards in southeastern Brazil. Although morphologically similar, two distinct phylogenetic lineages infecting erythrocytes and non-erythrocytic cells were found. The lineage found in the erythrocytes forms a monophyletic group with species from Colombia. However, the non-erythrocytic lineage shares a recent common ancestor with Plasmodium leucocytica , which infects leucocytes in lizards from the Caribbean islands. Here, Plasmodium ouropretensis n. sp. is described as a species that infects thrombocytes and lymphocyte-like cells.