Development of a novel molecular tool to study molecular ecology of Ornithomya (Hippoboscidae) avian louse flies.

Louse flies (Diptera: Hippoboscidae) are obligatory hematophagous ectoparasites of birds and mammals. These widely distributed parasitic flies may have a significant impact on wild and farm animals by feeding on their blood and transmitting bloodborne pathogens. However, despite their ecological importance, louse flies are clearly underrepresented in host-parasite research and implementation of genetic approaches in this group is generally hampered by lacking molecular tools. In addition, louse flies that parasitize long-distance migrants can travel long distances with their avian hosts, facilitating the large-scale spread of pathogens across landscapes and geographic regions. Given the wide diversity of louse flies that parasitize a variety of avian hosts, their direct negative impact on host survival, and their high potential to transmit bloodborne pathogens even along avian migration routes, it is surprising that our knowledge of louse fly ecology is rather modest and incomplete. Here, we aimed to develop a novel molecular tool for polyxenous avian louse flies from the genus Ornithomya, which are among the most common and widely distributed representatives of Hippoboscidae family, to improve research of their genetic population structure and molecular ecology. Using the Illumina Mi-seq sequencing, we conducted a genome-wide scan in Ornithomya avicularia to identify putative microsatellite markers. A panel of 26 markers was selected to develop amplification protocols and assess polymorphism in the Central European population of O. avicularia, as well as to test for cross-amplification in a congeneric species (O. chloropus). A genome-scan in O. avicularia identified over 12 thousand putative microsatellite markers. Among 26 markers selected for a population-wide screening; one did not amplify successfully and three were monomorphic. 22 markers were polymorphic with at least two alleles detected. Two markers showed presence of null alleles. A cross-amplification of microsatellite markers in O. chloropus revealed allelic polymorphism at 14 loci, with the mean allelic richness of 3.78 alleles per locus (range: 2-8). Our genome-wide scan in O. avicularia provides a novel and powerful tool for molecular research in Ornithomya louse flies. Our panel of polymorphic microsatellite loci should allow genotyping of louse flies from geographically distinct populations and from a wide spectrum of avian hosts, enhancing population genetic and phylogeographic research in Ornithomya.

Carry-Over Effects of Climate Variability at Breeding and Non-Breeding Grounds on Spring Migration in the European Wren Troglodytes troglodytes at the Baltic Coast.

Many studies have linked changes in avian phenology in Europe to the North Atlantic Oscillation (NAO), which serves as a proxy for conditions in western Europe. However, the effects of climate variation in other regions of Europe on the phenology of short-distance migrants with large non-breeding grounds remain unclear. We determined the combined influence of large-scale climate indices, NAO, the Mediterranean Oscillation Index (MOI), and the Scandinavian Pattern (SCAND), during the preceding year on spring migration timing of European wren at the southern Baltic coast during 1982-2021. We modelled the effects of these climate variables on the entire passage and subsequent percentiles of the wren's passage at Bukowo-Kopan and Hel ringing stations. Over 1982-2021, the start and median of migration shifted earlier at Hel, but the end of passage shifted later at both stations. In effect, the duration of passage at Hel was extended by 7.6 days. Early passage at Hel was related with high MOI in spring and the preceding autumn. Spring passage at Bukowo-Kopan was delayed after high NAO in the previous breeding season, and high winter and spring NAO. Late spring passage occurred at both stations following a high SCAND in the previous summer. At both locations, an early start or median of passage followed high local temperatures. We conclude that phenology of the wren's spring migration at the Baltic coast was shaped by conditions encountered at wintering quarters in western Europe, where NAO operates, and in the south-eastern Europe, where the MOI operates, in conjunction with conditions in Scandinavia during the previous breeding season. We demonstrated that climate variability in various parts of the migrants' range has combined carry-over effects on in migrants' phenology in Europe.

Divergent evolution drives high diversity of toll-like receptors (TLRs) in passerine birds: Buntings and finches.

Toll-like receptors (TLRs) form a key component of animal innate immunity, being responsible for recognition of conserved microbial structures. As such, TLRs may be subject to diversifying and balancing selection, which maintains allelic variation both within and between populations. However, most research on TLRs in non-model avian species is focused on bottlenecked populations with depleted genetic variation. Here, we assessed variation at the extracellular domains of three TLR genes (TLR1LA, TLR3, TLR4) across eleven species from two passerine families of buntings (Emberizidae) and finches (Fringillidae), all having large breeding population sizes (millions of individuals). We found extraordinary TLR polymorphism in our study taxa, with >100 alleles detected at TLR1LA and TLR4 across species and high haplotype diversity (>0.75) in several species. Despite recent species divergence, no nucleotide allelic variants were shared between species, suggesting rapid TLR evolution. Higher variation at TLR1LA and TLR4 than TLR3 was associated with a stronger signal of diversifying selection, as measured with nucleotide substitutions rates and the number of positively selected sites (PSS). Structural protein modelling of TLRs showed that some PSS detected within TLR1LA and TLR4 were previously recognized as functionally important sites or were located in their proximity, possibly affecting ligand recognition. Furthermore, we identified PSS responsible for major surface electrostatic charge clustering, which may indicate their adaptive importance. Our study provides compelling evidence for the divergent evolution of TLR genes in buntings and finches and indicates that high TLR variation may be adaptively maintained via diversifying selection acting on functional ligand binding sites.

Large-Scale Climatic Patterns Have Stronger Carry-Over Effects than Local Temperatures on Spring Phenology of Long-Distance Passerine Migrants between Europe and Africa.

Earlier springs in temperate regions since the 1980s, attributed to climate change, are thought to influence the earlier arrival of long-distance migrant passerines. However, this migration was initiated weeks earlier in Africa, where the Southern Oscillation, Indian Ocean Dipole, North Atlantic Oscillation drive climatic variability, and may additionally influence the migrants. Multiple regressions investigated whether 15 indices of climate in Africa and Europe explained the variability in timing of arrival for seven trans-Saharan migrants. Our response variable was Annual Anomaly (AA), derived from standardized mistnetting from 1982-2021 at Bukowo, Polish Baltic Sea. For each species, the best models explained a considerable part of the annual variation in the timing of spring's arrival by two to seven climate variables. For five species, the models included variables related to temperature or precipitation in the Sahel. Similarly, the models included variables related to the North Atlantic Oscillation (for four species), Indian Ocean Dipole (three), and Southern Oscillation (three). All included the Scandinavian Pattern in the previous summer. Our conclusion is that climate variables operating on long-distance migrants in the areas where they are present in the preceding year drive the phenological variation of spring migration. These results have implications for our understanding of carry-over effects.

Climate in Africa sequentially shapes spring passage of Willow Warbler Phylloscopus trochilus across the Baltic coast.

BACKGROUND: Many migrant birds have been returning to Europe earlier in spring since the 1980s. This has been attributed mostly to an earlier onset of spring in Europe, but we found the timing of Willow Warblers' passage to be influenced by climate indices for Africa as much as those for Europe. Willow Warblers' spring passage through northern Europe involves populations from different wintering quarters in Africa. We therefore expected that migration timing in the early, middle and late periods of spring would be influenced sequentially by climate indices operating in different parts of the winter range. METHODS: Using data from daily mistnetting in 1 April-15 May over 1982-2017 at Bukowo (Poland, Baltic Sea coast), we derived an Annual Anomaly (AA, in days) of Willow Warbler spring migration. We decomposed this anomaly into three main periods (1-26 April, 27 April-5 May, 6-15 May); one-third of migrants in each period. We modelled three sequential time series of spring passage using calendar year and 15 large-scale climate indices averaged over the months of Willow Warblers' life stages in the year preceding spring migration as explanatory variables in multiple regression models. Nine climate variables were selected in the best models. We used these nine explanatory variables and calculated their partial correlations in models for nine overlapping sub-periods of AA. The pattern of relationships between AA in these nine sub-periods of spring and the nine climate variables indicated how spring passage had responded to the climate. We recommend this method for the study of birds' phenological responses to climate change. RESULTS: The Southern Oscillation Index and Indian Ocean Dipole in Aug-Oct showed large partial correlations early in the passage, then faded in importance. For the Sahel Precipitation Index (PSAH) and Sahel Temperature Anomaly (TSAH) in Aug-Oct partial correlations occurred early then peaked in mid-passage; for PSAH (Nov-March) correlations peaked at the end of passage. NAO and local temperatures (April-May) showed low correlations till late April, which then increased. For the Scandinavian Index (Jun-Jul) partial correlations peaked in mid-passage. Year was not selected in any of the best models, indicating that the climate variables alone accounted for Willow Warblers' multiyear trend towards an earlier spring passage. DISCUSSION: Climate indices for southern and eastern Africa dominated relationships in early spring, but western African indices dominated in mid- and late spring. We thus concluded that Willow Warblers wintering in southern and eastern Africa dominated early arrivals, but those from western Africa dominated later. We suggest that drivers of phenological shifts in avian migration are related to changes in climate at remote wintering grounds and at stopovers, operating with climate change in the north, especially for species with complex and long-distance migration patterns.

Distinct evolutionary trajectories of MHC class I and class II genes in Old World finches and buntings.

Major histocompatibility complex (MHC) genes code for key proteins of the adaptive immune system, which present antigens from intra-cellular (MHC class I) and extra-cellular (MHC class II) pathogens. Because of their unprecedented diversity, MHC genes have long been an object of scientific interest, but due to methodological difficulties in genotyping of duplicated loci, our knowledge on the evolution of the MHC across different vertebrate lineages is still limited. Here, we compared the evolution of MHC class I and class II genes in three sister clades of common passerine birds, finches (Fringillinae and Carduelinae) and buntings (Emberizidae) using a uniform methodological (genotyping and data processing) approach and uniform sample sizes. Our analyses revealed contrasting evolutionary trajectories of the two MHC classes. We found a stronger signature of pervasive positive selection and higher allele diversity (allele numbers) at the MHC class I than class II. In contrast, MHC class II genes showed greater allele divergence (in terms of nucleotide diversity) and a much stronger recombination (gene conversion) signal. Gene copy numbers at both MHC class I and class II evolved via fluctuating selection and drift (Brownian Motion evolution), but the evolutionary rate was higher at class I. Our study constitutes one of few existing examples, where evolution of MHC class I and class II genes was directly compared using a multi-species approach. We recommend that re-focusing MHC research from single-species and single-class approaches towards multi-species analyses of both MHC classes can substantially increase our understanding MHC evolution in a broad phylogenetic context.

Individual variability and versatility in an eco-evolutionary model of avian migration.

Seasonal migration is a complex and variable behaviour with the potential to promote reproductive isolation. In Eurasian blackcaps (Sylvia atricapilla), a migratory divide in central Europe separating populations with southwest (SW) and southeast (SE) autumn routes may facilitate isolation, and individuals using new wintering areas in Britain show divergence from Mediterranean winterers. We tracked 100 blackcaps in the wild to characterize these strategies. Blackcaps to the west and east of the divide used predominantly SW and SE directions, respectively, but close to the contact zone many individuals took intermediate (S) routes. At 14.0° E, we documented a sharp transition from SW to SE migratory directions across only 27 (10-86) km, implying a strong selection gradient across the divide. Blackcaps wintering in Britain took northwesterly migration routes from continental European breeding grounds. They originated from a surprisingly extensive area, spanning 2000 km of the breeding range. British winterers bred in sympatry with SW-bound migrants but arrived 9.8 days earlier on the breeding grounds, suggesting some potential for assortative mating by timing. Overall, our data reveal complex variation in songbird migration and suggest that selection can maintain variation in migration direction across short distances while enabling the spread of a novel strategy across a wide range.

Age-dependent differences in iris colouration of passerines during autumn migration in Central Europe.

Avian eye colour changes with age, but many aspects of this transition are still insufficiently understood. We examined if an individual's sex, age, species and body condition are related to the iris colour in common migratory passerines during their autumn passage through Central Europe. A total of 1,399 individuals from nine numerous species were ringed and examined in late autumn in northern Poland. Each individual was sexed by plumage (if possible) and assigned to one of three classes of the iris colour-typical for immatures, typical for adults and intermediate. We found that the iris was typical in 97.7% cases of immatures and in 75.8% cases of adults and this difference was significant. Species, sex and body mass index (BMI) had no significant influence on the iris colour. We show that iris colour in passerines in late autumn is strongly age-dependent and thus can serve as a reliable feature for ageing in field studies, especially in species difficult to age by plumage.

Climatic variation in Africa and Europe has combined effects on timing of spring migration in a long-distance migrant Willow Warbler Phylloscopus trochilus.

BACKGROUND: The arrival of many species of migrant passerine in the European spring has shifted earlier over recent decades, attributed to climate change and rising temperatures in Europe and west Africa. Few studies have shown the effects of climate change in both hemispheres though many long-distance migrants use wintering grounds which span Africa. The migrants' arrival in Europe thus potentially reflects a combination of the conditions they experience across Africa. We examine if the timing of spring migration of a long-distance migrant, the Willow Warbler, is related to large-scale climate indices across Africa and Europe. METHODS: Using data from daily mistnetting from 1 April to 15 May in 1982-2017 at Bukowo (Poland, Baltic Sea coast), we developed an Annual Anomaly metric (AA, in days) to estimate how early or late Willow Warblers arrive each spring in relation to their multi-year average pattern. The Willow Warblers' spring passage advanced by 5.4 days over the 36 years. We modelled AA using 14 potential explanatory variables in multiple regression models. The variables were the calendar year and 13 large-scale indices of climate in Africa and Europe averaged over biologically meaningful periods of two to four months during the year before spring migration. RESULTS: The best model explained 59% of the variation in AA with seven variables: Northern Atlantic Oscillation (two periods), Indian Ocean Dipole, Southern Oscillation Index, Sahel Precipitation Anomaly, Scandinavian Index and local mean temperatures. The study also confirmed that a long-term trend for Willow Warblers to arrive earlier in spring continued up to 2017. DISCUSSION: Our results suggest that the timing of Willow Warbler spring migration at the Baltic Sea coast is related to a summation of the ecological conditions they had encountered over the previous year during breeding, migration south, wintering in Africa and migration north. We suggest these large-scale climate indices reflect ecological drivers for phenological changes in species with complex migration patterns and discuss the ways in which each of the seven climate indices could be related to spring migration at the Baltic Sea coast.

Sexual dimorphism in adult Little Stints (Calidris minuta) revealed by DNA sexing and discriminant analysis.

BACKGROUND: The sex of an individual organism plays such an important role in its life cycle that researchers must know a bird's sex to interpret key aspects of its biology. The sexes of dimorphic species can be easily distinguished, but sexing monomorphic bird species often requires expensive and time-consuming molecular methods. The Little Stint (Calidris minuta) is a numerous species, monomorphic in plumage but showing a small degree of reversed sexual size dimorphism. Females are larger than males but the ranges of their measurements overlap, making Little Stints difficult to sex in the field. Our aim was to develop reliable sexing criteria for Little Stints in different stages of primary moult during their stay on the non-breeding grounds in South Africa using DNA-sexed individuals and discriminant function analysis. METHODS: We caught 348 adult Little Stints in 2008-2016 on their non-breeding grounds at Barberspan Bird Sanctuary. To molecularly identify the birds' sex we used P2/P8 primers and DNA isolated from blood samples collected in the field. We used Storer's dimorphism index to assess the degree of sexual size dimorphism. Then we divided our sample into two groups: before or during and after primary moult. For each group we developed two functions: one using wing length only and the other a combination of morphometric features including wing, tarsus and total head length. Then we used a stepwise procedure to check which combination of measurements best discriminated sexes. To validate our result we used a jack-knife cross-validation procedure and Cohen-kappa statistics. RESULTS: All the morphometric features we measured were bigger in DNA-sexed females than in males. Birds with fresh primaries had on average 2.3 mm longer wings than those with worn primaries. A discriminant function using wing length (D1) correctly sexed 78.8% of individuals before moult, and a stepwise analysis showed that a combination of wing length and tarsus (D2) correctly identified the sex of 82.7% of these birds. For birds with freshly moulted primaries a function using wing length (D3) correctly classified 83.4% of the individuals, and a stepwise analysis revealed that wing and total head length (D4) classified 84.7%. DISCUSSION: Sexual size differences in Little Stints might be linked to their phylogenetics and breeding biology. Females are bigger, which increases their fecundity; males are smaller, which increases their manoeuverability during display flights and hence their mating success. Little Stints show an extreme lack of breeding site fidelity so we did not expect a geographical cline in their biometrics. Sexing criteria available for Little Stints in the literature were developed using museum specimens, which often shrink, leading to misclassification of live birds. The sexing criteria we developed can be used for studies on Little Stints at their non-breeding grounds and on past data, but should be applied cautiously because of the overlapping ranges.

Long-term changes in migration timing of Song Thrush Turdus philomelos at the southern Baltic coast in response to temperatures on route and at breeding grounds.

Climate warming causes the advancement of spring arrival of many migrant birds breeding in Europe, but the effects on their autumn migration are less known. We aimed to determine any changes in the timing of Song Thrush captured during spring and autumn migrations at the Polish Baltic coast from 1975 to 2014, and if these were related to long-term changes of temperature at their breeding grounds and migration routes. The timing of spring migration at Hel ringing station in 1975-2014 did not show long-term advance, but they had responded to environmental conditions on the year-to-year basis. The warmer the temperatures were in April on their migration route, the earlier were the dates of the median and the end of spring migration at Hel. The beginning of autumn migration at the Mierzeja Wislana ringing station advanced by 5 days between 1975 and 2014. The warmer the April on route, and the July at the Song Thrushes' breeding grounds, the earlier young birds began autumn migration across the Baltic coast. We suggest this was a combined effect of adults' migration and breeding early during warm springs and young birds getting ready faster for autumn migration during warm summers. The average time span of 90% of the autumn migration was extended by 5 days, probably because of early migration of young birds from first broods and late of those from second broods enabled by warm springs and summers. The response of Song Thrushes' migration timing to temperatures on route and at the breeding grounds indicated high plasticity in the species and suggested it might adapt well to climate changes.