Using a new RAD-sequencing approach to study the evolution of Micromeria in the Canary islands.

As found in other oceanic islands, the Canary Islands include a large number of single island endemic species, some of which form clades that are broadly distributed within the archipelago. The genus Micromeria (Lamiaceae), for instance, includes groups of morphologically similar but ecologically diverse species on each island, representing a great model to investigate niche shifts and adaptation within the Canary Archipelago. Previous attempts to reconstruct phylogenetic relationships within the genus did not lead to robust phylogenies, presumably due to introgression and/or incomplete lineage sorting. In this study, we use a newly developed RAD-sequencing method to improve phylogenetic resolution and to better understand relationships among the Canary Island endemic Micromeria. Overall, we obtained 3571 loci that were genotyped for a total of 46 individuals of Micromeria. Our data reconstructed a highly resolved phylogeny, and corroborated the latest species reclassification of the M. varia s.l. species complex, the taxonomically most complicated group within the genus. Furthermore, taxa occupying similar ecological conditions in different islands, were shown to be closely related. This is the case of taxa from the laurel forest from La Gomera and Gran Canaria, suggesting that the laurel forest likely worked as a filter, only allowing the establishment of colonizers already pre-adapted to these conditions. We also found introgression between these species so it is also possible that the genes that facilitated the adaptation to laurel forest were swapped between Gran Canaria and La Gomera. The observations obtained in this study also allowed us to explain the role of introgression in the origin of M. varia s.l. species complex.

A new amplicon based approach of whole mitogenome sequencing for phylogenetic and phylogeographic analysis: An example of East African white-eyes (Aves, Zosteropidae).

Classical Sanger sequencing is still frequently used to generate sequence data for phylogenetic and phylogeographic inference. In this contribution we present a novel approach to genotype whole mitogenomic haplotypes using Illumina MiSeq reads from indexed amplicons. Our new approach reduces preparation time by multiplexing loci within a single or few PCR reactions and by plate format library construction. The use of paired-end reads allows covering amplicons of about 0.5kb and thus no nebulisation and assembly are necessary. We tested the power and effectiveness of this technique by analysing the mitogenomic diversity of East African white-eye bird species (Zosteropidae), a taxonomically highly diverse and complex species flock found in various ecosystems spread across major parts of Africa. We compare the newly generated mitogenomic data set with published data of three mitochondrial genes for a similar set of populations and taxa. The comparison demonstrates that our new procedure represents a cost effective use of NGS for medium throughput phylogenetic analyses. Using this method, we were able to increase the amount of phylogenetic information significantly, while reducing the costs and effort in the laboratory. The mitogenomic data show a higher resolution than previous studies providing higher support and new insights in the relationships of Zosterops species. Our data suggest to split Z. poliogaster into four distinct species, three of which had previously been proposed: Z. silvanus, Z. mbulensis, Z. kikyuensis and Z. kulalensis. Our approach allows the genotyping of whole mitogenomes for a large number of individuals and thus allows more reliable reconstruction of phylogenetic and phylogeographic relationships - also for non-model organisms.

The large GTPase AtGBPL3 links nuclear envelope formation and morphogenesis to transcriptional repression.

Guanylate binding proteins (GBPs) are prominent regulators of immunity not known to be required for nuclear envelope formation and morphogenesis. Here we identify the Arabidopsis GBP orthologue AtGBPL3 as a lamina component with essential functions in mitotic nuclear envelope reformation, nuclear morphogenesis and transcriptional repression during interphase. AtGBPL3 is preferentially expressed in mitotically active root tips, accumulates at the nuclear envelope and interacts with centromeric chromatin as well as with lamina components transcriptionally repressing pericentromeric chromatin. Reduced expression of AtGBPL3 or associated lamina components similarly altered nuclear morphology and caused overlapping transcriptional deregulation. Investigating the dynamics of AtGBPL3-GFP and other nuclear markers during mitosis (1) revealed that AtGBPL3 accumulation on the surface of daughter nuclei precedes nuclear envelope reformation and (2) uncovered defects in this process in roots of AtGBPL3 mutants, which cause programmed cell death and impair growth. AtGBPL3 functions established by these observations are unique among dynamin-family large GTPases.

A next-generation dual-recombinase system for time- and host-specific targeting of pancreatic cancer.

Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre-loxP-based models. We have developed an inducible dual-recombinase system by combining flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carcinogenesis, genetic manipulation of tumor subpopulations (such as cancer stem cells), selective targeting of the tumor microenvironment and genetic validation of therapeutic targets in autochthonous tumors on a genome-wide scale. As a proof of concept, we performed tumor cell-autonomous and nonautonomous targeting, recapitulated hallmarks of human multistep carcinogenesis, validated genetic therapy by 3-phosphoinositide-dependent protein kinase inactivation as well as cancer cell depletion and show that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are dispensable for tumor formation.