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The importance of telomere biology in human disease is increasingly recognized and, in parallel, use of telomere length (TL) measures is proliferating in epidemiological and clinical studies. Such studies measure leukocyte TL (LTL) using several methodological approaches. Shorter LTL is associated with atherosclerosis1 and all-cause mortality.2 Given the increasingly recognized role of TL in human ageing and its related diseases, it is essential to know more about the reliability and validity of TL measurement methods, their comparability and which method is optimal for a specific epidemiological/clinical setting. In an effort to address this knowledge gap, Martin-Ruiz et al. (MR)3 studied the reliability of TL measurement techniques. They compared the popular qPCR method with the labour-intensive Southern blots (SBs) and single telomere length analysis (STELA). MR concluded that ‘neither technique nor laboratory had strong influence on result variation’, and that ‘Southern blotting and qPCR are similar in their reproducibility’. Unfortunately, for the following reasons we believe that for epidemiological studies neither conclusion is justified by the data. Reliability of LTL Most DNA samples (10/12) used by MR were obtained from human placenta, cell cultures and cancer cells. However, the inter-assay reliability of LTL is the pertinent parameter for epidemiological studies. MR included only two DNA samples from leukocytes and, because these were added in the second round of the study, they could not be used to measure inter-assay reliability of LTL. TL results for human placenta, cultured and cancer cells cannot be automatically generalized to LTL reliability, which is the primary concern of epidemiologists. Note also that MR used pooled leukocyte samples of multiple donors, and effects of pooling on assay reliability can therefore not be excluded. A previous comparison of LTL reliability has been done for the SB and the qPCR methods in a study4 cited by MR. The study reported a clear difference in inter-assay coefficient of variation (CV) between SB = 1.74% and qPCR = 6.54%, using 50 leukocyte DNA samples from individual donors. Moreover, Steenstrup et al.5 investigated whether LTL elongation in longitudinal studies can be attributed to measurement error vs a real biological phenomenon. They found little evidence for LTL elongation over and above the effects expected from measurement error. At the same time, the available data indicated a substantially larger proportion of individuals with an apparent LTL elongation in qPCR-based studies when compared with SB-based studies. In our view, the most parsimonious explanation for this finding is the higher measurement error of the qPCR method. MR observed that rank correlations between measurements obtained in different laboratories and with different methods were high, reflecting similar rank orders of the observations. Due to the inclusion of different cell types, the range of TLs in this study (4.7-9.2 kb) is much higher, however, than the age group-specific range (about 3 kb by direct SBs within age groups) used in most epidemiological studies of LTL. This will have inflated the rank correlation beyond what is relevant for LTL in epidemiological studies considerably, contributing to the erroneous conclusion that the SB and qPCR methods yielded similar results. Sample size and composition MR used 12 samples. These were measured by two laboratories using SBs, one laboratory using STELA and seven laboratories using qPCR. As both the number of samples and the number of laboratories using techniques other than qPCR were low, the statistical tests used by MR to infer no difference in reliability between methods are underpowered and consequently of limited value. We are thus left puzzled by the authors’ claim of > 95% power to detect the difference previously reported between inter-assay CVs for LTL using SBs and qPCR in 50 leukocyte DNA samples.4 MR provide no details of their calculation in support of this statement, nor on the exact difference between inter-assay CVs for which they calculated their statistical power. Furthermore, the authors combined the two SB and one STELA laboratories for comparisons of inter-laboratory CV across methods. We see little scientific justification for this choice, which in effect leaves one with no information specific to either the SB or STELA technique. For the two leukocyte samples, the inter-laboratory CVs were 6.2% and 6.5% for the SB/STELA laboratories vs 22.2% and 22.2% for the qPCR laboratories (samples K and L, Table 2, in erratum MR)6. These results, albeit from a tiny sample size, are consistent with higher measurement error of the qPCR over SB/STELA based-methods. This is not specific for the leukocyte samples; overall the inter-laboratory CVs were substantially higher when using qPCR (P = 0.001 according to MR). Finally, for the crucial analyses of the inter-assay and intra-assay CVs, the total number of DNA samples was restricted to 5 and 3, respectively, and none of these were from leukocytes. CV as a measure of reliability A characteristic of the CV is its dependence on the mean, and hence the implicit assumption when using the CV is heteroscedasticity, i.e. that the variance is proportional to the mean. We examined whether this assumption holds in the results presented by MR. Figure 1 suggests that it holds for SB. There is a negligible correlation between mean and CV, which is not surprising given the logarithmic nature of molecular size ladders on gels.7 By contrast, Figure 1 suggests that it does not hold for qPCR. There is a strong negative correlation between average TL and CV, which implies that the error made in qPCR-based TL measurements is not proportional to the mean, but instead is closer to a constant (assay-specific) value. Such a finding undermines the CV as a reliability measure for qPCR-based TL studies. Instead we recommend using the intra-class correlation coefficient, which yields an informative estimate, provided that the ‘test’ samples are similarly distributed as the samples in the investigated population. Figure 1. Coefficient of variation (CV%) between laboratories for SB/STELA vs qPCR plotted against telomere length. Telomere length was standardized per laboratory, dividing the results for all samples by the value obtained for sample G. The X-axis displays the ... Figure 1 also illustrates the larger range of values obtained with qPCR when compared with SB. MR suggest that the larger ‘dynamic range’ obtained with qPCR compensates for the lower precision of the method. However, when CV values are calculated for SB laboratories alone (i.e. ignoring the STELA results), the inter-laboratory CV is in fact over 40% higher for the qPCR laboratories (paired t-test, t = 2.39, df = 18, P

Male zebra finches display multiple secondary sexual traits such as song and red bill coloration. This color is dependent on carotenoids, which enhance immune function and are antioxidants. A red bill may thus function as an indicator signal. The zebra finch is extensively used in the study of carotenoid-dependent signaling. However, studies of female mate preferences for redder bills show mixed results. Here, we report a meta-analysis of mate-choice studies that reveals that female zebra finches do prefer males with redder bills (r = 0.61), except when there was reduced opportunity for imprinting or when bill color was experimentally manipulated, which both reduced preference for red bills to approximately zero. The latter may either be due to aspects of the experimental design or due to bill color being correlated with another trait such as song rate as was previously suggested. We show, however, in a separate meta-analysis on a different set of studies that the correlation between bill coloration and song rate (r = 0.14) was significantly lower than the r = 0.61 between bill color and attractiveness. We conclude, therefore, that the role of bill coloration in mate choice cannot be solely due to an association with song rate. Thus, we conclude that females do prefer males with redder bills when there was sufficient opportunity for sexual imprinting, but to what extent this is causally related to the bill color remains to be established. Copyright 2011, Oxford University Press.

We studied primary sex ratio of clutches in relation to social dominance for 6 years in a colony of free-living jackdaws, a small corvid. Social dominance was strongly associated with clutch sex ratio, with the difference in clutch sex ratio between the most and least dominant pairs being 30--40%. To our knowledge, this is the first demonstration of an association between social dominance and sex allocation in birds. However, the direction of this effect varied between years. Dominant jackdaws produced more sons during the first years of the study but fewer sons during the last years. Offspring sex was not related to laying order within a clutch, and the effect of social dominance on sex ratio was similar on eggs laid first, middle, or last. We investigated the effect of 2 factors (laying date and parental condition) that could have mediated the shift in the effect of social dominance on sex allocation in the course of the study. Laying date was positively associated with the proportion of males, but this effect was independent of social dominance. Maternal condition (residual mass over tarsus and egg volume) was related to social dominance but not to clutch sex ratio. Paternal condition (residual mass over tarsus) was not related to clutch sex ratio. We discuss how spatial or temporal variation in effects of variables such as social dominance on sex allocation can contribute to our understanding of the evolution of sex allocation in species with complex life histories. Copyright 2008, Oxford University Press.

Many studies have suggested that reproductive performance improves during the pair-bond, which might explain why individuals remate with the same partner in many species. However, discussion exists about whether the association between reproductive performance and pair-bond duration that is reported in these studies reflects a causal relationship. Usually it is unclear whether a positive association is caused by pairs improving during their pair-bond or by high-quality pairs staying together for longer. Furthermore, reproductive performance often also depends on the age or breeding experience of parents, which all covary with pair-bond duration. A much needed experimental approach is lacking so far. We investigated the effect of pair-bond duration on reproductive performance in a long-lived monogamous bird species based on natural as well as experimental variation. The duration of oystercatcher (Haematopus ostralegus) pair-bonds, which were followed for 21 years, strongly affected reproductive output, even after controlling for effects of age and breeding experience. Pairs improved during their pair-bond, and there were no indications of selective disappearance of low-quality pairs; however, pairs that stayed together for very long performed badly. Experimental removal of one partner showed that the reproductive cost of divorce depended on the pair-bond duration with the old partner. In addition, after remating, the newly formed pairs strongly improved again, independent of the age and breeding experience of the remated pair members. As such, this study provides the first experimental evidence of a causal effect of pairbond duration on reproductive performance. Key words: age-dependent reproduction, constraint, divorce, intrapair cooperation, long-term monogamy, restraint. [Behav Ecol]