Date of Award

January 2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Vasyl V. Tkach

Abstract

Birds possess the most diverse assemblage of haemosporidian parasites, including three genera, Plasmodium, Haemoproteus, and Leucocytozoon. Currently there are over 200 morphologically identified avian haemosporidian species, although the true diversity is unknown, due to high genetic diversity and insufficient sampling in highly diverse regions, such as the Neotropics. Brazil, specifically the Brazilian Amazon supports the world’s highest avian diversity and expected equally diverse yet undescribed community of avian haemosporidians. This study includes the largest sampling of avian haemosporidians in Brazil, and the first large scale survey of the Brazilian Amazon. A total of 4521 blood samples were collected from 447 host species, from 17 host orders and 49 host families. Samples were collected from five distinct Brazilian biomes, Amazonia (3381 samples), Atlantic Forest (39 samples), Caatinga (185 samples), Cerrado (790 samples), and Pantanal (126 samples). I developed a new real-time PCR assay to screen such large numbers of blood samples for the presence of avian haemosporidians. A 182 bp region of the conserved rDNA genes of avian haemosporidians was amplified. The real-time PCR assay proved as reliable as the two most widely used molecular screening methods, but has the additional benefit of screening for all three genera in a single reaction, saving time and expense. From positive samples a portion of the cytochrome b gene was amplified using two modified sets of nested PCR primers. One set amplified Haemoproteus/Plasmodium together and the

second set amplified Leucocytozoon. Sanger sequencing data was used to identify haemosporidian lineages for phylogenetic analysis. Of the 4521 samples screened, 730 were infected (16% prevalence) with Haemoproteus or Plasmodium. Due to expected low prevalence of Leucocytozoon, I attempted nested PCR amplification for only a subset of 1000 samples, and found no Leucocytozoon infections. More than three times as many blood samples were infected with Plasmodium (574 positive samples) than Haemoproteus (178 positive samples). These infections included individuals with coinfections of two lineages of Haemoproteus, two lineages of Plasmodium, or lineages of both Haemoproteus and Plasmodium. Haemosporidian prevalence differed between Brazilian biomes and avian host families. Haemosporidian diversity matched host diversity with 365 genetic lineages recovered, 86 Haemoproteus and 279 Plasmodium. More than 90% of these lineages (331) were novel lineages, never before described. The high number of novel lineages recovered from Brazil increases the known diversity of haemosporidian genetic lineages by 15 percent. An alignment containing these 365 newly discovered Brazilian lineages combined with all quality lineages from the MalAvi database was used for phylogenetic reconstruction. The Bayesian inference phylogeny produced showed a pattern of repeated lineage introduction into Brazil followed by diversification into unique lineages, endemic to Brazil. In the Amazonian biome, samples were collected from six distinct areas of avian endemism; Belém (323 samples), Guiana (353 samples), Imerí (164 samples), Inambari (1437 samples), Rondônia (1004 samples), and Tapajόs (100 samples). The areas of endemism in Amazonia directly affected haemosporidian parasite diversity and distribution. Infection prevalence varied significantly between areas of endemism, with higher prevalence south of the Amazon River. Compositional analysis on avian and parasite communities showed that parasite communities differ between areas of endemism and is attributed to differences in host communities. Areas of endemism with more similar host communities supported more similar parasite communities as well. Individual areas of endemism supported genetically more similar parasite communities, with a significant portion of genetic variation partitioned among areas of endemism. Haemosporidians are known to track host distribution, and analysis of genetic variation analysis showed that individual host families were infected by genetic lineages that were more genetically similar. Although area of endemism did not produce a significant phylogenetic signal in either Haemoproteus or Plasmodium, S-DIVA analysis did show a phylogeographic structuring in both genera, with the existence of area of endemism specific clades. This was especially true for Haemoproteus, where many lineages were concentrated within a Rondônia specific clade. The overall phylogeographic pattern was weaker for Plasmodium, but for several lineages area of endemism did appear to have phylogenetic signal. For Haemoproteus and Plasmodium, dispersal between areas of endemism was the most important event in their evolutionary history, likely due to lineages dispersing between avian hosts. Analysis of the effect of four host life history characteristics (nest height, nest type, foraging height, flocking behavior) on haemosporidian parasitism showed area of endemism as the only predictive variable when all samples were analyzed together. Only when each area of endemism was analyzed separately was host life history variation found to predict infection probability, although differing between areas of endemism. For Haemoproteus, nest height (Guiana, Rondônia), foraging height (Tapajόs), and flocking (Belém) were found to significantly predict the probability of infection, whereas for Plasmodium nest type (Inambari), foraging height (Guiana, Imerí) and flocking (Belém) were significant predictors. Host phylogenetic constraints on haemosporidian parasitism varied between areas of endemism. The 48 genetic lineages recovered from the Belém area of endemism were used for coevolutionary analysis of haemosporidian parasites and their avian hosts. Cost-event analysis showed that host switching was the most important event in the evolutionary history of haemosporidian parasites from the Belém area of endemism. Global cospeciation analysis showed a significant cospeciation signal between haemosporidian parasites and their avian hosts. The cospeciation signal was mostly due to strong coevolutionary links between Haemoproteus parasites and their non-passerine hosts. However, some Plasmodium lineages did show strong coevolutionary links with their passerine hosts, which contradicts what is known of the evolutionary history of avian Plasmodium parasites. Cospeciation analysis supports the presence of unique coevolutionary relationships between some haemosporidian parasites and their avian hosts. Along with rampant host switching, cospeciation has played a role in the highly diverse community of avian haemosporidians within Amazonian and throughout Brazil. Brazil supports a unique and diverse haemosporidian community, much of it contained within Amazonia, where unique biogeography has shaped the diversification and distribution of these parasites. The role of vectors is this region is unknown, since basic information on vector biology is lacking. Research is needed to determine the role that vectors have played in the distribution, and diversity of haemosporidian parasites within Amazonia and throughout Brazil.

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