Pieter Asselman

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 β-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.

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.

Phylogenetics, ancestral state reconstruction, and a new infrageneric classification of Scleria (Cyperaceae) based on three DNA markers

The genus Scleria (Cyperaceae), with ca. 250 species, is placed in the monotypic tribe Sclerieae. It has a primarily pantropical distribution. Previously, infrageneric classifications have been proposed but none has been based on molecular phylogenetic evidence and most studies have treated the genus from only one geographical region. In this study, DNA portions from 101 species of Scleria from across its distributional range and all infrageneric groups were extracted and amplified for three molecular markers: the coding chloroplast marker ndhF, the chloroplast intron rps16 and the nuclear ribosomal region ITS. Phylogenetic hypotheses were constructed using Bayesian inference and maximum likelihood approaches. Additionally, ancestral states of four morphological characters were reconstructed at important nodes using the program BayesTraits. A new infrageneric classification based on molecular evidence and supported by morphology is presented. Scleria is confirmed as monophyletic and sister to tribe Bisboeckelereae. A subdivision of Scleria into four subgenera (S. subg. Browniae, S. subg. Hypoporum, S. subg. Scleria, S. subg. Trachylomia) is supported by our results. In this paper, twelve major clades are recov- ered within Scleria subg. Scleria. Clear morphological diagnostic features match these clades. Ancestral state reconstruction indicates that androgynous spikelets, a zoniform hypogynium, a paniculate inflorescence with staminate and subandrogynous spikelets, and normal linear leaves are ancestral in Scleria. Androgynous spikelets are found in the three first-branching subgenera, while in S. subg. Scleria an evolutionary shift towards subandrogynous and strictly unisexual spikelets is noted. Hypogynia are generally well developed with the exception of S. subg. Hypoporum, where the hypogynium is reduced or even lost. Inflorescences in Scleria vary from short, densely paniculate and pyramidal to subcapitate, only in S. subg. Hypoporum a glomerate-spicate inflorescence is found. The pseudopremorse leaf habit arose only once within S. subg. Scleria. Scleria sect. Scleria shows a reversal to normal leaf tips.

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.

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.

Full sampling alignment Copiapoa phylogeny

Full sampling alignment including 109 sequences of 39 Copiapoa taxa and selected outgroup species. Often multiple accessions per Copiapoa taxon are included. Three cpDNA markers: ycf1 (coding), trnH-psbA & trnL-rpl32 (non-coding).