Multiple aspects of plasticity in clutch size vary among populations of a globally distributed songbird.

Plasticity in life-history characteristics can influence many ecological and evolutionary phenomena, including how invading organisms cope with novel conditions in new locations or how environmental change affects organisms in native locations. Variation in reaction norm attributes is a critical element to understanding plasticity in life history, yet we know relatively little about the ways in which reaction norms vary within and among populations. We amassed data on clutch size from marked females in eight populations of house sparrows (Passer domesticus) from North America and Europe. We exploited repeated measures of clutch size to assess both the extent of within-individual phenotypic plasticity and among-individual variation and to test alternative hypotheses about the underlying causes of reaction norm shape, particularly the decline in clutch size with date. Across all populations, females of this multibrooded species altered their clutch size with respect to date, attempt order, and the interaction of date and order, producing a reaction norm in multidimensional environmental space. The reaction norm fits that predicted by a model in which optimal clutch size is driven by a decline with date hatched in the ability of offspring to recruit. Our results do not fit those predicted for other proposed causes of a seasonal decline in clutch size. We also found significant differences between populations in response to date and the date by attempt order interaction. We tested the prediction that the relationship with date should be increasingly negative as breeding season becomes shorter but found steeper declines in clutch size with date in populations with longer seasons, contrary to the prediction. Populations also differed in the level of among-individual variation in reaction norm intercept, but we found no evidence of among-individual variation in reaction norm slope. We show that complex reaction norms in life-history characters exhibit within- and among-population variance. The nature of this variance is only partially consistent with current life-history theory and stimulates expansions of such theory to accommodate complexities in adaptive life history.

High-utility conserved avian microsatellite markers enable parentage and population studies across a wide range of species.

BACKGROUND: Microsatellites are widely used for many genetic studies. In contrast to single nucleotide polymorphism (SNP) and genotyping-by-sequencing methods, they are readily typed in samples of low DNA quality/concentration (e.g. museum/non-invasive samples), and enable the quick, cheap identification of species, hybrids, clones and ploidy. Microsatellites also have the highest cross-species utility of all types of markers used for genotyping, but, despite this, when isolated from a single species, only a relatively small proportion will be of utility. Marker development of any type requires skill and time. The availability of sufficient "off-the-shelf" markers that are suitable for genotyping a wide range of species would not only save resources but also uniquely enable new comparisons of diversity among taxa at the same set of loci. No other marker types are capable of enabling this. We therefore developed a set of avian microsatellite markers with enhanced cross-species utility. RESULTS: We selected highly-conserved sequences with a high number of repeat units in both of two genetically distant species. Twenty-four primer sets were designed from homologous sequences that possessed at least eight repeat units in both the zebra finch (Taeniopygia guttata) and chicken (Gallus gallus). Each primer sequence was a complete match to zebra finch and, after accounting for degenerate bases, at least 86% similar to chicken. We assessed primer-set utility by genotyping individuals belonging to eight passerine and four non-passerine species. The majority of the new Conserved Avian Microsatellite (CAM) markers amplified in all 12 species tested (on average, 94% in passerines and 95% in non-passerines). This new marker set is of especially high utility in passerines, with a mean 68% of loci polymorphic per species, compared with 42% in non-passerine species. CONCLUSIONS: When combined with previously described conserved loci, this new set of conserved markers will not only reduce the necessity and expense of microsatellite isolation for a wide range of genetic studies, including avian parentage and population analyses, but will also now enable comparisons of genetic diversity among different species (and populations) at the same set of loci, with no or reduced bias. Finally, the approach used here can be applied to other taxa in which appropriate genome sequences are available.

Microsatellite resources for Passeridae species: a predicted microsatellite map of the house sparrow Passer domesticus.

We identified microsatellite sequences of potential utility in the house sparrow (Passer domesticus) and assigned their predicted genome locations. These sequences included newly isolated house sparrow loci, which we fully characterized. Many of the newly isolated loci were polymorphic in two other species of Passeridae: Berthelot's pipit Anthus berthelotii and zebra finch Taeniopygia guttata. In total, we identified 179 microsatellite markers that were either isolated directly from, or are of known utility in, the house sparrow. Sixty-seven of these markers were designed from unique sequences that we isolated from a house sparrow genomic library. These new markers were combined with 36 house sparrow markers isolated by other studies and 76 markers isolated from other passerine species but known to be polymorphic in the house sparrow. We utilized sequence homology to assign chromosomal locations for these loci in the assembled zebra finch genome. One hundred and thirty-four loci were assigned to 25 different autosomes and eight loci to the Z chromosome. Examination of the genotypes of known-sex house sparrows for 37 of the new loci revealed a W-linked locus and an additional Z-linked locus. Locus Pdoµ2, previously reported as autosomal, was found to be Z-linked. These loci enable the creation of powerful and cost-effective house sparrow multiplex primer sets for population and parentage studies. They can be used to create a house sparrow linkage map and will aid the identification of quantitative trait loci in passerine species.

Heterozygosity predicts clutch and egg size but not plasticity in a house sparrow population with no evidence of inbreeding.

We investigated the link between heterozygosity and the reaction norm attributes of reproductive performance in female house sparrows (Passer domesticus). We collected data on clutch size, egg size, hatching success and nestling survival in 2816 nesting attempts made by 791 marked individuals over a 16-year period. Pedigree analysis revealed no evidence of inbreeding. Neither parent-offspring regression nor an animal model revealed significant heritability in clutch or egg size. We selected 42 females that laid at least seven clutches at our study site and used a survey of 21 autosomal microsatellite loci to estimate heterozygosity for each female. We controlled for phenotypic plasticity and found that both clutch and egg size showed significant positive correlations with heterozygosity. We found no evidence that heterozygosity influenced the slope of individual reaction norms. Further analysis suggested that clutch size was affected by heterozygosity across the genome, but egg size had more complex relationships, with evidence favouring the influence of multiple loci. Given the apparent lack of inbreeding and large population size, our results suggest associative overdominance as the likely mechanism for the impact of heterozygosity, but also created a puzzle about the process producing associations between neutral markers and the genes affecting clutch size or egg size. One possible explanation is a long-term residual effect of the historical bottleneck that occurred when house sparrows were introduced into North America. The existence of heterozygosity-fitness correlations in a population with considerable phenotypic plasticity and little inbreeding implies that the effects of heterozygosity may be more significant than previously thought.

New methods to identify conserved microsatellite loci and develop primer sets of high cross-species utility - as demonstrated for birds.

We have developed a new approach to create microsatellite primer sets that have high utility across a wide range of species. The success of this method was demonstrated using birds. We selected 35 avian EST microsatellite loci that had a high degree of sequence homology between the zebra finch Taeniopygia guttata and the chicken Gallus gallus and designed primer sets in which the primer bind sites were identical in both species. For 33 conserved primer sets, on average, 100% of loci amplified in each of 17 passerine species and 99% of loci in five non-passerine species. The genotyping of four individuals per species revealed that 24-76% (mean 48%) of loci were polymorphic in the passerines and 18-26% (mean 21%) in the non-passerines. When at least 17 individuals were genotyped per species for four Fringillidae finch species, 71-85% of loci were polymorphic, observed heterozygosity was above 0.50 for most loci and no locus deviated significantly from Hardy-Weinberg proportions. This new set of microsatellite markers is of higher cross-species utility than any set previously designed. The loci described are suitable for a range of applications that require polymorphic avian markers, including paternity and population studies. They will facilitate comparisons of bird genome organization, including genome mapping and studies of recombination, and allow comparisons of genetic variability between species whilst avoiding ascertainment bias. The costs and time to develop new loci can now be avoided for many applications in numerous species. Furthermore, our method can be readily used to develop microsatellite markers of high utility across other taxa.

Complex interactions among temporal variables affect the plasticity of clutch size in a multi-brooded bird.

Phenotypic plasticity is a widespread phenomenon and may have important influences on evolutionary processes. Multidimensional plasticity, in which multiple environmental variables affect a phenotype, is especially interesting if there are interactions among these variables. We used a long-term data set from House Sparrows (Passer domesticus), a multi-brooded passerine bird, to test several predictions from life-history theory regarding the shape of optimal reaction norms for clutch size. The best-fit model for variation in clutch size included three temporal variables (the order of attempt within a season, the date of those attempts, and the age of the female). Clutch size was also sensitive to the quadratics of date and female age, both of which had negative coefficients. Finally, we found that the relationship between date and clutch size became more negative as attempt order increased. These results suggest that female sparrows have a multidimensional reaction norm for clutch size that matches predictions of life-history theory but also implicates more complexity than can be captured by any single model. Analysis of the sources of variation in reaction norm height and slope was complicated by the additional environmental dimensions. We found significant individual variation in mean clutch size in all analyses, indicating that individuals differed in the height of their clutch size reaction norm. By contrast, we found no evidence of significant individual heterogeneity in the slopes of several dimensions. We assess the possible mechanisms producing this reaction norm and discuss their implications for understanding complex plasticity.