Including the invisible fraction in whole population studies: a guide to the genetic sampling of unhatched bird eggs

1. Early embryo mortality has recently been proven to be a significant component of avian reproductive failure. Due to the difficulty in distinguishing eggs, which have suffered early embryo mortality from unfertilised eggs, this cause of reproductive failure has historically been underestimated and overlooked. 2. We describe methods for recognising and collecting early failed, unhatched eggs from wild bird populations, identifying and isolating embryonic material in unhatched eggs, and efficiently extracting DNA from those samples. We test these methods on unhatched hihi (Notiomystis cincta) eggs collected from the field, which have undergone postmortem incubation. 3. We obtained DNA yields from early-stage embryos that are sufficient for a wide range of molecular techniques, including microsatellite genotyping for parentage analysis and sex-typing. The type of tissue sample taken from the egg affected downstream DNA yields and microsatellite amplification rates. Species-specific microsatellite markers had higher amplification success rates than cross-species markers. We make key recommendations for each stage of the sampling and extraction process and suggest potential protocol improvements and modifications. 4. Genetic and possibly genomic analysis of embryos that die early in development has the potential to advance many fields. The methods described here will allow a more in-depth exploration of the previously overlooked causes of early embryo mortality in wild populations of birds, including threatened species