Variation and trade-offs in life history traits of the protist parasite Monocystis perplexa (Apicomplexa) in its earthworm host Amynthas agrestis.

The life history of a parasite describes its partitioning of assimilated resources into growth, reproduction, and transmission effort, and its precise timing of developmental events. The life cycle, in contrast, charts the sequence of morphological stages from feeding to the transmission forms. Phenotypic plasticity in life history traits can reveal how parasites confront variable environments within hosts. Within the protist phylum Apicomplexa major clades include the malaria parasites, coccidians, and most diverse, the gregarines (with likely millions of species). Studies on life history variation of gregarines are rare. Therefore, life history traits were examined for the gregarine Monocystis perplexa in its host, the invasive earthworm Amynthas agrestis at three sites in northern Vermont, United States of America. An important value of this system is the short life-span of the hosts, with only seven months from hatching to mass mortality; we were thus able to examine life history variation during the entire life cycle of both host and parasite. Earthworms were collected (N = 968 over 33 sample periods during one host season), then parasites of all life stages were counted, and sexual and transmission stages measured, for each earthworm. All traits varied substantially among individual earthworm hosts and across the sites. Across sites, timing of first appearance of infected earthworms, date when transmission stage (oocysts packed within gametocysts) appeared, date when number of both feeding (trophic) cells and gametocysts were at maximum, and date when 100% of earthworms were infected differed from 2-8 weeks, surprising variation for a short season available for parasite development. The maximal size of mating cells varied among hosts and across sites and this is reflected in the number of oocysts produced by the gametocyst. A negative trade-off was observed for the number of oocysts and their size. Several patterns were striking: (1) Prevalence reached 100% at all sites by mid season, only one to three weeks after parasites first appeared in the earthworms. (2) The number of parasites per host was large, reaching 300 × 103 cells in some hosts, and such high numbers were present even when parasites first appeared in the host. (3) At one site, few infected earthworms produced any oocysts. (4) The transmission rate to reach such high density of parasites in hosts needed to be very high for a microbe, from >0.33% to >34.3% across the three sites. Monocystis was one of the first protist parasites to have its life cycle described (early 19th century), but these results suggest the long-accepted life cycle of Monocystis could be incomplete, such that the parasites may be transmitted vertically (within the earthworm's eggs) as well as horizontally (leading to 100% prevalence) and merogony (asexual replication) could be present, not recognized for Monocystis, leading to high parasitemia even very early in the host's season.

APOLOCYSTIS BOSANQUETI N. SP. (APICOMPLEXA: EUGREGARINORIDA) FROM THE INVASIVE EARTHWORM AMYNTHAS AGRESTIS (ANNELIDA: MEGASCOLECIDAE), WITH SIGNIFICANCE FOR THE MONOPHYLY OF THE FAMILY MONOCYSTIDAE.

Apolocystis bosanqueti n. sp., a parasite of an important invasive earthworm in North America, Amynthas agrestis, is described from a site in northern Vermont. The earthworm host follows an annual life cycle in Vermont, so the entire life cycle of the parasite can be observed in 7 mo. In spring, the parasites were first seen in juvenile worms as paired gamonts (suggesting precocious association). These paired gamonts mature into gametocytes that form an opaque structure, with a thick gelatinous envelope (epicyst), that becomes full of zygotes. The resulting gametocyst becomes packed with ∼105 fusiform oocysts. The mature orbicular gametocysts are large (∼1 mm in diameter) and visible to the naked eye through the body wall of the host's anterior segments. The new species most resembles Apolocystis herculea described from many lumbricid earthworm species in Europe but differs from that parasite because Ap. herculea infects the intestinal wall in the posterior of the host rather than the anterior segments. A survey of 9 other earthworm species sympatric with Am. agrestis revealed that only Amynthas tokioensis, also an invasive species, was infected with Ap. bosanqueti, albeit much less commonly. Diagnosis for the family Monocystidae is problematic because cardinal characters are lacking, and the commonly cited character, a trophozoite with no anterior differentiation, is violated in most genera placed in the family. For the first time, a molecular phylogeny is presented that includes 3 genera of monocystids with diverse cell morphology (including the new species) and supports the monophyly of the family. The only morphological character that may be used to diagnose the Monocystidae is the morphology of oocysts, which are fusiform with extended terminal tips. A comparison of oocysts from 7 parasites recovered from local earthworms, including from 3 monocystid species in the phylogeny, confirms the utility of this diagnostic trait. The 2 hosts of the new species were most likely introduced from Japan, so the range of Apolocystis likely extends into East Asia.

Genetic population structure and reproductive system of two invasive Asian earthworms, Amynthas tokioensis and Amynthas agrestis.

The invasive Asian earthworms, Amynthas tokioensis and A. agrestis, have been successful in entering North American forests in recent decades, with significant damage to both soils and above-ground environments. This success could be driven in part by a polyploid genetic system and parthenogenetic reproduction, often suggested as benefits for invasive species. Therefore, we assessed the genetic population structure, genetic diversity, and reproductive system of both species using morphological traits and panels of microsatellite markers. A total of 216 A. tokioensis and 196 A. agrestis from six sites in Vermont USA were analyzed. Although all worms were morphologically hermaphroditic, all the A. agrestis lacked the male pore (the structure allowing pass of sperm between individuals), and only 19% of the A. tokioensis possessed the male pore. All A. tokioensis earthworms were triploid (scored for three alleles for at least 1 locus, and usually several), and A. agrestis was a mix of triploid and diploid individuals. Notable was the high proportion (80%) of A. agrestis earthworms that were diploid at one site. There was clearly clonal reproduction, with identical seven- locus genotypes observed for earthworms from each site, with as many as 45 individuals with the identical genotype at one site. However, the earthworms were also genetically diverse, with 14 genotypes observed for A. tokioensis and 54 for A. agrestis, and with many singleton genotypes (a single individual). Most genotypes (71% for A. tokioensis and 92% for A. agrestis) were found at a single site. The greatest number of genotypes was found at a commercial nursery where fully 23/26 A. agrestis earthworms were singleton genotypes. As expected for the pattern of private clone alleles at sites, several measures of geographic genetic differentiation were positive, and as expected for triploid systems, an AMOVA analysis showed high within-individual genetic diversity. The paradox of clear clonal reproduction, but with a great number of genotypes for each species, and the mix of triploid and diploid individuals could be explained if the worms have been sexually reproductive, with the switch to the uniparental system only recently (or even if sexual reproduction is episodic). Last, a large number of microsatellite loci were recovered for each species and there sequence and suggested PCR primers are provided for free use by other researchers.

A New Species of Monocystis (Apicomplexa: Gregarina: Monocystidae) from the Asian Invasive Earthworm Amynthas agrestis (Megascolecidae), with an Improved Standard for Monocystis Species Descriptions.

Monocystis perplexa n. sp., a parasite of an important invasive Japanese earthworm in North America, Amynthas agrestis, is described from a site in Vermont. An improved standard for Monocystis species descriptions is proposed including a standard nomenclature to reduce synonymies, a standard set of biometrics and shape descriptions for living cells, and a DNA genomic sequence for the 18S rRNA (∼1,700 base pairs). Comparing morphologies of Monocystis parasites in sympatric earthworm species indicates that M. perplexa is specific to A. agrestis in the study region. Also, polymerase chain reaction primers specific to M. perplexa amplified samples of A. agrestis earthworms taken from several sites in Japan. This suggests the parasite entered North America from Japan, the origin of the invasive Amynthas earthworm, and thus M. perplexa would be the first Monocystis described from the diverse Japanese Amynthas earthworms and the first from East Asia. Monocystis perplexa was found in every population of A. agrestis surveyed in Vermont, always reaching 100% prevalence by late summer (the host has an annual life cycle in Vermont). The 18S gene sequence differed from that of Monocystis agilis from the sympatric earthworm Lumbricus terrestris (the only other sequence available for Monocystis), and a genetic similarity tree places them closest among other gregarines. Many of the 95 described species of Monocystis are very similar in morphology (based on species descriptions), so the 18S gene can act as a barcode for Monocystis species and thus will help to eliminate both synonymies and reveal cryptic species.

The drivers and consequences of unstable Plasmodium dynamics: a long-term study of three malaria parasite species infecting a tropical lizard.

Understanding the consequences of environmental fluctuations for parasite dynamics requires a long-term view stretching over many transmission cycles. Here we studied the dynamics of three malaria parasites (Plasmodium azurophilum, P. leucocytica and P. floridense) infecting the lizard Anolis gundlachi, in the rainforest of Puerto Rico. In this malaria-anole system we evaluated temporal fluctuations in individual probability of infection, the environmental drivers of observed variation and consequences for host body condition and Plasmodium parasites assemblage. We conducted a total of 15 surveys including 10 from 1990 to 2002 and five from 2015 to 2017. During the early years, a lizard's probability of infection by all Plasmodium species appeared stable despite disturbances ranging from two hurricanes to short droughts. Over a longer timescale, probability of infection and overall prevalence varied significantly, following non-linear relationships with temperature and rainfall such that highest prevalence is expected at intermediate climate measures. A perplexing result was that host body condition was maximized at intermediate levels of rainfall and/or temperature (when risk of infection was highest), yet we found no significant decreases in body condition due to infection. Plasmodium parasite species composition varied through time with a reduction and near local extinction of P. floridense. Our results emphasize the need for long-term studies to reveal host-parasite dynamics, their drivers and consequences.

Genetic differentiation over a small spatial scale of the sand fly Lutzomyia vexator (Diptera: Psychodidae).

BACKGROUND: The geographic scale and degree of genetic differentiation for arthropod vectors that transmit parasites play an important role in the distribution, prevalence and coevolution of pathogens of human and wildlife significance. We determined the genetic diversity and population structure of the sand fly Lutzomyia vexator over spatial scales from 0.56 to 3.79 km at a study region in northern California. The study was provoked by observations of differentiation at fine spatial scales of a lizard malaria parasite vectored by Lu. vexator. METHODS: A microsatellite enrichment/next-generation sequencing protocol was used to identify variable microsatellite loci within the genome of Lu. vexator. Alleles present at these loci were examined in four populations of Lu. vexator in Hopland, CA. Population differentiation was assessed using Fst and D (of Cavalli-Sforza and Edwards), and the program Structure was used to determine the degree of subdivision present. The effective population size for the sand fly populations was also calculated. RESULTS: Eight microsatellite markers were characterized and revealed high genetic diversity (uHe = 0.79-0.92, Na = 12-24) and slight but significant differentiation across the fine spatial scale examined (average pairwise D = 0.327; F ST = 0.0185 (95 % bootstrapped CI: 0.0102-0.0264). Even though the insects are difficult to capture using standard methods, the estimated population size was thousands per local site. CONCLUSIONS: The results argue that Lu. vexator at the study sites are abundant and not highly mobile, which may influence the overall transmission dynamics of the lizard malaria parasite, Plasmodium mexicanum, and other parasites transmitted by this species.

Dynamics of clonal diversity in natural infections of the malaria parasite Plasmodium mexicanum in its free-ranging lizard host.

Within mixed-genotype infections of malaria parasites (Plasmodium), the number of genetic clones present is associated with variation in important life history traits of the infection, including virulence. Although the number of clones present is important, how the proportion of those clones varies over time is poorly known. Clonal proportions of the lizard malaria parasite, Plasmodium mexicanum, were assessed in naturally infected free-ranging lizards followed in a mark-recapture program over as long as two warm seasons, the typical life span of the lizard. Clonal proportions were determined by amplifying two microsatellite markers, a method previously verified for accuracy. Most blood samples had been stored for over a decade, so a verification test determined that these samples had not degraded. Although the environment experienced by the parasite (its host) varies over the seasons and transmission occurs over the entire warm season, 68% of infections were stable over time, harboring a single clone (37% of infections) or multiple clones changing only 1-12% maximum comparing any two samples (31% of infections). The maximum change seen in any infection (comparing any two sample periods) was only 30%. A new clone entered three infections (only once successfully), and a clone was lost in only three infections. These results mirror those seen for a previous study of experimentally induced infections that showed little change in relative proportions over time. The results of this study, the first look at how clonal proportions vary over time for any malaria parasite of a nonhuman vertebrate host for natural infections, were surprising because experimental studies show clones of P. mexicanum appear to interact, yet relative proportions of clones typically remain constant over time.

Testing sex ratio theory with the malaria parasite Plasmodium mexicanum in natural and experimental infections.

The malaria parasite (Plasmodium) life history accords well with the assumptions of local mate competition (LMC) of sex ratio theory. Within a single meal of the blood-feeding vector, sexually dimorphic gametocyte cells produce gametes (females produce one, males several) that mate and undergo sexual recombination. The theory posits several factors drive the Plasmodium sex ratio: male fecundity (gametes/male gametocyte), number and relative abundance of parasite clones, and gametocyte density. We measured these traits for the lizard malaria parasite, Plasmodium mexicanum, with a large sample of natural infections and infections from experiments that manipulated clonal diversity. Sex ratio in single-clone infections was slightly female-biased, but matched predictions of theory for this low-fecundity species. Sex ratio was less female-biased in clonally diverse infections as predicted by LMC for the experimental, but not natural infections. Gametocyte density was not positively related to sex ratio. These results are explained by the P. mexicanum life history of naturally low clonal diversity and high gametocyte production. This is the first study of a natural malaria system that examines all traits relevant to LMC in individual vertebrate hosts and suggests a striking example of sex ratio theory having significance for human public health.

Establishment efficiency among clones of the malaria parasite, Plasmodium mexicanum, for mixed-clone infections in its natural lizard host.

Within genetically diverse infections of malaria parasites ( Plasmodium spp.), the relative proportions of genetic clones in the vertebrate host's blood can influence clonal competition, transmission success, gametocyte sex ratio, and virulence. Clonal proportions depend on establishment success of each clone when they enter a new host and on subsequent differences in rates of asexual replication and clearance. Both of these life history traits could be influenced by clone genotype. To assess genetic (clonal) influences on both establishment success and later changes in relative proportion for the lizard malaria parasite Plasmodium mexicanum , 7 naturally infected fence lizards harboring a single clone of P. mexicanum served as donors to initiate replicate experimental infections containing each of the clones and combinations of 2 clones. Measured were relative establishment success of each clone, change in relative proportions over time, and rate of increase of parasite density and total parasitemia. Relative clonal proportions were determined using microsatellite markers. Rates of increase in the parasitemia and degree of change in relative proportions were not correlated, so both rapidly and slowly growing infections could show either little or substantial change in clonal proportions over time. There was a significant clone effect on establishment efficiency but not on later changes in relative proportions. These results argue for a combination of genetic and environmental (host) effects on the success of P. mexicanum clones in genetically complex infections. The maintenance of genetic variation for establishment success, but not subsequent replication rate or shifts in relative proportion, suggests trade-offs between these traits during life history evolution of malaria parasites.

Relative clonal proportions over time in mixed-genotype infections of the lizard malaria parasite Plasmodium mexicanum.

Vertebrate hosts of malaria parasites (Plasmodium) often harbour two or more genetically distinct clones of a single species, and interaction among these co-existing clones can play an important role in Plasmodium biology. However, how relative clonal proportions vary over time in a host is still poorly known. Experimental mixed-clone infections of the lizard malaria parasite, Plasmodium mexicanum, were followed in its natural host, the western fence lizard using microsatellite markers to determine the relative proportions of two to five co-existing clones over time (2-3 months). Results for two markers, and two PCR primer pairs for one of those, matched very closely, supporting the efficacy of the method. Of the 54 infections, 67% displayed stable relative clonal proportions, with the others showing a shift in proportions, usually with one clone outpacing the others. Infections with rapidly increasing or slowly increasing parasitemia were stable, showing that all clones within these infections reproduced at the same rapid or slow rate. Replicate infections containing the same clones did not always reveal the same growth rate, final parasitemia or dominant clone; thus there was no clone effect for these life history measures. The rate of increase in parasitemia was not associated with stable versus unstable relative proportions, but infections with four to five clones were more likely to be unstable than those with two to three clones. This rare look into events in genetically complex Plasmodium infections suggests that parasite clones may be interacting in complex and unexpected ways.

Virulence of a malaria parasite, Plasmodium mexicanum, for its sand fly vectors, Lutzomyia vexator and Lutzomyia stewarti (Diptera: Psychodidae).

Evolutionary theory predicts that virulence of parasites for mobile vector insects will be low for natural parasite-host associations that have coevolved. I determined virulence of the malaria parasite of lizards, Plasmodium mexicanum, for its vectors, two species of sand fly (Diptera: Psychodidae), Lutzomyia vexator (Coquillett 1907) and Lutzomyia stewarti (Mangabeira Fo & Galindo 1944), by measuring several life history traits. Developmental rate from egg to eclosion differed for the two species when noninfected. For both sand fly species, developmental rate for each stage (egg to larval hatching, larval period, pupal period) and life span were not altered by infection. Infected sand flies, however, produced fewer eggs. This reduction in fecundity may be a result of lower quality of the blood meal taken from infected lizards (lower concentration of hemoglobin). This report is the first measure of virulence of Plasmodium for an insect vector other than a mosquito and concords with both expectations of theory and previous studies on natural parasite-host associations that revealed low virulence.

Relative clonal density of malaria parasites in mixed-genotype infections: validation of a technique using microsatellite markers for Plasmodium falciparum and P. mexicanum.

Quantifying the relative proportion of coexisting genotypes (clones) of a malaria parasite within its vertebrate host's blood would provide insights into critical features of the biology of the parasite, including competition among clones, gametocyte sex ratio, and virulence. However, no technique has been available to extract such data for natural parasite-host systems when the number of clones cycling in the overall parasite population is likely to be large. Recent studies find that data from genetic analyzer instruments for microsatellite markers allow measuring clonal proportions. We conducted a validation study for Plasmodium mexicanum and Plasmodium falciparum by mixing DNA from single-clone infections to simulate mixed infections of each species with known proportions of clones. Results for any mixture of DNA gave highly reproducible results. The relationship between known and measured relative proportions of clones was linear, with high regression r² values. Known and measured clone proportions for simulated infections followed over time (mixtures) were compared with 3 methods: using uncorrected data, with uncorrected data and confidence intervals constructed from observed experimental error, and using a baseline mixture of equal proportions to calibrate all other results. All 3 methods demonstrated value in studies of mixed-genotype infections sampled a single time or followed over time. Thus, the method should open new windows into the biology of malaria parasites.

Lack of sequence variation of the mitochondrial cytochrome b gene from a malaria parasite, Plasmodium mexicanum.

Very slight sequence differences in the mitochondrial cytochrome b gene, even single nucleotide substitutions, have been proposed as indicative of different species of avian malaria parasites. However, few studies have examined within-species variation in that gene for Plasmodium or related genera. We examined sequences for the entire cytochrome b gene from Plasmodium mexicanum , a parasite of lizards, for sites where microsatellite markers revealed substantial genetic diversity. For sites where the parasite is geographically genetically differentiated, and may have been isolated for thousands of years, there was no sequence variation (1,153 nucleotides) for >160 infections studied. The low degree of variation found in the cytochrome b gene for two human malaria parasites world-wide, as well as the lack of variation for P. mexicanum , contrast with the substantial variation found in surveys of bird malaria parasites, even in restricted geographic regions.

Geographic genetic differentiation of a malaria parasite, Plasmodium mexicanum, and its lizard host, Sceloporus occidentalis.

Gene flow, and resulting degree of genetic differentiation among populations, will shape geographic genetic patterns and possibly local adaptation of parasites and their hosts. Some studies of Plasmodium falciparum in humans show substantial differentiation of the parasite in locations separated by only a few kilometers, a paradoxical finding for a parasite in a large, mobile host. We examined genetic differentiation of the malaria parasite Plasmodium mexicanum, and its lizard host, Sceloporus occidentalis, at 8 sites in northern California, with the use of variable microsatellite markers for both species. These lizards are small and highly territorial, so we expected local genetic differentiation of both parasite and lizard. Populations of P. mexicanum were found to be differentiated by analysis of 5 markers (F(st) values >0.05-0.10) over distances as short as 230-400 m, and greatly differentiated (F(st) values >0.25) for sites separated by approximately 10 km. In contrast, the lizard host had no, or very low, levels of differentiation for 3 markers, even for sites >40 km distant. Thus, gene flow for the lizard was great, but despite the mobility of the vertebrate host, the parasite was locally genetically distinct. This discrepancy could result if infected lizards move little, but their noninfected relatives were more mobile. Previous studies on the virulence of P. mexicanum for fence lizards support this hypothesis. However, changing prevalence of the parasite, without changes in density of the lizard, could also result in this pattern.

Detecting number of clones, and their relative abundance, of a malaria parasite (Plasmodium mexicanum) infecting its vertebrate host.

Microsatellites, short tandem repeats of nucleotides in the genome, are useful markers to detect clonal diversity within Plasmodium infections. However, accuracy in determining number of clones and their relative proportions based on standard genetic analyzer instruments is poorly known. DNA extracted from lizards infected with a malaria parasite, Plasmodium mexicanum, provided template to genotype the parasite based on three microsatellite markers. Replicate genotyping of the same natural infections demonstrated strong repeatability of data from the instrument. Mixing DNA extracted from several infected lizards simulated mixed-clone infections with known clonal diversity and relative proportions of clones (N = 56 simulations). The instrument readily detected at least four alleles (clones), even when DNA concentrations among clones differed up to tenfold, but alleles of similar size can be missed because they fall within the "stutter" artifact, and rarely does an allele fail to be detected. For simulations of infections that changed their relative proportions over time, changes in relative peak heights on the instrument output closely followed the known changes in relative proportions. Such data are useful for a broad range of studies on the ecology of malaria parasites.

Clonal diversity alters the infection dynamics of a malaria parasite (Plasmodium mexicanum) in its vertebrate host.

Ecological and evolutionary theory predicts that genetic diversity of microparasites within infected hosts will influence the parasite replication rate, parasitemia, transmission strategy, and virulence. We manipulated clonal diversity (number of genotypes) of the malaria parasite, Plasmodium mexicanum, in its natural lizard host and measured important features of the infection dynamics, the first such study for any natural Plasmodium-host association. Hosts harboring either a single P. mexicanum clone or various combinations of clones (scored via three microsatellite markers) were established. Production of asexually replicating stages (meronts) and maximal meront parasitemia did not differ by clonal diversity, nor did timing of first production of transmission stages (gametocytes). However, mean rate of gametocyte increase and maximal gametocyte parasitemia were greater for hosts with mixed-clone infections. Characteristics of infections were more variable in hosts with mixed-clone infections than with single-clone infections except for first production of gametocytes. One or more of the parasite reproductive traits were extreme in 20 of 52 hosts with mixed-clone infections. This was not associated with specific clones, but diversity itself. The overall pattern from studies of clonal diversity for human, rodent, and now reptile malaria parasites confirms that the genetic diversity of infections in the vertebrate host is of central importance for the ecology of Plasmodium.

Do malaria parasites follow the algebra of sex ratio theory?

The ratio of male to female gametocytes seen in infections of Plasmodium and related haemosporidian parasites varies substantially, both within and among parasite species. Sex ratio theory, a mainstay of evolutionary biology, accounts for this variation. The theory provides an algebraic solution for the optimal sex ratio that will maximize parasite fitness. A crucial term in this solution is the probability of selfing by clone-mates within the vector (based on the clone number and their relative abundance). Definitive tests of the theory have proven elusive because of technical challenges in measuring clonal diversity within infections. Newly developed molecular methods now provide opportunities to test the theory with an exquisite precision.

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.

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.