Frankia strains induce the formation of nitrogen-fixing nodules on roots of actinorhizal plants. Phylogenetically, Frankia strains can be grouped in four clusters. The earliest divergent cluster, cluster-2, has a particularly wide host range. The analysis of cluster-2 strains has been hampered by the fact that with two exceptions, they could never be cultured. In this study, 12 Frankia-enriched metagenomes of Frankia cluster-2 strains or strain assemblages were sequenced based on seven inoculum sources. Sequences obtained via DNA isolated from whole nodules were compared with those of DNA isolated from fractionated preparations enhanced in the Frankia symbiotic structures. The results show that cluster-2 inocula represent groups of strains, and that strains not represented in symbiotic structures, that is, unable to perform symbiotic nitrogen fixation, may still be able to colonize nodules. Transposase gene abundance was compared in the different Frankia-enriched metagenomes with the result that North American strains contain more transposase genes than Eurasian strains. An analysis of the evolution and distribution of the host plants indicated that bursts of transposition may have coincided with niche competition with other cluster-2 Frankia strains. The first genome of an inoculum from the Southern Hemisphere, obtained from nodules of Coriaria papuana in Papua New Guinea, represents a novel species, postulated as Candidatus Frankia meridionalis. All Frankia-enriched metagenomes obtained in this study contained homologs of the canonical nod genes nodABC; the North American genomes also contained the sulfotransferase gene nodH, while the genome from the Southern Hemisphere only contained nodC and a truncated copy of nodB.

BackgroundThe ability to establish root nodule symbioses is restricted to four different plant orders. Soil actinobacteria of the genus Frankia can establish a symbiotic relationship with a diverse group of plants within eight different families from three different orders, the Cucurbitales, Fagales and Rosales. Phylogenetically, Frankia strains can be divided into four clusters, three of which (I, II, III) contain symbiotic strains. Members of Cluster II nodulate the broadest range of host plants with species from four families from two different orders, growing on six continents. Two Cluster II genomes were sequenced thus far, both from Asia.ResultsIn this paper we present the first Frankia cluster II genome from North America (California), Dg2, which represents a metagenome of two major and one minor strains. A phylogenetic analysis of the core genomes of 16 Frankia strains shows that Cluster II the ancestral group in the genus, also ancestral to the non-symbiotic Cluster IV. Dg2 contains the canonical nod genes nodABC for the production of lipochitooligosaccharide Nod factors, but also two copies of the sulfotransferase gene nodH. In rhizobial systems, sulfation of Nod factors affects their host specificity and their stability.ConclusionsA comparison with the nod gene region of the previously sequenced Dg1 genome from a Cluster II strain from Pakistan shows that the common ancestor of both strains should have contained nodABC and nodH. Phylogenetically, Dg2 NodH proteins are sister to rhizobial NodH proteins. A glnA-based phylogenetic analysis of all Cluster II strains sampled thus far supports the hypothesis that Cluster II Frankia strains came to North America with Datisca glomerata following the Madrean-Tethyan pattern.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3140-1) contains supplementary material, which is available to authorized users.

Aim Unsustainable hunting is leading to widespread defaunation across the tropics. To mitigate against this threat with limited conservation resources, stakeholders must make decisions on where to focus anti‐poaching activities. Identifying priority areas in a robust way allows decision‐makers to target areas of conservation importance, therefore maximizing the impact of conservation interventions. Location Annamite mountains, Vietnam and Laos. Methods We conducted systematic landscape‐scale surveys across five study sites (four protected areas, one unprotected area) using camera‐trapping and leech‐derived environmental DNA. We analysed detections within a Bayesian multispecies occupancy framework to evaluate species responses to environmental and anthropogenic influences. Species responses were then used to predict occurrence to unsampled regions. We used predicted species richness maps and occurrence of endemic species to identify areas of conservation importance for targeted conservation interventions. Results Analyses showed that habitat‐based covariates were uninformative. Our final model therefore incorporated three anthropogenic covariates as well as elevation, which reflects both ecological and anthropogenic factors. Conservation‐priority species tended to found in areas that are more remote now or have been less accessible in the past, and at higher elevations. Predicted species richness was low and broadly similar across the sites, but slightly higher in the more remote site. Occupancy of the three endemic species showed a similar trend. Main conclusion Identifying spatial patterns of biodiversity in heavily defaunated landscapes may require novel methodological and analytical approaches. Our results indicate that to build robust prediction maps it is beneficial to sample over large spatial scales, use multiple detection methods to increase detections for rare species, include anthropogenic covariates that capture different aspects of hunting pressure and analyse data within a Bayesian multispecies framework. Our models further suggest that more remote areas should be prioritized for anti‐poaching efforts to prevent the loss of rare and endemic species.