High throughput sequencing technologies are revolutionizing genetic research. With this “rise of the machines”, genomic sequences can be obtained even for unknown genomes within a short time and for reasonable costs. This has enabled evolutionary biologists studying genetically unexplored species to identify molecular markers or genomic regions of interest (e.g. micro- and minisatellites, mitochondrial and nuclear genes) by sequencing only a fraction of the genome. However, when using such datasets from non-model species, it is possible that DNA from non-target contaminant species such as bacteria, viruses, fungi, or other eukaryotic organisms may complicate the interpretation of the results. In this study we analysed 14 genomic pyrosequencing libraries of aquatic non-model taxa from four major evolutionary lineages. We quantified the amount of suitable micro- and minisatellites, mitochondrial genomes, known nuclear genes and transposable elements and searched for contamination from various sources using bioinformatic approaches. Our results show that in all sequence libraries with estimated coverage of about 0.02–25%, many appropriate micro- and minisatellites, mitochondrial gene sequences and nuclear genes from different KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways could be identified and characterized. These can serve as markers for phylogenetic and population genetic analyses. A central finding of our study is that several genomic libraries suffered from different biases owing to non-target DNA or mobile elements. In particular, viruses, bacteria or eukaryote endosymbionts contributed significantly (up to 10%) to some of the libraries analysed. If not identified as such, genetic markers developed from high-throughput sequencing data for non-model organisms may bias evolutionary studies or fail completely in experimental tests. In conclusion, our study demonstrates the enormous potential of low-coverage genome survey sequences and suggests bioinformatic analysis workflows. The results also advise a more sophisticated filtering for problematic sequences and non-target genome sequences prior to developing markers.

BackgroundPlants are traditionally used for medicinal treatment of numerous human disorders including infectious diseases caused by microorganisms. Due to the increasing resistance of many pathogens to commonly used antimicrobial agents, there is an urgent need for novel antimicrobial compounds. Plants of the genus Rhododendron belong to the woody representatives of the family Ericaceae, which are typically used in a range of ethno-medical applications. There are more than one thousand Rhododendron species worldwide. The Rhododendron-Park Bremen grows plants representing approximately 600 of the known Rhododendron species, and thus enables research involving almost two thirds of all known Rhododendron species.MethodsTwenty-six bacterial species representing different taxonomic clades have been used to study the antimicrobial potential of Rhododendron leaf extracts. Agar diffusion assay were conducted using 80% methanol crude extracts derived from 120 Rhododendron species. Data were analyzed using principal component analysis and the plant-borne antibacterial activities grouped according the first and second principal components.ResultsThe leaf extracts of 17 Rhododendron species exhibited significant growth-inhibiting activities against Gram-positive bacteria. In contrast, only very few of the leaf extracts affected the growth of Gram-negative bacteria. All leaf extracts with antimicrobial bioactivity were extracted from representatives of the subgenus Rhododendron, with 15 from the sub-section Rhododendron and two belonging to the section Pogonanthum. The use of bacterial multidrug efflux pump mutants revealed remarkable differences in the susceptibility towards Rhododendron leaf extract treatment.ConclusionsFor the first time, our comprehensive study demonstrated that compounds with antimicrobial activities accumulate in the leaves of certain Rhododendron species, which mainly belong to a particular subgenus. The results suggested that common genetic traits are responsible for the production of bioactive secondary metabolite(s) which act primarily on Gram-positive organisms, and which may affect Gram-negative bacteria in dependence of the activity of multidrug efflux pumps in their cell envelope.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0596-5) contains supplementary material, which is available to authorized users.

Premise: Classification of taxa depends on the quality of inferred phylogenies. Rhododendron, a highly species-rich genus (>1156 species) of woody plants, has a highly debated infrageneric classification, due to its huge diversity, homoplasy in key characters, and incongruence among data sets. We provide a broad coverage of representative species to resolve Rhododendron infrageneric phylogeny and highlight the areas of incongruence. We further investigate the effect of polyploidy and genome size evolution on diversification of Rhododendron. Methods: We generated two plastid and two nuclear loci for 260 Rhododendron species. We analyzed the loci separately as well as concatenated, utilizing both likelihood and Bayesian methods. We tested incongruence both among the data sets and with previous studies. We estimated genome sizes for 125 species through flow cytometry. Results: Our results suggest stronger support for larger subgenera; however, the smaller subgenera pose several problems; for example, R. tomentosum (former genus Ledum) occupies incongruent positions based on different DNA regions. The main shift to higher diversification in the genus occurs in the Himalayan/Southeast Asian clade of R. subg. Hymenanthes. We found that polyploidy occurs in almost all subgenera but most frequently within R. subg. Rhododendron sections Rhododendron and Schistanthe. Conclusions: We endorse the recognition of five major clades at the subgeneric level, but a number of species cannot be confidently assigned to these clades due to incongruency. With regard to genome size evolution, results support previous reports that genome sizes of tropical plants are lower than those of colder and temperate regions and that genome downsizing promotes diversification.

PREMISERhododendron L. (Ericaceae Juss.), is the most species-rich genus of woody flowering plants with > 1000 species. Despite the interest in the genus and numerous previous phylogenetic analysis, the infrageneric classification for the genus is still debated, partly due to its huge diversity, partly due to homoplasy in key characters and partly due to incongruence between phylogenetic markers. Here, we provide a broad coverage of representative species of all Rhododendron subgenera, sections, and most subsections to resolve its infrageneric phylogeny or highlight areas of incongruence, support previous analyses of diversification patterns and establish a relationship between genome size evolution and its diversification.METHODSWe generated sequences of two plastid (trnK and trnL-F) and two nuclear (ITS and rpb2-i) markers for a total of 259 Rhododendron species, and used likelihood and Bayesian statistics to analyze the data. We analyzed the markers separately to discuss and understand incongruence among the data sets and among previous studies.RESULTSWe found that the larger a subgenus, the more strongly it is supported as monophyletic. However, the smaller subgenera pose several problems, e.g., R. subgen. Azaleastrum consists of two sections inferred to be polyphyletic. The main shift to higher diversification in the genus occurred in the Himalayan/SE Asian clade of R. subgen. Hymenanthes. We found that polyploidy occurs in almost all subgenera but most polyploid species are within R. subgen. Rhododendron sections Rhododendron and Schistanthe.CONCLUSIONWhereas previous reports stated that genome sizes of tropical plants are lower than those of colder and temperate regions in angiosperms in general, our study provides evidence for such a shift to small genome-tropical species within a genus. Taken together, we see the merit in the recognition of the five major clades at the sub generic level but given the amount of incongruence a large amount of species cannot be confidently assigned to one of these five clades. Further, genome-wide data will be necessary to assess whether these currently unassignable taxa are independent taxa, assignable to one of the five major clades or whether they are inter-subgeneric hybrids.