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1. Natal body mass is a key predictor of viability and fitness in many animals. While variation in body mass and therefore viability of juveniles may be explained by genetic and environmental factors, emerging evidence points to the gut microbiota as an important factor influencing host health. The gut microbiota is known to change during development, but it remains unclear whether the microbiome predicts fitness, and if it does, at which developmental stage it affects fitness traits. 2. We collected data on two traits associated with fitness in wild nestling great tits (Parus major): weight and survival to fledging. We characterised the gut microbiome using 16S rRNA sequencing from nestling faeces and investigated temporal associations between the gut microbiome and fitness traits across development at day 8 (D8) and day 15 (D15) post-hatching. We also explored whether particular microbial taxa were indicator species that reflected whether nestlings survived or not. 3. There was no link between mass and microbial diversity on D8 or D15. However, we detected a time-lagged relationship whereby the microbial diversity at D8 was negatively associated with weight at D15, while controlling for weight at D8, therefore reflecting weight gain over the intervening period. Indicator species analysis revealed that while several taxa were unique to birds that either survived or did not survive, there were no universal taxa that were consistently found across all birds within either survival group. This suggests that the presence of particular bacterial taxa may be sufficient, but not necessary for determining future survival, perhaps owing to functional overlap in microbiota. 4. We highlight that measuring microbiome-fitness relationships at just one time point may be misleading, especially early in life. Instead, microbial-host fitness effects should be investigated longitudinally as there may be critical development windows in which key microbiota are established and prime host traits associated with nestling growth. Pinpointing which features of the gut microbial community impact on host fitness, and when during development this occurs, will shed light on population level processes and has the potential to support conservation.

The gut microbiota have important consequences for host biological processes and there is some evidence that they also affect fitness. However, the complex, interactive nature of ecological factors that influence the gut microbiota has scarcely been investigated in natural populations. We sampled the gut microbiota of wild great tits (Parus major) at different life stages allowing us to evaluate how microbiota varied with respect to a diverse range of key ecological factors of two broad types: (1) host state, namely age and sex, and the life history variables, timing of breeding, fecundity and reproductive success; and (2) the environment, including habitat type, the distance of the nest to the woodland edge, and the general nest and woodland site environments.The gut microbiota varied with life history and the environment in many ways that were largely dependent on age. Nestlings were far more sensitive to environmental variation than adults, pointing to a high degree of flexibility at an important time in development. As nestlings developed their microbiota from one to two weeks of life, they retained consistent (i.e., repeatable) among-individual differences. However these apparent individual differences were driven entirely by the effect of sharing the same nest. Our findings point to important early windows during development in which the gut microbiota are most sensitive to a variety of environmental drivers at multiple scales, and suggest reproductive timing, and hence potentially parental quality or food availability, are linked with the microbiota. Identifying and explicating the

The gut microbiome has important consequences for fitness, yet the complex, interactive nature of ecological factors that influence the gut microbiome has scarcely been investigated in natural populations. We sampled the gut microbiota of wild great tits (Parus major) at different life stages and across multiple conifer and mixed woodland fragments, allowing us to evaluate multiple factors that relate to within-individual gut microbiota acquisition, including habitat type, nest position and life history traits. The gut microbiota varied with both environment and life-history in ways that were largely dependent on age. Notably, it was the nestling, as opposed to the adult gut microbiota that was most sensitive to ecological variation, pointing to a high degree of developmental plasticity. Individual nestling differences in gut microbiota were consistently different (repeatable) from one to two weeks of life, driven entirely by the effect of sharing the same nest. Our findings point to important early developmental windows in which the gut microbiota are most sensitive to environmental variation and suggest reproductive timing, and hence parental quality or food availability, interact with the microbiome.