Contrasting drivers of diversity in hosts and parasites across the tropical Andes.

Geographic turnover in community composition is created and maintained by eco-evolutionary forces that limit the ranges of species. One such force may be antagonistic interactions among hosts and parasites, but its general importance is unknown. Understanding the processes that underpin turnover requires distinguishing the contributions of key abiotic and biotic drivers over a range of spatial and temporal scales. Here, we address these challenges using flexible, nonlinear models to identify the factors that underlie richness (alpha diversity) and turnover (beta diversity) patterns of interacting host and parasite communities in a global biodiversity hot spot. We sampled 18 communities in the Peruvian Andes, encompassing ∼1,350 bird species and ∼400 hemosporidian parasite lineages, and spanning broad ranges of elevation, climate, primary productivity, and species richness. Turnover in both parasite and host communities was most strongly predicted by variation in precipitation, but secondary predictors differed between parasites and hosts, and between contemporary and phylogenetic timescales. Host communities shaped parasite diversity patterns, but there was little evidence for reciprocal effects. The results for parasite communities contradicted the prevailing view that biotic interactions filter communities at local scales while environmental filtering and dispersal barriers shape regional communities. Rather, subtle differences in precipitation had strong, fine-scale effects on parasite turnover while host-community effects only manifested at broad scales. We used these models to map bird and parasite turnover onto the ecological gradients of the Andean landscape, illustrating beta-diversity hot spots and their mechanistic underpinnings.

Coinfection rates of avian blood parasites increase with latitude in parapatric host species.

Animals are frequently coinfected with multiple parasites concurrently, and advances in our sampling of these complex intra-host parasite communities have revealed important ecological impacts on their hosts. However, the spatial distributions and environmental determinants of parasite coinfection remain infrequently studied. Here, we investigated the drivers of haemosporidian blood parasite coinfection in the Bicknell's thrush (Catharus bicknelli) and grey-cheeked thrush (Catharus minimus), parapatric sister species that occur across a broad latitudinal range in northern North America. Using 298 samples from across the distributions of these species, we found high overall infection (86%) and coinfection (41%) rates within host populations. Coinfection rates within populations were highly variable across sampling sites, ranging from 7 to 75%. Latitude was a more important predictor of coinfection frequency than host species identity, with coinfections becoming more abundant at higher latitudes. The 2 host species exhibited similar parasite faunas, and an analysis of the co-occurrence patterns among haemosporidians showed that host species identity was largely not a factor in structuring which parasites were found within coinfections. To our knowledge, this is the first study to illustrate a reverse latitudinal gradient in coinfection frequency in a eukaryotic parasite system. Further work is necessary to determine whether vector ecology or some other factor is the primary proximate driver of this pattern.

Parasite-associated mortality in birds: the roles of specialist parasites and host evolutionary distance.

The factors that influence whether a parasite is likely to cause death in a given host species are not well known. Generalist parasites with high local abundances, broad distributions and the ability to infect a wide phylogenetic diversity of hosts are often considered especially dangerous for host populations, though comparatively little research has been done on the potential for specialist parasites to cause host mortality. Here, using a novel database of avian mortality records, we tested whether phylogenetic host specialist or host generalist haemosporidian blood parasites were associated with avian host deaths based on infection records from over 81 000 examined hosts. In support of the hypothesis that host specialist parasites can be highly virulent in novel hosts, we found that the parasites that were associated with avian host mortality predominantly infected more closely related host species than expected under a null model. Hosts that died tended to be distantly related to the host species that a parasite lineage typically infects, illustrating that specialist parasites can cause death outside of their limited host range. Overall, this study highlights the overlooked potential for host specialist parasites to cause host mortality despite their constrained ecological niches.

Detecting turnover among complex communities using null models: a case study with sky-island haemosporidian parasites.

Turnover in species composition between sites, or beta diversity, is a critical component of species diversity that is typically influenced by geography, environment, and biotic interactions. Quantifying turnover is particularly challenging, however, in multi-host, multi-parasite assemblages where undersampling is unavoidable, resulting in inflated estimates of turnover and uncertainty about its spatial scale. We developed and implemented a framework using null models to test for community turnover in avian haemosporidian communities of three sky islands in the southwestern United States. We screened 776 birds for haemosporidian parasites from three genera (Parahaemoproteus, Plasmodium, and Leucocytozoon) by amplifying and sequencing a mitochondrial DNA barcode. We detected infections in 280 birds (36.1%), sequenced 357 infections, and found a total of 99 parasite haplotypes. When compared to communities simulated from a regional pool, we observed more unique, single-mountain haplotypes and fewer haplotypes shared among three mountain ranges than expected, indicating that haemosporidian communities differ to some degree among adjacent mountain ranges. These results were robust even after pruning datasets to include only identical sets of host species, and they were consistent for two of the three haemosporidian genera. The two more distant mountain ranges were more similar to each other than the one located centrally, suggesting that the differences we detected were due to stochastic colonization-extirpation dynamics. These results demonstrate that avian haemosporidian communities of temperate-zone forests differ on relatively fine spatial scales between adjacent sky islands. Null models are essential tools for testing the spatial scale of turnover in complex, undersampled, and poorly known systems.

An inverse latitudinal gradient in infection probability and phylogenetic diversity for Leucocytozoon blood parasites in New World birds.

Geographic variation in environmental conditions as well as host traits that promote parasite transmission may impact infection rates and community assembly of vector-transmitted parasites. Identifying the ecological, environmental and historical determinants of parasite distributions and diversity is therefore necessary to understand disease outbreaks under changing environments. Here, we identified the predictors and contributions of infection probability and phylogenetic diversity of Leucocytozoon (an avian blood parasite) at site and species levels across the New World. To explore spatial patterns in infection probability and lineage diversity for Leucocytozoon parasites, we surveyed 69 bird communities from Alaska to Patagonia. Using phylogenetic Bayesian hierarchical models and high-resolution satellite remote-sensing data, we determined the relative influence of climate, landscape, geography and host phylogeny on regional parasite community assembly. Infection rates and parasite diversity exhibited considerable variation across regions in the Americas. In opposition to the latitudinal gradient hypothesis, both the diversity and prevalence of Leucocytozoon parasites decreased towards the equator. Host relatedness and traits known to promote vector exposure neither predicted infection probability nor parasite diversity. Instead, the probability of a bird being infected with Leucocytozoon increased with increasing vegetation cover (NDVI) and moisture levels (NDWI), whereas the diversity of parasite lineages decreased with increasing NDVI. Infection rates and parasite diversity also tended to be higher in cooler regions and higher latitudes. Whereas temperature partially constrains Leucocytozoon diversity and infection rates, landscape features, such as vegetation cover and water body availability, play a significant role in modulating the probability of a bird being infected. This suggests that, for Leucocytozoon, the barriers to host shifting and parasite host range expansion are jointly determined by environmental filtering and landscape, but not by host phylogeny. Our results show that integrating host traits, host ancestry, bioclimatic data and microhabitat characteristics that are important for vector reproduction are imperative to understand and predict infection prevalence and diversity of vector-transmitted parasites. Unlike other vector-transmitted diseases, our results show that Leucocytozoon diversity and prevalence will likely decrease with warming temperatures.

Deeply conserved susceptibility in a multi-host, multi-parasite system.

Variation in susceptibility is ubiquitous in multi-host, multi-parasite assemblages, and can have profound implications for ecology and evolution in these systems. The extent to which susceptibility to parasites is phylogenetically conserved among hosts can be revealed by analysing diverse regional communities. We screened for haemosporidian parasites in 3983 birds representing 40 families and 523 species, spanning ~ 4500 m elevation in the tropical Andes. To quantify the influence of host phylogeny on infection status, we applied Bayesian phylogenetic multilevel models that included a suite of environmental, spatial, temporal, life history and ecological predictors. We found evidence of deeply conserved susceptibility across the avian tree; host phylogeny explained substantial variation in infection status, and results were robust to phylogenetic uncertainty. Our study suggests that susceptibility is governed, in part, by conserved, latent aspects of anti-parasite defence. This demonstrates the importance of deep phylogeny for understanding present-day ecological interactions.

Evolutionary lability of host associations promotes phylogenetic overdispersion of co-infecting blood parasites.

Co-infections with multiple parasite taxa are ubiquitous in nature and have the potential to impact the co-evolutionary dynamics between host and parasite, though patterns of phylogenetic community structure of co-infecting parasites and the processes that generate these patterns have rarely been studied across diverse host-parasite communities. Here, we tested for the roles of host and parasite evolutionary history as well as environmental variables as drivers of phylogenetic community structure among co-infecting haemosporidian (malaria) parasites and their avian hosts in the North American boreal forest, a region characterized by an extraordinarily high blood parasite co-infection rate. We used multiple methods to identify non-random patterns of co-infection among parasite species and determined whether these patterns were influenced more by co-evolutionary host associations or environmental variables. We used model-based approaches to test whether parasites that occurred together in a single host individual exhibited phylogenetic clustering or overdispersion. Lastly, we tested whether the observed phylogenetic community structure could be explained by parasites having convergently evolved similar patterns of host associations. We found that haemosporidian parasite co-infections occurred at a high frequency in the boreal forest system and that parasite taxa co-occurred in significantly non-random patterns within host individuals and among host species. Parasite taxa that occurred in co-infections tended to be phylogenetically overdispersed. We show that this pattern of phylogenetic overdispersion can be attributed largely to the effect of evolutionarily labile, convergent host associations that have resulted in the pool of parasites that have the potential to infect a given host consisting nearly exclusively of distantly related lineages. Our findings illustrate that environmental filtering of convergent traits can produce phylogenetically overdispersed communities, even at the level of co-infecting parasites within an individual host organism. Broadly, this analysis illustrates how co-evolutionary history can have a strong influence on the modern phylogenetic community assembly of diverse host-symbiont communities.

Integrating coalescent species delimitation with analysis of host specificity reveals extensive cryptic diversity despite minimal mitochondrial divergence in the malaria parasite genus Leucocytozoon.

BACKGROUND: Coalescent methods that use multi-locus sequence data are powerful tools for identifying putatively reproductively isolated lineages, though this approach has rarely been used for the study of microbial groups that are likely to harbor many unrecognized species. Among microbial symbionts, integrating genetic species delimitation methods with trait data that could indicate reproductive isolation, such as host specificity data, has rarely been used despite its potential to inform species limits. Here we test the ability of an integrative approach combining genetic and host specificity data to delimit species within the avian malaria parasite genus Leucocytozoon in central Alaska. RESULTS: We sequenced seven nuclear loci for 69 Leucocytozoon samples and used multiple species delimitation methods (GMYC and BPP models), tested for differences in host infection patterns among putative species based on 406 individual infections, and characterized parasite morphology. We found that cryptic morphology has masked a highly diverse Leucocytozoon assemblage, with most species delimitation methods recovering support for at least 21 separate species that occur sympatrically and have divergent host infection patterns. Reproductive isolation among putative species appears to have evolved despite low mtDNA divergence, and in one instance two Leucocytozoon cytb haplotypes that differed by a single base pair (~ 0.2% divergence) were supported as separate species. However, there was no consistent association between mtDNA divergence and species limits. Among cytb haplotypes that differed by one to three base pairs we observed idiosyncratic patterns of nuclear and ecological divergence, with cytb haplotype pairs found to be either conspecific, reproductively isolated with no divergence in host specificity, or reproductively isolated with divergent patterns of host specialization. CONCLUSION: Integrating multi-locus genetic species delimitation methods and non-traditional ecological data types such as host specificity provide a novel view of the diversity of avian malaria parasites that has been missed previously using morphology and mtDNA barcodes. Species delimitation methods show that Leucocytozoon is highly species-rich in Alaska, and the genus is likely to harbor extraordinary species-level diversity worldwide. Integrating genetic and ecological data will be an important approach for understanding the diversity and evolutionary history of microbial symbionts moving forward.

Why are tropical mountain passes "low" for some species? Genetic and stable-isotope tests for differentiation, migration and expansion in elevational generalist songbirds.

Most tropical bird species have narrow elevational ranges, likely reflecting climatic specialization. This is consistent with Janzen's Rule, the tendency for mountain passes to be effectively "higher" in the tropics. Hence, those few tropical species that occur across broad elevational gradients (elevational generalists) represent a contradiction to Janzen's Rule. Here, we aim to address the following questions. Are elevational generalists being sundered by diversifying selection along the gradient? Does elevational movement cause these species to resist diversification or specialization? Have they recently expanded, suggesting that elevational generalism is short-lived in geological time? To answer these questions, we tested for differentiation, movement and expansion in four elevational generalist songbird species on the Andean west slope. We used morphology and mtDNA to test for genetic differentiation between high- and low-elevation populations. To test for elevational movements, we measured hydrogen isotope (δ2 H) values of metabolically inert feathers and metabolically active liver. Morphology differed for House Wren (Troglodytes aedon) and Hooded Siskin (Spinus magellanicus), but not for Cinereous Conebill (Conirostrum cinereum) and Rufous-collared Sparrow (Zonotrichia capensis) respectively. mtDNA was structured by elevation only in Z. capensis. δ2 H data indicated elevational movements by two tree- and shrub-foraging species with moderate-to-high vagility (C. cinereum and S. magellanicus), and sedentary behaviour by two terrestrial-foraging species with low-to-moderate vagility (T. aedon and Z. capensis). In S. magellanicus, elevational movements and lack of mtDNA structure contrast with striking morphological divergence, suggesting strong diversifying selection on body proportions across the c. 50 km gradient. All species except C. cinereum exhibited mtDNA haplotype variation consistent with recent population expansion across the elevational gradient, potentially concurrent with Holocene anthropogenic habitat conversion for agriculture. In different ways, each species defies the tendency for tropical birds to have long-term stable distributions and sedentary habits. We conclude that tropical elevational generalism is rare due to evolutionary instability.

Contribution of a mutational hot spot to hemoglobin adaptation in high-altitude Andean house wrens.

A key question in evolutionary genetics is why certain mutations or certain types of mutation make disproportionate contributions to adaptive phenotypic evolution. In principle, the preferential fixation of particular mutations could stem directly from variation in the underlying rate of mutation to function-altering alleles. However, the influence of mutation bias on the genetic architecture of phenotypic evolution is difficult to evaluate because data on rates of mutation to function-altering alleles are seldom available. Here, we report the discovery that a single point mutation at a highly mutable site in the ß(A)-globin gene has contributed to an evolutionary change in hemoglobin (Hb) function in high-altitude Andean house wrens (Troglodytes aedon). Results of experiments on native Hb variants and engineered, recombinant Hb mutants demonstrate that a nonsynonymous mutation at a CpG dinucleotide in the ß(A)-globin gene is responsible for an evolved difference in Hb-O2 affinity between high- and low-altitude house wren populations. Moreover, patterns of genomic differentiation between high- and low-altitude populations suggest that altitudinal differentiation in allele frequencies at the causal amino acid polymorphism reflects a history of spatially varying selection. The experimental results highlight the influence of mutation rate on the genetic basis of phenotypic evolution by demonstrating that a large-effect allele at a highly mutable CpG site has promoted physiological differentiation in blood O2 transport capacity between house wren populations that are native to different elevations.

Cascading effects of habitat loss on ectoparasite-associated bacterial microbiomes.

Suitable habitat fragment size, isolation, and distance from a source are important variables influencing community composition of plants and animals, but the role of these environmental factors in determining composition and variation of host-associated microbial communities is poorly known. In parasite-associated microbial communities, it is hypothesized that evolution and ecology of an arthropod parasite will influence its microbiome more than broader environmental factors, but this hypothesis has not been extensively tested. To examine the influence of the broader environment on the parasite microbiome, we applied high-throughput sequencing of the V4 region of 16S rRNA to characterize the microbiome of 222 obligate ectoparasitic bat flies (Streblidae and Nycteribiidae) collected from 155 bats (representing six species) from ten habitat fragments in the Atlantic Forest of Brazil. Parasite species identity is the strongest driver of microbiome composition. To a lesser extent, reduction in habitat fragment area, but not isolation, is associated with an increase in connectance and betweenness centrality of bacterial association networks driven by changes in the diversity of the parasite community. Controlling for the parasite community, bacterial network topology covaries with habitat patch area and exhibits parasite-species specific responses to environmental change. Taken together, habitat loss may have cascading consequences for communities of interacting macro- and microorgansims.

Phylogenomics from transcriptomic "bycatch" clarify the origins and diversity of avian trypanosomes in North America.

The eukaryotic blood parasite genus Trypanosoma includes several important pathogens of humans and livestock, but has been understudied in wildlife broadly. The trypanosomes that infect birds are in particular need of increased attention, as these parasites are abundant and globally distributed, yet few studies have addressed their evolutionary origins and diversity using modern molecular and analytical approaches. Of specific interest are the deep evolutionary relationships of the avian trypanosomes relative to the trypanosome species that are pathogenic in humans, as well as their species level diversity in regions where they have been understudied such as North America. Here, we address these unresolved areas of study using phylogenomic data for two species of avian trypanosomes that were isolated as "bycatch" from host transcriptome assemblies, as well as a large 18S DNA barcode sequence dataset that includes 143 novel avian Trypanosoma 18S sequences from North America. Using a phylogenomic approach, we find that the avian trypanosomes are nested within a clade of primarily mammalian trypanosomes that includes the human pathogen Trypanosoma cruzi, and are paraphyletic with respect to the ruminant trypanosome Trypanosoma theileri. DNA barcode sequences showed that T. avium and an unidentified small, non-striated trypanosome that was morphologically similar to T. everetti are each represented by highly abundant and divergent 18S haplotypes in North America. Community-level sampling revealed that additional species-level Trypanosoma lineages exist in this region. We compared the newly sequenced DNA barcodes from North America to a global database, and found that avian Trypanosoma 18S haplotypes generally exhibited a marked lack of host specificity with at least one T. avium haplotype having an intercontinental distribution. This highly abundant T. avium haplotype appears to have a remarkably high dispersal ability and cosmopolitan capacity to evade avian host immune defenses, which warrant further study.

Metatranscriptomics yields new genomic resources and sensitive detection of infections for diverse blood parasites.

Metatranscriptomics is a powerful method for studying the composition and function of complex microbial communities. The application of metatranscriptomics to multispecies parasite infections is of particular interest, as research on parasite evolution and diversification has been hampered by technical challenges to genome-scale DNA sequencing. In particular, blood parasites of vertebrates are abundant and diverse although they often occur at low infection intensities and exist as multispecies infections, rendering the isolation of genomic sequence data challenging. Here, we use birds and their diverse haemosporidian parasites to illustrate the potential for metatranscriptome sequencing to generate large quantities of genome-wide sequence data from multiple blood parasite species simultaneously. We used RNA-sequencing of 24 blood samples from songbirds in North America to show that metatranscriptomes can yield large proportions of haemosporidian protein-coding gene repertoires even when infections are of low intensity (<0.1% red blood cells infected) and consist of multiple parasite taxa. By bioinformatically separating host and parasite transcripts and assigning them to the haemosporidian genus of origin, we found that transcriptomes detected ~23% more total parasite infections across all samples than were identified using microscopy and DNA barcoding. For single-species infections, we obtained data for >1,300 loci from samples with as low as 0.03% parasitaemia, with the number of loci increasing with infection intensity. In total, we provide data for 1,502 single-copy orthologous loci from a phylogenetically diverse set of 33 haemosporidian mitochondrial lineages. The metatranscriptomic approach described here has the potential to accelerate ecological and evolutionary research on haemosporidians and other diverse parasites.

Ecology, not distance, explains community composition in parasites of sky-island Audubon's Warblers.

Haemosporidian parasites of birds are ubiquitous in terrestrial ecosystems, but their coevolutionary dynamics remain poorly understood. If species turnover in parasites occurs at a finer scale than turnover in hosts, widespread hosts would encounter diverse parasites, potentially diversifying as a result. Previous studies have shown that some wide-ranging hosts encounter varied haemosporidian communities throughout their range, and vice-versa. More surveys are needed to elucidate mechanisms that underpin spatial patterns of diversity in this complex multi-host multi-parasite system. We sought to understand how and why a community of avian haemosporidian parasites varies in abundance and composition across elevational transects in eight sky islands in southwestern North America. We tested whether bird community composition, environment, or geographic distance explain haemosporidian parasite species turnover in a widespread host that harbors a diverse haemosporidian community, the Audubon's Warbler (Setophaga auduboni). We tested predictors of infection using generalized linear models, and predictors of bird and parasite community dissimilarity using generalized dissimilarity modeling. Predictors of infection differed by parasite genus: Parahaemoproteus was predicted by elevation and climate, Leucocytozoon varied idiosyncratically among mountains, and Plasmodium was unpredictable, but rare. Parasite turnover was nearly three-fold higher than bird turnover and was predicted by elevation, climate, and bird community composition, but not geographic distance. Haemosporidian communities vary strikingly at fine spatial scales (hundreds of kilometers), across which the bird community varies only subtly. The finer scale of turnover among parasites implies that their ranges may be smaller than those of their hosts. Avian host species should encounter different parasite species in different parts of their ranges, resulting in spatially varying selection on host immune systems. The fact that parasite turnover was predicted by bird turnover, even when considering environmental characteristics, implies that host species or their phylogenetic history plays a role in determining which parasite species will be present in a community.

The polyphyly of Plasmodium: comprehensive phylogenetic analyses of the malaria parasites (order Haemosporida) reveal widespread taxonomic conflict.

The evolutionary relationships among the apicomplexan blood pathogens known as the malaria parasites (order Haemosporida), some of which infect nearly 200 million humans each year, has remained a vexing phylogenetic problem due to limitations in taxon sampling, character sampling and the extreme nucleotide base composition biases that are characteristic of this clade. Previous phylogenetic work on the malaria parasites has often lacked sufficient representation of the broad taxonomic diversity within the Haemosporida or the multi-locus sequence data needed to resolve deep evolutionary relationships, rendering our understanding of haemosporidian life-history evolution and the origin of the human malaria parasites incomplete. Here we present the most comprehensive phylogenetic analysis of the malaria parasites conducted to date, using samples from a broad diversity of vertebrate hosts that includes numerous enigmatic and poorly known haemosporidian lineages in addition to genome-wide multi-locus sequence data. We find that if base composition differences were corrected for during phylogenetic analysis, we recovered a well-supported topology indicating that the evolutionary history of the malaria parasites was characterized by a complex series of transitions in life-history strategies and host usage. Notably we find that Plasmodium, the malaria parasite genus that includes the species of human medical concern, is polyphyletic with the life-history traits characteristic of this genus having evolved in a dynamic manner across the phylogeny. We find support for multiple instances of gain and loss of asexual proliferation in host blood cells and production of haemozoin pigment, two traits that have been used for taxonomic classification as well as considered to be important factors for parasite virulence and used as drug targets. Lastly, our analysis illustrates the need for a widespread reassessment of malaria parasite taxonomy.

Contrasting molecular and morphological evidence for the identification of an anomalous Buteo: a cautionary tale for hybrid diagnosis.

An adult Buteo was found dead as a road-kill south of Sacramento, California, and was thought to represent the first state record of the eastern Red-shouldered Hawk (B. lineatus lineatus;). It is now a specimen in the Museum of Wildlife and Fisheries Biology (WFB 4816) at the University of California, Davis. We examined this specimen and found that many of its plumage characters differed from all other adult Red-shouldered Hawks examined, including nominate adults. Plumage markings and measurements were intermediate between Red-tailed Hawk (Buteo jamaicensis, ssp calurus) and Red-shouldered Hawk (ssp elegans), leading us to hypothesize that the bird was a hybrid. However, mtDNA sequences and nuDNA microsatellites proved definitively that the bird was a Red-shouldered Hawk, most likely of eastern origin. This case illustrates that apparent hybrids or apparent vagrants could be individuals with anomalous phenotypes caused by rare genetic variation or novel epigenetic effects.