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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., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

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.

Changes in population allele frequencies may be driven by several forces, including selection and drift, and are revealed only by sampling over many generations. Such studies, however, are rare for protist parasites. Microsatellite allele frequencies for 4 loci were followed in a population of Plasmodium mexicanum, a malaria parasite of lizards in California USA at 1 site from 1978 to 2010. Rapid turnover of the lizards indicates the parasite was studied for a minimum of 33 transmission cycles and possibly twice that number. Sample sizes ranged from 841 to 956 scored parasite clones per locus. DNA was extracted from frozen dried blood and blood removed from stained blood smears from the earliest years, and a verification study demonstrated DNA from the blood smears provided valid genetic data. Parasite prevalence and effective population size (Ne) dropped after 2000, remaining lower for the next decade. For 2 loci, allele frequencies appeared stable for the first 2 decades of the study, but changed more rapidly after the decline in prevalence. Allele frequencies changed more gradually for the other 2 loci. Genetic drift could account for changes in allele frequencies, especially after the drop in prevalence and Ne, but the force of selection could also have driven the observed patterns.

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.

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.

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.

Malaria parasites in the genus Plasmodium are now placed within 11 subgenera based on morphology under the light microscope, life-history traits, and host taxon. The phylogenetic significance of these characters, however, is problematic because the observed variation could be homoplasious. Using Plasmodium infections found in 2632 birds of many avian families collected in the USA, and several samples from other locations, we compared identifications to subgenus based on morphology in blood smears with a 2-gene molecular phylogeny (the first for avian Plasmodium) to determine if the 5 avian Plasmodium subgenera represent monophyletic groups. Phylogenetic trees recovered by parsimony, likelihood, and Bayesian methods presented nearly identical topologies. The analysis allowed testing the hypothesis of monophyly for the subgenera. Monophyly of the subgenera Haemamoeba, Huffia, and Bennettinia was supported by the analysis. The distinctive morphology of Haemamoeba species appears to have evolved once. Most samples identified to Novyella also fell within a monophyletic clade with the exception of 2 samples that fell basal to all other avian Plasmodium. Samples of the subgenus Giovannolaia did not form a monophyletic group. Thus, the characters used by parasitologists for over a century to define subgenera of Plasmodium vary in their phylogenetic significance.