Identifying microbiota community patterns important for plant protection using synthetic communities and machine learning.

Plant-associated microbiomes contribute to important ecosystem functions such as host resistance to biotic and abiotic stresses. The factors that determine such community outcomes are inherently difficult to identify under complex environmental conditions. In this study, we present an experimental and analytical approach to explore microbiota properties relevant for a microbiota-conferred host phenotype, here plant protection, in a reductionist system. We screened 136 randomly assembled synthetic communities (SynComs) of five bacterial strains each, followed by classification and regression analyses as well as empirical validation to test potential explanatory factors of community structure and composition, including evenness, total commensal colonization, phylogenetic diversity, and strain identity. We find strain identity to be the most important predictor of pathogen reduction, with machine learning algorithms improving performances compared to random classifications (94-100% versus 32% recall) and non-modelled predictions (0.79-1.06 versus 1.5 RMSE). Further experimental validation confirms three strains as the main drivers of pathogen reduction and two additional strains that confer protection in combination. Beyond the specific application presented in our study, we provide a framework that can be adapted to help determine features relevant for microbiota function in other biological systems.

Capacity of soil bacteria to reach the phyllosphere and convergence of floral communities despite soil microbiota variation.

Leaves and flowers are colonized by diverse bacteria that impact plant fitness and evolution. Although the structure of these microbial communities is becoming well-characterized, various aspects of their environmental origin and selection by plants remain uncertain, such as the relative proportion of soilborne bacteria in phyllosphere communities. Here, to address this issue and to provide experimental support for bacteria being filtered by flowers, we conducted common-garden experiments outside and under gnotobiotic conditions. We grew Arabidopsis thaliana in a soil substitute and added two microbial communities from natural soils. We estimated that at least 25% of the phyllosphere bacteria collected from the plants grown in the open environment were also detected in the controlled conditions, in which bacteria could reach leaves and flowers only from the soil. These taxa represented more than 40% of the communities based on amplicon sequencing. Unsupervised hierarchical clustering approaches supported the convergence of all floral microbiota, and 24 of the 28 bacteria responsible for this pattern belonged to the Burkholderiaceae family, which includes known plant pathogens and plant growth-promoting members. We anticipate that our study will foster future investigations regarding the routes used by soil microbes to reach leaves and flowers, the ubiquity of the environmental filtering of Burkholderiaceae across plant species and environments, and the potential functional effects of the accumulation of these bacteria in the reproductive organs of flowering plants.

Consistent host and organ occupancy of phyllosphere bacteria in a community of wild herbaceous plant species.

Bacteria colonizing the aerial parts of plants (phyllosphere) are linked to the biology of their host. They impact plant-pathogen interactions and may influence plant reproduction. Past studies have shown differences in composition and structure of the leaf, flower, and host microbiota, but an investigation of the impact of individual taxa on these variations remains to be tested. Such information will help to evaluate disparities and to better understand the biology and evolution of the plant-microbe associations. In the present study, we investigated the community structure, occupancy of host and organ, and the prevalence of phyllosphere bacteria from three host species collected at the same location. Almost all (98%) of bacterial taxa detected in the phyllosphere were not only shared across leaves and flowers, or different plant species but also had a conserved prevalence across sub-environments of the phyllosphere. We also found nonrandom associations of the phylogenetic diversity of phyllosphere bacteria. These results suggest that the phyllosphere microbiota is more conserved than previously acknowledged, and dominated by generalist bacteria adapted to environmental heterogeneity through evolutionary conserved traits.

The ancestral flower of angiosperms and its early diversification.

Recent advances in molecular phylogenetics and a series of important palaeobotanical discoveries have revolutionized our understanding of angiosperm diversification. Yet, the origin and early evolution of their most characteristic feature, the flower, remains poorly understood. In particular, the structure of the ancestral flower of all living angiosperms is still uncertain. Here we report model-based reconstructions for ancestral flowers at the deepest nodes in the phylogeny of angiosperms, using the largest data set of floral traits ever assembled. We reconstruct the ancestral angiosperm flower as bisexual and radially symmetric, with more than two whorls of three separate perianth organs each (undifferentiated tepals), more than two whorls of three separate stamens each, and more than five spirally arranged separate carpels. Although uncertainty remains for some of the characters, our reconstruction allows us to propose a new plausible scenario for the early diversification of flowers, leading to new testable hypotheses for future research on angiosperms.

Five major shifts of diversification through the long evolutionary history of Magnoliidae (angiosperms).

BACKGROUND: With 10,000 species, Magnoliidae are the largest clade of flowering plants outside monocots and eudicots. Despite an ancient and rich fossil history, the tempo and mode of diversification of Magnoliidae remain poorly known. Using a molecular data set of 12 markers and 220 species (representing >75% of genera in Magnoliidae) and six robust, internal fossil age constraints, we estimate divergence times and significant shifts of diversification across the clade. In addition, we test the sensitivity of magnoliid divergence times to the choice of relaxed clock model and various maximum age constraints for the angiosperms. RESULTS: Compared with previous work, our study tends to push back in time the age of the crown node of Magnoliidae (178.78-126.82 million years, Myr), and of the four orders, Canellales (143.18-125.90 Myr), Piperales (158.11-88.15 Myr), Laurales (165.62-112.05 Myr), and Magnoliales (164.09-114.75 Myr). Although families vary in crown ages, Magnoliidae appear to have diversified into most extant families by the end of the Cretaceous. The strongly imbalanced distribution of extant diversity within Magnoliidae appears to be best explained by models of diversification with 6 to 13 shifts in net diversification rates. Significant increases are inferred within Piperaceae and Annonaceae, while the low species richness of Calycanthaceae, Degeneriaceae, and Himantandraceae appears to be the result of decreases in both speciation and extinction rates. CONCLUSIONS: This study provides a new time scale for the evolutionary history of an important, but underexplored, part of the tree of angiosperms. The ages of the main clades of Magnoliidae (above the family level) are older than previously thought, and in several lineages, there were significant increases and decreases in net diversification rates. This study is a new robust framework for future investigations of trait evolution and of factors influencing diversification in this group as well as angiosperms as a whole.

Increased sampling of both genes and taxa improves resolution of phylogenetic relationships within Magnoliidae, a large and early-diverging clade of angiosperms.

Magnoliidae have been supported as a clade in the majority of large-scale molecular phylogenetic studies of angiosperms. This group consists of about 10,000 species assigned to 20 families and four orders, Canellales, Piperales, Laurales, and Magnoliales. Some relationships among the families are still largely debated. Here, we reconstruct the phylogenetic relationships of Magnoliidae as a whole, sampling 199 species (representing ca. 75% of genera) and 12 molecular markers from the three genomes (plastid atpB, matK, trnL intron, trnL-trnF spacer, ndhF, rbcL; mitochondrial atp1, matR, mtSSU, mtLSU; nuclear 18s rDNA, 26S rDNA). Maximum likelihood, Bayesian and maximum parsimony analyses yielded congruent trees, with good resolution and high support values for higher-level relationships. This study further confirms, with greater levels of support, two major clades in Magnoliidae: Canellales+Piperales and Laurales+Magnoliales. Relationships among the 20 families are, in general, well resolved and supported. Several previously ambiguous relationships are now well supported. For instance, the Aristolochiaceae s.l. (incl. Asaroideae, Aristolochioideae, and Lactoris) are monophyletic with high support when Hydnoraceae are excluded. The latter family was not included in most previous studies because of the lack of suitable plastid sequences, a consequence of the parasitic habit of its species. Here, we confirm that it belongs in Aristolochiaceae. Our analyses also provide moderate support for a sister group relationship between Lauraceae and Monimiaceae. Conversely, the exact position of Magnoliaceae remains very difficult to determine. This study provides a robust phylogenetic background to address the evolutionary history of an important and highly diverse clade of early-diverging angiosperms.

Development of Graphidium strigosum (Nematoda, Haemonchidae) in its natural host, the rabbit (Oryctolagus cuniculus) and comparison with several Haemonchidae parasites of ruminants.

The morphogenesis (studied for the first time) and the chronology of the life cycle of Graphidium strigosum (Dujardin, 1845) were studied in detail in its natural host, Oryctolagus cuniculus. Naive rabbits were each infected per os with G. strigosum infective larvae (L3). Animals were euthanized each day for the first 10 days after infection (DAI), then every 2 days from 12 to 40 DAI. The free living period lasted 5-8 days at 24°C. By 1 DAI, all the larvae were exsheathed in the stomach. The third molt occurred between 9 and 17 DAI. The last molt occurred between 24 and 32 DAI. The prepatent period lasted 42-44 DAI, while the patent period lasted at least 13 months. For each experiment, the morphology of the different stages of the life cycle was described. The chronology of the G. strigosum life cycle and its morphogenesis were compared to those of different Haemonchidae parasites of ruminants (Ostertagia ostertagi, Teladorsagia circumcincta, Haemonchus contortus, and Haemonchus placei) in their natural hosts.