Plastid phylogenomics of the Sansevieria Clade of Dracaena (Asparagaceae) resolves a recent radiation.

Best known as low maintenance houseplants, sansevierias are a diverse group of flowering plants native to Africa, Madagascar, the Arabian Peninsula, and the Indian subcontinent. Traditionally recognised as a distinct genus, Sansevieria was recently merged with the larger genus Dracaena based on molecular phylogenetic data. Within the Sansevieria Clade of Dracaena, taxonomic uncertainties remain despite attempts to unravel the relationships between the species. To investigate the evolutionary relationships, morphological evolution and biogeographical history in the group, we aim to reconstruct a robust dated phylogenetic hypothesis. Using genome skimming, a chloroplast genome (cpDNA) dataset and a nuclear ribosomal (nrDNA) dataset were generated. The sampling included representatives of all sections and informal groups previously described in Sansevieria based on morphology. Analysis of the cpDNA dataset using a maximum likelihood approach resulted in a well-supported phylogeny. The time-calibrated phylogeny indicated a recent radiation with five main clades emerging in the Pliocene. Two strongly supported clades align with previously defined groups, i.e., Sansevieria section Dracomima, characterised by the Dracomima-type inflorescence, and the Zeylanica informal group, native to the Indian subcontinent. Other previously defined groups were shown to be polyphyletic; a result of convergent evolution of the identifying characters. Switches between flat and cylindrical leaves occurred multiple times in the evolution of the Sansevieria Clade. Similarly, the Cephalantha-type inflorescence has originated multiple times from an ancestor with a Sansevieria-type inflorescence. Analysis of the nrDNA dataset resulted in a phylogenetic hypothesis with low resolution, yet it supported the same two groups confirmed by the cpDNA dataset. This study furthers our understanding of the evolution of the Sansevieria Clade, which will benefit taxonomic and applied research, and aid conservation efforts.

The evolutionary history of the Caribbean magnolias (Magnoliaceae): Testing species delimitations and biogeographical hypotheses using molecular data.

The Caribbean islands provide an ideal setting for studying biodiversity, given their complex geological and environmental history, and their historical and current geographical proximity to the American mainland. Magnolia, a flagship tree genus that has 15 endemic and threatened taxa (12 species and 3 subspecies) on the Caribbean islands, offers an excellent case study to empirically test Caribbean biogeographical hypotheses. We constructed phylogenetic hypotheses to: (1) reveal their evolutionary history, (2) test the current largely morphology-based classification and assess species limits, and (3) investigate major biogeographic hypotheses proposed for the region. Nuclear and chloroplast DNA sequence data of all 15 Caribbean Magnolia taxa are included, supplemented by a selection of American mainland species, and species representing most major clades of the Magnoliaceae family. We constructed phylogenetic hypotheses in a time-calibrated Bayesian framework, supplemented with haplotype network analyses and ancestral range estimations. Genetic synapomorphies in the studied markers confirm the species limits of 14 out of 15 morphologically recognizable Caribbean Magnolia taxa. There is evidence for four colonization events of Magnolia into the Caribbean from the American mainland, which most likely occurred by overwater dispersal, given age estimates of maximum 16 mya for their presence on the Caribbean islands.

Genetic patterns in Neotropical Magnolias (Magnoliaceae) using de novo developed microsatellite markers.

Conserving tree populations safeguards forests since they represent key elements of the ecosystem. The genetic characteristics underlying the evolutionary success of the tree growth form: high genetic diversity, extensive gene flow and strong species integrity, contribute to their survival in terms of adaptability. However, different biological and landscape contexts challenge these characteristics. This study employs 63 de novo developed microsatellite or SSR (Single Sequence Repeat) markers in different datasets of nine Neotropical Magnolia species. The genetic patterns of these protogynous, insect-pollinated tree species occurring in fragmented, highly-disturbed landscapes were investigated. Datasets containing a total of 340 individuals were tested for their genetic structure and degree of inbreeding. Analyses for genetic structure depicted structuring between species, i.e. strong species integrity. Within the species, all but one population pair were considered moderate to highly differentiated, i.e. no indication of extensive gene flow between populations. No overall correlation was observed between genetic and geographic distance of the pairwise species' populations. In contrast to the pronounced genetic structure, there was no evidence of inbreeding within the populations, suggesting mechanisms favouring cross pollination and/or selection for more genetically diverse, heterozygous offspring. In conclusion, the data illustrate that the Neotropical Magnolias in the context of a fragmented landscape still have ample gene flow within populations, yet little gene flow between populations.

Development of microsatellite markers using next-generation sequencing for the columnar cactus Echinopsis chiloensis (Cactaceae).

The aim of this study was to develop microsatellite markers as a tool to study population structure, genetic diversity and effective population size of Echinopsis chiloensis, an endemic cactus from arid and semiarid regions of Central Chile. We developed 12 polymorphic microsatellite markers for E. chiloensis using next-generation sequencing and tested them in 60 individuals from six sites, covering all the latitudinal range of this species. The number of alleles per locus ranged from 3 to 8, while the observed (Ho) and expected (He) heterozygosity ranged from 0.0 to 0.80 and from 0.10 to 0.76, respectively. We also detected significant differences between sites, with FST values ranging from 0.05 to 0.29. Microsatellite markers will enable us to estimate genetic diversity and population structure of E. chiloensis in future ecological and phylogeographic studies.

An integrative approach to understanding the evolution and diversity of Copiapoa (Cactaceae), a threatened endemic Chilean genus from the Atacama Desert.

PREMISE OF THE STUDY: Species of the endemic Chilean cactus genus Copiapoa have cylindrical or (sub)globose stems that are solitary or form (large) clusters and typically yellow flowers. Many species are threatened with extinction. Despite being icons of the Atacama Desert and well loved by cactus enthusiasts, the evolution and diversity of Copiapoa has not yet been studied using a molecular approach. METHODS: Sequence data of three plastid DNA markers (rpl32-trnL, trnH-psbA, ycf1) of 39 Copiapoa taxa were analyzed using maximum likelihood and Bayesian inference approaches. Species distributions were modeled based on geo-referenced localities and climatic data. Evolution of character states of four characters (root morphology, stem branching, stem shape, and stem diameter) as well as ancestral areas were reconstructed using a Bayesian and maximum likelihood framework, respectively. KEY RESULTS: Clades of species are revealed. Though 32 morphologically defined species can be recognized, genetic diversity between some species and infraspecific taxa is too low to delimit their boundaries using plastid DNA markers. Recovered relationships are often supported by morphological and biogeographical patterns. The origin of Copiapoa likely lies between southern Peru and the extreme north of Chile. The Copiapó Valley limited colonization between two biogeographical areas. CONCLUSIONS: Copiapoa is here defined to include 32 species and five heterotypic subspecies. Thirty species are classified into four sections and two subsections, while two species remain unplaced. A better understanding of evolution and diversity of Copiapoa will allow allocating conservation resources to the most threatened lineages and focusing conservation action on real biodiversity.

Metagenomics of African Empogona and Tricalysia (Rubiaceae) reveals the presence of leaf endophytes.

Background: Leaf symbiosis is a phenomenon in which host plants of Rubiaceae interact with bacterial endophytes within their leaves. To date, it has been found in around 650 species belonging to eight genera in four tribes; however, the true extent in Rubiaceae remains unknown. Our aim is to investigate the possible occurrence of leaf endophytes in the African plant genera Empogona and Tricalysia and, if present, to establish their identity. Methods: Total DNA was extracted from the leaves of four species of the Coffeeae tribe (Empogona congesta, Tricalysia hensii, T. lasiodelphys, and T. semidecidua) and sequenced. Bacterial reads were filtered out and assembled. Phylogenetic analysis of the endophytes was used to reveal their identity and their relationship with known symbionts. Results: All four species have non-nodulated leaf endophytes, which are identified as Caballeronia. The endophytes are distinct from each other but related to other nodulated and non-nodulated endophytes. An apparent phylogenetic or geographic pattern appears to be absent in endophytes or host plants. Caballeronia endophytes are present in the leaves of Empogona and Tricalysia, two genera not previously implicated in leaf symbiosis. This interaction is likely to be more widespread, and future discoveries are inevitable.

Comparison of Magnoliaceae Plastomes: Adding Neotropical Magnolia to the Discussion.

Chloroplast genomes are considered to be highly conserved. Nevertheless, differences in their sequences are an important source of phylogenetically informative data. Chloroplast genomes are increasingly applied in evolutionary studies of angiosperms, including Magnoliaceae. Recent studies have focused on resolving the previously debated classification of the family using a phylogenomic approach and chloroplast genome data. However, most Neotropical clades and recently described species have not yet been included in molecular studies. We performed sequencing, assembly, and annotation of 15 chloroplast genomes from Neotropical Magnoliaceae species. We compared the newly assembled chloroplast genomes with 22 chloroplast genomes from across the family, including representatives from each genus and section. Family-wide, the chloroplast genomes presented a length of about 160 kb. The gene content in all species was constant, with 145 genes. The intergenic regions showed a higher level of nucleotide diversity than the coding regions. Differences were higher among genera than within genera. The phylogenetic analysis in Magnolia showed two main clades and corroborated that the current infrageneric classification does not represent natural groups. Although chloroplast genomes are highly conserved in Magnoliaceae, the high level of diversity of the intergenic regions still resulted in an important source of phylogenetically informative data, even for closely related taxa.

Sorting out the plants responsible for a contamination with pyrrolizidine alkaloids in spice seeds by means of LC-MS/MS and DNA barcoding: Proof of principle with cumin and anise spice seeds.

High value commodities such as spices suffer from occasional contaminations of both chemical and biological origin. Consequently, quality control and safety monitoring has become a pressing issue for the spice industry. Two recent independent studies showed that at least one third of the analyzed cumin and green anise spice seeds samples surpassed the by the European Union recently established threshold value for toxic pyrrolizidine alkaloids (PAs) and their corresponding N-oxides (PANOs). These heterocyclic secondary plant metabolites are produced by a large number of different plant families. In those spice seeds, it was found by means of DNA metabarcoding, that predominant contamination was due to the presence of herbal material from the Heliotropium genus (Boraginaceae). Unfortunately, the use of this specific type of DNA-based identification remains controversial for the majority of the official instances and preference is still given to the use of more tangible classical approaches, including microscopy and chemical analysis. However, these methodologies often suffer from inherent drawbacks. Here we demonstrate that at least for spice seeds, a combinatory approach of microscopy, chemical analysis and classical DNA barcoding of the isolated contaminants using the matK and trnH-psbA loci, provides qualitative and quantitative information on the amount of plant material responsible for the contaminations and the extent of the contamination. The generated data also demonstrates that the presence of a very limited number of Heliotropium sp. seeds in a standard commercially available canister is sufficient to surpass the allowed threshold value, illustrating once more the importance of weed control.

Genome skimming reveals novel plastid markers for the molecular identification of illegally logged African timber species.

Tropical forests represent vast carbon stocks and continue to be key carbon sinks and buffer climate changes. The international policy constructed several mechanisms aiming at conservation and sustainable use of these forests. Illegal logging is an important threat of forests, especially in the tropics. Several laws and regulations have been set up to combat illegal timber trade. Despite significant enforcement efforts of these regulations, illegal logging continues to be a serious problem and impacts for the functioning of the forest ecosystem and global biodiversity in the tropics. Microscopic analysis of wood samples and the use of conventional plant DNA barcodes often do not allow to distinguish closely-related species. The use of novel molecular technologies could make an important contribution for the identification of tree species. In this study, we used high-throughput sequencing technologies and bioinformatics tools to obtain the complete de-novo chloroplast genome of 62 commercial African timber species using the genome skimming method. Then, we performed a comparative genomic analysis that revealed new candidate genetic regions for the discrimination of closely-related species. We concluded that genome skimming is a promising method for the development of plant genetic markers to combat illegal logging activities supporting CITES, FLEGT and the EU Timber Regulation.

Enlightening the black and white: species delimitation and UNITE species hypothesis testing in the Russula albonigra species complex.

Russula albonigra is considered a well-known species, morphologically delimited by the context of the basidiomata blackening without intermediate reddening, and the menthol-cooling taste of the lamellae. It is supposed to have a broad ecological range and a large distribution area. A thorough molecular analysis based on four nuclear markers (ITS, LSU, RPB2 and TEF1-α) shows this traditional concept of R. albonigra s. lat. represents a species complex consisting of at least five European, three North American, and one Chinese species. Morphological study shows traditional characters used to delimit R. albonigra are not always reliable. Therefore, a new delimitation of the R. albonigra complex is proposed and a key to the described European species of R. subgen. Compactae is presented. A lectotype and an epitype are designated for R. albonigra and three new European species are described: R. ambusta, R. nigrifacta, and R. ustulata. Different thresholds of UNITE species hypotheses were tested against the taxonomic data. The distance threshold of 0.5% gives a perfect match to the phylogenetically defined species within the R. albonigra complex. Publicly available sequence data can contribute to species delimitation and increase our knowledge on ecology and distribution, but the pitfalls are short and low quality sequences.

Chloroplast genomes of Rubiaceae: Comparative genomics and molecular phylogeny in subfamily Ixoroideae.

In Rubiaceae phylogenetics, the number of markers often proved a limitation with authors failing to provide well-supported trees at tribal and generic levels. A robust phylogeny is a prerequisite to study the evolutionary patterns of traits at different taxonomic levels. Advances in next-generation sequencing technologies have revolutionized biology by providing, at reduced cost, huge amounts of data for an increased number of species. Due to their highly conserved structure, generally recombination-free, and mostly uniparental inheritance, chloroplast DNA sequences have long been used as choice markers for plant phylogeny reconstruction. The main objectives of this study are: 1) to gain insight in chloroplast genome evolution in the Rubiaceae (Ixoroideae) through efficient methodology for de novo assembly of plastid genomes; and, 2) to test the efficiency of mining SNPs in the nuclear genome of Ixoroideae based on the use of a coffee reference genome to produce well-supported nuclear trees. We assembled whole chloroplast genome sequences for 27 species of the Rubiaceae subfamily Ixoroideae using next-generation sequences. Analysis of the plastid genome structure reveals a relatively good conservation of gene content and order. Generally, low variation was observed between taxa in the boundary regions with the exception of the inverted repeat at both the large and short single copy junctions for some taxa. An average of 79% of the SNP determined in the Coffea genus are transferable to Ixoroideae, with variation ranging from 35% to 96%. In general, the plastid and the nuclear genome phylogenies are congruent with each other. They are well-resolved with well-supported branches. Generally, the tribes form well-identified clades but the tribe Sherbournieae is shown to be polyphyletic. The results are discussed relative to the methodology used and the chloroplast genome features in Rubiaceae and compared to previous Rubiaceae phylogenies.

Molecular phylogenetic study of Scleria subgenus Hypoporum (Sclerieae, Cyperoideae, Cyperaceae) reveals several species new to science.

Scleria subgen. Hypoporum (Cyperaceae), with 68 species, is the second largest subgenus in Scleria. Species of this pantropically distributed subgenus generally occur in seasonally or permanently wet grasslands or on shallow soils over sandstone or lateritic outcrops, less often they can be found in (open) woodlands. Previous studies established the monophyly of the subgenus, but the relationships between the species remained uncertain. In this study, DNA sequence data of 61 taxa of Scleria subgen. Hypoporum, where possible represented by multiple accessions from across their distributional range, were obtained for four molecular markers: the coding chloroplast marker ndhF, the chloroplast intron rps16 and the nuclear ribosomal regions ETS and ITS. Phylogenetic trees were constructed using Bayesian inference and maximum likelihood approaches. A species tree was constructed to summarise the results. The results indicate the existence of three sections: the monotypic, pantropically occurring, Scleria sect. Lithospermae, a new section from central and south America containing two species, and Scleria sect. Hypoporum, also pantropically distributed, containing the remainder of the species of the subgenus. Relationships in the latter section are not fully resolved. However, three or four different clades can be distinguished supported by some morphological characters. Our results indicate at least six new species in Scleria sect. Hypoporum. The new section and species are described in a taxonomical treatment. Their morphology is compared with (morphologically) closely related species.

Development and application of novel constructs to score C:G-to-T:A transitions and homologous recombination in Arabidopsis.

We report on the development of five missense mutants and one recombination substrate of the beta-glucuronidase (GUS)-encoding gene of Escherichia coli and their use for detecting mutation and recombination events in transgenic Arabidopsis (Arabidopsis thaliana) plants by reactivation of GUS activity in clonal sectors. The missense mutants were designed to find C:G-to-T:A transitions in a symmetrical sequence context and are in that respect complementary to previously published GUS point mutants. Small peptide tags (hemagglutinin tag and Strep tag II) and green fluorescent protein were translationally fused to GUS, which offers possibilities to check for mutant GUS production levels. We show that spontaneous mutation and recombination events took place. Mutagenic treatment of the plants with ethyl methanesulfonate and ultraviolet-C increased the number of mutations, validating the use of these constructs to measure mutation and recombination frequencies in plants exposed to biotic or abiotic stress conditions, or in response to different genetic backgrounds. Plants were also subjected to heavy metals, methyl jasmonate, salicylic acid, and heat stress, for which no effect could be seen. Together with an ethyl methanesulfonate mutation induction level much higher than previously described, the need is illustrated for many available scoring systems in parallel. Because all GUS missense mutants were cloned in a bacterial expression vector, they can also be used to score mutation events in E. coli.