Shifts in Bacterial Communities of Eggshells and Antimicrobial Activities in Eggs during Incubation in a Ground-Nesting Passerine

Microbial invasion of egg contents is a cause of embryonic death. To counter infection risks, the embryo is protected physically by the eggshell and chemically by antimicrobial proteins. If microbial pressure drives embryo mortality, then females may have evolved, through natural selection, to adapt their immune investment into eggs. Although frequently hypothesized, this match between immune allocation and microorganisms has not been explored yet. To examine if correlations between microbes on eggs and immunity in eggs exist, we collected eggs from red-capped larks (Calandrella cinerea) and simultaneously examined their bacterial communities and antimicrobial components—pH, lysozyme and ovotransferrin—during natural incubation. Using molecular techniques, we find that bacterial communities are highly dynamic: bacterial abundance increases from the onset to late incubation, Shannon’s α-diversity index increases during early incubation stages, and β-diversity analysis shows that communities from 1 day-old clutches are phylogenetically more similar to each other than the older ones. Regarding the antimicrobials, we notice a decrease of pH and lysozyme concentration, while ovotransferrin concentration increases during incubation. Interestingly, we show that two eggs of the same clutch share equivalent immune protection, independent of clutch age. Lastly, our results provide limited evidence of significant correlation between antimicrobial compounds and bacterial communities. Our study examined simultaneously, for the first time in a wild bird, the dynamics of bacterial communities present on eggshells and of albumen-associated antimicrobial components during incubation and investigated their relationship. However, the link between microorganisms and immunity of eggs remains to be elucidated further. Identifying invading microbes and their roles in embryo mortality, as well as understanding the role of the eggshell microbiome, might be key to better understand avian strategies of immune maternal investment.     

Avian Incubation Inhibits Growth and Diversification of Bacterial Assemblages on Eggs

Microbial infection is a critical source of mortality for early life stages of oviparous vertebrates, but parental defenses against infection are less well known. Avian incubation has been hypothesized to reduce the risk of trans-shell infection by limiting microbial growth of pathogenic bacteria on eggshells, while enhancing growth of commensal or beneficial bacteria that inhibit or competitively exclude pathogens. We tested this hypothesis by comparing bacterial assemblages on naturally incubated and experimentally unincubated eggs at laying and late incubation using a universal 16S rRNA microarray containing probes for over 8000 bacterial taxa. Before treatment, bacterial assemblages on individual eggs from both treatment groups were dissimilar to one another, as measured by clustering in non-metric dimensional scaling (NMDS) ordination space. After treatment, assemblages of unincubated eggs were similar to one another, but those of incubated eggs were not. Furthermore, assemblages of unincubated eggs were characterized by high abundance of six indicator species while incubated eggs had no indicator species. Bacterial taxon richness remained static on incubated eggs, but increased significantly on unincubated eggs, especially in several families of Gram-negative bacteria. The relative abundance of individual bacterial taxa did not change on incubated eggs, but that of 82 bacterial taxa, including some known to infect the interior of eggs, increased on unincubated eggs. Thus, incubation inhibits all of the relatively few bacteria that grow on eggshells, and does not appear to promote growth of any bacteria.     

Effect of Incubation on Bacterial Communities of Eggshells in a Temperate Bird,the Eurasian Magpie (Pica pica)

Inhibitory effect of incubation on microbial growth has extensively been studied in wild bird populations using culture-based methods and conflicting results exist on whether incubation selectively affects the growth of microbes on the egg surface. In this study, we employed culture-independent methods, quantitative PCR and 16S rRNA gene pyrosequencing, to elucidate the effect of incubation on the bacterial abundance and bacterial community composition on the eggshells of the Eurasian Magpie (Pica pica). We found that total bacterial abundance increased and diversity decreased on incubated eggs while there were no changes on non-incubated eggs. Interestingly, Gram-positive Bacillus, which include mostly harmless species, became dominant and genus Pseudomonas, which include opportunistic avian egg pathogens, were significantly reduced after incubation. These results suggest that avian incubation in temperate regions may promote the growth of harmless (or benevolent) bacteria and suppress the growth of pathogenic bacterial taxa and consequently reduce the diversity of microbes on the egg surface. We hypothesize that this may occur due to difference in sensitivity to dehydration on the egg surface among microbes, combined with the introduction of Bacillus from bird feathers and due to the presence of antibiotics that certain bacteria produce.     

Effects of host genetics and environment on egg‐associated microbiotas in brown trout (Salmo trutta)

Recent studies found fish egg‐specific bacterial communities that changed over the course of embryogenesis, suggesting an interaction between the developing host and its microbiota. Indeed, single‐strain infections demonstrated that the virulence of opportunistic bacteria is influenced by environmental factors and host immune genes. However, the interplay between a fish embryo host and its microbiota has not been studied yet at the community level. To test whether host genetics affects the assemblage of egg‐associated bacteria, adult brown trout (Salmo trutta) were sampled from a natural population. Their gametes were used for full‐factorial in vitro fertilizations to separate sire from dam effects. In total, 2520 embryos were singly raised under experimental conditions that differently support microbial growth. High‐throughput 16S rRNA amplicon sequencing was applied to characterize bacterial communities on milt and fertilized eggs across treatments. Dam and sire identity influenced embryo mortality, time until hatching and composition of egg‐associated microbiotas, but no link between bacterial communities on milt and on fertilized eggs could be found. Elevated resources increased embryo mortality and modified bacterial communities with a shift in their putative functional potential. Resource availability did not significantly affect any parental effects on embryo performance. Sire identity affected bacterial diversity that turned out to be a significant predictor of hatching time: embryos associated with high bacterial diversity hatched later. We conclude that both host genetics and the availability of resources define diversity and composition of egg‐associated bacterial communities that then affect the life history of their hosts.     

沙坡头地区凋落物分解及其对土壤微生物群落的影响

以腾格里沙漠东南缘沙坡头人工固沙植被区典型植物种凋落物(小画眉草、藓类、油蒿叶片)为对象,运用凋落物分解袋法和高通量测序技术,分析了3种植物凋落物分解特征及其对土壤微生物群落的影响。结果表明: 分解时间和凋落物类型均显著影响分解速率,藓类分解最慢,13个月后质量损失比仅为15.4%,油蒿叶片和小画眉草的平均分解速率分别是藓类的4.9和3.4倍。经过11个月的分解,细菌群落的优势菌门为放线菌门和变形菌门,真菌群落的优势菌门是子囊菌门;藓类分解过程中,拟杆菌门和绿弯菌门的相对丰度显著增加,担子菌门的相对丰度显著降低。凋落物分解后,细菌和真菌群落物种多样性和丰富度显著增加,细菌群落组成在凋落物间变化不显著,真菌群落变化显著。凋落物的分解速率与细菌和真菌群落多样性及丰富度均呈负线性变化。植物多糖、全磷和土壤pH、微生物生物量氮、铵态氮含量是影响微生物群落结构的主要因子。凋落物分解改变了土壤微生物群落物种组成和种间相似性,显著增加了土壤中微生物群落的多样性和丰富度,促进了土壤生境的恢复。     

Effect of long‐term mineral fertilisation on soil microbial abundance,community structure and diversity in a Typic Hapludoll under intensive farming systems

Fertiliser application can not only influence plant communities, but also the soil microbial community dynamics, and consequently soil quality. Specifically, mineral fertilisation can directly or indirectly affect soil chemical properties, microbial abundance and, the structure and diversity of soil microbial communities. We investigated the impact of six different mineral fertiliser regimes in a maize/soybean rotation system: control (CK, without fertilisation), PS (application of phosphorus plus sulphur), NS (application of nitrogen plus S), NP (application of N plus P), NPS (application of N, P plus S) and NPSm (application of N, P, S plus micronutrients). Soil samples were collected at the physiological maturity stage of maize and soybean in March of 2013 and 2014, respectively. Overall, mineral fertilisation resulted in significantly decreased soil pH and increased total organic carbon compared with the control (CK). The analysis of terminal restriction fragment length polymorphism (T‐RFLP) revealed that mineral fertilisers caused a shift in the composition of both bacterial and fungal communities. In 2013, the highest value of Shannon diversity of bacterial terminal restriction fragments (TRFs) was found in control soils. In 2014, NPSm treated soils showed the lowest values of diversity for both bacterial and fungal TRFs. In both crop growing seasons, the analysis of phospholipid fatty acid (PLFA) detected the lowest value of total microbial biomass under CK. As PLFA analysis can be used to evaluate total microbial community, this result suggests that fertilisation increased total microbial biomass. When the bacterial and fungal abundance were examined using real time polymerase chain reaction, the results revealed that mineral fertilisation led to decreased bacterial abundance (16S rRNA), while fungal abundance (18S rRNA) was found to be increased in both crop growing seasons. Our results show that mineral fertiliser application has a significant impact on soil properties, bacterial and fungal abundance and microbial diversity. However, further studies are needed to better understand the mechanisms involved in the changes to microbial communities as a consequence of mineral fertilisation.     

Emergence Shapes the Structure of the Seed Microbiota

Seeds carry complex microbial communities, which may exert beneficial or deleterious effects on plant growth and plant health. To date, the composition of microbial communities associated with seeds has been explored mainly through culture-based diversity studies and therefore remains largely unknown. In this work, we analyzed the structures of the seed microbiotas of different plants from the family Brassicaceae and their dynamics during germination and emergence through sequencing of three molecular markers: the ITS1 region of the fungal internal transcribed spacer, the V4 region of 16S rRNA gene, and a species-specific bacterial marker based on a fragment of gyrB. Sequence analyses revealed important variations in microbial community composition between seed samples. Moreover, we found that emergence strongly influences the structure of the microbiota, with a marked reduction of bacterial and fungal diversity. This shift in the microbial community composition is mostly due to an increase in the relative abundance of some bacterial and fungal taxa possessing fast-growing abilities. Altogether, our results provide an estimation of the role of the seed as a source of inoculum for the seedling, which is crucial for practical applications in developing new strategies of inoculation for disease prevention.     

Prevalence of potentially pathogenic culturable bacteria on eggshells and in cloacae of female Pied Flycatchers in a temperate habitat in central Spain

ABSTRACT Bacteria grow on avian eggshells and thus can potentially cause diseases in developing embryos. Little is known about culturable bacteria colonizing avian eggshells in free‐living birds, with most studies restricted to poultry. Our objective was to examine the culturable bacterial array growing on eggshells during incubation that could negatively affect hatching success of Pied Flycatchers (Ficedula hypoleuca) in a temperate montane habitat in central Spain. Cloacal culturable bacteria of females were also analyzed because bacteria can be vertically transmitted from females to eggs. We used fecal samples as surrogates of cloacal samples due to the small size of sampled birds. We found that eggshells and female cloacae of Pied Flycatchers harbored 24 and 40 bacterial families and species, respectively, but only a few in each clutch and each cloaca. Rod‐shaped gram‐negative bacteria and bacteria in the family Pseudomonadaceae were the most common bacteria on eggshells during early and late incubation and in female cloacae. Although based on small sample sizes, we found that females with rod‐shaped gram‐negative bacteria in their cloacae laid eggs that also had these bacteria, providing possible evidence for vertical transmission. We found no evidence for vertical transmission of Pseudomonadaceae, suggesting a possible environmental source for these bacteria. The prevalence of bacterial morphological types and major taxonomical categories on eggshells did not vary from early to late stages of incubation, providing support for the hypothesis that incubation may have bacteriostatic effects on bacterial proliferation on eggshells. Despite being primary egg invaders in poultry, we detected no effects of culturable Pseudomonadaceae or Pseudomonas luteola on hatching success. Our study represents the first to examine the culturable bacteria growing on the eggshells of a wild bird in a temperate habitat and additional studies based on culture‐independent techniques are required to confirm our results.     

Diversity and dynamics of microbial communities in brown planthopper at different developmental stages revealed by high‐throughput amplicon sequencing

The microbiome associated with brown planthopper (BPH) plays an important role in mediating host health and fitness. Characterization of the microbial community and its structure is prerequisite for understanding the intricate symbiotic relationships between microbes and host insect. Here, we investigated the bacterial and fungal communities of BPH at different developmental stages using high‐throughput amplicon sequencing. Our results revealed that both the bacterial and fungal communities were diverse and dynamic during BPH development. The bacterial communities were generally richer than fungi in each developmental stage. At 97% similarly, 19 phyla and 278 genera of bacteria were annotated, while five fungal phyla comprising 80 genera were assigned. The highest species richness for the bacterial communities was detected in the nymphal stage. The taxonomic diversity of the fungal communities in female adults was generally at a relatively higher level when compared to other developmental stages. The most dominant phylum of bacteria and fungi at each developmental stage all belonged to Proteobacteria and Ascomycota, respectively. A significantly lower abundance of bacterial genus Acinetobacter was recorded in the egg stage when compared to other developmental stages, while the dominant fungal genus Wallemia was more abundant in the nymph and adult stages than in the egg stage. Additionally, the microbial composition differed between male and female adults, suggesting that the microbial communities in BPH were gender‐dependent. Overall, our study enriches our knowledge on the microbial communities associated with BPH and will provide clues to develop potential biocontrol techniques against this rice pest.     

不同树盘覆盖对矮砧苹果园土壤微生物群落结构和多样性的影响

以7年生矮化中间砧苹果为试材,研究了不同材质树盘覆盖对土壤微生物丰度、群落结构和多样性的影响。高通量测序结果表明,防水园艺地布和塑膜覆盖处理提高了0-20 cm土层变形菌门的相对丰度和20-40 cm土层变形菌门的相对丰度,防水园艺地布处理还显著提高了土壤细菌Ace和Simpson指数。相较于防水、塑膜覆盖处理,透水园艺地布不仅提高了0-20 cm土层变形菌门、接合菌门的相对丰度,而且提高了土壤细菌Ace、Chao1和Simpson指数,降低了土壤真菌Ace、Chao1和Simpson指数。树状聚类图结果显示,树盘覆盖显著改变了土壤细菌和真菌群落结构,以透水园艺地布覆盖处理的改变最为显著。综合分析认为,不同覆盖措施均有利于提高土壤细菌群落的丰富度和多样性,降低土壤真菌群落的丰富度和多样性,优化土壤微生物群落结构,其中以透水园艺地布覆盖的效果最好。     

Experimental evidence of microbial inheritance in plants and transmission routes from seed to phyllosphere and root

While the environment is considered the primary origin of the plant microbiome, the potential role of seeds as a source of transmitting microorganisms has not received much attention. Here we tested the hypothesis that the plant microbiome is partially inherited through vertical transmission. An experimental culturing device was constructed to grow oak seedlings in a microbe-free environment while keeping belowground and aboveground tissues separated. The microbial communities associated with the acorn's embryo and pericarp and the developing seeding's phyllosphere and root systems were analysed using amplicon sequencing of fungal ITS and bacterial 16S rDNA. Results showed that the seed microbiome is diverse and non-randomly distributed within an acorn. The microbial composition of the phyllosphere was diverse and strongly resembled the composition found in the embryo, whereas the roots and pericarp each had a less diverse and distinct microbial community. Our findings demonstrate a high level of microbial diversity and spatial partitioning of the fungal and bacterial community within both seed and seedling, indicating inheritance, niche differentiation and divergent transmission routes for the establishment of root and phyllosphere communities.     

Historical forest disturbance mediates soil microbial community responses to drought

Despite the abundance of studies demonstrating the effects of drought on soil microbial communities, the role of land use legacies in mediating these drought effects is unclear. To assess historical land use influences on microbial drought responses, we conducted a drought-rewetting experiment in soils from two adjacent and currently forested watersheds with distinct land use histories: an undisturbed ‘reference’ site and a ‘disturbed’ site that was clear-cut and converted to agriculture ~60 years prior. We incubated intact soil cores at either constant moisture or under a drought-rewet treatment and characterized bacterial and fungal communities using amplicon sequencing throughout the experiment. Bacterial alpha diversity decreased following drought-rewetting while fungal diversity increased. Bacterial beta diversity also changed markedly following drought-rewetting, especially in historically disturbed soils, while fungal beta diversity exhibited little response. Additionally, bacterial beta diversity in disturbed soils recovered less from drought-rewetting compared with reference soils. Disturbed soil communities also exhibited notable reductions in nitrifying taxa, increases in putative r-selected bacteria, and reductions in network connectivity following drought-rewetting. Overall, our study reveals historical land use to be important in mediating responses of soil bacterial communities to drought, which will influence the ecosystem-scale trajectories of these environments under ongoing and future climate change.     

Incubation reduces microbial growth on eggshells and the opportunity for trans-shell infection

Avian eggshells harbour microbes shortly after laying, and under appropriate ambient conditions they can multiply rapidly, penetrate through shell pores, infect egg contents and cause embryo mortality. We experimentally examined how incubation affects bacterial processes on the eggshells of pearl-eyed thrashers Margarops fuscatus nesting in tropical montane and lowland forests in Puerto Rico. Bacteria and fungi grew rapidly on shells of newly laid, unincubated eggs exposed to ambient conditions, but declined to low levels on shells of eggs incubated by thrashers. Divergence in bacterial growth between incubated and exposed eggs was more marked at the montane forest than at the lowland site. Pathogenic microorganisms became increasingly dominant on shells of exposed eggs, but these groups were relatively rare on incubated eggs, where more benign, less invasive groups prevailed. Some incubation during laying may be necessary to decrease the probability of trans-shell infection by reducing the growth of harmful bacteria and fungi on eggshells, although it may increase hatching asynchrony and the likelihood of brood reduction.     

Drying eggs to inhibit bacteria: Incubation during laying in a cavity nesting passerine

Early incubation has been suggested as a defensive adaptation against potentially pathogenic bacteria colonizing avian eggshells in the wild. The inhibitory mechanisms underlying this adaptation are poorly understood and only recent experimental evidence demonstrates that keeping eggs dry is a proximate mechanism for the antimicrobial effects of avian incubation. We estimated partial incubation (the bouts of incubation that some birds perform during the egg-laying period, days of lay 3-5 in our population) intensity of female pied flycatchers breeding in nest-boxes using data loggers that allowed a precise measurement of temperature just between the eggs in the nest-cup. We also measured relative humidity within the nest-boxes and related it to incubation intensity, showing that more intense incubation during laying contributes to drying the air near the eggs. We analyzed separately the effects of incubation and of relative humidity on loads of three types of culturable bacteria known to be present on eggshells, heterotrophic bacteria, Gram-negative enterics and pseudomonads. Our results show an association of early incubation with an inhibition of bacterial proliferation through a drying effect on eggshells, as we found that incubation intensity was negatively and relative humidity positively associated with eggshell bacterial loads for heterotrophic bacteria, Gram-negative bacteria and pseudomonads, although the significance of these associations varied between bacterial groups. These results point to microclimatically driven effects of incubation on bacterial proliferation on eggshells during laying in a temperate cavity nesting passerine.     

Warming and drying suppress microbial activity and carbon cycling in boreal forest soils

Climate warming is expected to have particularly strong effects on tundra and boreal ecosystems, yet relatively few studies have examined soil responses to temperature change in these systems. We used closed‐top greenhouses to examine the response of soil respiration, nutrient availability, microbial abundance, and active fungal communities to soil warming in an Alaskan boreal forest dominated by mature black spruce. This treatment raised soil temperature by 0.5 °C and also resulted in a 22% decline in soil water content. We hypothesized that microbial abundance and activity would increase with the greenhouse treatment. Instead, we found that bacterial and fungal abundance declined by over 50%, and there was a trend toward lower activity of the chitin‐degrading enzyme N‐acetyl‐glucosaminidase. Soil respiration also declined by up to 50%, but only late in the growing season. These changes were accompanied by significant shifts in the community structure of active fungi, with decreased relative abundance of a dominant Thelephoroid fungus and increased relative abundance of Ascomycetes and Zygomycetes in response to warming. In line with our hypothesis, we found that warming marginally increased soil ammonium and nitrate availability as well as the overall diversity of active fungi. Our results indicate that rising temperatures in northern‐latitude ecosystems may not always cause a positive feedback to the soil carbon cycle, particularly in boreal forests with drier soils. Models of carbon cycle‐climate feedbacks could increase their predictive power by incorporating heterogeneity in soil properties and microbial communities across the boreal zone.     

The evolution of size of the uropygial gland: mutualistic feather mites and uropygial secretion reduce bacterial loads of eggshells and hatching failures of European birds

Potentially, pathogenic bacteria are one of the main infective agents against which a battery of chemical and physical barriers has evolved in animals. Among these are the secretions by the exocrine uropygial gland in birds. The antimicrobial properties of uropygial secretions may prevent colonization and growth of microorganisms on feathers, skin and eggshells. However, uropygial gland secretions also favour the proliferation of feather mites that feed on secretions and microorganisms living on feathers that would otherwise reach eggshells during incubation if not consumed by feather mites. Therefore, at the interspecific level, uropygial gland size (as an index of volume of uropygial secretion) should be positively related to eggshell bacterial load (i.e. the risk of egg infection), whereas eggshell bacterial loads may be negatively related to abundance of feather mites eating bacteria. Here, we explore these previously untested predictions in a comparative framework using information on eggshell bacterial loads, uropygial gland size, diversity and abundance of feather mites and hatching success of 22 species of birds. The size of the uropygial gland was positively related to eggshell bacterial loads (mesophilic bacteria and Enterobacteriaceae), and bird species with higher diversity and abundance of feather mites harboured lower bacterial density on their eggshells (Enterococcus and Staphylococcus), in accordance with the hypothesis. Importantly, eggshell bacterial loads of mesophilic bacteria, Enterococcus and Enterobacteriaceae were negatively associated with hatching success, allowing us to interpret these interspecific relationships in a functional scenario, where both uropygial glands and mutualistic feather mites independently reduce the negative effects of pathogenic bacteria on avian fitness.     

Eggshells as hosts of bacterial communities: An experimental test of the antimicrobial egg coloration hypothesis

Oviparous animals have evolved multiple defenses to prevent microbes from penetrating their eggs and causing embryo mortality. In birds, egg constituents such as lysozyme and antibodies defend against microbial infestation, but eggshell pigments might also impact survival of bacteria. If so, microbes could exert an important selective pressure on the evolution of eggshell coloration. In a previous lab experiment, eggshell protoporphyrin caused drastic mortality in cultures of Gram positive, but not Gram negative, bacteria when exposed to light. Here, we test this “photodynamic antimicrobial hypothesis” in a field experiment. In a paired experimental design, we placed sanitized brown, protoporphyrin‐rich chicken eggs alongside white eggs that lack protoporphyrin. We deployed eggs for 48 hr without incubation, as can occur between laying and incubation, when microbial infection risk is highest. Eggs were placed on the open ground exposed to sunlight and in dark underground storm‐petrel burrows. We predicted that the proportion of Gram‐positive bacteria on brown eggs should be lower when exposed to sunlight than when kept in the dark, but we expected no such difference for white eggs. Although our data revealed variation in bacterial community composition, the proportion of Gram‐positive bacteria on eggshells did not vary by egg color, and there was no interaction between egg color and location. Instead, Gram‐positive bacteria were proportionally more common on eggs on the ground than eggs in burrows. Overall, our experiment did not support the photodynamic antimicrobial hypothesis. The diverse range of avian egg colors is generated by just two pigments, but over 10 hypotheses have been proposed for the evolution of eggshell color. If our results are generalizable, eggshell protoporphyrin might not play a substantial role in defending eggs against microbes, which narrows the field of candidate hypotheses for the evolution of avian eggshell coloration.     

Environmental and biotic drivers of soil microbial β‐diversity across spatial and phylogenetic scales

Soil microbial communities play a key role in ecosystem functioning but still little is known about the processes that determine their turnover (β‐diversity) along ecological gradients. Here, we characterize soil microbial β‐diversity at two spatial scales and at multiple phylogenetic grains to ask how archaeal, bacterial and fungal communities are shaped by abiotic processes and biotic interactions with plants. We characterized microbial and plant communities using DNA metabarcoding of soil samples distributed across and within eighteen plots along an elevation gradient in the French Alps. The recovered taxa were placed onto phylogenies to estimate microbial and plant β‐diversity at different phylogenetic grains (i.e. resolution). We then modeled microbial β‐diversities with respect to plant β‐diversities and environmental dissimilarities across plots (landscape scale) and with respect to plant β‐diversities and spatial distances within plots (plot scale). At the landscape scale, fungal and archaeal β‐diversities were mostly related to plant β‐diversity, while bacterial β‐diversities were mostly related to environmental dissimilarities. At the plot scale, we detected a modest covariation of bacterial and fungal β‐diversities with plant β‐diversity; as well as a distance–decay relationship that suggested the influence of ecological drift on microbial communities. In addition, the covariation between fungal and plant β‐diversity at the plot scale was highest at fine or intermediate phylogenetic grains hinting that biotic interactions between those clades depends on early‐evolved traits. Altogether, we show how multiple ecological processes determine soil microbial community assembly at different spatial scales and how the strength of these processes change among microbial clades. In addition, we emphasized the imprint of microbial and plant evolutionary history on today's microbial community structure.     

Diversity and stability of egg‐bacterial assemblages: The role of paternal care in the glassfrog Hyalinobatrachium colymbiphyllum

Embryos of oviparous organisms must cope with harsh environments and are especially susceptible to disease, considering that many immune mechanisms do not develop until later in life. Parents may transmit symbiotic microflora to eggs, which can contribute to embryo immune defense. Despite the importance of symbiotic microbes for immune function and survival of adult amphibians, vertical transfer of symbionts in amphibians has received less attention than in other taxa. Here, we test the role of male‐only parental care in establishing and maintaining the diversity of egg‐bacterial assemblages in a Neotropical glassfrog (Centrolenidae). Previous research suggests that father Hyalinobatrachium colymbiphyllum may transfer bacterial symbionts to their eggs. We combined a male‐removal experiment in situ with 16S rRNA gene amplicon sequencing to determine whether egg attendance by father H. colymbiphyllum influences the bacterial community and survival of eggs. We found that eggs harbor a diverse and stable bacterial assemblage. Despite different host environments, we found that adult skin and eggs supported very similar bacterial assemblages—even after removing fathers. While we found overlap in the bacteria present on eggs and their fathers, our experiment reveals that extended male care does not contribute to the maintenance of egg‐bacterial communities, so there may be other potential routes of transfer. This study contributes to our understanding of the diversity and maintenance of egg microbiomes, and motivates further research on how initial bacteria are acquired and the ontogenetic development of host–symbiont communities.     

Decline of soil microbial diversity does not influence the resistance and resilience of key soil microbial functional groups following a model disturbance

Analysing the consequences of the decrease in biodiversity for ecosystem functioning and stability has been a major concern in ecology. However, the impact of decline in soil microbial diversity on ecosystem sustainability remains largely unknown. This has been assessed for decomposition, which is insured by a large proportion of the soil microbial community, but not for more specialized and less diverse microbial groups. We determined the impact of a decrease in soil microbial diversity on the stability (i.e. resistance and resilience following disturbance) of two more specialized bacterial functional groups: denitrifiers and nitrite oxidizers. Soil microbial diversity was reduced using serial dilutions of a suspension obtained from a non-sterile soil that led to loss of species with low cell abundance, inoculation of microcosms of the same sterile soil with these serial dilutions, and subsequent incubation to enable establishment of similar cell abundances between treatments. The structure, cell abundance and activity of denitrifying and nitrite-oxidizing communities were characterized after incubation. Increasing dilution led to a progressive decrease in community diversity as assessed by the number of denaturating gradient gel electrophoresis (DGGE) bands, while community functioning was not impaired when cell abundance recovered after incubation. The microcosms were then subjected to a model disturbance: heating to 42 degrees C for 24 h. Abundance, structure and activity of each community were measured 3 h after completion of the disturbance to assess resistance, and after incubation of microcosms for 1 month to assess resilience. Resistance and resilience to the disturbance differed between the two communities, nitrite oxidizers being more affected. However, reducing the diversity of the two microbial functional groups did not impair either their resistance or their resilience following the disturbance. These results demonstrate the low sensitivity of the resistance and resilience of both microbial groups to diversity decline provided that cell abundance is similar between treatments.