Multiple cryptic species of sympatric generalists within the avian blood parasite Haemoproteus majoris.

The avian haemosporidian parasite Haemoproteus majoris has been reported to infect a wide range of passerine birds throughout the Holarctic ecozone. Five cytochrome b (cyt b) lineages have been described as belonging to the morphological species H. majoris, and these form a tight phylogenetic cluster together with 13 undescribed lineages that differ from each other by < 1.2% in sequence divergence. Records in a database (MalAvi) that contains global findings of haemosporidian lineages generated by universal primers suggest that these lineages vary substantially in host distribution. We confirm this pattern in a data set collected at Lake Kvismaren, Sweden, where three of the generalist lineages have local transmission. However, whether these lineages represent intraspecific mitochondrial diversity or clusters of cryptic species has previously not been examined. In this study, we developed novel molecular markers to amplify the partial segments of four nuclear genes to determine the level of genetic diversity and gene phylogenies among the five morphologically described cyt b lineages of H. majoris. All five cyt b lineages were strongly associated with unique nuclear alleles at all four nuclear loci, indicating that each mitochondrial lineage represents a distinct biological species. Within lineages, there was no apparent association between nuclear alleles and host species, indicating that they form genetically unstructured populations across multiple host species.

Urban environment shortens telomere length in nestling great tits, Parus major.

Urban environments are expanding rapidly, and with urbanization come both challenges and opportunities for wildlife. Challenges include combating the anthropogenic disturbances such as light, noise and air pollution and lower availability of natural food sources. The benefits are many, including the availability of anthropogenic food sources, breeding boxes and warmer temperatures. Thus, depending on the context, urbanization can have both positive and negative effects on fitness related traits. It is well known that early-life conditions can have lifelong implications on fitness; little is however known about development in urban environments. We reciprocally cross-fostered urban and rural nestling great tits (Parus major L.) to study how growing up in an urban versus rural habitat affected telomere length (TL)-a suggested biomarker of longevity. We show, for the first time, that growing up in an urban environment significantly shortens TL, independently of natal origin (i.e. urban or rural). This implies that the urban environment imposes a challenge to developing birds, with potentially irreversible effects on lifespan.

Transcontinental migratory connectivity predicts parasite prevalence in breeding populations of the European barn swallow.

Parasites exert a major impact on the eco-evolutionary dynamics of their hosts and the associated biotic environment. Migration constitutes an effective means for long-distance invasions of vector-borne parasites and promotes their rapid spread. Yet, ecological and spatial information on population-specific host-parasite connectivity is essentially lacking. Here, we address this question in a system consisting of a transcontinental migrant species, the European barn swallow (Hirundo rustica) which serves as a vector for avian endoparasites in the genera Plasmodium, Haemoproteus and Leucocytozoon. Using feather stable isotope ratios as geographically informative markers, we first assessed migratory connectivity in the host: Northern European breeding populations predominantly overwintered in dry, savannah-like habitats in Southern Africa, whereas Southern European populations were associated with wetland habitats in Western Central Africa. Wintering areas of swallows breeding in Central Europe indicated a migratory divide with both migratory programmes occurring within the same breeding population. Subsequent genetic screens of parasites in the breeding populations revealed a link between the host's migratory programme and its parasitic repertoire: controlling for effects of local breeding location, prevalence of Africa-transmitted Plasmodium lineages was significantly higher in individuals overwintering in the moist habitats of Western Central Africa, even among sympatrically breeding individuals with different overwintering locations. For the rarer Haemoproteus parasites, prevalence was best explained by breeding location alone, whereas no clear pattern emerged for the least abundant parasite Leucocytozoon. These results have implications for our understanding of spatio-temporal host-parasite dynamics in migratory species and the spread of avian borne diseases.

Host preferences of ornithophilic biting midges of the genus Culicoides in the Eastern Balkans.

Many biting midges of the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) are competent vectors of a diverse number of pathogens. The identification of their feeding behaviour and of vector-host associations is essential for understanding their transmission capacity. By applying two different nested polymerase chain reaction (PCR) assays, of which one targeted the avian cyt b gene and the other targeted the COI gene of a wide range of vertebrates, we identified the blood hosts of six biting midge species including Culicoides circumscriptus, Culicoides festivipennis, Culicoides punctatus, Culicoides pictipennis, Culicoides alazanicus and Culicoides cf. griseidorsum, the latter two of which are reported in Bulgaria for the first time. Bird DNA was found in 50.6% of 95 investigated bloodmeals, whereas mammalian DNA was identified in 13.7%. Two Culicoides species were found to feed on both birds and mammals. There was remarkable diversity in the range of avian hosts: 23 species from four orders were identified in the abdomens of four Culicoides species. The most common bird species identified was the magpie, Pica pica (n = 7), which was registered in all four ornithophilic biting midge species. Six bloodmeals from the great tit, Parus major, were recorded only in C. alazanicus. None of the studied species of Culicoides appeared to be restricted to a single avian host.

Circannual variation in blood parasitism in a sub-Saharan migrant passerine bird, the garden warbler.

Knowing the natural dynamics of pathogens in migratory birds is important, for example, to understand the factors that influence the transport of pathogens to and their transmission in new geographical areas, whereas the transmission of other pathogens might be restricted to a specific area. We studied haemosporidian blood parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon in a migratory bird, the garden warbler Sylvia borin. Birds were sampled in spring, summer and early autumn at breeding grounds in Sweden, on migration at Capri, Italy and on arrival and departure from wintering staging areas in West Africa: mapping recoveries of garden warblers ringed in Fennoscandia and Capri showed that these sites are most probably on the migratory flyway of garden warblers breeding at Kvismaren. Overall, haemosporidian prevalence was 39%, involving 24 different parasite lineages. Prevalence varied significantly over the migratory cycle, with relatively high prevalence of blood parasites in the population on breeding grounds and at the onset of autumn migration, followed by marked declines in prevalence during migration both on spring and autumn passage. Importantly, we found that when examining circannual variation in the different lineages, significantly different prevalence profiles emerged both between and within genera. Our results suggest that differences in prevalence profiles are the result of either different parasite transmission strategies or coevolution between the host and the various parasite lineages. When separating parasites into common vs. rare lineages, we found that two peaks in the prevalence of rare parasites occur; on arrival at Swedish breeding grounds, and after the wintering period in Africa. Our results stress the importance of appropriate taxonomic resolution when examining host-parasite interactions, as variation in prevalence both between and within parasite genera can show markedly different patterns.

Molecular identification of bloodmeals and species composition in Culicoides biting midges.

Investigations of host preferences in haematophagous insects, including Culicoides biting midges (Diptera: Ceratopogonidae), are critical in order to assess transmission routes of vector-borne diseases. In this study, we collected and morphologically identified 164 blood-engorged Culicoides females caught in both light traps and permanent 12-m high suction traps during 2008-2010 in Sweden. Molecular analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene in the biting midges was performed to verify species classification, discern phylogenetic relationships and uncover possible cryptic species. Bloodmeal analysis using universal vertebrate cytochrome b primers revealed a clear distinction in host selection between mammalophilic and ornithophilic Culicoides species. Host sequences found matches in horse (n = 59), sheep (n = 39), cattle (n = 26), Eurasian elk (n = 1) and 10 different bird species (n = 18). We identified 15 Culicoides species previously recorded in Scandinavia and four additional species haplotypes that were distinctly different from the described species. All ornithophilic individuals (n = 23) were caught exclusively in the suction traps, as were, interestingly, almost all mammalophilic species (n = 41), indicating that many biting midge species may be able to cover long distances after completing a bloodmeal. These results add new information on the composition of Culicoides species and their host preferences and their potential long-distance dispersal while blood-engorged.

Evidence of a neo-sex chromosome in birds.

Neo-sex chromosomes often originate from sex chromosome-autosome fusions and constitute an important basis for the study of gene degeneration and expression in a sex chromosomal context. Neo-sex chromosomes are known from many animal and plant lineages, but have not been reported in birds, a group in which genome organization seems particularly stable. Following indications of sex linkage and unexpected sex-biased gene expression in warblers (Sylvioidea; Passeriformes), we have conducted an extensive marker analysis targeting 31 orthologues of loci on zebra finch chromosome 4a in five species, representative of independent branches of Passerida. We identified a region of sex linkage covering approximately the first half (10 Mb) of chromosome 4a, and associated to both Z and W chromosomes, in three Sylvioidea passerine species. Linkage analysis in an extended pedigree of one species additionally confirmed the association between this part of chromosome 4a and the Z chromosome. Markers located between 10 and 21 Mb of chromosome 4a showed no signs of sex linkage, suggesting that only half of the chromosome was involved in this transition. No sex linkage was observed in non-Sylvioidea passerines, indicating that the neo-sex chromosome arose at the base of the Sylvioidea branch of the avian phylogeny, at 47.4-37.6 millions years ago (MYA), substantially later than the ancestral sex chromosomes (150 MYA). We hypothesize that the gene content of chromosome 4a might be relevant in its transition to a sex chromosome, based on the presence of genes (for example, the androgen receptor) that could offer a selective advantage when associated to Z-linked sex determination loci.

Broad-scale latitudinal patterns of genetic diversity among native European and introduced house sparrow (Passer domesticus) populations.

Introduced species offer unique opportunities to study evolution in new environments, and some provide opportunities for understanding the mechanisms underlying macroecological patterns. We sought to determine how introduction history impacted genetic diversity and differentiation of the house sparrow (Passer domesticus), one of the most broadly distributed bird species. We screened eight microsatellite loci in 316 individuals from 16 locations in the native and introduced ranges. Significant population structure occurred between native than introduced house sparrows. Introduced house sparrows were distinguished into one North American group and a highly differentiated Kenyan group. Genetic differentiation estimates identified a high magnitude of differentiation between Kenya and all other populations, but demonstrated that European and North American samples were differentiated too. Our results support previous claims that introduced North American populations likely had few source populations, and indicate house sparrows established populations after introduction. Genetic diversity also differed among native, introduced North American, and Kenyan populations with Kenyan birds being least diverse. In some cases, house sparrow populations appeared to maintain or recover genetic diversity relatively rapidly after range expansion (<50 years; Mexico and Panama), but in others (Kenya) the effect of introduction persisted over the same period. In both native and introduced populations, genetic diversity exhibited large-scale geographic patterns, increasing towards the equator. Such patterns of genetic diversity are concordant with two previously described models of genetic diversity, the latitudinal model and the species diversity model.

Characterization of a divergent chromosome region in the willow warbler Phylloscopus trochilus using avian genomic resources.

Genome scans have made it possible to find outlier markers thought to have been influenced by divergent selection in almost any wild population. However, the lack of genomic information in nonmodel species often makes it difficult to associate these markers with certain genes or chromosome regions. Furthermore, the extent of linkage disequilibrium (LD) in the genome will determine the density of markers required to identify the genes under selection. In this study, we investigated a chromosome region in the willow warbler Phylloscopus trochilus surrounding a single marker previously identified in a genome scan. We first located the marker in the assembled genome of another species, the zebra finch Taeniopygia guttata, and amplified surrounding sequences in Fennoscandian willow warblers. Within an investigated chromosome region of 7.3 Mb as mapped to the zebra finch genome, we observed elevated genetic differentiation between a southern and a northern population across a 2.5-Mb interval comprising numerous coding genes. Within the southern and northern populations, higher values of LD were mostly found between SNPs within the same locus, but extended across distantly situated loci when the analyses were restricted to sampling sites showing intermediate allele frequencies of southern and northern alleles. Our study shows that cross-species genome information is a useful resource to obtain candidate sequences adjacent to outlier markers in nonmodel species.

Parallelism and historical contingency during rapid ecotype divergence in an isopod.

Recent studies on parallel evolution have focused on the relative role of selection and historical contingency during adaptive divergence. Here, we study geographically separate and genetically independent lake populations of a freshwater isopod (Asellus aquaticus) in southern Sweden. In two of these lakes, a novel habitat was rapidly colonized by isopods from a source habitat. Rapid phenotypic changes in pigmentation, size and sexual behaviour have occurred, presumably in response to different predatory regimes. We partitioned the phenotypic variation arising from habitat ('selection': 81­94%), lake ('history': 0.1­6%) and lake × habitat interaction ('unique diversification': 0.4­13%) for several traits. There was a limited role for historical contingency but a strong signature of selection. We also found higher phenotypic variation in the source populations. Phenotype sorting during colonization and strong divergent selection might have contributed to these rapid changes. Consequently, phenotypic divergence was only weakly influenced by historical contingency.

Isospora hypoleucae sp. n. (Apicomplexa: Eimeriidae), a new coccidian parasite found in the Pied Flycatcher (Ficedula hypoleuca).

A new Coccidia species is reported from the natural population of Pied Flycatcher (Ficedula hypoleuca) in northern Germany. Sporulated oocysts were found in faeces from 6 of 8 sampled adults. The spherical oocysts of the new Isospora species have a brownish, smooth, bi-layered wall. Average size of sporulated oocysts was 19.4 x 19.3 microm (17.5-22.8 microm x 17.5-22.8 microm ) with a shape index (length/width) of 1.0. The sporulated oocysts have no micropyle or residuum, but enclose several small polar granules that often cluster into 2-3 dumbbell-shaped formations. Sporocysts are slightly elongated, rounded at the end opposite the Stieda body, 15.3 microm x 9.2 microm in size (13.8-16.1 microm x 8.5-10.3 microm ), and have a shape index of 1.7 (1.6-1.8). The Stieda body has a prominent knob-like cap, whereas the substieda body is absent. Sporocysts contain a small compact sporocyst residuum and 4 sporozoites. COI haplotypes identical to those isolated from faecal oocysts were PCR amplified from the blood of 13-day-old nestlings, suggesting that the newly described species has extra-intestinal stages in blood. This represents the first description of a new avian Isospora species supported by molecular sequence data from the same oocysts that are described morphologically.

Bird hosts, blood parasites and their vectors--associations uncovered by molecular analyses of blackfly blood meals.

The level of host specificity of blood-sucking invertebrates may have both ecological and evolutionary implications for the parasites they are transmitting. We used blood meals from wild-caught blackflies for molecular identification of parasites and hosts to examine patterns of host specificity and how these may affect the transmission of avian blood parasites of the genus Leucocytozoon. We found that five different species of ornithophilic blackflies preferred different species of birds when taking their blood meals. Of the blackflies that contained avian blood meals, 62% were infected with Leucocytozoon parasites, consisting of 15 different parasite lineages. For the blackfly species, there was a significant association between the host width (measured as the genetic differentiation between the used hosts) and the genetic similarity of the parasites in their blood meals. The absence of similar parasite in blood meals from blackflies with different host preferences is interpreted as a result of the vector-host associations. The observed associations between blackfly species and host species are therefore likely to hinder parasites to be transmitted between different host-groups, resulting in ecologically driven associations between certain parasite lineages and hosts species.

Patterns of differentiation in a colour polymorphism and in neutral markers reveal rapid genetic changes in natural damselfly populations.

The existence and mode of selection operating on heritable adaptive traits can be inferred by comparing population differentiation in neutral genetic variation between populations (often using F(ST) values) with the corresponding estimates for adaptive traits. Such comparisons indicate if selection acts in a diversifying way between populations, in which case differentiation in selected traits is expected to exceed differentiation in neutral markers [F(ST )(selected) > F(ST )(neutral)], or if negative frequency-dependent selection maintains genetic polymorphisms and pulls populations towards a common stable equilibrium [F(ST) (selected) < F(ST) (neutral)]. Here, we compared F(ST) values for putatively neutral data (obtained using amplified fragment length polymorphism) with estimates of differentiation in morph frequencies in the colour-polymorphic damselfly Ischnura elegans. We found that in the first year (2000), population differentiation in morph frequencies was significantly greater than differentiation in neutral loci, while in 2002 (only 2 years and 2 generations later), population differentiation in morph frequencies had decreased to a level significantly lower than differentiation in neutral loci. Genetic drift as an explanation for population differentiation in morph frequencies could thus be rejected in both years. These results indicate that the type and/or strength of selection on morph frequencies in this system can change substantially between years. We suggest that an approach to a common equilibrium morph frequency across all populations, driven by negative frequency-dependent selection, is the cause of these temporal changes. We conclude that inferences about selection obtained by comparing F(ST) values from neutral and adaptive genetic variation are most useful when spatial and temporal data are available from several populations and time points and when such information is combined with other ecological sources of data.

Effects of malaria double infection in birds: one plus one is not two.

Avian malaria parasites are supposed to exert negative effects on host fitness because these intracellular parasites affect host metabolism. Recent advances in molecular genotyping and microscopy have revealed that coinfections with multiple parasites are frequent in bird-malaria parasite systems. However, studies of the fitness consequences of such double infections are scarce and inconclusive. We tested if the infection with two malaria parasite lineages has more negative effects than single infection using 6 years of data from a natural population of house martins. Survival was negatively affected by both types of infections. We found an additive cost from single to double infection in body condition, but not in reproductive parameters (double-infected had higher reproductive success). These results demonstrate that malaria infections decrease survival, but also have different consequences on the breeding performance of single- and double-infected wild birds.

Haemosporidian blood parasites in European birds of prey and owls.

Avian blood parasites have been intensively studied using morphological methods with limited information on their host specificity and species taxonomic status. Now the analysis of gene sequences, especially the mitochondrial cytochrome b gene of the avian haemosporidian species of Haemoproteus, Plasmodium, and Leucocytozoon, offers a new tool to review the parasite specificity and status. By comparing morphological and genetic techniques, we observed nearly the same overall prevalence of haemosporidian parasites by microscopy (19.8%) and polymerase chain reaction (PCR) (21.8%) analyses. However, in contrast to the single valid Leucocytozoon species (L. toddi) in the Falconiformes we detected 4 clearly distinctive strains by PCR screening. In the Strigiformes, where the only valid Leucocytozoon species is L. danilewskyi, we detected 3 genetically different strains of Leucocytozoon spp. Two strains of Haemoproteus spp. were detected in the birds of prey and owls examined, whereas the strain found in the tawny owl belonged to the morphospecies Haemoproteus noctuae. Three Plasmodium spp. strains that had already been found in Passeriformes were also detected in the birds of prey and owls examined here, supporting previous findings indicating a broad and nonspecific host spectrum bridging different bird orders.

Chronic infection. Hidden costs of infection: chronic malaria accelerates telomere degradation and senescence in wild birds.

Recovery from infection is not always complete, and mild chronic infection may persist. Although the direct costs of such infections are apparently small, the potential for any long-term effects on Darwinian fitness is poorly understood. In a wild population of great reed warblers, we found that low-level chronic malaria infection reduced life span as well as the lifetime number and quality of offspring. These delayed fitness effects of malaria appear to be mediated by telomere degradation, a result supported by controlled infection experiments on birds in captivity. The results of this study imply that chronic infection may be causing a series of small adverse effects that accumulate and eventually impair phenotypic quality and Darwinian fitness.

Nestling growth and song repertoire size in great reed warblers: evidence for song learning as an indicator mechanism in mate choice.

Females of many songbird species show a preference for mating with males that have larger song repertoires, but the advantages associated with this preference are uncertain. We tested the hypothesis that song complexity can serve as an indicator of male quality because the development of the brain regions underlying song learning and production occurs when young birds typically face nutritional and other stresses, so that song reflects how well a male fared during post-hatch development. A key prediction of this hypothesis is that variation in nestling condition should correspond to variation in the adult song repertoires of individuals. We used data from a long-term study of the great reed warbler (Acrocephalus arundinaceus) to test this prediction, correlating two measures of nestling development with subsequent repertoire size of males. We found that the length of the innermost primary feather, a standard measure of development, significantly predicted first-year repertoire size. The relationship between repertoire size and body mass was nearly significant, in spite of the large variance inherent in this measure. These data support the idea that song may provide females with information about a male's response to developmental stress, which in turn is expected to correlate with indirect or direct benefits she might receive.

Microsatellite diversity predicts recruitment of sibling great reed warblers.

Inbreeding increases the level of homozygosity, which in turn might depress fitness. In addition, individuals having the same inbreeding coefficient (e.g. siblings) vary in homozygosity. The potential fitness effects of variation in homozygosity that is unrelated to the inbreeding coefficient have seldom been examined. Here, we present evidence from wild birds that genetic variation at five microsatellite loci predicts the recruitment success of siblings. Dyads of full-sibling great reed warblers (Acrocephalus arundinaceus), one individual of which became a recruit to the natal population while the other did not return, were selected for the analysis. Each dyad was matched for sex and size. Local recruitment is strongly tied to fitness in great reed warblers as the majority of offspring die before adulthood, philopatry predominates among surviving individuals and emigrants have lower lifetime fitness. Paired tests showed that recruited individuals had higher individual heterozygosity and higher genetic diversity, which was measured as the mean squared distance between microsatellite alleles (mean d(2)), than their non-recruited siblings. These relationships suggest that the microsatellite markers, which are generally assumed to be neutral, cosegregated with genes exhibiting genetic variation for fitness.

Observation of a ZZW female in a natural population: implications for avian sex determination.

Avian sex determination is chromosomal; however, the underlying mechanisms are not yet understood. There is no conclusive evidence for either of two proposed mechanisms: a dominant genetic switch or a dosage mechanism. No dominant sex-determining gene on the female-specific W chromosome has been found. Birds lack inactivation of one of the Z chromosomes in males, but seem to compensate for a double dose of Z-linked genes by other mechanisms. Recent studies showing female-specific expression of two genes may support an active role of the W chromosome. To resolve the question of avian sex determination the investigation of birds with a 2A: ZZW or 2A: ZO genotype would be decisive. Here, we report the case of an apparent 2A: ZZW great reed warbler (Acrocephalus arundinaceus) female breeding in a natural population, which was detected using Z-linked microsatellites. Our data strongly suggest a role of W-linked genes in avian sex determination.

Stable isotopes examined across a migratory divide in Scandinavian willow warblers (Phylloscopus trochilus trochilus and Phylloscopus trochilus acredula) reflect their African winter quarters.

The C and N isotopes of feathers from two subspecies of willow warblers Phylloscopus trochilus trochilus and Phylloscopus trochilus acredula) are isotopically distinct. Our analysis of 138 adult males from 14 sites distributed across Sweden shows that the mean delta15N and delta13C values of subspecies acredula (from latitudes above 63 degrees N) were significantly higher than the mean delta15N and delta13C values of subspecies trochilus (from latitudes below 61 degrees N). The analysed willow warbler feathers had been moulted in the winter quarters and the observed isotopic signatures should thus reflect the isotopic pattern of food assimilated in Africa. The isotopic data observed in Sweden match the cline in morphology, both showing abrupt changes around 62 degrees N. This result agrees with data from ringing recoveries indicating that the two subspecies occupy geographically and isotopically distinct wintering grounds in Africa. Our isotopic data suggest that analysis of stable isotopes of C and N is a promising method to track wintering quarters of European birds that migrate to Africa.