Migration direction in a songbird explained by two loci

Migratory routes and remote wintering quarters in birds are often species and even population specific. It has been known for decades that songbirds mainly migrate solitarily, and that the migration direction is genetically controlled. Yet, the underlying genetic mechanisms remain unknown. To investigate the genetic basis of migration direction, we track genotyped willow warblers Phylloscopus trochilus from a migratory divide in Sweden, where South-West migrating, and South-East migrating subspecies form a hybrid swarm. We find evidence that migration direction follows a dominant inheritance pattern with epistatic interaction between two loci explaining 74% of variation. Consequently, most hybrids migrate similarly to one of the parental subspecies, and therefore do not suffer from the cost of following an inferior, intermediate route. This has significant implications for understanding the selection processes that maintain narrow migratory divides.

We deployed a total of 466 Migrate Technology Ltd geolocators (Intigeo-W30Z11-DIP 12x5x4mm, 0.32 g) and recaptured a total of 73, of which one logger malfunctioned thus had a final data set of 72 migration tracks and DNA samples of all these birds. We chose to deploy 466 units because that is how many we could afford. From those we deployed 60 in northern Sweden and 50 in southern Sweden to get meaningful number of reference tracks of alopatric acredula and trochilus. We deployed more in northern Sweden because return rates of northern populations are smaller and more difficult to predict ( We retrieved total of 73 loggers. One malfunctioned so we had 72 tracks in total. %Five of these tracks were from norhtern Sweden (acredula) and 16 of trochilus from southern Sweden. Remaining 51 tracks were form migratory divide in central Sweden. Upon recapture we collected blood samples of each of the tracked birds for DNA analyzis.
We tracked only adult males, i.e. birds that were in their 2nd calendar year or older. It is not possible to age willow warblers more precisely than that.
We were primarily interested in migratory phenotypes and how the asociate with various genotypes in birds from a migratory divide. Since none of the hybrid genotypes in willow warblers can be identified by morphometrics we deployed as many geolocators as we could afford to increase the chance of tagging as many interesting genotypes as possible.
Birds were captured from late May to late June (from 2018 to 2021) in their breeding territories using mistnets and playback of a song. Geolocators were deployed with leg loop harness and blood was collected with a syringe from brachial vein.
As a first step we purchased 349 loggers of which we deployed 60 in northern Sweden (between lat 65 and 68) 50 in southern Sweden (between Lat 55 and 58) and 239 in the migratory divide in central Sweden (between lat 61 and 64). In 2019 we retreived 16 in soputhern Sweden, 5 in northern Sweden and 36 in central Sweden. We carried out analyzis of genotype association and migratory direction on these samples. However the sample size from the migratory divide was too small to draw robust conlcusions. Therefore in 2020 we purchased 117 more geolocators (as many as we could afford at that point in time) and deployed them in one single site in migratory divide in central Sweden where we knew from previosu work that there are many hybrids breeding. In 2021 we retreived ata from 16 more birds from migratoru divide. This was great addition and this boost in samples size allowed us to draw solid conclusions. Work was carried out from mid may to late June of each respective year.
No data was excluded from the analyzis.
All the resutls we have reported can be reproduced with the data we provide laongside wioth this manuscript. We have reported exact locations of the study sites and used standard methods for processing geolocator data. All molecular work was carried out according to two protocols previously published and also described in this study. Raw geolocator data is uploaded and publicly accessible in Dryad https://datadryad.org/stash/share/tCEX5YTwodjkplmwPnPgX8FuVlKaYZxNVRoKgKguMWQ. Genotyping results

March 2021
Randomization Blinding Did the study involve field work? Wild animals are reported in Sourca data file. Blood samples andd extracted DNA sampels are store at -80 C freezer at Lund university and can be reanalyzed if necessery. Every step of this work can be reproduced.
We deployed a subset of loggers in northern and southern Sweden where pure allopatric subspecies that migrate SE and SW respectively breed to obtain reference data on pure subspecies. The remaining loggers were randomly distributed on willow warblers in the migratory divide in central Sweden between latitudes 61 and 64°N Not relevant to the present study because it is an observational study.
All field work was carried out in willow warbler breeding habitats in Sweden during May-June of 2018, 19, 20 and 21. We never worked if i it was raining because capturing birds during rain is harmful to them. Weather conditions in Sweden in Spring/early summer are very variable. We chose to handle birds only when it was sufficianetly warm (above +10°C and not rainy). All field sites were accessed byt driving and walking.