Indigenous ecological calendars and seasonal vector-borne diseases in the Colombian Amazon: an intercultural and interdisciplinary approach / Calendarios ecológicos indígenas y enfermedades estacionales transmitidas por vectores en la Amazonia colombiana: un enfoque intercultural e interdisciplinario

Traditional ecological knowledge of indigenous groups in the southeastern Colombian Amazon coincides in identifying the two main hydrological transition periods (wet-dry: August-November; dry-wet: March-April) as those with greater susceptibility to disease in humans. Here we analyze the association between indigenous knowledge about these two periods and the incidence of two vector-borne diseases: malaria and dengue. We researched seven "ecological calendars" from three regions in the Colombian Amazon, malaria and dengue cases reported from 2007 to 2019 by the Colombian National Institute of Health, and daily temperature and precipitation data from eight meteorological stations in the region from 1990-2019 (a climatological normal). Malaria and dengue follow a seasonal pattern: malaria has a peak from August to November, corresponding with the wet-dry transition (the "season of the worms" in the indigenous calendars), and dengue has a peak in March and April, coinciding with the dry-wet transition. Previous studies have shown a positive correlation between rainfall and dengue and a negative correlation between rainfall and malaria. However, as the indigenous ecological knowledge codified in the calendars suggests, disease prediction cannot be reduced to a linear correlation with a single environmental variable. Our data show that two major aspects of the indigenous calendars (the time of friaje as a critical marker of the year and the hydrological transition periods as periods of greater susceptibility to diseases) are supported by meteorological data and by the available information about the incidence of malaria and dengue.(AU)

Los conocimientos ecológicos tradicionales de grupos indígenas del sureste de la Amazonia colombiana coinciden en identificar dos principales periodos de transición hidrológica (seco-húmedo: agosto-noviembre; húmedo-seco: marzo-abril) como los de mayor susceptibilidad a enfermedades en humanos. Aquí analizamos la asociación entre el conocimiento indígena sobre estos dos periodos y la incidencia de dos enfermedades transmitidas por vectores: malaria y dengue. Investigamos siete calendarios ecológicos de tres regiones en la Amazonia colombiana, casos de dengue y malaria reportados de 2007 hasta 2019 por el Instituto Nacional de Salud de Colombia y datos diarios de temperatura y precipitación de ocho estaciones meteorológicas en la región, de 1990 a 2019 (una normal climatológica). Malaria y dengue siguen un patrón estacional, la malaria tiene un pico de agosto a noviembre, correspondiendo con la transición húmedo-seco (el "tiempo de gusano" según los calendarios indígenas), mientras que dengue tiene un pico de marzo a abril, coincidiendo con la transición seco-húmedo. Estudios previos mostraron una correlación positiva entre precipitación y dengue, y una correlación negativa entre precipitación y malaria. Sin embargo, como lo sugiere el conocimiento ecológico codificado en los calendarios indígenas, la predicción de enfermedades no puede reducirse a una correlación lineal con una sola variable medioambiental. Nuestros datos muestran que dos aspectos principales de los calendarios indígenas (el tiempo de friaje como un marcador crítico anual y los periodos de transición hidrológica como épocas de mayor susceptibilidad a enfermedades) están soportados por datos meteorológicos e información disponible acerca de la incidencia de malaria y dengue.(AU)

Haemosporidian parasites in the ash-breasted Sierra finch (Geospizopsis plebejus): insights from an Andean dry forest population.

Haemosporidian genera Plasmodium, Haemoproteus and Leucocytozoon, responsible for avian malarial infections, are highly diverse and have a wide range of health effects and predictors, depending on the host and its environmental context. Here, we present, for the first time, detailed information on the identity, prevalence and parasitaemia of haemosporidians and other haemoparasites that infect the ash-breasted Sierra finch, Geospizopsis plebejus, in an Andean dry forest. We study the consequences of infection in the host body and health conditions and explore the environmental and intrinsic factors that influence infection status and parasitaemia. We conducted diagnoses by cytochrome b (cytb) sequencing and morphological identification, and estimated the levels of parasitaemia based on microscopy. We identified 6 cytb lineages infecting G. plebejus. Two of them were new lineages: Haemoproteus sp. GEPLE01 and GEPLE02. We also detected Haemoproteus sp. ZOCAP08, Haemoproteus sp. AMAVIR01, Plasmodium homopolare BAEBIC02 and Plasmodium cathemerium ZONCAP15. By microscopy, we detected Haemoproteus coatneyi, Haemoproteus erythrogravidus, P. homopolare and other unidentified species of Haemoproteus, Plasmodium, Babesia sp. and 1 microfilaria. We found no evidence of Leucocytozoon. Additionally, we detected several coinfections by sequencing and microscopy. The prevalence of haemosporidian infections was high (87.7%), and the mean parasitaemia was 61.65 infected cells per 10 000 erythrocytes examined. Prevalence and parasitaemia were higher for Haemoproteus than for Plasmodium. Haemoproteus sp. AMAVIR01 showed the highest prevalence (43.1%) and mean parasitaemia (94.39/10 000 erythrocytes) and might be associated with H. coatneyi. Immature individuals showed a lower prevalence than adults, supporting previous findings.

Is Haemoproteus gabaldoni a valid species? An approach from morphology and molecular tools applied to parasites of Anseriformes.

Currently, there are three recognized species of haemoproteids infecting Anseriformes: Haemoproteus nettionis, H. macrovacuolatus, and H. greineri. Unfortunately, genetic information associated with a morphotype is available only for H. macrovacuolatus. We recently found a parasite morphologically compatible with Haemoproteus gabaldoni, a species Bennet (1993) described in a Cairina moschata (Muscovy duck) from Venezuela. This species was synonymized to H. nettionis by Valkiunas (2005), arguing not enough morphological differentiation between them; it was said that H. greineri could be as well a synonym of H. nettionis. In this study, we aimed to provide evidence to determine if Haemoproteus gabaldoni is a different species of H. nettionis and help to clarify other species status. We first performed morphological and morphometrical analyses and compared this information against the parahapantotypes of H. greineri, H. gabaldoni and material diagnosed as H. nettionis provided by the International Reference center for Avian Haematozoa (IRCAH), and H. macrovacuolatus from the Host-Parasite Relationship Study Group (GERPH, in Spanish Grupo de Estudio Relación Parásito Hospedero) biological collection. We used Principal Component Analysis (PCA) of dimensionless standard morphometrical variables from gametocytes. Furthermore, we amplified a small fragment of cytochrome b (cyt b) to compare the sequence with information in GenBank and Malavi through phylogenetic analyses and haplotype networks. PCA analyses revealed the presence of three distinct groups in the samples studied, supported in the morphological traits of each parasite species analyzed; phylogenetic analyses grouped parasite lineages separately according to the host and continent of provenance. Such results indicate that, H. gabaldoni, is a different species from H. nettionis. One more time, it is demonstrated the importance of linking barcode surveys to morphological studies. Finally, it is highlighted the importance of biological collections as repositories of worldwide biodiversity.

Avian haemosporidian (Haemosporida: Plasmodium and Haemoproteus) in the department of Arauca, Colombian Orinoquia region.

Avian haemosporidian from the genera Haemoproteus and Plasmodium are a diverse and widely distributed group of vector-borne blood parasites. These parasites can have negative effects on bird survival by influencing several aspects of their life cycle, causing different clinical signs and even death. Colombia has the widest range of bird richness throughout the globe; however, the associations between haemosporidian parasites and wild birds in different ecosystems remain poorly explored. Within this frame of reference, the objective of the present study was to identify and understand haemosporidian associated with resident and migratory wild birds and their lineages in northeast Orinoquia region, Colombia. Birds were captured in 8 localities and blood samples were collected, identifying the presence of haemosporidian parasites through morphological and molecular analysis of the mitochondrial cytochrome b gene. Blood samples from 233 wild birds (86 species) were analyzed for haemosporidian parasites. Sixteen individuals (6.9%) from 15 resident and migratory species were positive for Haemoproteus or Plasmodium. Fourteen haemosporidian lineages were identified, five of them reported for the first time. These new lineages are reported in four resident birds and one boreal migratory bird (Parkesia noveboracensis). This study is the first developed in the department of Arauca and contributes to the knowledge of haemosporidian lineages and their interaction with wild birds in the Colombian Orinoquia region and South America.

Plasmodium parasites in reptiles from the Colombia Orinoco-Amazon basin: a re-description of Plasmodium kentropyxi Lainson R, Landau I, Paperna I, 2001 and Plasmodium carmelinoi Lainson R, Franco CM, da Matta R, 2010.

Colombia is a megadiverse country with about 600 species of reptiles; however, there are few studies on species of hemoparasites found in this taxonomic group. Here, we document the presence of Plasmodium spp. in four species of reptiles from the northern part of the Orinoco-Amazon region in Colombia. Individuals analyzed in this study were captured in localities between 200 and 500 m altitude, in the department of Guaviare. Each sample was screened for haemosporidian parasites by using morphology and a nested polymerase chain reaction (PCR) protocol that targets the mitochondrial cytochrome b (cytb) gene. Four morphotypes of the genus Plasmodium were found; two of these species are re-described using morphological and molecular data (cytb). For the other two morphotypes, it was not possible to assign a described species. Among those, Plasmodium screened one species was only detected by microscopy. Considering the potential species diversity, it is possible that commonly used primers may not detect all species, reinforcing the importance of using microscopy in haematozoa surveys. There was no correspondence between the morphological traits associated with the subgenera and the phylogenetic relationships that we found in our analyses. Additionally, we found an expansion in the geographical distribution of these two species, and a new host for P. kentropyxi, demonstrating that studies of tropical herpetofauna and their parasites deserve more attention.

Haemoproteus erythrogravidus n. sp. (Haemosporida, Haemoproteidae): Description and molecular characterization of a widespread blood parasite of birds in South America.

The great diversity of birds and ecosystems in the Andean mountains has been understudied in terms of their parasite species. We describe a new Haemoproteus parasite, H. (Parahaemoproteus) erythrogravidus infecting Zonotrichia capensis (Rufous-Collared Sparrow) in South America. The description of this blood parasite species is supported by morphological and molecular data based on a fragment of cytochrome b gene (cyt b) and complete mitochondrial genome sequences. The new species is closely related to H. (Parahaemoproteus) coatneyi, and it can be readily distinguished from the latter parasite due to morphology of its blood stages, particularly 1) the formation of a marked protrusion on envelope of infected erythrocytes by the majority of developing gametocytes, a feature which is unique for this Haemoproteus species and 2) the extremely attenuated width of the growing dumbbell-shaped macro- and microgametocytes. Additionally, Haemoproteus erythrogravidus is shown to be a monophyletic taxon that diverges from Haemoproteus coatneyi at the molecular level. We provide the complete mitochondrial DNA genome for both H. coatneyi and H. erythrogravidus. Molecular and morphological evidences indicate that H. erythrogravidus is present in Ecuador and Colombia, and genetic lineages with 100% of identity for the cyt b gene were reported in Chile, Perú, and Venezuela. Our study also indicates that H. erythrogravidus and H. coatneyi are sympatric sister taxa sharing Z. capensis as a host species across its distribution, which could be the result of sympatric speciation or complex biogeographic processes. Further studies on the distribution and evolutionary history of Z. capensis and its parasites H. erythrogravidus and H. coatneyi insight for our better understanding of the factors and dynamics driving parasite speciation.

Leucocytozoon Diversity and Possible Vectors in the Neotropical highlands of Colombia.

Studies of the lowland avifauna in the Neotropical Region have shown a paucity of Leucocytozoon species. However, surveys conducted in the Colombian highlands revealed a great diversity of these parasites infecting resident birds. To further investigate the relationship between Leucocytozoon diversity, the potential vectors, and altitudinal distribution, birds from 41 families were sampled at low and high elevations in Colombia. Blood samples were screened by microscopy, and a fragment of cytochrome b was amplified from Leucocytozoon-positive samples. The complete mitochondrial genome was also obtained for each morphospecies of Leucocytozoon. Leucocytozoon species were detected in resident birds, with various degrees of host specificity, at elevations from 2,400 to 3,950 meters above sea level, where five new host-parasite associations were discovered. Phylogenetic analysis based on the cytochrome b fragment suggested that two nominal taxa, L. fringillinarum and L. majoris, are species complexes. Blood sources of Simuliidae revealed generalist-feeding habits that included avian and mammalian hosts. Molecular analysis of parasites in black flies indicated a close relationship with the parasites found in birds. Our investigation provides further evidence that the distribution and transmission of Leucocytozoon species in the Neotropics are influenced by elevation, with the highest prevalence between 2,400 and 3,200 m asl.

In vitro development of Haemoproteus columbae (Haemosporida: Haemoproteidae), with perspectives for genomic studies of avian haemosporidian parasites.

The evolutionary origin of wildlife and human malaria parasites (Plasmodium spp.) has been discussed for several decades. The lack of genomic data about species of wildlife haemosporidian parasites related to Plasmodium limits the number of taxa available for phylogenetic analysis. Genomic data about avian parasites of the genus Haemoproteus parasites, the sister genus to Plasmodium are still not available, mainly due to difficulties in obtaining pure DNA of parasites inhabiting nucleated avian host cells. Recent studies show that microgametes of Haemoproteus (Parahaemoproteus) spp. develop in vitro and can be isolated by simple centrifugation, allowing the isolation of pure parasite DNA for genomic studies. However, in vitro development of Haemoproteus (Haemoproteus) spp. has not been investigated, and it is unclear if microgametes of these parasites also can be obtained under in vitro conditions. Here, we provide the first data about the in vitro development of Haemoproteus (Haemoproteus) columbae, a widespread avian haemosporidian parasite, which is specific to pigeons and doves (Columbiformes) and is transmitted by hippoboscid flies (Diptera, Hippoboscidae). In vitro gametogenesis and ookinete development of H. columbae were studied using a strain isolated from a feral Rock Pigeon (Columba livia) in Bogotá-Colombia. The morphological events leading to exflagellation, fertilization and ookinete formation, as well as the rate of development of these stages were followed in vitro at 40 °C, 19 °C and 15 °C for 48 h. Macrogametes, microgametes, zygotes and initial stages of ookinete development were observed in all temperatures, but mature ookinetes were seen only at 40 °C. The largest diversity of sporogonic stages of H. columbae were present at 40 °C however, exflagellation, fertilization of macrogametes and development of immature ookinetes were also observed at 15 °C and 19 °C. Morphological and morphometric features of these stages in vitro were described and illustrated. This study demonstrates a requirement of high temperature for the successful development of mature ookinetes of H. columbae, but not gametes. We show that 1) parasites of the H. (Haemoproteus) subgenus exflagellate in vitro at 15-19 °C, as is the case in H. (Parahaemoproteus) spp. and 2) in vitro exflagellation can be used to obtain pure DNA for genomic studies.

Avian haemosporidians from Neotropical highlands: Evidence from morphological and molecular data.

Avian haemosporidian parasites have been scarcely studied in the Neotropical highlands despite the high avian diversity reported and the uniqueness of these ecosystems. The aims of this study were to examine Haemoproteus and Plasmodium diversity based on morphological and molecular data, as well as to explore the concordance between these two approaches, when identifying species. We sampled 1487 birds belonging to 166 species, in localities of the Colombian Andean region at elevations ranging from 2100 to 4000 m above sea level. Here, we report twelve morphological parasite species, of which five are undescribed. Thirty parasite cytochrome b lineages are reported, 17 of which for the first time. We provide morphological information and illustrations, as well as, cytochrome b lineages for six morphospecies: Haemoproteus columbae, Haemoproteus witti, Haemoproteus coatneyi, Haemoproteus vireonis, Plasmodium lutzi, and Plasmodium unalis. This is the first report to provide a linkage between morphology and a molecular lineage for H. witti. Cytochrome b gene proved to be useful for species determination as DNA barcoding. Differences in parasite composition between lowlands and highlands in Colombia suggest a replacement of avian Plasmodium fauna. Parasite lineages restricted to either Colombian resident or Nearctic migratory birds were found; but a single lineage common in both has not been recorded in Nearctic non-migratory birds. We generated valuable information by using both morphological and molecular data representing competent host-parasite relationships which are based on observation of gametocytes in circulation; and increased the taxon sampling of avian haemosporidian.

Leucocytozoon pterotenuis sp. nov. (Haemosporida, Leucocytozoidae): description of the morphologically unique species from the Grallariidae birds, with remarks on the distribution of Leucocytozoon parasites in the Neotropics.

We describe morphologically unique Leucocytozoon pterotenuis sp. nov. (Haemosporida, Leucocytozoidae), the first reported leucocytozoid species developing in fusiform host cell found in a Neotropical passeriform bird. The type host of this parasite is the Chestnut-crowned Antpitta (Grallaria ruficapilla, Grallariidae), an elusive native passerine bird whose natural history remains, to a large degree, unexplored. This bird was captured in Palacio forest in the damping zone of Chingaza National Natural Park, Cundinamarca, Colombia, at 2900 m above sea level (asl). Gametocytes of the new species develop both in roundish and fusiform host cells. This parasite is readily morphologically distinguishable from the described Leucocytozoon species because its host cells possess the narrow (needle-like) spindle-shaped processes, which length markedly exceeds their width. Additionally, the host cell nucleus markedly extends into the processes. Phylogenetic relationships were constructed based on a fragment of the mitochondrial cytochrome b gene and the complete mitochondrial genome. Phylogenetic analysis placed the lineage of L. pterotenuis in different positions depending on the length of the sequence analyzed that is likely due to poor sampling of Leucocytozoon species, especially from rare or non-passerine hosts, as well as a paucity of complete mitochondrial sequences of these parasites. Available data indicate that Leucocytozoon parasites are distributed mainly in mountain regions of the Neotropics where unique morphological forms have been recently discovered. To a better knowledge of the diversity of Leucocytozoon spp. and their host-vector-parasite interactions in Neotropical countries, additional deep and intensive samplings are needed, particularly in orders different to Passeriformes.

Description, molecular characterization, and patterns of distribution of a widespread New World avian malaria parasite (Haemosporida: Plasmodiidae), Plasmodium (Novyella) homopolare sp. nov.

Plasmodium (Novyella) homopolare, a newly described Plasmodium species, was found in a wide range of Passeriformes species in California, USA, and Colombia. This parasite infected more than 20% of the sampled bird community (N = 399) in California and was found in 3.6% of birds sampled (N = 493) in Colombia. Thus far, it has been confirmed in North and South America where it is present in numerous species of migratory and resident birds from six families. Based on 100% matches, or near-100% matches (i.e., ≤2-nucleotide difference), to DNA sequences previously deposited in GenBank, this parasite is likely also distributed in the Eastern USA, Central America, and the Caribbean. Here, we describe the blood stages of P. homopolare and its mtDNA cytochrome b sequence. P. homopolare belongs to the subgenus Novyella and can be readily distinguished from the majority of other Novyella species, primarily, by the strictly polar or subpolar position of meronts and advanced trophozoites in infected erythrocytes. We explore possible reasons why this widespread parasite has not been described in earlier studies. Natural malarial parasitemias are usually light and co-infections predominate, making the parasites difficult to detect and identify to species when relying exclusively on microscopic examination of blood films. The combined application of sequence data and digital microscopy techniques, such as those used in this study, provides identifying markers that will facilitate the diagnosis of this parasite in natural avian populations. We also address the evolutionary relationship of this parasite to other species of Plasmodium using phylogenetic reconstruction.

Mixed species flock, nest height, and elevation partially explain avian haemoparasite prevalence in Colombia.

The high avian biodiversity present in the Neotropical region offers a great opportunity to explore the ecology of host-parasite relationships. We present a survey of avian haemoparasites in a megadiverse country and explore how parasite prevalences are related to physical and ecological host characteristics. Using light microscopy, we documented the presence of haemoparasites in over 2000 individuals belonging to 246 species of wild birds, from nine localities and several ecosystems of Colombia. We analysed the prevalence of six avian haemoparasite taxa in relation to elevation and the following host traits: nest height, nest type, foraging strata, primary diet, sociality, migratory behaviour, and participation in mixed species flocks. Our analyses indicate significant associations between both mixed species flocks and nest height and Haemoproteus and Leucocytozoon prevalence. The prevalence of Leucocytozoon increased with elevation, whereas the prevalence of Trypanosoma and microfilariae decreased. Plasmodium and Haemoproteus prevalence did not vary significantly with elevation; in fact, both parasites were found up to 3300 m above sea level. The distribution of parasite prevalence across the phylogeny of bird species included in this study showed little host phylogenetic signal indicating that infection rates in this system are evolutionarily labile. Vector distribution as well as the biology of transmission and the maintenance of populations of avian haemoparasites deserve more detailed study in this system.

Description of Leucocytozoon quynzae sp. nov. (Haemosporida, Leucocytozoidae) from hummingbirds, with remarks on distribution and possible vectors of leucocytozoids in South America.

We describe Leucocytozoon quynzae sp. nov. (Haemosporida, Leucocytozoidae), which is the first Leucocytozoon parasite identified to species level in hummingbirds. It was found in the Amethyst-throated Sunangel (Heliangelus amethysticollis, Trochilidae, Apodiformes) captured in the Palacio Forest, which belongs to the damping zone of Chingaza National Natural Park, Cundinamarca, Colombia, at 2,900 m above sea level where the transmission occurs; the new species were found both in the high Andean forest and Paramo ecosystem. This parasite is described based on the morphology of its blood stages, a fragment of the mitochondrial cytochrome b gene, and the complete mitochondrial genome. Illustrations of blood stages of the new species are given, and the phylogenetic analysis places this lineage in a well-supported clade with other lineages of unidentified to species level leucocytozoids reported in the Trochilidae birds elsewhere. The new species possess gametocytes in roundish host cells; it can be readily distinguished from other similar leucocytozoids, primarily due to (1) a comma-like shape of the host cell nucleus, which extended one half or less of the circumference of the gametocyte and (2) a large number of prominent volutin granules in the cytoplasm. Identical mitochondrial cytochrome b sequence of Leucocytozoon quynzae was found in different hummingbird species at the type locality and also was reported in one passerine bird at the highlands of Peru. Leucocytozoon quynzae is the first leucocytozoid parasite described from South American birds; its transmission occurs both at low temperatures and high elevations. We discuss some patterns of distribution of avian leucocytozoids in South America and the role of Gigantodax spp. (Diptera, Simuliidae) as potential vectors of Leucocytozoon parasites in the Andean Region.

Description and molecular characterization of Plasmodium (Novyella) unalis sp. nov. from the Great Thrush (Turdus fuscater) in highland of Colombia.

Plasmodium (Novyella) unalis sp. nov. was found in the Great Thrush, Turdus fuscater (Passeriformes, Turdidae) in Bogotá, Colombia, at 2,560 m above sea level where the active transmission occurs. This parasite is described based on the morphology of its blood stages and a fragment of the mitochondrial cytochrome b gene (lineage UN227). Illustrations of blood stages of new species are given, and the phylogenetic analysis identifies closely related species and lineages of avian malaria parasites. The new species is most similar to Plasmodium (Novyella) vaughani (lineage SYAT05), a cosmopolitan avian malaria parasite; these parasites are also closely related genetically, with a genetic difference of 3.2% between them. P. unalis can be readily distinguished from the latter species morphologically, primarily due to the (1) presence of a single large, circular shaped pigment granule in the erythrocytic trophozoites and meronts; (2) presence of prominent vacuoles in trophozoites and growing meronts; and (3) presence of predominantly fan-like shaped erythrocytic meronts. Cytochrome b lineages with high similarity to the new species have been reported in Costa Rica, Brazil, Chile, and USA. It is probable that the new species of malaria parasite is widely distributed in the New World. This parasite has been reported only in the Great Thrush at the study site and might have a narrow range of avian hosts. Records of P. unalis are of particular theoretical interest due to its active transmission at highlands in Andes. Possible influence of urbanization on transmission of this malaria parasite in Bogotá is discussed.

Identification of Plasmodium (Haemamoeba) lutzi (Lucena, 1939) from Turdus fuscater (Great Thrush) in Colombia.

This study reports a broadening of the altitudinal range and a new host for Plasmodium (Haemamoeba) lutzi in Colombia. The study was conducted in the city of Bogotá, located in the Eastern Cordillera of Colombia at 2,560 m asl (meters above sea level) with an average annual temperature of 15 C. In total, 156 specimens of birds belonging to 25 species and 14 families were captured using mist nets. The blood samples were collected through venipuncture and analyzed by light microscopy. Plasmodium (H.) lutzi was only found in 2 individuals of Turdus fuscater (Great Thrush). This parasite has previously been reported in Aramides cajaneus (before: Aramides cajanea) (Grey-Necked Wood Rail), a bird found in the lowlands of Brazil, Venezuela, and Colombia. This finding provides evidence for a broad host range for P. lutzi that include 2 different orders, Gruiformes and Passeriformes, and also altitudinal expansion of its distribution. The blood stages were compared with the parasite's original descriptions, and the sequence of the parasite's mitochondrial genome (mtDNA) confirms that P. lutzi is a sister taxa of Plasmodium relictum, as previously proposed.