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Population assignment of willow warblers (Phylloscopus trochilus) based on qPCR-based SNP genotyping

Zhao, Tianhao (2019) BION02 20172
Degree Projects in Biology
Abstract
The fact that solitary migratory birds can find their migration routes has indicated the possibility that a genetic basis may be involved in determining the route of migration. Researchers have also found evidence to investigate this hypothesis in various ways. The focal species of this study is the willow warbler (Phylloscopus trochilus), which has three described subspecies (trochilus, acredula and yakutensis) with different migration routes and wintering quarters. The areas where the subspecies trochilus and acredula come into contact are called “migratory divides” as these geographical areas are associated with drastic changes in their migratory behavior. Migratory divides provide opportunities to study the associations between genetic... (More)
The fact that solitary migratory birds can find their migration routes has indicated the possibility that a genetic basis may be involved in determining the route of migration. Researchers have also found evidence to investigate this hypothesis in various ways. The focal species of this study is the willow warbler (Phylloscopus trochilus), which has three described subspecies (trochilus, acredula and yakutensis) with different migration routes and wintering quarters. The areas where the subspecies trochilus and acredula come into contact are called “migratory divides” as these geographical areas are associated with drastic changes in their migratory behavior. Migratory divides provide opportunities to study the associations between genetic differences and migratory behaviors. Nevertheless, the morphological difference between the three subspecies are small and overlapping, hence difficult to identify in the field. Several highly fixed SNPs between breeding populations of the willow warbler have been found and they hold the potential to genotype and assign the unknown individuals to different breeding populations. To realize this purpose, qPCR is an efficient and precise method for SNP genotyping. In this study we selected two SNPs on each of the chromosome 1, 3 and 5, respectively to separate the subspecies trochilus (southern population) and acredula (northern population) breeding in Scandinavia. Besides, one SNP was selected on chromosome 6 to separate the eastern populations (the subspecies yakutensis) breeding in Siberia from the western populations (the subspecies trochilus and acredula). We used breeding individuals of southern (n=66) and northern populations (n=60) far away from the contact zone, as well as breeders in the contact zone (n=209) between the two populations to examine the reliability of the selected SNPs on chromosome 1,3 and 5. In addition, we included 41 eastern samples to test the SNP on chromosome 6 for its assignment power. After determining the allele frequencies in the breeding populations, we examined unknown individuals sampled from autumn migration in Europe as well as from wintering sites in Africa. We used specially designed models to assign them to breeding populations based on their genotypes of the SNPs on chromosome 1 and 5. In agreement with data from ringing recoveries we found that the subspecies trochilus dominated the western migration route (Portugal) and western Africa as its wintering range, whereas acredula dominated the eastern migration route (Bulgaria) and eastern Africa as its wintering range. In between the western and eastern migration routes (Italy), a substantial proportion (50%) of the passing individuals were assigned to originate from, or possibly originated from the contact zone between the northern and southern populations. Besides, we did not find any confirmed yakutensis individual in our sample collections, but 3 heterozygotes (2 from Bulgaria and 1 from Kenya). In conclusion, our results matched with the previous knowledge of the migratory divide in willow warblers and found further evidence indicating the hybrid offspring tend to go intermediate routes. We also found convenient way to distinguish the three subspecies of willow warblers by their genotypes. (Less)
Popular Abstract
ID willow warblers to illustrate their migration patterns

Everybody knows migration. The image you may come up with can be the mammals’ migration on the savanna in Africa, or the elegant Arctic tern travel back and forth between Arctic and Antarctic every year. But have you heard of a tiny common bird in Europe, the willow warbler (Phylloscopus trochilus), is a crazy migratory species? Do you know they can be far to South Africa for winter but returning to northern Scandinavia for breeding?

With the impressive migration trait, willow warblers have drawn much attention from the researchers. It has 3 subspecies, the trochilus breeding in southern Scandinavia and western Europe, and will migrate through Iberia to western Africa; the... (More)
ID willow warblers to illustrate their migration patterns

Everybody knows migration. The image you may come up with can be the mammals’ migration on the savanna in Africa, or the elegant Arctic tern travel back and forth between Arctic and Antarctic every year. But have you heard of a tiny common bird in Europe, the willow warbler (Phylloscopus trochilus), is a crazy migratory species? Do you know they can be far to South Africa for winter but returning to northern Scandinavia for breeding?

With the impressive migration trait, willow warblers have drawn much attention from the researchers. It has 3 subspecies, the trochilus breeding in southern Scandinavia and western Europe, and will migrate through Iberia to western Africa; the acredula breeding in northern Scandinavia and eastern Europe, and will migrate through eastern Europe and overwinter in eastern Africa; the other one, yakutensis, is breeding throughout Siberia and will also migrate to eastern Africa. Due to the differences among their migration trait, researchers have been trying to study in genomic ways to find out what genes are responsible for such differences. But when it comes to reality, how can we tell these subspecies apart?

You must be surprised: How come I can ask this question? Shouldn’t it be easy? But unfortunately, they are warblers! Being a warbler means that it is tricky to identify an individual even on the species level, not to mention between subspecies. The fact is , there are slight differences on the body color among subspecies, but it is the morphological traits are overlapped, not to mention there are hybrids between subspecies and we would like to find them out.

For the purpose of identification, using genetic markers is a good way. In our genome, there are numerous markers, and the combination makes individual unique. Further, if we select a certain combo of markers which is population-specific, then it will become a “tag” from which we can tell the origin of an individual easily.

SNP (Single Nucleotide Polymorphism) is a good choice as genetic markers on population level. For instance, in population A almost all individuals have A-T allele on one nucleotide locus, whereas in population B all of them have C-G allele. If we can detect the allele type of an individual, we can say this individual is from A or B regardless of its morphology.

To do so, qPCR (quantitative Polymerase Chain Reaction) is capable for detecting SNP allele with high precision and efficiency. In brief, for a given SNP, each of its allele has a linkage with a type of fluorescence; we can determine if an individual is homozygous for an allele or a heterozygous by detecting which type of fluorescence(s) there is in the system.

When it comes back to our willow warblers, we did have some candidate SNPs available to facilitate this strategy to differentiate the 3 subspecies. My wonderful colleague Max Lundberg has found several SNPs in the genome to differ trochilus and acredula, and also one SNP to differ yakutensis from the other two subspecies.

Now that we have this handy strategy ready, we can then take the advantage of it and examine some willow warbler samples without confirmed identities, and use this information on migration studies of the willow warbler.

We first tested autumn migration samples from western Europe (Portugal) and eastern Europe (Bulgaria). Consistent as our previous knowledge, trochilus were found to dominate the western route of migration and acredula dominates the east migration population Furthermore, we tested the winter samples from western Africa (Ivory Coast and Cameron) and eastern Africa (Zambia, Kenya, Tanzania, South Africa): again, rochilus are in the west and acredula are in the east.

Then we tested autumn migration samples from Italy. Intriguingly, we found 6 hybrid (trochilus X acredula) individuals out of 44 samples here. In theory, Italy is located on the intermediate route between the two major migration routes of trochilus and acredula, and we expect the hybrids between these two subspecies to go in this way as a hypothesis based on the genetic migration theory. Given that this “intermediate route” is suboptimal for willow warblers, during which one need to fly through the Mediterranean Sea, and then the Sahara Desert, both with rare chances to have a stopover to rest and refuel during the journey, hybrids are supposed to suffer higher fatality rate on migration.

Our another attempt was for finding yakutensis individuals. This is the subspecies that we have very limited knowledge of, particularly its migration pattern, for instance, where they migrate and where they overwinter. Now that we have a great assistant, the SNP marker, we can examine through our collections and see if we happen to miss any yakutensis in acredula. Unfortunately, we haven’t yet found any, but some suspicious “hybrids” between acredula and yakutensis. In theory, the populations of these two subspecies meet in western Siberia and from where can hybrids be originated. Still, we need more information to support this hypothesis.

In all, similar as the job of detectives, researchers collect clues and hints for solving puzzles. This SNP genotyping strategy can be a handy tool to help us on the study of willow warblers’ migration and facilitates many related projects.

Master´s Degree Project in Biology 45 credits 2019

Supervisor: Prof. Staffan Bensch
Department of Biology
Lund University (Less)
Please use this url to cite or link to this publication:
author
Zhao, Tianhao
supervisor
organization
course
BION02 20172
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
8967700
date added to LUP
2019-01-23 14:18:32
date last changed
2019-01-23 14:31:15
@misc{8967700,
  abstract     = {The fact that solitary migratory birds can find their migration routes has indicated the possibility that a genetic basis may be involved in determining the route of migration. Researchers have also found evidence to investigate this hypothesis in various ways. The focal species of this study is the willow warbler (Phylloscopus trochilus), which has three described subspecies (trochilus, acredula and yakutensis) with different migration routes and wintering quarters. The areas where the subspecies trochilus and acredula come into contact are called “migratory divides” as these geographical areas are associated with drastic changes in their migratory behavior. Migratory divides provide opportunities to study the associations between genetic differences and migratory behaviors. Nevertheless, the morphological difference between the three subspecies are small and overlapping, hence difficult to identify in the field. Several highly fixed SNPs between breeding populations of the willow warbler have been found and they hold the potential to genotype and assign the unknown individuals to different breeding populations. To realize this purpose, qPCR is an efficient and precise method for SNP genotyping. In this study we selected two SNPs on each of the chromosome 1, 3 and 5, respectively to separate the subspecies trochilus (southern population) and acredula (northern population) breeding in Scandinavia. Besides, one SNP was selected on chromosome 6 to separate the eastern populations (the subspecies yakutensis) breeding in Siberia from the western populations (the subspecies trochilus and acredula). We used breeding individuals of southern (n=66) and northern populations (n=60) far away from the contact zone, as well as breeders in the contact zone (n=209) between the two populations to examine the reliability of the selected SNPs on chromosome 1,3 and 5. In addition, we included 41 eastern samples to test the SNP on chromosome 6 for its assignment power. After determining the allele frequencies in the breeding populations, we examined unknown individuals sampled from autumn migration in Europe as well as from wintering sites in Africa. We used specially designed models to assign them to breeding populations based on their genotypes of the SNPs on chromosome 1 and 5. In agreement with data from ringing recoveries we found that the subspecies trochilus dominated the western migration route (Portugal) and western Africa as its wintering range, whereas acredula dominated the eastern migration route (Bulgaria) and eastern Africa as its wintering range. In between the western and eastern migration routes (Italy), a substantial proportion (50%) of the passing individuals were assigned to originate from, or possibly originated from the contact zone between the northern and southern populations. Besides, we did not find any confirmed yakutensis individual in our sample collections, but 3 heterozygotes (2 from Bulgaria and 1 from Kenya). In conclusion, our results matched with the previous knowledge of the migratory divide in willow warblers and found further evidence indicating the hybrid offspring tend to go intermediate routes. We also found convenient way to distinguish the three subspecies of willow warblers by their genotypes.},
  author       = {Zhao, Tianhao},
  language     = {eng},
  note         = {Student Paper},
  title        = {Population assignment of willow warblers (Phylloscopus trochilus) based on qPCR-based SNP genotyping},
  year         = {2019},
}