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Diet and host genetic or evolutionary history are considered the two main factors determining gut microbiota of animals, although studies are scarce in natural populations. The system of great spotted cuckoos (Clamator glandarius) parasitizing magpies (Pica pica) is ideal to study both effects since magpie adults feed cuckoo and magpie nestlings with the same diet and, consequently, differences in gut microbiota of nestlings of these two species will mainly reflect the importance of genetic components. Moreover, the diet of adults and of nestling cuckoos drastically differ from each other and, thus, differences and similarities in their microbiotas would respectively reflect the effect of environmental and genetic factors. We used next-generation sequencing technologies to analyze the gut microbiota of cuckoo adults and nestlings and of magpie nestlings. The highest α-diversity estimates appeared in nestling cuckoos and the lowest in nestling magpies. Moreover, despite the greatest differences in the microbiome composition of magpies and cuckoos of both ages, cuckoo nestlings harbored a mixture of the Operational Taxonomic Units (OTUs) present in adult cuckoos and nestling magpies. We identified the bacterial taxa responsible for such results. These results suggest important phylogenetic components determining gut microbiome of nestlings, and that diet might be responsible for similarities between gut microbiome of cuckoo and magpie nestlings that allow cuckoos to digest food provided by magpie adults., This work was supported by the Ministerio de Ciencia, Innovacion y Universidades and European (FEDER) funds (CGL2017-83103-P)., Peer reviewed

The uropygial gland of hoopoe nestlings and nesting females hosts bacterial symbionts that cause changes in the characteristics of its secretion, including an increase of its antimicrobial activity. These changes occur only in nesting individuals during the breeding season, possibly associated with the high infection risk experienced during the stay in the hole-nests. However, the knowledge on hoopoes uropygial gland microbial community dynamics is quite limited and based so far on culture-dependent and molecular fingerprinting studies. In this work, we sampled wild and captive hoopoes of different sex, age, and reproductive status, and studied their microbiota using quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization (FISH) and pyrosequencing. Surprisingly, we found a complex bacterial community in all individuals (including non-nesting ones) during the breeding season. Nevertheless, dark secretions from nesting hoopoes harbored significantly higher bacterial density than white secretions from breeding males and both sexes in winter. We hypothesize that bacterial proliferation may be host-regulated in phases of high infection risk (i.e., nesting). We also highlight the importance of specific antimicrobial-producing bacteria present only in dark secretions that may be key in this defensive symbiosis. Finally, we discuss the possible role of environmental conditions in shaping the uropygial microbiota, based on differences found between wild and captive hoopoes., This work was supported by the Spanish Ministry of Economy and Competitiveness and European
(FEDER) funds (CGL2013-48193-C3-1-P/BOS, CGL2013-48193-C3-2-P/BOS, CGL2017-83103-P), and the Junta de Andalucía (RNM 339, RNM 340). S. M. Rodríguez-Ruano received a grant from the Spanish Ministry of Science and Innovation (FPI program)., Peer reviewed

Diet and host genetic or evolutionary history are considered the two main factors determining gut microbiota of animals, although studies are scarce in natural populations. The system of great spotted cuckoos (Clamator glandarius) parasitizing magpies (Pica pica) is ideal to study both effects since magpie adults feed cuckoo and magpie nestlings with the same diet and, consequently, differences in gut microbiota of nestlings of these two species will mainly reflect the importance of genetic components. Moreover, the diet of adults and of nestling cuckoos drastically differ from each other and, thus, differences and similarities in their microbiotas would respectively reflect the effect of environmental and genetic factors. We used next-generation sequencing technologies to analyze the gut microbiota of cuckoo adults and nestlings and of magpie nestlings. The highest α-diversity estimates appeared in nestling cuckoos and the lowest in nestling magpies. Moreover, despite the greatest differences in the microbiome composition of magpies and cuckoos of both ages, cuckoo nestlings harbored a mixture of the Operational Taxonomic Units (OTUs) present in adult cuckoos and nestling magpies. We identified the bacterial taxa responsible for such results. These results suggest important phylogenetic components determining gut microbiome of nestlings, and that diet might be responsible for similarities between gut microbiome of cuckoo and magpie nestlings that allow cuckoos to digest food provided by magpie adults., This work was supported by the Ministerio de Ciencia, Innovacion y Universidades and European (FEDER) funds (CGL2017-83103-P)., Peer reviewed

Nestlings of most avian species produce faecal sacs, which facilitate the removal of nestlings’ excrements by parents, thereby reducing proliferation of potentially pathogenic microorganisms and/or detectability by predators and parasites. The nest microbial environment that birds experience during early life might also affect their development and thus, faecal sacs facilitating parental removal may be a strategy to decrease bacterial contamination of nests that could harm developing nestlings. Here, we tested this hypothesis by experimentally broken faecal sacs and spreading them in nests of spotless starlings (Sturnus unicolor), thereby avoiding their removal by adults. In accordance with the hypothesis, experimental nests harboured higher bacterial density than control nests. Nestlings in experimental nests were of smaller size (tarsus length) and experienced lower probability of survival (predation) than those in control nests. Moreover, nestlings in experimental nests tended to suffer more from ectoparasites than those in control nests. We discuss the possible pivotal role of bacteria producing chemical volatiles that ectoparasites and predators might use to find avian nests, and that could explain our experimental results in starlings, Ministerio de Ciencia, Innovación y Universidades and European (FEDER) funds (CGL2013-48193-C3-1-P, CGL2017-83103-P), Peer reviewed

Factors affecting the evolution of plumage conspicuousness in females are nowadays the focus of debate, and here we explore the possibility that the conspicuousness of female plumage and male participation in nest building are associated in birds. We hypothesize that males that participate in nest building will gain higher fecundity from high-quality ornamented females, whereas ornamented females will adjust fecundity to the costly nest-building behaviour of males. Large-sized species might experience higher costs of nest building and, thus, body size should affect the scenario described above. We used information on male contribution to nest construction (yes/no), male and female conspicuousness (conspicuous or cryptic plumage) and body size of Western Palaearctic passerines. In accordance with the hypothesis, we found that female conspicuousness, in interaction with body mass, was strongly associated with male participation in nest building. For large-sized species, female conspicuousness was positively associated with male participation in nest building. Discrete analyses of correlated evolution rendered evidence of female conspicuousness determining the evolution of male contributions to nest building, with the loss of female conspicuousness occurring more frequently before the loss of male participation in nest building. We discuss possible adaptive scenarios explaining the detected evidence because of mutual sexual selection in males and females, The study was financed by the Ministerio de Economía y Competitividad MINECO
(CGL2013-48193-C3-1-P, CGL2013-48193-C3-2-P, CGL2013-48193-C3-3-P and CGL2017-83103-P) and the European Regional Development Fund (FEDER), Peer reviewed

Nestlings of most avian species produce faecal sacs, which facilitate the removal of nestlings’
excrements by parents, thereby reducing proliferation of potentially pathogenic
microorganisms and/or detectability by predators and parasites. The nest microbial
environment that birds experience during early life might also affect their development
and thus, faecal sacs facilitating parental removal may be a strategy to decrease bacterial
contamination of nests that could harm developing nestlings. Here, we tested this
hypothesis by experimentally broken faecal sacs and spreading them in nests of spotless
starlings Sturnus unicolor, thereby avoiding their removal by adults. In accordance with
the hypothesis, experimental nests harboured higher bacterial density than control
nests. Nestlings in experimental nests were of smaller size (tarsus length) and experienced
lower probability of survival (predation) than those in control nests. Moreover,
nestlings in experimental nests tended to suffer more from ectoparasites than those
in control nests. We discuss the possible pivotal role of bacteria producing chemical
volatiles that ectoparasites and predators might use to find avian nests, and that could
explain our experimental results in starlings., This work was supported by the currently named
Ministerio de Ciencia, Innovación y Universidades and European
(FEDER) funds (CGL2013-48193-C3-1-P, CGL2017-83103-P).
MAG was financed by a predoctoral contract (BES-2014-068661)
from the Spanish Ministerio de Economía y Competitividad
(CGL2013-48193-C3-1-P)., Peer reviewed

The offspring of many animals are conspicuous during parental dependence, despite juveniles generally suffering from high predation risk. However, to date, it is unclear whether offspring structural ornaments play a role in intrafamily communication. This is the case of conspicuous plumage in young birds, which is worn unchanged during a long period after fledging, when they still depend on their parents. If plumage color facilitates intrafamily interactions, its role should be more important in large-brooded species, where the strength of intrafamily conflict is potentially stronger. We therefore performed a comparative study in 210 passerine bird species to test whether an offspring structural trait, white plumage, evolves more frequently in lineages with larger clutches. We also explored the number of broods raised per year as another source of intrafamily conflict. First, we found that juvenile whiteness was more frequent in open-nesting species. Moreover, in agreement with our prediction, the presence of juvenile white tail/wing patches was strongly and positively associated with clutch size. This relationship was not due to the strong resemblance between offspring and adult plumage, which was controlled for in the statistical analyses. Moreover, the association remained significant after taking into account predation risk, for which there was information for a subset of species. In contrast, juvenile whiteness was not associated with the number of broods raised per year. These results may suggest that the evolution of juvenile conspicuousness is favored in species with potentially stronger intrabrood sibling conflict., This work was financed by the Ministerio de Economía, Industria y
Competitividad MINECO (CGL2016-79390-P, CGL2017‐83103‐P,
CGL2017-83843-C2-1-P and CGL2013-48193-C3-1-P) and the European
Regional Development Fund (FEDER)., Peer reviewed

Data file includes (i) average values of melanised (M) and non-melanised (NM) feather wear of nestling birds that grew in different nests. The file also included (ii) information of feather mass degradability of nestlings and adult birds from different nests and species estimated as the olipeptide concentration (μg x mL1) of tubes containing 4mL of PBS buffer and a colony Bacillus licheniformis D13 after 21 days of incubation at 37 ºC in constant agitation at 120 rpm in an orbital agitator (VWR, Spain). The third excel sheet contain information of bacterial loads in terms of terms of mesophilic bacteria (TSA), Enterobacteriaceae (HK), Staphylococcus (VJ), and Enterococcus (KF), and average wear of melanised (M) and non-melanised (NM) wing feathers of nestlings that grew in the same nests where bacterial loads were estimated. The last excel file contain information on feather wear of melanised and non-melanised feathers of each sampled nestlings and adult. All these data were collected the Guadix region (southern Spain) and used to perform the statistical analyses in Tables 2 and 3 in Azcárate et al. 2020 (Doi: 10.1111/jav.02320)., This work was supported by the currently named Ministerio de Ciencia, Innovación y Universidades and European (FEDER) funds (CGL2013-48193-C3-1-P, CGL2013-48193-C3-2-P, CGL2013-48193-C3-3-P, CGL2017-83103-P). MAG and SDL were financed by predoctoral contracts (BES-2014-068661, BES-2014-069116) from the Spanish Ministerio de Economía y Competitividad., Peer reviewed

Feathers are essential for avian life, and factors affecting their integrity are important to understand their evolution. These factors should depend on, among other traits, species‐specific bacterial environments and life‐history characteristics. However, interspecific variation in feather deterioration, feather susceptibility to degradation by keratinolytic bacteria (degradability), and bacterial environment, have rarely been quantified. Here, we did so by measuring deterioration and degradability of wing feathers of fledglings in 16 bird species, and characterizing the bacterial environment where they developed. We found statistically significant interspecific variation for all considered variables. On average, non‐melanised were more deteriorated than melanised feathers, but differences depended on the species. Moreover, nest bacterial loads were related to feathers wear, but the sign of the association depended on the bacterial group considered and on feather pigmentation. We also found a positive association of feather degradability with wear of non‐melanised feathers, and with bacterial loads. These results suggest that bacterial environments determine the integrity of fledgling feathers as well as their resistance to bacterial degradation, which implies a preponderant role of bacteria in driving the evolution of avian feathers., This work was supported by the currently named Ministerio de Ciencia, Innovación y Universidades and European (FEDER) funds (CGL2013-48193-C3-1-P, CGL2013-48193-C3-2-P, CGL2013-48193-C3-3-P, CGL2017-83103-P). MAG and SDL were financed by predoctoral contracts (BES-2014-068661, BES-2014-069116) from the Spanish Ministerio de Economía y Competitividad., Peer reviewed

Nest re-use in birds has the potential cost of infection by parasites and pathogens but may also be a source of beneficial symbiotic bacteria transmitted horizontally. Eurasian hoopoes Upupa epops host antibiotic-producing bacteria in their uropygial gland but only while breeding, which suggests that the nest-hole may be a source of those symbionts. Interestingly, hoopoes do not build nests, thus might prefer for reproduction nest holes with soft materials from previous reproductions. Here, we tested experimentally this preference by installing in the field new nest boxes that were left empty or filled with either sawdust or a mixture of sawdust and hoopoe's nest material from the previous year. We explored the experimental effect on the composition of the uropygial secretion bacterial community, on eggshell bacterial loads, and on several proxies of reproductive success. Hoopoes bred significantly more often in nest boxes with nest material than in empty ones, but the type of nest material did not affect nest box occupancy. Eggs in nest boxes with old-soft material harbored higher bacterial density on their shells, and the microbiota of the uropygial secretion of nestlings and females in these nest boxes differed from those in nest boxes without old-soft material. Moreover, although the experiment did not affect breeding success or related proxies, several operational taxonomic units from female uropygial secretions were positively associated with hatching success. This is the first experimental evidence showing that re-used nest material affects the bacterial community of the uropygial secretions of hoopoe females. This suggests that the nest material can be a source of strains for their incorporation to both the uropygial gland and eggshell communities, highlighting a possible advantage of nest re-use previously unconsidered., Silvia Díaz Lora was financed by a predoctoral contract (BES‐2014‐069116) and research by three projects from the Spanish Ministerio de Economía y Competitividad, Ministerio de Ciencia, Innovacion y Universidades, and European (FEDER) funds (CGL2013‐48193‐C3‐1‐P, CGL2013‐48193‐C3‐2‐P and CGL2017‐83103‐P).