Climate change has been driving long-distance migratory birds to alter their schedules under the threat of being mismatched with their food peak at the breeding grounds. It is important to study the relative contribution of environmental, genetic and ontogenetic components in various spring timing traits in the wild in order to predict the true potential for migratory birds to adapt to the changing environment. We aimed to detect if heritable and ontogenetic components can explain variation in the timing of spring migration and breeding in pied flycatchers (Ficedula hypoleuca). Geolocator tracks of 44 locally hatched birds deployed during 2016-2019 in the Netherlands and the United Kingdom were used to investigate the role of early-life traits in the pre-fledging phase, as well as parental timings, in contributing to individual differences in the timing of spring migration and breeding in adulthood. We found a positive relationship between an individuals’ birth date and spring departure date from Africa in adulthood, but not for breeding arrival or laying date. Variation in spring departure date could not be explained by any other early-life trait in the pre-fledging phase, yet was well explained by the arrival dates of its parents in its birth year. This suggests that under natural conditions, individual differences in spring departure timing have a strong heritable component (in the broad sense), but that environmental conditions experienced along the migratory route and at breeding sites are partially masking this expression in arrival and laying schedules in these early breeding populations. Such environmental masking may reduce heritability in the timing of arrival and laying, thereby slowing down climatic adaptation towards earlier time schedules in pied flycatchers.

Ecological research is often hampered by the inability to quantify animal diets. Diet composition can be tracked through DNA metabarcoding of faecal samples, but whether (complex) diets can be quantitatively determined with metabarcoding is still debated and needs validation using free-living animals. This study validates that DNA metabarcoding of faeces can retrieve actual ingested taxa, and most importantly, that read numbers retrieved from sequencing can also be used to quantify relative abundances of dietary taxa. Validation was done with the hole-nesting insectivorous Pied Flycatcher whose diet was quantified using camera footage. Size-adjusted counts of food items delivered to nestlings were used to approximate provided biomass of prey orders and families and subsequently nestling faeces were assessed through DNA metabarcoding. To explore potential effects of digestion, stomach and lower intestine samples of freshly collected birds were subjected to DNA metabarcoding. For metabarcoding with Cytochrome Oxidase subunit I (COI), we modified published invertebrate COI primers LCO1490 and HCO1777, which reduced host reads to 0.03%, and amplified Arachnida DNA without significant changing the recovery of other arthropod taxa. DNA metabarcoding retrieved all commonly camera-recorded taxa. Overall, and in each replicate year (N = 3), the relative abundances of size-adjusted prey counts and COI read numbers correlated at R=0.85 (CI:0.68-0.94) at order level and at R=0.75 (CI:0.67-0.82) at family level. Similarity in arthropod community composition between stomach and intestines suggested limited digestive bias. This DNA metabarcoding validation demonstrates that quantitative analyses of arthropod diet is possible. We discuss the ecological applications for insectivorous birds.