Genotyping blastoderms of avian eggs.

Undeveloped eggs occur frequently in birds and are often considered infertile, and discarded. However, the majority of undeveloped eggs may in fact have been fertilised and embryos might have died at an early stage. Such eggs contain valuable information, for example about offspring sex and paternity, and level of inbreeding. Obtaining such information may also give insight into the patterns and causes of early embryo mortality. Here we describe a simple technique for removing embryo cells from the blastoderm to obtain DNA to genotype the offspring and unequivocally ascertain fertilisation status, while retaining the overlying perivitelline layer (PVL) for sperm counts over the entire membrane. We tested this method on freshly collected eggs (high-quality material), as well as on eggs from abandoned clutches and unhatched eggs (potentially deteriorated material) of blue tits (Cyanistes caeruleus). We sampled a total of 707 eggs from a wild population of blue tits, extracted DNA from the eggs' blastoderm using a Qiagen kit, and genotyped the samples with 14 polymorphic microsatellite markers, plus one sexing marker. Overall, we successfully genotyped 97% of all eggs. Our study is the most extensive dataset of genotyped undeveloped eggs to date and demonstrates that one can reliably genotype undeveloped fertile eggs as well as retain the PVL for observations of sperm and embryo cells.

Occasional paternal inheritance of the germline-restricted chromosome in songbirds.

Songbirds have one special accessory chromosome, the so-called germline-restricted chromosome (GRC), which is only present in germline cells and absent from all somatic tissues. Earlier work on the zebra finch (Taeniopygia guttata castanotis) showed that the GRC is inherited only through the female line-like the mitochondria-and is eliminated from the sperm during spermatogenesis. Here, we show that the GRC has the potential to be paternally inherited. Confocal microscopy using GRC-specific fluorescent in situ hybridization probes indicated that a considerable fraction of sperm heads (1 to 19%) in zebra finch ejaculates still contained the GRC. In line with these cytogenetic data, sequencing of ejaculates revealed that individual males from two families differed strongly and consistently in the number of GRCs in their ejaculates. Examining a captive-bred male hybrid of the two zebra finch subspecies (T. g. guttata and T. g. castanotis) revealed that the mitochondria originated from a castanotis mother, whereas the GRC came from a guttata father. Moreover, analyzing GRC haplotypes across nine castanotis matrilines, estimated to have diverged for up to 250,000 y, showed surprisingly little variability among GRCs. This suggests that a single GRC haplotype has spread relatively recently across all examined matrilines. A few diagnostic GRC mutations that arose since this inferred spreading suggest that the GRC has continued to jump across matriline boundaries. Our findings raise the possibility that certain GRC haplotypes could selfishly spread through the population via occasional paternal transmission, thereby outcompeting other GRC haplotypes that were limited to strict maternal inheritance, even if this was partly detrimental to organismal fitness.

Sperm morphology and evidence for sperm competition among parrots.

Sperm competition is an important component of post-copulatory sexual selection that has shaped the evolution of sperm morphology. Previous studies have reported that sperm competition has a concurrently directional and stabilizing effect on sperm size. For example, bird species that show higher levels of extrapair paternity and larger testes (proxies for the intensity of sperm competition) have longer sperm and lower coefficients of variation in sperm length, both within and between males. For this reason, these sperm traits have been proposed as indexes to estimate the level of sperm competition in species for which other measures are not available. The relationship between sperm competition and sperm morphology has been explored mostly for bird species that breed in temperate zones, with the main focus on passerine birds. We measured sperm morphology in 62 parrot species that breed mainly in the tropics and related variation in sperm length to life-history traits potentially indicative of the level of sperm competition. We showed that sperm length negatively correlated with the within-male coefficient of variation in sperm length and positively with testes mass. We also showed that sperm is longer in sexually dichromatic and in gregarious species. Our results support the general validity of the hypothesis that sperm competition drives variation in sperm morphology. Our analyses suggest that post-copulatory sexual selection is also important in tropical species, with more intense sperm competition among sexually dichromatic species and among species that breed at higher densities.

Life history shapes variation in egg composition in the blue tit Cyanistes caeruleus.

Maternal investment directly shapes early developmental conditions and therefore has long-term fitness consequences for the offspring. In oviparous species prenatal maternal investment is fixed at the time of laying. To ensure the best survival chances for most of their offspring, females must equip their eggs with the resources required to perform well under various circumstances, yet the actual mechanisms remain unknown. Here we describe the blue tit egg albumen and yolk proteomes and evaluate their potential to mediate maternal effects. We show that variation in egg composition (proteins, lipids, carotenoids) primarily depends on laying order and female age. Egg proteomic profiles are mainly driven by laying order, and investment in the egg proteome is functionally biased among eggs. Our results suggest that maternal effects on egg composition result from both passive and active (partly compensatory) mechanisms, and that variation in egg composition creates diverse biochemical environments for embryonic development.

Variation in fine-scale genetic structure and local dispersal patterns between peripheral populations of a South American passerine bird.

The distribution of suitable habitat influences natal and breeding dispersal at small spatial scales, resulting in strong microgeographic genetic structure. Although environmental variation can promote interpopulation differences in dispersal behavior and local spatial patterns, the effects of distinct ecological conditions on within-species variation in dispersal strategies and in fine-scale genetic structure remain poorly understood. We studied local dispersal and fine-scale genetic structure in the thorn-tailed rayadito (Aphrastura spinicauda), a South American bird that breeds along a wide latitudinal gradient. We combine capture-mark-recapture data from eight breeding seasons and molecular genetics to compare two peripheral populations with contrasting environments in Chile: Navarino Island, a continuous and low density habitat, and Fray Jorge National Park, a fragmented, densely populated and more stressful environment. Natal dispersal showed no sex bias in Navarino but was female-biased in the more dense population in Fray Jorge. In the latter, male movements were restricted, and some birds seemed to skip breeding in their first year, suggesting habitat saturation. Breeding dispersal was limited in both populations, with males being more philopatric than females. Spatial genetic autocorrelation analyzes using 13 polymorphic microsatellite loci confirmed the observed dispersal patterns: a fine-scale genetic structure was only detectable for males in Fray Jorge for distances up to 450 m. Furthermore, two-dimensional autocorrelation analyzes and estimates of genetic relatedness indicated that related males tended to be spatially clustered in this population. Our study shows evidence for context-dependent variation in natal dispersal and corresponding local genetic structure in peripheral populations of this bird. It seems likely that the costs of dispersal are higher in the fragmented and higher density environment in Fray Jorge, particularly for males. The observed differences in microgeographic genetic structure for rayaditos might reflect the genetic consequences of population-specific responses to contrasting environmental pressures near the range limits of its distribution.