PCR cycles above routine numbers do not compromise high-throughput DNA barcoding results.

High-throughput DNA barcoding has become essential in ecology and evolution, but some technical questions still remain. Increasing the number of PCR cycles above the routine 20-30 cycles is a common practice when working with old-type specimens, which provide little amounts of DNA, or when facing annealing issues with the primers. However, increasing the number of cycles can raise the number of artificial mutations due to polymerase errors. In this work, we sequenced 20 COI libraries in the Illumina MiSeq platform. Libraries were prepared with 40, 45, 50, 55, and 60 PCR cycles from four individuals belonging to four species of four genera of cephalopods. We found no relationship between the number of PCR cycles and the number of mutations despite using a nonproofreading polymerase. Moreover, even when using a high number of PCR cycles, the resulting number of mutations was low enough not to be an issue in the context of high-throughput DNA barcoding (but may still remain an issue in DNA metabarcoding due to chimera formation). We conclude that the common practice of increasing the number of PCR cycles should not negatively impact the outcome of a high-throughput DNA barcoding study in terms of the occurrence of point mutations.

NGS metabarcoding proves successful for quantitative assessment of symbiont abundance: the case of feather mites on birds.

Understanding the ecological function of species and the structure of communities is crucial in the study of ecological interactions among species. For this purpose, not only the occurrence of particular species but also their abundance in ecological communities is required. However, abundance quantification of species through morphological characters is often difficult or time/money consuming when dealing with elusive or small taxa. Here we tested the use of next-generation sequencing (NGS) for abundance estimation of two species of feather mites (Proctophyllodes stylifer and Pteronyssoides parinus) under five proportions (16:1, 16:4, 16:16, 16:64, and 16:256 mites) against a mock community composed by Proctophyllodes clavatus and Proctophyllodes sylviae. In all mixtures, we retrieved sequence reads from all species. We found a strong linear relationship between 454 reads and the real proportion of individuals in the mixture for both focal species. The slope for Pr. stylifer was close to one (0.904), and the intercept close to zero (-0.007), thus showing an almost perfect correspondence between real and estimated proportions. The slope for Pt. parinus was 0.351 and the intercept 0.307, showing that while the estimated proportion increased linearly relative to real proportions of individuals in the samples, proportions were overestimated at low real proportions and underestimated at larger ones. Additionally, pyrosequencing replicates from each DNA extraction were highly repeatable (R = 0.920 and 0.972, respectively), showing that the quantification method is highly consistent given a DNA extract. Our study suggests that NGS is a promising tool for abundance estimation of feather mites' communities in birds.

Resource unpredictability promotes species diversity and coexistence in an avian scavenger guild: a field experiment.

Chance per se plays a key role in ecology and evolution, e.g., genetic mutation, resource spatiotemporal unpredictability. In community ecology, chance is recognized as a key factor in community assemblage, but less is known about its role in intraguild processes leading to species coexistence. Here we study the relevance of resource unpredictability per se as a promoter of intraguild positive interspecific interactions and as a biodiversity enhancer in an Old World avian scavenger guild, which has evolved to feed upon spatially and temporally unpredictable resources, i.e., carcasses. We performed a large-scale field experiment in which 58 carcasses were disposed of and observed until complete consumption, either in continuously active supplementary feeding stations (predictable carcasses) or disposed of at random in the field (unpredictable carcasses). Richness of scavenger species was similar at unpredictable and predictable carcasses, but their relative abundances were highly uneven at predictable carcasses leading to higher scavenger diversity (Shannon index) at unpredictable carcasses. Facilitatory interspecific processes only occurred at unpredictable resources but were disrupted in predictable conditions because the dominant specialist species (in our case, the Griffon Vulture Gyps fulvus) arrived earlier and in larger numbers, monopolizing the resource. Small, endangered scavengers congregated at supplementary feeding stations but profited less compared to unpredictable carcasses, suggesting that they could constitute an ecological trap. Our findings offer new insights into the relevance of unpredictability of trophic resources in promoting both positive facilitatory interspecific interactions and species diversity and thus maintaining the function of guilds. Finally, the preservation of randomness in resource availability and the processes associated with its exploitation should be a major goal of conservation strategies aimed to preserve scavenger guilds evolved under naturally unpredictable trophic resources.

Adaptive allocation of stress-induced deformities on bird feathers.

Physiological stress during ontogeny is known to cause abnormalities in keratin structures of vertebrates, but little is known about if and how organisms have evolved mechanisms to reduce the negative effects of these abnormalities. Stress experienced during avian feather growth is known to lead to the formation of fault bars, and thereby to the weakening of feathers because of shortage and slimming of barbules. Here we propose and test a new hypothesis (the 'fault bar allocation hypothesis') according to which birds should have evolved adaptive strategies to counteract this evolutionary pressure. In particular, we predicted and tested the idea that in flying birds, natural selection should have selected for mechanisms to reduce fault bar load on feathers with high strength requirements during flight. Data on the growth of feathers of nestling white storks (Ciconia ciconia) revealed a consistent allocation of more, and more intense, fault bars in innermost than in outermost wing feathers as predicted by our hypothesis. Moreover, the same pattern emerged from feathers of adult storks. We discuss the generality of our results, and suggest avenues for further investigations in this area.

Are hippoboscid flies a major mode of transmission of feather mites?

Feather mites (Astigmata) are distributed around the world, living on the feathers of birds, but their mechanisms for transmission among hosts are not fully understood. There is anecdotal evidence of feather mites attached to louseflies (Diptera: Hippoboscidae), suggesting that feather mites may use these flies as a mode of phoretic transmission among birds. Two bird-lousefly associations (alpine swift Apus melba-Crataerina melbae and feral pigeon Columba livia-Pseudolynchia canariensis) were inspected to test the hypothesis that feather mites use hippoboscid flies as major mode of transmission. Both bird species showed a high prevalence and abundance of feather mites and louseflies. However, no feather mites were found attached to the 405 louseflies inspected, although skin mites (Epidermoptidae and Cheyletiellidae) were found on louseflies collected from feral pigeons. This study suggests that feather mites do not use hippoboscid flies as a major mode of transmission among birds.

Sources of variability in aggregation and sex ratios of Crataerina melbae (Diptera: Hippoboscidae) among adult colonial alpine swifts.

Aggregation of Crataerina melbae flies on breeding adult alpine swifts (Apus melba) was low when compared with other host-parasite systems and varied with sampling date, year, and sex of the flies. Generalized linear models were performed to ascertain which factors, extrinsic and/or intrinsic to the host, explained variability in the number of louse flies present on a single host, i.e., abundance. Overall abundance was unrelated to any host characteristic but varied slightly among years. Abundance of female flies varied among years, but also with date of sampling, the number of females increasing as the breeding season advanced. In contrast, abundance of males decreased as the season progressed, independently of host characteristics. Despite these different patterns, the number of flies of each sex on a given host was strongly intercorrelated. These results suggest that mate attraction may explain aggregation patterns in this louse fly species. Overall sex ratio of louse flies did not differ from unity. However, the proportion of males decreased during the breeding season, as a consequence of the opposite sex-related seasonal patterns in parasite abundance. Sex-ratio variability was not related to host characteristics or to infrapopulation sizes.