Prevalence and diversity patterns of avian blood parasites in degraded African rainforest habitats

Land use changes including deforestation, road construction and agricultural encroachments have been linked to the increased prevalence of several infectious diseases. In order to better understand how deforestation affects the prevalence of vector-borne infectious diseases in wildlife, nine paired sites were sampled (disturbed vs. undisturbed habitats) in Southern Cameroon. We studied the diversity, prevalence and distribution of avian malaria parasites ( Plasmodium spp.) and other related haemosporidians (species of Haemoproteus and Leucocytozoon ) from these sites in two widespread species of African rainforest birds, the yellow-whiskered greenbul ( Andropadus latirostris , Pycnonotidae) and the olive sunbird ( Cyanomitra olivacea , Nectariniidae). Twenty-six mitochondrial cytochrome b lineages were identified: 20 Plasmodium lineages and 6 Haemoproteus lineages. These lineages showed no geographic specificity, nor significant differences in lineage diversity between habitat types. However, we found that the prevalence of Leucocytozoon and Haemoproteus infections were significantly higher in undisturbed than in deforested habitats ( Leucocytozoon spp. 50.3% vs. 35.8%, Haemoproteus spp. 16.3% vs. 10.8%). We also found higher prevalence for all haemosporidian parasites in C. olivacea than in A. latirostris species (70.2% vs. 58.2%). Interestingly, we found one morphospecies of Plasmodium in C. olivacea , as represented by a clade of related lineages, showed increased prevalence at disturbed sites, while another showed a decrease, testifying to different patterns of transmission, even among closely related lineages of avian malaria, in relation to deforestation. Our work demonstrates that anthropogenic habitat change can affect host–parasite systems and result in opposing trends in prevalence of haemosporidian parasites in wild bird populations.     

Haemosporidian blood parasites in European birds of prey and owls

Avian blood parasites have been intensively studied using morphological methods with limited information on their host specificity and species taxonomic status. Now the analysis of gene sequences, especially the mitochondrial cytochrome b gene of the avian haemosporidian species of Haemoproteus, Plasmodium, and Leucocytozoon, offers a new tool to review the parasite specificity and status. By comparing morphological and genetic techniques, we observed nearly the same overall prevalence of haemosporidian parasites by microscopy (19.8%) and polymerase chain reaction (PCR) (21.8%) analyses. However, in contrast to the single valid Leucocytozoon species (L. toddi) in the Falconiformes we detected 4 clearly distinctive strains by PCR screening. In the Strigiformes, where the only valid Leucocytozoon species is L. danilewskyi, we detected 3 genetically different strains of Leucocytozoon spp. Two strains of Haemoproteus spp. were detected in the birds of prey and owls examined, whereas the strain found in the tawny owl belonged to the morphospecies Haemoproteus noctuae. Three Plasmodium spp. strains that had already been found in Passeriformes were also detected in the birds of prey and owls examined here, supporting previous findings indicating a broad and nonspecific host spectrum bridging different bird orders.     

Nested cytochrome b polymerase chain reaction diagnostics underestimate mixed infections of avian blood haemosporidian parasites: microscopy is still essential

Numerous polymerase chain reaction (PCR)-based methods have been developed and used increasingly to screen vertebrate blood samples for the diagnosis of haemosporidian blood parasites (Sporozoa, Haemosporida), but a rigorous evaluation of the sensitivity of these methods for detecting mixed infections of different haemosporidian species belonging to the same and different genera and subgenera is lacking. This study links the information obtained by nested cytochrome b PCR and traditional microscopy in determining mixed haemosporidian infections in naturally infected birds. Samples from 83 individual passerine birds with single infections of Haemoproteus or Plasmodium spp., as determined by mitochondrial DNA amplification, also were investigated by microscopic examination of stained blood films. Thirty-six samples (43%) were found to harbor mixed Haemoproteus, or Plasmodium spp. infections, or both. Thus, the PCR assays alone underestimate the occurrence of mixed infections of haemosporidian parasites in naturally infected birds. To determine the true species composition of the haemosporidians in each individual host, PCR diagnostics need to be improved. Specific primers for Haemoproteus spp. and Plasmodium spp. should be developed. Ideally, a combination of the approaches of both microscopy and PCR-based methods is recommended for this purpose.     

High lineage diversity and host sharing of malarial parasites in a local avian assemblage

Bird populations often have high prevalences of the haemosporidians Haemoproteus spp. and Plasmodium spp., but the extent of host sharing and host switching among these parasite lineages and their avian hosts is not well known. While sampling within a small geographic region in which host individuals are likely to have been exposed to the same potential parasite lineages, we surveyed highly variable mitochondrial DNA from haemosporidians isolated from 14 host taxa representing 4 avian families (Hirundinidae, Parulidae, Emberizidae, and Fringillidae). Analyses of cytochrome b sequences from 83 independent infections identified 29 unique haplotypes, representing 2 well-differentiated Haemoproteus spp. lineages and 6 differentiated Plasmodium spp. lineages. A phylogenetic reconstruction of relationships among these lineages provided evidence against host specificity at the species and family levels, as all haemosporidian lineages recovered from 2 or more host individuals (2 Haemoproteus and 3 Plasmodium lineages) were found in at least 2 host families. We detected a similar high level of host sharing; the 3 most intensively sampled host species each harbored 4 highly differentiated haemosporidian lineages. These results indicate that some Haemoproteus spp. and Plasmodium spp. lineages exhibit a low degree of host specificity, a phenomenon with implications for ecological and evolutionary interactions among these parasites and their hosts.     

Cross-species infection of blood parasites between resident and migratory songbirds in Africa

We studied the phylogeny of avian haemosporidian parasites, Haemoproteus and Plasmodium, in a number of African resident and European migratory songbird species sampled during spring and autumn in northern Nigeria. The phylogeny of the parasites was constructed through sequencing part of their mitochondrial cytochrome b gene. We found eight parasite lineages, five Haemoproteus and three Plasmodium, infecting multiple host species. Thus, 44% of the 18 haemospiridian lineages found in this study were detected in more than one host species, indicating that host sharing is a more common feature than previously thought. Furthermore, one of the Plasmodium lineages infected species from different host families, Sylviidae and Ploceidae, expressing exceptionally large host range. We mapped transmission events, e.g. the occurrence of the parasite lineages in resident bird species in Europe or Africa, onto a phylogenetic tree. This yielded three clades, two Plasmodium and one Haemoproteus, in which transmission seems to occur solely in Africa. One Plasmodium clade showed European transmission, whereas the remaining two Haemoproteus clades contained mixes of lineages of African, European or unknown transmission. The mix of areas of transmission in several branches of the phylogenetic tree suggests that transmission of haemosporidian parasites to songbirds has arisen repeatedly in Africa and Europe. Blood parasites could be viewed as a cost of migration, as migratory species in several cases were infected with parasite lineages from African resident species. This cost of migration could have considerable impact on the evolution of migration and patterns of winter distribution in migrating birds.     

Prevalence and differential host-specificity of two avian blood parasite genera in the Australo-Papuan region

The degree to which widespread avian blood parasites in the genera Plasmodium and Haemoproteus pose a threat to novel hosts depends in part on the degree to which they are constrained to a particular host or host family. We examined the host distribution and host-specificity of these parasites in birds from two relatively understudied and isolated locations: Australia and Papua New Guinea. Using polymerase chain reaction (PCR), we detected infection in 69 of 105 species, representing 44% of individuals surveyed (n = 428). Across host families, prevalence of Haemoproteus ranged from 13% (Acanthizidae) to 56% (Petroicidae) while prevalence of Plasmodium ranged from 3% (Petroicidae) to 47% (Ptilonorhynchidae). We recovered 78 unique mitochondrial lineages from 155 sequences. Related lineages of Haemoproteus were more likely to derive from the same host family than predicted by chance at shallow (average LogDet genetic distance = 0, n = 12, P = 0.001) and greater depths (average distance = 0.014, n = 11, P < 0.001) within the parasite phylogeny. Within two major Haemoproteus subclades identified in a maximum likelihood phylogeny, host-specificity was evident up to parasite genetic distances of 0.029 and 0.007 based on logistic regression. We found no significant host relationship among lineages of Plasmodium by any method of analysis. These results support previous evidence of strong host-family specificity in Haemoproteus and suggest that lineages of Plasmodium are more likely to form evolutionarily-stable associations with novel hosts.     

A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood

Many bird species host several lineages of apicomplexan blood parasites (Protista spp., Haemosporida spp.), some of which are shared across different host species. To understand such complex systems, it is essential to consider the fact that different lineages, species, and families of parasites can occur in the same population, as well as in the same individual bird, and that these parasites may compete or interact with each other. In this study, we present a new polymerase chain reaction (PCR) protocol that, for the first time, enables simultaneous typing of species from the 3 most common avian blood parasite genera (Haemoproteus, Plasmodium, and Leucocytozoon). By combining the high detection rate of a nested PCR with another PCR step to separate species of Plasmodium and Haemoproteus from Leucocytozoon, this procedure provides an easy, rapid, and accurate method to separate and investigate these parasites within a blood sample. We have applied this method to bird species with known infections of Leucocytozoon spp., Plasmodium spp., and Haemoproteus spp. To obtain a higher number of parasite lineages and to test the repeatability of the method, we also applied it to blood samples from bluethroats (Luscinia svecica), for which we had no prior knowledge regarding the blood parasite infections. Although only a small number of different bird species were investigated (6 passerine species), we found 22 different parasite species lineages (4 Haemoproteus, 8 Plasmodium, and 10 Leucocytozoon).     

Low haemosporidian diversity and one key-host species in a bird malaria community on a mid-Atlantic island (São Miguel, Azores)

When host species colonize new areas, the parasite assemblage infecting the hosts might change, with some parasite species being lost and others newly acquired. These changes would likely lead to novel selective forces on both host and its parasites. We investigated the avian blood parasites in the passerine bird community on the mid-Atlantic island of S?o Miguel, Azores, a bird community originating from continental Europe. The presence of haemosporidian blood parasites belonging to the genera Haemoproteus, Plasmodium, and Leucocytozoon was assessed using polymerase chain reaction. We found two Plasmodium lineages and two Leucocytozoon lineages in 11 bird species (84% of all breeding passerine species) on the island. These lineages were unevenly distributed across bird species. The Eurasian Blackbird (Turdus merula) was the key-host species (total parasite prevalence of 57%), harboring the main proportion of parasite infections. Except for Eurasian Blackbirds, all bird species had significantly lower prevalence and parasite diversity compared to their continental populations. We propose that in evolutionary novel bird communities, single species may act as key hosts by harboring the main part of the parasite fauna from which parasites "leak" into the other species. This would create very different host-parasite associations in areas recently colonized by hosts as compared to in their source populations.     

A molecular phylogeny of malarial parasites recovered from cytochrome b gene sequences

A phylogeny of haemosporidian parasites (phylum Apicomplexa, family Plasmodiidae) was recovered using mitochondrial cytochrome b gene sequences from 52 species in 4 genera (Plasmodium, Hepatocystis, Haemoproteus, and Leucocytozoon), including parasite species infecting mammals, birds, and reptiles from over a wide geographic range. Leucocytozoon species emerged as an appropriate out-group for the other malarial parasites. Both parsimony and maximum-likelihood analyses produced similar phylogenetic trees. Life-history traits and parasite morphology, traditionally used as taxonomic characters, are largely phylogenetically uninformative. The Plasmodium and Hepatocystis species of mammalian hosts form 1 well-supported clade, and the Plasmodium and Haemoproteus species of birds and lizards form a second. Within this second clade, the relationships between taxa are more complex. Although jackknife support is weak, the Plasmodium of birds may form 1 clade and the Haemoproteus of birds another clade, but the parasites of lizards fall into several clusters, suggesting a more ancient and complex evolutionary history. The parasites currently placed within the genus Haemoproteus may not be monophyletic. Plasmodium falciparum of humans was not derived from an avian malarial ancestor and, except for its close sister species, P. reichenowi, is only distantly related to haemospordian parasites of all other mammals. Plasmodium is paraphyletic with respect to 2 other genera of malarial parasites, Haemoproteus and Hepatocystis. Explicit hypothesis testing supported these conclusions.     

High prevalence and diversity of blood parasites of passerine birds in Southern Turkmenistan

Thirty nine specimens of passerine birds belonging to 19 species and eight families were investigated by blood smear technique in four localities of Southern Turkmenistan in 3-18 August 1991. The overall prevalence of infection was 59%. Protists from the orders Haemosporida (genera Haemoproteus, Plasmodium, Leucocytozoon), Kinetoplastida (Trypanosoma), and Adeleida (Hepatozoon), as well as Microfilaria were found. Haemoproteids (the prevalence of infection is 44%), leucocytozoids (23%), malarial parasites (13%) and trypanosomes (13%) were most frequently recorded. Only low chronic infections (< 1% of infected cells for the great majority of intracellular parasites, and a few trypanosomes and Microfilaria in each blood smear) were seen. Haemoproteus belopolskyi, H. balmorali, H. dolniki, H. magnus, H. minutus, H. fringillae, H. majoris, Leucocytozoon dubreuili, and Trypanosoma avium were recorded for the first time in Turkmenistan. The former five above-mentioned species of haemoproteids are new records for the fauna of Middle Asia. Gametocytes of leucocytozoids in fusiform host cells were found for the first time in passerine birds in the Holarctic. The host is Parus bokharensis. Due to the wide distribution and the opportunity to collect a large parasitological material using harmless for hosts methods, bird haemosporidian parasites can be used as convenient models for ecological and evolutionary biology studies in South Turkmenistan. The heavily infected Orphean Warbler Sylvia hortensis is an especially convenient host for such purposes.     

A restriction enzyme-based assay to distinguish between avian hemosporidians

We describe a reliable and relatively inexpensive method for detecting and differentiating between the commonly studied avian blood parasite genera Haemoproteus, Plasmodium, and Leucocytozoon. The assay takes advantage of a Haemoproteus-specific restriction site identified by sequencing full mitochondrial genomes from two Haemoproteus and three Plasmodium lineages and an adjacent, genus-specific restriction site identified in Leucocytozoon spp. The assay was sensitive to simulated parasitemias of approximately 8 x 10(-6) per erythrocyte and was 100% accurate in differentiating between parasite genera isolated from a broad geographical and taxonomic sampling of infected hosts.     

Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds

A fragment of the mitochondrial cytochrome b gene of avian malaria (genera Haemoproteus and Plasmodium) was amplified from blood samples of 12 species of passerine birds from the genera Acrocephalus, Phylloscopus and Parus. By sequencing 478 nucleotides of the obtained fragments, we found 17 different mitochondrial haplotypes of Haemoproteus or Plasmodium among the 12 bird species investigated. Only one out of the 17 haplotypes was found in more than one host species, this exception being a haplotype detected in both blue tits (Parus caeruleus) and great tits (Parus major). The phylogenetic tree which was constructed grouped the sequences into two clades, most probably representing Haemoproteus and Plasmodium, respectively. We found two to four different parasite mitochondrial DNA (mtDNA) haplotypes in four bird species. The phylogenetic tree obtained from the mtDNA of the parasites matched the phylogenetic tree of the bird hosts poorly. For example, the two tit species and the willow warbler (Phylloscopus trochilus) carried parasites differing by only 0.6% sequence divergence, suggesting that Haemoproteus shift both between species within the same genus and also between species in different families. Hence, host shifts seem to have occurred repeatedly in this parasite host system. We discuss this in terms of the possible evolutionary consequences for these bird species.     

Avian hemosporidian parasites from northern California oak woodland and chaparral habitats

During spring-summer 2003-2004, the avian community was surveyed for hemosporidian parasites in an oak (Quercus spp.) and madrone (Arbutus spp.) woodland bordering grassland and chaparral habitats at a site in northern California, a geographic location and in habitat types not previously sampled for these parasites. Of 324 birds from 46 species (21 families) sampled (including four species not previously examined for hemosporidians), 126 (39%) were infected with parasites identified as species of one or more of the genera Plasmodium (3% of birds sampled), Haemoproteus (30%), and Leucocytozoon (11%). Species of parasite were identified by morphology in stained blood smears and were consistent with one species of Plasmodium, 11 species of Haemoproteus, and four species of Leucocytozoon. We document the presence of one of the parasite genera in seven new host species and discovered 12 new parasite species-host species associations. Hatching-year birds were found infected with parasites of all three genera. Prevalence of parasites for each genus differed significantly for the entire sample, and prevalence of parasites for the most common genus, Haemoproteus, differed significantly among bird families. Among families with substantial sample sizes, the Vireonidae (63%) and Emberizidae (70%) were most often infected with Haemoproteus spp. No evidence for parasite between-genus interaction, either positive or negative, was found. Overall prevalence of hemosporidians at the northern California sites and predominance of Haemoproteus spp. was similar to that reported in most other surveys for the USA, Canada, and the Caribbean islands.     

Prevalence and lineage diversity of avian haemosporidians from three distinct cerrado habitats in Brazil

Habitat alteration can disrupt host-parasite interactions and lead to the emergence of new diseases in wild populations. The cerrado habitat of Brazil is being fragmented and degraded rapidly by agriculture and urbanization. We screened 676 wild birds from three habitats (intact cerrado, disturbed cerrado and transition area Amazonian rainforest-cerrado) for the presence of haemosporidian parasites (Plasmodium and Haemoproteus) to determine whether different habitats were associated with differences in the prevalence and diversity of infectious diseases in natural populations. Twenty one mitochondrial lineages, including 11 from Plasmodium and 10 from Haemoproteus were identified. Neither prevalence nor diversity of infections by Plasmodium spp. or Haemoproteus spp. differed significantly among the three habitats. However, 15 of the parasite lineages had not been previously described and might be restricted to these habitats or to the region. Six haemosporidian lineages previously known from other regions, particularly the Caribbean Basin, comprised 50-80% of the infections in each of the samples, indicating a regional relationship between parasite distribution and abundance.     

Host phylogeography and beta diversity in avian haemosporidian (Plasmodiidae) assemblages of the Lesser Antilles

1. We estimated the correlation between host phylogeographical structure and beta diversity of avian haemosporidian assemblages of passerine birds to determine the degree to which parasite communities change with host evolution, expressed as genetic divergence between island populations, and we investigated whether differences among islands in the haemosporidia of a particular host species reflect beta diversity in the entire parasite assemblage, beta diversity in vectors, turnover of bird species and/or geographical distance. 2. We used Mantel tests to assess the significance of partial correlations between host nucleotide difference (based on cytochrome b) and haemosporidian (Haemoproteus spp. and Plasmodium spp.) mitochondrial lineage beta diversity within a given host species and between Plasmodium mitochondrial lineage beta diversity and mosquito and bird species beta diversity (or turnover). Three abundant and widespread host species (Tiaris bicolor, Coereba flaveola and Loxigilla noctis/barbadensis) were included in the study. Haemosporidian lineage beta diversity among nine islands was assessed using the Chao-Jaccard, Chao-S?rensen and Morisita-Horn indices of community similarity. Beta diversity indices of mosquito species and turnover of bird species were calculated from data in published records and field guides. 3. In Loxigilla spp., we found a positive correlation with geographical distance and an unexpected negative correlation between haemosporidian beta diversity and host genetic distance. Tiaris bicolor exhibited a significant positive correlation between haemosporidian beta diversity and beta diversity within the entire parasite assemblage. We did not find significant correlations between parasite beta diversity and mosquito beta diversity or bird species turnover. 4. Host phylogeographical structure does not appear to drive within-host beta diversity of haemosporidian lineages. Instead, the array of parasites on one host can reflect the haemosporidian assemblage on other hosts.     

Blood parasites in owls with conservation implications for the Spotted Owl (Strix occidentalis)

The three subspecies of Spotted Owl (Northern, Strix occidentalis caurina; California, S. o. occidentalis; and Mexican, S. o. lucida) are all threatened by habitat loss and range expansion of the Barred Owl (S. varia). An unaddressed threat is whether Barred Owls could be a source of novel strains of disease such as avian malaria (Plasmodium spp.) or other blood parasites potentially harmful for Spotted Owls. Although Barred Owls commonly harbor Plasmodium infections, these parasites have not been documented in the Spotted Owl. We screened 111 Spotted Owls, 44 Barred Owls, and 387 owls of nine other species for haemosporidian parasites (Leucocytozoon, Plasmodium, and Haemoproteus spp.). California Spotted Owls had the greatest number of simultaneous multi-species infections (44%). Additionally, sequencing results revealed that the Northern and California Spotted Owl subspecies together had the highest number of Leucocytozoon parasite lineages (n = 17) and unique lineages (n = 12). This high level of sequence diversity is significant because only one Leucocytozoon species (L. danilewskyi) has been accepted as valid among all owls, suggesting that L. danilewskyi is a cryptic species. Furthermore, a Plasmodium parasite was documented in a Northern Spotted Owl for the first time. West Coast Barred Owls had a lower prevalence of infection (15%) when compared to sympatric Spotted Owls (S. o. caurina 52%, S. o. occidentalis 79%) and Barred Owls from the historic range (61%). Consequently, Barred Owls on the West Coast may have a competitive advantage over the potentially immune compromised Spotted Owls.     

Avian hematozoa of some birds from Tchad.

A total of 389 birds of 32 species representing 14 families from Tchad were examined for blood parasites. Eighty-nine (22.9%) harbored infections of Haemoproteus (64%), Plasmodium (12.4%), Trypanosoma (1.1%), Atoxoplasma (=Lankesterella) (5.6%), and microfilaria (28.1%). Species of Leucocytozoon were not observed in the present study. The occurrence of the different genera differed markedly between bird families; members of the Ploceidae comprised 85% of the infected birds.     

Presence of Plasmodium and Haemoproteus in breeding prothonotary warblers (Protonotaria citrea: Parulidae): temporal and spatial trends in infection prevalence

Prothonotary warbler (Protonotaria citrea) has shown a long-term decline in abundance in the United States. As a long-range migrant, these warblers are exposed to parasites in both tropical and temperate regions. The focus of this study was to use molecular techniques to examine the temporal prevalence patterns of heamosopridian parasites Plasmodium and Haemoproteus in breeding prothonotary warblers. The prevalence (presence or absence) of Plasmodium and Haemoproteus species was assayed using primer sets for the cytochrome b gene of the mitochondrial DNA. Blood samples were obtained from 187 adult prothonotary warblers collected at 3 central Virginia, U.S.A., breeding sites. The relationship between haemosporidian parasite infections and reproductive success also was examined. We found that 71% of captured prothonotary warblers were infected with haemosporidian parasites, specifically, with 36% prevalence for Haemoproteus spp. and 44% prevalence for Plasmodium spp., during the 2008 breeding season; for both parasites, prevalence increased throughout the season. We found significant variation in haemosporidian parasite prevalence across the breeding season that was strongly site specific. Conversely, we found no significant effects of haemosporidian parasite infections on the reproductive success of prothonotary warblers. This is in sharp contrast to recent reports suggesting considerable effects of these parasites on the reproductive success of wild birds.     

Prevalence and diversity of avian hematozoan parasites in Asia: a regional survey

Tissue samples from 699 birds from three regions of Asia (Myanmar, India, and South Korea) were screened for evidence of infection by avian parasites in the genera Plasmodium and Haemoproteus. Samples were collected from November 1994 to October 2004. We identified 241 infected birds (34.0%). Base-on-sequence data for the cytochrome b gene from 221 positive samples, 34 distinct lineages of Plasmodium, and 41 of Haemoproteus were detected. Parasite diversity was highest in Myanmar followed by India and South Korea. Parasite prevalence differed among regions but not among host families. There were four lineages of Plasmodium and one of Haemoproteus shared between Myanmar and India and only one lineage of Plasmodium shared between Myanmar and South Korea. No lineages were shared between India and South Korea, although an equal number of distinct lineages were recovered from each region. Migratory birds in South Korea and India originate from two different migratory flyways; therefore cross-transmission of parasite lineages may be less likely. India and Myanmar shared more host species and habitat types compared to South Korea. Comparison between low-elevation habitat in India and Myanmar showed a difference in prevalence of haematozoans.     

A three-genome phylogeny of malaria parasites (Plasmodium and closely related genera): evolution of life-history traits and host switches

Phylogenetic analysis of genomic data allows insights into the evolutionary history of pathogens, especially the events leading to host switching and diversification, as well as alterations of the life cycle (life-history traits). Hundreds, perhaps thousands, of malaria parasite species exploit squamate reptiles, birds, and mammals as vertebrate hosts as well as many genera of dipteran vectors, but the evolutionary and ecological events that led to this diversification and success remain unresolved. For a century, systematic parasitologists classified malaria parasites into genera based on morphology, life cycle, and vertebrate and insect host taxa. Molecular systematic studies based on single genes challenged the phylogenetic significance of these characters, but several significant nodes were not well supported. We recovered the first well resolved large phylogeny of Plasmodium and related haemosporidian parasites using sequence data for four genes from the parasites' three genomes by combining all data, correcting for variable rates of substitution by gene and site, and using both Bayesian and maximum parsimony analyses. Major clades are associated with vector shifts into different dipteran families, with other characters used in traditional parasitological studies, such as morphology and life-history traits, having variable phylogenetic significance. The common parasites of birds now placed into the genus Haemoproteus are found in two divergent clades, and the genus Plasmodium is paraphyletic with respect to Hepatocystis, a group of species with very different life history and morphology. The Plasmodium of mammal hosts form a well supported clade (including Plasmodium falciparum, the most important human malaria parasite), and this clade is associated with specialization to Anopheles mosquito vectors. The Plasmodium of birds and squamate reptiles all fall within a single clade, with evidence for repeated switching between birds and squamate hosts.