The association between haemosporidian infection and non-breeding moult location in great reed warblers revisited by combining feather stable isotope profiles and geolocator data.

Stable isotope analysis provides valuable insights into the ecology of long-distance migratory birds during periods spent away from a specific study site. In a previous study, Swedish great reed warblers (Acrocephalus arundinaceus) infected with haemosporidian parasites differed in feather isotope ratios compared to non-infected birds, suggesting that infected and non-infected birds spent the non-breeding season in different locations or habitats. Here, we use a novel dataset comprising geolocator data, isotopes, and haemosporidian infection status of 92 individuals from four Eurasian populations to investigate whether parasite transmission varies with geography or habitats. We found that the probability of harbouring Plasmodium and Leucocytozoon parasites was higher in birds moulting in the eastern region of the non-breeding grounds. However, no geographic pattern occurred for Haemoproteus infections or overall infection status. In contrast to the previous study, we did not find any relationship between feather isotope ratios and overall haemosporidian infection for the entire current dataset. Plasmodium-infected birds had lower feather δ15N values indicating that they occupied more mesic habitats. Leucocytozoon-infected birds had higher feather δ34S values suggesting more coastal sites or wetlands with anoxic sulphate reduction. As the composition and prevalence of haemosporidian parasites differed between the old and the current dataset, we suggest that the differences might be a consequence of temporal dynamics of haemosporidian parasites. Our results emphasize the importance of replicating studies conducted on a single population over a restricted time period, as the patterns can become more complex for data from wider geographical areas and different time periods.

Host shift and natural long-distance dispersal to an oceanic island of a host-specific parasite.

Parasite dispersal and host-switching may be better understood by knowing when they occurred. We estimated when the ancestor of a parasite of great reed warblers (Acrocephalus arundinaceus) dispersed to the Seychelles and began infecting the endemic Seychelles warbler (A. sechellensis). We used mitochondrial genomes and published molecular divergence rates to estimate the date of divergence between mitochondrial haplotypes of the parasite Haemoproteus nucleocondensis (lineage GRW01) in the great reed warbler and the Seychelles warbler. We also constructed a time-calibrated phylogeny of the hosts and their relatives to determine when the ancestor of the Seychelles warbler dispersed to the Seychelles. The two GRW01 lineages diverged ca 20-451 kya, long after the ancestor of the Seychelles warbler colonized the Seychelles ca 1.76-4.36 Mya. GRW01 rarely infects other species despite apparent opportunity. Humans were likely not involved in the dispersal of this parasite because humans settled the Seychelles long after the parasite diverged from its mainland relative. Furthermore, introduced birds are unlikely hosts of GRW01. Instead, the ancestor of GRW01 may have dispersed to the Seychelles with an errant migrating great reed warbler. Our results indicate that even specialized parasites can naturally disperse long distances to become emerging infectious diseases.

Transposable elements mark a repeat-rich region associated with migratory phenotypes of willow warblers (Phylloscopus trochilus).

The genetic basis of bird migration has been the focus of several studies. Two willow warbler subspecies (Phylloscopus trochilus trochilus and Phylloscopus trochilus acredula) follow different migratory routes to wintering grounds in Africa. Their breeding populations overlap in contact areas or "migratory divides" located in central Scandinavia and in eastern Poland. Earlier analyses demonstrated that the genetic differences between these two migratory phenotypes are few and cluster on chromosomes 1 and 5. In addition, an amplified fragment length polymorphism-derived biallelic marker (known as WW2) presents steep clines across both migratory divides but failed to be mapped in the genome. Here, we characterize the WW2 marker and describe its two variants (WW2 ancestral and WW2 derived) as portions of long terminal repeat retrotransposons originating from an ancient infection by an endogenous retrovirus. We used quantitative polymerase chain reaction techniques to quantify copy numbers of the WW2 derived variant in the two subspecies and their hybrids. This, together with genome analyses revealed that WW2 derived variants are much more abundant in P. t. acredula and appear embedded in a large repeat-rich region (>12 Mbp), not associated with the divergent regions of chromosomes 1 or 5. However, it might interact with genetic elements controlling migration direction. Testing this hypothesis further will require knowing the exact location of this region, such as by obtaining more complete genome assemblies preferably in combination with techniques like fluorescence in situ hybridization applied to a willow warbler karyotype, and finally to investigate the copy number of this marker in hybrids with known migratory tracks.

A highly invasive malaria parasite has expanded its range to non-migratory birds in North America.

Parasite range expansions are a direct consequence of globalization and are an increasing threat to biodiversity. Here, we report a recent range expansion of the SGS1 strain of a highly invasive parasite, Plasmodium relictum, to two non-migratory passerines in North America. Plasmodium relictum is considered one of the world's most invasive parasites and causes the disease avian malaria: this is the first reported case of SGS1 in wild non-migratory birds on the continent. Using a long-term database where researchers report avian malaria parasite infections, we summarized our current understanding of the geographical range of SGS1 and its known hosts. We also identified the most likely geographical region of this introduction event using the MSP1 allele. We hypothesize that this introduction resulted from movements of captive birds and subsequent spillover to native bird populations, via the presence of competent vectors and ecological fitting. Further work should be conducted to determine the extent to which SGS1 has spread following its introduction in North America.

Evolution of vector transmitted parasites by host switching revealed through sequencing of Haemoproteus parasite mitochondrial genomes.

Parasite species evolve by switching to new hosts, cospeciating with their current hosts, or speciating on their current hosts. Vector transmitted parasites are expected to speciate by host switching, but confirming this hypothesis has proved challenging. Parasite DNA can be difficult to sequence, thus well resolved parasite phylogenies that are needed to distinguish modes of parasite speciation are often lacking. Here, we studied speciation in vector transmitted avian haemosporidian parasites in the genus Haemoproteus and their warbler hosts (family Acrocephalidae). We overcome the difficulty of generating parasite genetic data by combining nested long-range PCR with next generation sequencing to sequence whole mitochondrial genomes from 19 parasite haplotypes confined to Acrocephalidae warblers, resulting in a well-supported parasite phylogeny. We also generated a well-supported host phylogeny using five genes from published sources. Our phylogenetic analyses confirm that these parasites have speciated by host switching. We also found that closely related host species shared parasites which themselves were not closely related. Sharing of parasites by closely related host species is not due to host geographic range overlap, but may be the result of phylogenetically conserved host immune systems.

The global biogeography of avian haemosporidian parasites is characterized by local diversification and intercontinental dispersal.

The biogeographic histories of parasites and pathogens are infrequently compared with those of free-living species, including their hosts. Documenting the frequency with which parasites and pathogens disperse across geographic regions contributes to understanding not only their evolution, but also the likelihood that they may become emerging infectious diseases. Haemosporidian parasites of birds (parasite genera Plasmodium, Haemoproteus and Leucocytozoon) are globally distributed, dipteran-vectored parasites. To date, over 2000 avian haemosporidian lineages have been designated by molecular barcoding methods. To achieve their current distributions, some lineages must have dispersed long distances, often over water. Here we quantify such events using the global avian haemosporidian database MalAvi and additional records primarily from the Americas. We scored lineages as belonging to one or more global biogeographic regions based on infection records. Most lineages were restricted to a single region but some were globally distributed. We also used part of the cytochrome b gene to create genus-level parasite phylogenies and scored well-supported nodes as having descendant lineages in regional sympatry or allopatry. Descendant sister lineages of Plasmodium, Haemoproteus and Leucocytozoon were distributed in allopatry in 11, 16 and 15% of investigated nodes, respectively. Although a small but significant fraction of the molecular variance in cytochrome b of all three genera could be explained by biogeographic region, global parasite dispersal likely contributed to the majority of the unexplained variance. Our results suggest that avian haemosporidian parasites have faced few geographic barriers to dispersal over their evolutionary history.

A new one-step multiplex PCR assay for simultaneous detection and identification of avian haemosporidian parasites.

Accurate detection and identification are essential components for epidemiological, ecological, and evolutionary surveys of avian haemosporidian parasites. Microscopy has been used for more than 100 years to detect and identify these parasites; however, this technique requires considerable training and high-level expertise. Several PCR methods with highly sensitive and specific detection capabilities have now been developed in addition to microscopic examination. However, recent studies have shown that these molecular protocols are insufficient at detecting mixed infections of different haemosporidian parasite species and genetic lineages. In this study, we developed a simple, sensitive, and specific multiplex PCR assay for simultaneous detection and discrimination of parasites of the genera Plasmodium, Haemoproteus, and Leucocytozoon in single and mixed infections. Relative quantification of parasite DNA using qPCR showed that the multiplex PCR can amplify parasite DNA ranging in concentration over several orders of magnitude. The detection specificity and sensitivity of this new multiplex PCR assay were also tested in two different laboratories using previously screened natural single and mixed infections. These findings show that the multiplex PCR designed here is highly effective at identifying both single and mixed infections from all three genera of avian haemosporidian parasites. We predict that this one-step multiplex PCR assay, being convenient and inexpensive, will become a widely used method for molecular screening of avian haemosporidian parasites.

Delineation of the Genera Haemoproteus and Plasmodium Using RNA-Seq and Multi-gene Phylogenetics.

Members of the order Haemosporida are protist parasites that infect mammals, reptiles and birds. This group includes the causal agents of malaria, Plasmodium parasites, the genera Leucocytozoon and Fallisia, as well as the species rich genus Haemoproteus with its two subgenera Haemoproteus and Parahaemoproteus. Some species of Haemoproteus cause severe disease in avian hosts, and these parasites display high levels of diversity worldwide. This diversity emphasizes the need for accurate evolutionary information. Most molecular studies of wildlife haemosporidians use a bar coding approach by sequencing a fragment of the mitochondrial cytochrome b gene. This method is efficient at differentiating parasite lineages but insufficient for accurate phylogenetic inferences in highly diverse taxa such as haemosporidians. Recent studies have utilized multiple mitochondrial genes (cyt b, cox1 and cox3), sometimes combined with a few apicoplast and nuclear genes. These studies have been highly successful with one notable exception: the evolutionary relationships of the genus Haemoproteus remain unresolved. Here we describe the transcriptome of Haemoproteus columbae and investigate its phylogenetic position recovered from a multi-gene dataset (600 genes). This genomic approach restricts the taxon sampling to 18 species of apicomplexan parasites. We employed Bayesian inference and maximum likelihood methods of phylogenetic analyses and found H. columbae and a representative from the subgenus Parahaemoproteus to be sister taxa. This result strengthens the hypothesis of genus Haemoproteus being monophyletic; however, resolving this question will require sequences of orthologs from, in particular, representatives of Leucocytozoon species.

Generalist haemosporidian parasites are better adapted to a subset of host species in a multiple host community.

Parasites that can infect multiple host species are considered to be host generalists with low host specificity. However, whether generalist parasites are better adapted to a subset of their host species remains unknown. To elucidate this possibility, we compared the variation in prevalence and infection intensity among host species of three generalist parasite lineages belonging to the morphological species Haemoproteus majoris, in a natural bird community in southern Sweden. Prevalence in each host species was confirmed by nested PCR and DNA sequencing, and infection intensities were quantified using lineage-specific real-time qPCR. For two of the three lineages, we detected positive correlations between prevalence and infection intensity, indicating that these generalist parasites are better adapted to a subset of host species, which may have been more frequently encountered during the evolution of the parasite; we refer to these as main host species. For both lineages, the main host species were more phylogenetically related than expected by chance as revealed by strong phylogenetic signal in prevalence among hosts. By comparing our results with previous records of these parasites, we found that the host range of a generalist parasite can vary among different communities and may partly be shaped by the presence of other parasites. Our study reveals that generalist parasites may be specialized on a subset of their host species and it highlights the importance of considering infection intensity and host phylogeny when determining the host specificity of a parasite.

Ecological determinants of avian malaria infections: An integrative analysis at landscape, mosquito and vertebrate community levels.

Vector and host communities, as well as habitat characteristics, may have important but different impacts on the prevalence, richness and evenness of vector-borne parasites. We investigated the relative importance of (1) the mosquito community composition, (2) the vertebrate community composition and (3) landscape characteristics on the prevalence, richness and evenness of avian Plasmodium. We hypothesized that parasite prevalence will be more affected by vector-related parameters, while host parameters should be also important to explain Plasmodium richness and evenness. We sampled 2,588 wild house sparrows (Passer domesticus) and 340,829 mosquitoes, and we performed vertebrate censuses at 45 localities in the Southwest of Spain. These localities included urban, rural and natural landscapes that were characterized by several habitat variables. Twelve Plasmodium lineages were identified in house sparrows corresponding to three major clades. Variation partitioning showed that landscape characteristics explained the highest fraction of variation in all response variables (21.0%-44.8%). Plasmodium prevalence was in addition explained by vector-related variables (5.4%) and its interaction with landscape (10.2%). Parasite richness and evenness were mostly explained by vertebrate community-related variables. The structuring role of landscape characteristics in vector and host communities was a key factor in determining parasite prevalence, richness and evenness, although the role of each factor differed according to the parasite parameters studied. These results show that the biotic and abiotic contexts are important to explain the transmission dynamics of mosquito-borne pathogens in the wild.

De novo synthesis of thiamine (vitamin B1) is the ancestral state in Plasmodium parasites - evidence from avian haemosporidians.

Parasites often have reduced genomes as their own genes become redundant when utilizing their host as a source of metabolites, thus losing their own de novo production of metabolites. Primate malaria parasites can synthesize vitamin B1 (thiamine) de novo but rodent malaria and other genome-sequenced apicomplexans cannot, as the three essential genes responsible for this pathway are absent in their genomes. The unique presence of functional thiamine synthesis genes in primate malaria parasites and their sequence similarities to bacterial orthologues, have led to speculations that this pathway was horizontally acquired from bacteria. Here we show that the genes essential for the de novo synthesis of thiamine are found also in avian Plasmodium species. Importantly, they are also present in species phylogenetically basal to all mammalian and avian Plasmodium parasites, i.e. Haemoproteus. Furthermore, we found that these genes are expressed during the blood stage of the avian malaria infection, indicating that this metabolic pathway is actively transcribed. We conclude that the ability to synthesize thiamine is widespread among haemosporidians, with a recent loss in the rodent malaria species.

Pale and dark morphs of tawny owls show different patterns of telomere dynamics in relation to disease status.

Parasites are expected to exert long-term costs on host fecundity and longevity. Understanding the consequences of heritable polymorphic variation in disease defence in wild populations is essential in order to predict evolutionary responses to changes in disease risk. Telomeres have been found to shorten faster in malaria-diseased individuals compared with healthy ones with negative effects on longevity and thereby fitness. Here, we study the impact of haemosporidian blood parasites on telomere dynamics in tawny owls, which display a highly heritable plumage colour polymorphism. Previously, it has been shown that blood parasites have morph-specific impact on body mass maintenance. Here, we show that telomeres shortened faster in individuals with shorter breeding lifespan. Telomere length was negatively associated with the degree of pheomelanic brown coloration and shorter in infected than uninfected individuals. The rate of telomere shortening between breeding seasons was faster in darker pheomelanic individuals and suppression of parasite intensity between seasons was associated with faster telomere shortening in the paler individuals but not in darker ones. We propose that morph-specific physiological profiles cause differential telomere shortening and that this is likely to be a mechanism involved in previously documented environment-driven survival selection against the pheomelanic morph in this population.

Selective disappearance of great tits with short telomeres in urban areas.

Urban environments pose novel challenges, as well as opportunities, for urban-dwelling wildlife. Although differences have been reported in several phenotypic traits (e.g. morphology, physiology and behaviour) between urban and rural populations, it is poorly understood whether this affects individual fitness. Telomere dynamics are posited as one possible mechanism underlying senescence and mortality. It was recently shown that telomere shortening is accelerated when growing up in an urban, compared with a rural, environment. However, the implications of accelerated telomere attrition for fitness are still unclear. Here, we examine the relationship between telomere length (TL) and survival in a bird common to urban and rural environments, and during both early and later life. The results reveal that TL is a strong predictor of post-fledging survival and recruitment in both habitats but, crucially, selective disappearance of individuals with short telomeres early in life is more pronounced in the urban environment, resulting in a longer average TL among the adult population. However, following recruitment, we found no difference in the relationship between TL and survival between the urban and rural environments. This indicates that the urban environment has negative effects in early life, while during later life the benefits could potentially outweigh the costs.

Parallel telomere shortening in multiple body tissues owing to malaria infection.

Several studies have shown associations between shorter telomere length in blood and weakened immune function, susceptibility to infections, and increased risk of morbidity and mortality. Recently, we have shown that malaria accelerates telomere attrition in blood cells and shortens lifespan in birds. However, the impact of infections on telomere attrition in different body tissues within an individual is unknown. Here, we tested whether malarial infection leads to parallel telomere shortening in blood and tissue samples from different organs. We experimentally infected siskins (Spinus spinus) with the avian malaria parasite Plasmodium ashfordi, and used real-time quantitative polymerase chain reaction (PCR) to measure telomere length in control and experimentally infected siskins. We found that experimentally infected birds showed faster telomere attrition in blood over the course of infection compared with control individuals (repeatedly measured over 105 days post-infection (DPI)). Shorter telomeres were also found in the tissue of all six major organs investigated (liver, lungs, spleen, heart, kidney, and brain) in infected birds compared with controls at 105 DPI. To the best of our knowledge, this is the first study showing that an infectious disease results in synchronous telomere shortening in the blood and tissue cells of internal organs within individuals, implying that the infection induces systemic stress. Our results have far-reaching implications for understanding how the short-term effects of an infection can translate into long-term costs, such as organ dysfunction, degenerative diseases, and ageing.

Genetic rescue in a severely inbred wolf population.

Natural populations are becoming increasingly fragmented which is expected to affect their viability due to inbreeding depression, reduced genetic diversity and increased sensitivity to demographic and environmental stochasticity. In small and highly inbred populations, the introduction of only a few immigrants may increase vital rates significantly. However, very few studies have quantified the long-term success of immigrants and inbred individuals in natural populations. Following an episode of natural immigration to the isolated, severely inbred Scandinavian wolf (Canis lupus) population, we demonstrate significantly higher pairing and breeding success for offspring to immigrants compared to offspring from native, inbred pairs. We argue that inbreeding depression is the underlying mechanism for the profound difference in breeding success. Highly inbred wolves may have lower survival during natal dispersal as well as competitive disadvantage to find a partner. Our study is one of the first to quantify and compare the reproductive success of first-generation offspring from migrants vs. native, inbred individuals in a natural population. Indeed, our data demonstrate the profound impact single immigrants can have in small, inbred populations, and represent one of very few documented cases of genetic rescue in a population of large carnivores.

Multiple instances of paraphyletic species and cryptic taxa revealed by mitochondrial and nuclear RAD data for Calandrella larks (Aves: Alaudidae).

The avian genus Calandrella (larks) was recently suggested to be non-monophyletic, and was divided into two genera, of which Calandrella sensu stricto comprises 4-5 species in Eurasia and Africa. We analysed mitochondrial cytochrome b (cytb) and nuclear Restriction-site Associated DNA (RAD) sequences from all species, and for cytb we studied 21 of the 22 recognised subspecies, with the aim to clarify the phylogenetic relationships within the genus and to compare large-scale nuclear sequence patterns with a widely used mitochondrial marker. Cytb indicated deep splits among the currently recognised species, although it failed to support the interrelationships among most of these. It also revealed unexpected deep divergences within C. brachydactyla, C. blanfordi/C. erlangeri, C. cinerea, and C. acutirostris. It also suggested that both C. brachydactyla and C. blanfordi, as presently circumscribed, are paraphyletic. In contrast, most of the many subspecies of C. brachydactyla and C. cinerea were unsupported by cytb, although two populations of C. cinerea were found to be genetically distinct. The RAD data corroborated the cytb tree (for the smaller number of taxa analysed) and recovered strongly supported interspecific relationships. However, coalescence analyses of the RAD data, analysed in SNAPP both with and without an outgroup, received equally strong support for two conflicting topologies. We suggest that the tree rooted with an outgroup - which is not recommended for SNAPP - is more trustworthy, and suggest that the reliability of analyses performed without any outgroup species should be thoroughly evaluated. We also demonstrate that degraded museum samples can be phylogenetically informative in RAD analyses following careful bioinformatic treatment. We note that the genus Calandrella is in need of taxonomic revision.

Maternal and genetic factors determine early life telomere length.

In a broad range of species--including humans--it has been demonstrated that telomere length declines throughout life and that it may be involved in cell and organismal senescence. This potential link to ageing and thus to fitness has triggered recent interest in understanding how variation in telomere length is inherited and maintained. However, previous studies suffer from two main drawbacks that limit the possibility of understanding the relative importance of genetic, parental and environmental influences on telomere length variation. These studies have been based on (i) telomere lengths measured at different time points in different individuals, despite the fact that telomere length changes over life, and (ii) parent-offspring regression techniques, which do not enable differentiation between genetic and parental components of inheritance. To overcome these drawbacks, in our study of a songbird, the great reed warbler, we have analysed telomere length measured early in life in both parents and offspring and applied statistical models (so-called 'animal models') that are based on long-term pedigree data. Our results showed a significant heritability of telomere length on the maternal but not on the paternal side, and that the mother's age was positively correlated with their offspring's telomere length. Furthermore, the pedigree-based analyses revealed a significant heritability and an equally large maternal effect. Our study demonstrates strong maternal influence on telomere length and future studies now need to elucidate possible underlying factors, including which types of maternal effects are involved.

Disentangling the complex evolutionary history of the Western Palearctic blue tits (Cyanistes spp.) - phylogenomic analyses suggest radiation by multiple colonization events and subsequent isolation.

Isolated islands and their often unique biota continue to play key roles for understanding the importance of drift, genetic variation and adaptation in the process of population differentiation and speciation. One island system that has inspired and intrigued evolutionary biologists is the blue tit complex (Cyanistes spp.) in Europe and Africa, in particular the complex evolutionary history of the multiple genetically distinct taxa of the Canary Islands. Understanding Afrocanarian colonization events is of particular importance because of recent unconventional suggestions that these island populations acted as source of the widespread population in mainland Africa. We investigated the relationship between mainland and island blue tits using a combination of Sanger sequencing at a population level (20 loci; 12 500 nucleotides) and next-generation sequencing of single population representatives (>3 200 000 nucleotides), analysed in coalescence and phylogenetic frameworks. We found (i) that Afrocanarian blue tits are monophyletic and represent four major clades, (ii) that the blue tit complex has a continental origin and that the Canary Islands were colonized three times, (iii) that all island populations have low genetic variation, indicating low long-term effective population sizes and (iv) that populations on La Palma and in Libya represent relicts of an ancestral North African population. Further, demographic reconstructions revealed (v) that the Canary Islands, conforming to traditional views, hold sink populations, which have not served as source for back colonization of the African mainland. Our study demonstrates the importance of complete taxon sampling and an extensive multimarker study design to obtain robust phylogeographical inferences.

Prevalence and diversity of Plasmodium and Haemoproteus parasites in the globally-threatened Aquatic Warbler Acrocephalus paludicola.

The diversity and prevalence of malaria parasites of the genera Plasmodium and Haemoproteus were determined in the globally-threatened Aquatic Warbler Acrocephalus paludicola. Birds were sampled during migration in Portugal and at the wintering quarters in Senegal and parasites were detected using molecular methods. Only three generalist parasite lineages (Plasmodium) were found. There were no significant differences in the prevalence of parasites between sexes in Europe, but adults had higher prevalence than first-year birds, and birds in Europe had higher prevalence than those captured in Africa. When comparing with other Acrocephalus species and taking sample size into account, Aquatic Warblers had the lowest prevalence and, together with another threatened species, the Seychelles Warbler Acrocephalus sechellensis, the lowest diversity of malaria parasites. We hypothesize that the low diversity of parasites and absence of specialist lineages of Aquatic Warblers are caused by its small population size and fragmented distribution. Furthermore, Aquatic Warblers' extreme habitat specialization may decrease their exposure to malaria parasites, but other explanations such as high mortality (which would constraint the sampling of infected birds) or, in contrast, very efficient immunological system in clearing the infections cannot be ruled out. This study contributes to explain variation in prevalence and diversity of malaria parasites among hosts.