The role of Mallard (Anas platyrhynchos) in the spread of avian influenza: genomics, population genetics, and flyways

Birds, in particular poultry and ducks, are a source of many infectious diseases, such as those caused by influenza viruses. These viruses are a threat not only to the birds themselves but also to poultry farming and human health, as forms that can infect humans are known to have evolved. It is believed that migratory birds in general play an important role in the global spread of avian influenza (AI). However, it is still debated how large this role precisely is and whether other modes of spread may be more important. The mallard (Anas platyrhynchos) is the world’s most abundant and well-studied waterfowl species. Besides being an important game and agricultural species, it is also a flagship species in wetland conservation and restoration. Waterfowl (Anseriformes: Anatidae) and especially ducks currently are the focal bird group in long distance dispersal of Avian Influenza in the wild, and the mallard has been identified as the most likely species to transport this virus. In my thesis I report aspects of the biology of this important host species of AI by molecular ecological means. As molecular marker system I established a genome-wide set of more than 100,000 SNPs of which I developed a subset of 384 SNPs into an assay to genotype about 1,000 ducks. This subset was employed to study the evolutionary history and speciation processes in the Anas genus. Further investigations into the world-wide mallard population structure on a species level were based not only on this set of 384 SNPs but also on mitochondrial DNA sequences. Last but not last, I investigated an option of AI sampling and detection from duck faeces by technology that is safe from a biohazard perspective, and solves transportation issues related to cold chains. The main results of my thesis include the development of a generally applicable improved analysis pipeline to develop genome-wide SNP sets for non-model organisms. Further, my results show that, from a migration system perspective, mallard flyways/populations can hardly be delineated from a biological point of view. Detailed phylogenetic, population genetic and coalescent analyses of a data set of samples spanning the whole northern hemisphere leads me to conclude that the only firm population boundaries that I can draw are between Eurasia and North America, within which panmixia is almost achieved. Mallards’ and other Anas-ducks’ whole continental to global distribution brings them together in sympatry. I can show that a combination of sympatric distribution, conflicting genetically determined and learned mate recognition mechanisms, and genomic compatibility between species helps to explain the long-standing puzzle of waterfowl hybridisation and introgression of genes from one duck species into another. Besides obvious management implications I propose that this fact can be part of the explanation why ducks are so well adaptable and successful, as well as why they show extraordinary abilities to withstand AI infections, or its consequences for health status. </p

Identifying hybridizing taxa within the Daphnia longispina species complex: a comparison of genetic methods and phenotypic approaches

Daphnia galeata Sars, D. longispina O. F. Muller and D. cucullata Sars (Crustacea: Cladocera) are closely related species which often produce interspecific hybrids in natural populations. Several marker systems are available for taxon determination in this hybridizing complex, but their performance and reliability has not been systematically assessed. We compared results from identifications by three molecular methods. More than 1,200 individuals from 10 localities in the Czech Republic were identified as parental species or hybrids by allozyme electrophoresis and the analysis of the restriction fragment length polymorphism of the internal transcribed spacer (ITS-RFLP); over 440 of them were additionally analyzed and identified by 12 microsatellite loci. Identification by microsatellite markers corresponded well with allozyme analyses. However, consistent discrepancies between ITS-RFLP and other markers were observed in two out of 10 studied localities. Although some marker discrepancies may have been caused by occasional recent introgression, consistent deviations between ITS-RFLP and other markers suggest a long-term maintenance of introgressed alleles. These results warn against its use as a sole identification method in field studies. Additionally, we quantitatively evaluated the discriminatory power of geometric morphometric (elliptic Fourier) analysis of body shapes based on photos of over 1,300 individuals pre-classified by allozyme markers. Furthermore, a randomly selected subset of 240 individuals was independently determined from photos by several experts. Despite a tendency for morphological divergence among parental Daphnia species, some taxa (especially D. galeata, D. longispina, and their hybrids) substantially overlapped in their body shapes. This was reflected in different determination success for particular species and hybrids in discriminant analysis based on shape data as well as from photograph

Discrimination of hybrid classes using cross-species amplification of microsatellite loci: methodological challenges and solutions in Daphnia

Microsatellite markers are important tools in population, conservation and forensic studies and are frequently used for species delineation, the detection of hybridization and introgression. Therefore, marker sets that amplify variable DNA regions in two species are required; however, cross-species amplification is often difficult, as genotyping errors such as null alleles may occur. To estimate the level of potential misidentifications based on genotyping errors, we compared the occurrence of parental alleles in laboratory and natural Daphnia hybrids (Daphnia longispina group). We tested a set of 12 microsatellite loci with regard to their suitability for unambiguous species and hybrid class identification using F1 hybrids bred in the laboratory. Further, a large set of 44 natural populations of D. cucullata, D. galeata and D. longispina (1715 individuals) as well as their interspecific hybrids were genotyped to validate the discriminatory power of different marker combinations. Species delineation using microsatellite multilocus genotypes produced reliable results for all three studied species using assignment tests. Daphnia galeata × cucullata hybrid detection was limited due to three loci exhibiting D. cucullata-specific null alleles, which most likely are caused by differences in primer-binding sites of parental species. Overall, discriminatory power in hybrid detection was improved when a subset of markers was identified that amplifies equally well in both species

Potential barriers to gene flow in the endangered European wildcat (Felis silvestris)

The European wildcat (Felis silvestris silvestris) is a focal species for conservation in many European countries. After a severe population decline during the 19th century, many populations became extinct or isolated. Within Germany, suitable wildcat habitat is assumed to be highly fragmented. We thus investigated fine-scale genetic structure of wildcat populations in Central Germany across two major potential barriers, the Rhine River with its valley and a major highway. We analyzed 260 hair and tissue samples collected between 2006 and 2011 in the Taunus and Hunsrück mountain ranges (3,500 km2 study area). We identified 188 individuals by genotyping 14 microsatellite loci, and found significant genetic substructure in the study area. Both the Rhine River and the highway were identified as significant barrier to gene flow. While the long-term effect of the river has led to stronger genetic differentiation in the river compared to the highway, estimates of current gene flow and relatedness across barriers indicated a similar or even stronger barrier effect to ongoing wildcat dispersal of the highway. Despite these barrier effects, we found evidence for the presence of recent migration across both the river and the highway. Our study thus suggests that although wildcats have the capability of dispersal across major anthropogenic and natural landscape barriers, these structures still lead to an effective isolation of populations as reflected by genetic analysis. The results strengthen the need for currently ongoing national strategies of wildcat conservation aiming for large scale habitat connectivity

Hair trapping with valerian-treated lure sticks as a tool for genetic wildcat monitoring in low-density habitats

Wildcats are among the most elusive and least investigated carnivores in Central Europe. Here, we propose a hair-trapping method that allows reliable detection of wildcat presence even in low-density habitats. The trap is simple, consisting of a wooden stick with valerian as cat attractant. We performed non-invasive genetic wildcat monitoring in the Kellerwald-Edersee National Park, Germany, between 2007 and 2011. Our results provide the first evidence of wildcat presence in this region. Microsatellite analysis and mtDNA sequencing of hair samples furthermore confirm the existence of at least six individuals (males and females) in the study region. Four individuals were detected over two consecutive years, suggesting the resident status of wildcats in this area. Our results show that the lure stick method releases its full potential when combined with genetic analysis and is a sensitive tool which not only enables the detection of wildcat presence but also provides individual identification, even in recently colonised low-density areas

Global lack of flyway structure in a cosmopolitan bird revealed by a genome wide survey of single nucleotide polymorphisms

Knowledge about population structure and connectivity of waterfowl species, especially mallards (Anas platyrhynchos), is a priority because of recent outbreaks of avian influenza. Ringing studies that trace large-scale movement patterns have to date been unable to detect clearly delineated mallard populations. We employed 363 single nucleotide polymorphism markers in combination with population genetics and phylogeographical approaches to conduct a population genomic test of panmixia in 801 mallards from 45 locations worldwide. Basic population genetic and phylogenetic methods suggest no or very little population structure on continental scales. Nor could individual-based structuring algorithms discern geographical structuring. Model-based coalescent analyses for testing models of population structure pointed to strong genetic connectivity among the world's mallard population. These diverse approaches all support the conclusion that there is a lack of clear population structure, suggesting that the world's mallards, perhaps with minor exceptions, form a single large, mainly interbreeding population

An additional field method to sex adult Barn Swallows during the non-breeding season in Zambia: white spot length in the outer tail feather

Adult Barn Swallows Hirundo rustica exhibit strong sexual size dimorphism in the length of the outermost tail feathers, which are longer in males compared with females. This trait is traditionally used to sex adult Barn Swallows in the field. However, due to the wear and breakage of the tips of the outer tail feather and tail moult during the non-breeding season, sexing becomes unreliable or even impossible. We therefore tested whether the length of the white spot on the outer tail feather is sexually dimorphic, and whether it can be used as an additional sexing method for adult Barn Swallows. The white spot length was sexually dimorphic, based on DNA analysis of 101 adult individuals caught at their roost during the non-breeding season in Zambia. Accuracy in sex determination of 95% could be obtained by classifying individuals with a white spot length <17.5 mm as females and those with a white spot length >29.5 mm as males. When applying the length of the white spot as an additional method to sex adult Barn Swallows on all birds caught in Zambia during the study period (N = 759), the percentage of birds that could successfully be sexed increased to more than 55%. Therefore we emphasise the importance of measuring the white spot length in addition to the tail fork depth and tail length to sex adult Barn Swallows in the non-breeding season.

An additional field method to sex adult Barn Swallows during the non-breeding season in Zambia: white spot length in the outer tail feather

Adult Barn Swallows Hirundo rustica exhibit strong sexual size dimorphism in the length of the outermost tail feathers, which are longer in males compared with females. This trait is traditionally used to sex adult Barn Swallows in the field. However, due to the wear and breakage of the tips of the outer tail feather and tail moult during the non-breeding season, sexing becomes unreliable or even impossible. We therefore tested whether the length of the white spot on the outer tail feather is sexually dimorphic, and whether it can be used as an additional sexing method for adult Barn Swallows. The white spot length was sexually dimorphic, based on DNA analysis of 101 adult individuals caught at their roost during the non-breeding season in Zambia. Accuracy in sex determination of 95% could be obtained by classifying individuals with a white spot length 29.5 mm as males. When applying the length of the white spot as an additional method to sex adult Barn Swallows on all birds caught in Zambia during the study period (N = 759), the percentage of birds that could successfully be sexed increased to more than 55%. Therefore we emphasise the importance of measuring the white spot length in addition to the tail fork depth and tail length to sex adult Barn Swallows in the non-breeding season